tjh-2012-4

Review

DOI: 10.5505/tjh.2012.39205

The Molecular Genetics of von Willebrand Disease Von Willebrand Hastalığı’nın Moleküler Genetiği Ergül Berber İstanbul Arel University, Department of Molecular Biology and Genetics, İstanbul, Turkey

Abstract Quantitative and/or qualitative deficiency of von Willebrand factor (vWF) is associated with the most common inherited bleeding disease von Willebrand disease (vWD). vWD is a complex disease with clinical and genetic heterogeneity. Incomplete penetrance and variable expression due to genetic and environmental factors contribute to its complexity. vWD also has a complex molecular pathogenesis. Some vWF gene mutations are associated with the affected vWF biosynthesis and multimerization, whereas others are associated with increased clearance and functional impairment. Moreover, in addition to a particular mutation, type O blood may result in the more severe phenotype. The present review aimed to provide a summary of the current literature on the molecular genetics of vWD.

Key Words: Von Willebrand factor, Von Willebrand disease, Von Willebrand factor biosynthesis

Özet Von Willebrand Faktöründe (vWF) görülen kantitatif azalma veya kalitatif bozukluklar bir kanama diyatezi olan ve oldukça sık rastlanan von Willebrand Hastalığının (VWH) oluşumuna neden olur. VWH klinik ve genetik heterojenite gösteren karmaşık bir hastalıktır. Genetik ve çevresel nedenlere ikincil eksik penetrans ve VWF düzeyinde olan değişiklikler olması VWH’nın karmaşık bir yapı göstermesine neden olan faktörlerdir. VWF genindeki mutasyonlardan bazıları biyosentezi ve multimerizasyonu etkilerken, diğer mutasyonlar vWF’nin dolaşımdan daha erken uzaklaştırılmasına neden olarak fonksiyonunu etkiler. Belirli bir mutasyon ile birlikte kan grubunun da O olması fenotipin daha ağır olmasına neden olabilir. Bu derlemenin amacı vWH’nın moleküler genetiği ile ilgili güncel yayınları inceleyerek onların bir özetini yapmaktır.

Anahtar Sözcükler: Von Willebrand faktör, Von Willebrand hastalığı, Von Willebrand faktör biyosentezi Introduction Von Willebrand disease (vWD) was first described by Erik von Willebrand in a large Aland Islands (Swedish-speaking region of Finland) family in 1924 and was reported to be the most common inherited bleeding disorder in 1926 [1,2]. Its prevalence is estimated to be 0.6%-1.3%; however, 1 in 10,000 patients have significant bleeding [3-5]. The characteristic clinical symptoms

of vWD affect both males and females, and include mucosal bleeding (including epistaxis), menorrhagia, and prolonged bleeding following trauma or surgery. Severely affected patients may also bleed into soft tissues and joints [6]. vWD is associated with qualitative and quantitative deficiency of von Willebrand factor (vWF). Clinical diagnosis of vWD is based on von Willebrand antigen (vWF:Ag),

Address for Correspondence: Ergül BERBER, PhD, İstanbul Arel Üniversitesi, Moleküler Biyoloji ve Genetik Bölümü, İstanbul, Turkey Phone: +90 212 860 04 80 E-mail: ergulberber@arel.edu.tr Received/Geliş tarihi : October 14, 2011 Accepted/Kabul tarihi : April 24, 2012

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factor (F) VIII clotting activity (FVIII:C), and von Willebrand ristocetin cofactor activity (vWF:RCo). According to The International Society of Thrombosis and Haemostasis (ISTH), vWD is classified as type 1, type 2, and type 3 (Table 1). Type 1 and type 3 vWD are characterized by partial and complete deficiency of vWF, respectively. Functional deficiency of vWF is characteristic of type 2 vWD, which is further classified as 2A, 2B, and 2M based on defective interaction with platelets, and as 2N based on defective binding to the FVIII molecule. Although ISTH classification of 1994 indicated that vWD is a disease associated with vWF gene mutations, the 2006 revised ISTH classification states that, “additional genes might influence the biosynthesis and stability of plasma vWF” [7].

Table 1: vWD types.

Partial quantitative deficiency of vWF and a proportional decrease in FVIII. Defective vWF-platelet binding, and a Type 2A decrease in HMWM—both in plasma and platelets. Increased vWF-platelet Gp1b binding and a Type 2B decrease in HMWM in plasma. Defective vWF-platelet binding and normal, Type 2M but dysfunctional HMWM in plasma. Lack of or markedly reduced vWF affinity Type 2N for FVIII binding. Complete deficiency of vWF and severely Type 3 reduced FVIII. Type 1

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von Willebrand Factor vWF is a multimeric plasma glycoprotein essential for primary hemostasis that mediates platelet plug formation via adhesion at the site of injury. In addition, vWF protects FVIII in plasma from proteolytic degradation by noncovalently binding to it and transports FVIII to the site of coagulation [8]. vWF is produced in endothelial cells and megakaryocytes as a pre-pro-polypeptide (pre-pro-vWF) that is 2813 amino acids long. Pre-pro-vWF is composed of a signal sequence that is 22 amino acids long, a pro-peptide sequence 741 amino acids long, and a vWF monomer sequence 2050 amino acids long (Figure 1) [9-11]. During vWF biosynthesis the signal sequence is cleaved off by a signal peptidase after it is directed to the endoplasmic reticulum. Pro-vWF monomer undergoes dimerization at the CK domain via disulfide bonding between cysteine residues. In addition to dimerization, vWF is glycosylated in the endoplasmic reticulum. The dimers are then transported to the Golgi apparatus where they undergo amino terminal multimerization, sulfation, and further glycosylation, and change carbohydrates into high mannose oligosaccharides. Finally, propeptide is cleaved from the vWF multimers by furin (PACE [paired basic amino acid cleaving enzyme]) and remains non-covalently associated with the vWF multimers; 95% of vWF multimers are secreted constitutively and 5% of plasma vWF multimers are stored in the Weibel-Palade bodies (WPBs) of endothelial cells and are secreted in a regulated manner upon stimulation by thrombin, fibrin, and histidine [4,9,11-16].

Figure 1: Structure and functional domains of pre-pro-VWF.

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Research has shown that the pro-peptide sequence acts as a chaperone to direct vWF multimers to WPBs [17]. Glycosylation of vWF is an important post-translational modification that protects vWF from proteolytic destruction, affects plasma clearance, and maintains vWF’s multimeric structure and interaction with platelets and collagen [18-20]. vWF multimers range in size from a dimer (~500 kDa) to ultra large multimers (>10 x 106 kDa). vWF multimers released from WPBs are ultra large (UL-vWF) and are the most effective for maintaining hemostasis at the site of injury. Pro-peptide’s Cys-X-X-Cys sequence, similar to the active site of protein disulfide isomerases, is thought to be important for vWF multimer formation [20,21]. UL-vWF multimers are proteolytically cleaved by vWF cleaving protease (ADAMTS-13) to physiologically active plasma vWF multimer size within the A2 domain (Y1605-M1606) [22, 23]. The A2 domain serves as a shear sensor and its unfolding is necessary for proteolytic cleavage to expose the cleavage site [24]. In addition to the importance of the cleavage site in the A2 domain for ADAMTS-13 proteolysis, the residues C1669 and C1670 that form a disulfide bond are important for A2 domain folding [25]. Furthermore, studies have shown that polymorphisms in the A1 and A2 domains affect the efficiency of ADAMTS-13 cleavage [26]. The vWF gene spans a 178-kb genomic region with 52 exons; exon 28 is 1379 bp long and is the largest exon. Exon 28 encodes the domains involved in FVIII binding (D3), platelet binding (A1), collagen binding (A3), and ADAMTS-13 cleavage (A2). There is a highly homologous pseudo-gene containing the vWF gene region from exon 23 to 34 on chromosome 22 (22q11.2) [27,28]. The half-life of vWF in circulation is 12-20 h. The vWF plasma level ranges between 50 IU/dL and 200 IU/dL in the general population [29]. Twin studies reported that genetic factors are responsible for 60% of the variation in

Berber E, : The Molecular Genetics of von Willebrand Disease

the vWF plasma level, which is also affected by a variety of other factors, including age, blood type, stress, thyroid hormone, pregnancy, single-nucleotide polymorphisms (SNPs) in the vWF gene—including the promoter region and other genetic loci [30-33]. Recent studies reported that macrophages are involved in the removal of vWF/ FVIII complex in the liver and spleen, and that D’-D3 domains are implicated in the clearance, although the precise mechanism of vWF clearance remains unknown [3435]. Blood type is a major genetic determinant of the vWF plasma level [30]; it was reported that individuals with type O blood have 25% less vWF due to increased susceptibility to cleavage by ADAMTS-13 [36]. Type 3 vWD Type 3 vWD is characterized by the virtual absence of plasma vWF and a consequent decrease in the FVIII level to 10%. The frequency of type 3 vWD is between 0.5 and 5.3/1,000,000 individuals [37,38]. Type 3 vWF gene mutations are recessive and such patients are homozygous or compound heterozygous for the vWF gene mutation that creates a null allele [39,40]. Nonsense mutations are the most common of the wide range of type 3 mutations scattered throughout the gene, most of which are in exon 28 (Figure 2). The most common nonsense mutation is R1659X in exon 28 [41]. Nonsense-mediated decay of the allele-specific mRNA is thought to be the molecular mechanism of the nonsense mutations [42]. Missense mutations are the second most common mutations, some of which result in the replacement of cysteine residues that might cause multimerization and secretion defects [37,43,44]. Deletions resulting from recombination events include a single exon deletion, multiple exon deletion, and whole gene deletion. The most common deletion in the vWF mutation database is c.2435delC in exon 18. Deletion of exons 4 and 5 is reported to be a recurrent deletion in the UK, and is associated with a

Figure 2: Types and distribution of the VWF gene mutations identified in Type 3 VWD patients [39,41]

315 Figure 2. Types and distribution of the VWF gene mutations identified in Type 3

Berber E, : The Molecular Genetics of von Willebrand Disease

Table 2: vWF gene deletions and inhibitor development.

Deletion

Inhibitor development

Exons 1-3 Exons 4-5 Exons 14-52 Exons 22-43 Exons 23-52 Exons 26-34 Exons 33-38 Exon 42 Exons 1-52

No inhibitor No inhibitor Inhibitor Inhibitor Inhibitor No Inhibitor Inhibitor Inhibitor Inhibitor

common haplotype and founder effect [45]. Although it is a rare complication, development of alloantibodies against vWF is observed in association with large deletions and whole gene deletion (Table 2) [39,46,47]. Alu (short stretch of DNA)-mediated recombination, impaired secretion, and multimerization are the causative mechanisms for some of the deletions. In addition, gene conversion events between the vWF pseudogene and the vWF gene that result in multiple substitutions and a stop codon in the vWF gene are reported to be a common pathogenic mechanism in type 3 vWD patients [48,49]. Type 2 vWD There is a functional deficiency of vWF in patients with type 2 vWD, which is further classified as 2A, 2B, and 2M based on defective interaction with platelets, and as 2N based on defective binding to the FVIII molecule. Type 2A vWD Type 2A vWD is characterized by defective platelet binding due to the absence of high molecular weight vWF

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(HMW-vWF) multimers in both plasma and platelets. Type 2A vWD patients have a low vWF:RCo to vWF:Ag ratio (<0.6) [37]. Type 2A mutations include missense, deletion, insertion, and frameshift mutations; 73% of these mutations are located within exon 28 (Figure 3) and 90% of these mutations are missense mutations that are either recessive or dominant [40]. D2 domain mutations are recessive and prevent multimer formation; patients are either homozygous or compound heterozygous with a null allele. Mutations in the D3, A2, A1, and CK domains are dominant mutations. D3 and CK mutations inhibit multimerization and dimerization, respectively [50,51]. A2 and A1 mutations result in an increase in susceptibility to ADAMTS-13 proteolysis, defective biosynthesis, or intracellular retention [52,53]. Type 2A vWD mutations that cause defective biosynthesis, intracellular storage, and intracellular retention (e.g. N528S) are group I mutations [54-56]; other mutations that increase ADAMTS-13 sensitivity are group II mutations (e.g. L1505R) [57]. Group I mutations result in a more severe phenotype than do group II mutations and patients respond better to DDAVP treatment [58]. Type 2B vWD Spontaneous and increased binding of vWF to GpIbÎą receptors on platelets due to dominant gain-of-function A1 domain mutations and the absence of HMW-vWF multimers in plasma are characteristic of type 2B vWD. Due to spontaneous platelet binding HMW-vWF multimers in plasma are proteolyzed by ADAMTS-13 [7,59]. Patients have a low VWF:RCo to VWF:Ag ratio (<0.6) and an increase in ristocetin-induced platelet aggregation (RIPA) at low dose ristocetin [37,60]. Thrombocytopenia is also observed in some type 2B vWD patients under stress conditions, such as pregnancy or infection, and after DDAVP use. Patients may have giant platelets and also suffer from

Figure 3: Types and distribution of the VWF gene mutations identified in Type 2A VWD patients [39,41]

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Berber E, : The Molecular Genetics of von Willebrand Disease

Figure 4: Types and distribution of the VWF gene mutations identified in Type 2B VWD patients [39,41]

Figure 5: Types and distribution of the VWF gene mutations identified in Type 2M VWD patients [39,41]

spontaneous platelet agglutination [61-65]. Clinical manifestation of type 2B vWD varies and patients with thrombocytopenia have the most severe phenotype [66]. Furthermore, mouse models of type 2B mutations R2306Q and V1316M show that both mutations and ADAMTS-13 determine the phenotype [59]. There are >50 type 2B mutation submissions in the vWF mutation database. Type 2B mutations are highly penetrant and are detected only between codons 1266 and 1461—the region of exon 28 of the vWF gene encoding A1 domain; 96% of these mutations are missense mutations, most of which are observed at mutation hotspot arginine codons at positions 1306 (R1306W/Q/L), 1308 (R1308C/P), and 1341 (R1341Q/P/L) (Figure 4) [41]. A recent type 2B genotype-phenotype study reported that V1316M mutation is associated with the most severe bleeding score, as compared to other type 2B mutations [66,67]. Moreover, some type 2B mutations are associated with dynamic changes in the vWF level in association with the platelet count. For example, it was reported that a patient with R1306W mutation had normalized high molecular weight multimers (HMWM), but severe thrombocytopenia and a decrease in HMWM after correction of the platelet count [68]. In contrast to classical type 2B mutations, P1266Q/L (New York/MalmÜ) and R1308L mutations do not affect multimer size and do not cause thrombocytopenia [66].

Type 2M vWD Type 2M vWD is characterized by a defect in vWFplatelet binding due to dysfunctional HMW-vWF caused by vWF gene mutations, despite a quantitatively normal vWF multimeric structure. Type 2M mutations are dominant loss-of-function mutations predominantly located within the platelet GP1b binding A1 domain [7,37]; 93% of these mutations are missense mutations and the remainder are small in-frame deletions [41]. Type 2M mutations are fully penetrant and 75% occur in exon 28 of the vWF gene (Figure 5). A Canadian cohort study reported that a vWF:RCo to vWF:Ag ratio <0.4 in type 2M vWD patients was strongly associated with A1 domain mutations [69]. There are also a small number of mutations in the A3 domain (S1731T, W1745C, and S1738A) that affect collagen attachment and cause mild bleeding. Despite the fact that type 2M vWD patients respond poorly to DDAVP treatment, patients with A3 domain mutations respond well to DDAVP [70,71]. Type 2N vWD Type 2N vWD is characterized by markedly reduced or lack of vWF affinity FVIII binding. Recessive mutations in the vWF-FVIII binding domain result in the lack of FVIII binding and a disproportionate decrease in the FVIII:C level to between 0.05 and 0.30 IU/mL. The type 2N vWD phenotype is observed in patients that are homo-

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mutation is associated with a less severe phenotype and a FVIII level of 20 IU/mL, and such patients do respond to DDAVP [78].

zygous for the same FVIII binding mutation, compound heterozygous for 2 different FVIII binding mutations, or compound heterozygous for a FVIII binding mutation and a vWF null allele [37,72-74]. vWF binds to FVIII through its D’ domain and part of the D3 domain between residues Ser764 and Arg1035 encoded by exons 18-23 in the vWF gene [75]; however, mutations beyond the FVIII binding regions (from exon 23 to 27) are also associated with decreased FVIII binding (e.g., Q1053H and C1060R) [76,77].

Type 1 vWD Type 1 vWD is characterized by partial quantitative deficiency of functionally normal vWF. The level of vWF is reduced to between 5 and 50 IU/dL, without significant abnormalities in multimer structure [5]. It is generally inherited as autosomal dominant; however, its clinical diagnosis is complicated due to incomplete penetrance and variable expression of the vWF gene [79]. In addition, compound heterozygosity for type 3 or type 2N mutations influence the severity of the disease. Recent studies performed in the European Union, the UK, and Canada have provided some data on the molecular pathology of type 1 vWD and established that there is a genotype-phenotype correlation [80-82]; the vWF gene was analyzed in ~300 type 1 vWD patients in the 3 studies, which demonstrated that both allelic and locus heterogeneity should be considered to play a role in the molecular pathogenesis of type 1 vWD.

In addition to FVIII binding impairment, type 2N mutations might also cause secretion and multimerization defects, especially cysteine mutations (C788R/Y, Y795C, and C804F) [78]. Moreover, the 2 pro-peptide mutations R760C and R763G sterically inhibit FVIII binding by preventing furin cleavage of pro-peptide, resulting in the formation of UL-vWF multimers [37,74]. Type 2N mutations occur primarily between exon 18 and 20 [41]; 95% of the mutations are missense mutations (Figure 6). R816W and R854Q are the most common type 2N mutations. Type 2N mutations are highly penetrant and the level of FVIII in type 2N vWD patients is associated with the specific mutation. For example, R816W mutation leads to a severe decrease in the FVIII level (<10 IU/mL) and patients do not respond to DDAVP, whereas R854Q

Many candidate mutations, including promoter, splice site, nonsense, missense, and small insertions, as well as deletions have been identified; 80% of the mutations

Figure 6: Types and distribution of the VWF gene mutations identified in Type 2N VWD patients [39,41] Type 1 VWD VWF gene mutation distribution

Transcription

Small deletion Nonsense Small insertion

Mutation number

30 Splice site

25 20 15 10 5

Missense 52 0‐ 
4

40 5‐ 
3

35 
2

9‐

28

27 0‐ 
2

20 5‐ 
1

0‐

0 
1

‐1 
2

Pr om

15

0

ot er

80%

VWF gene exons

Figure 7. Types and distribution of the VWF gene mutations identified in Type 1 VWD patients [38,40]

318

Figure 7: Types and distribution of the VWF gene mutations identified in Type 1 VWD patients [39,41]

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are missense mutations and mutations primarily occur in exon 28 (Figure 7). In addition, some patients have more than vWF gene mutations. One of the major findings of these studies is that type 1 vWD is not always related to vWF gene mutations. Candidate mutations were identified only in 65% of patients and are more likely in patients with vWF:Ag <30 IU/dL. Moreover, mutation penetrance increases as the vWF plasma level decreases [37,81]. Type O blood type is associated with type 1 vWD in patients lacking any identified vWF gene mutation [39,81]. Studies that examined expression of the candidate mutations noted 2 primary pathogenetic mechanisms in type 1 vWD. The first mechanism is intracellular retention of the mutant vWF gene. Some vWF gene mutations, such as C1149R, were shown to dominantly impair vWF secretion [83,84]. Type 1 mutations that cause a loss or increase in cysteine residue (C2257S or G2441C) affect biosynthesis by causing significant intracellular retention and loss of multimeric structure. In contrast to this, D4 domain mutation L2207P caused similar significant intracellular retention and multimer loss to the mutations that involve Cys residue [85]. The second pathogenic mechanism is accelerated vWF clearance, which causes a very brief response to DDAVP in patients (<4 h), as compared to healthy individuals (6-9 h), and an increase in the vWF pro-peptide (vWFpp) to vWF:Ag ratio. Due to the clearance this type of vWD is also known as type 1C vWD. The mutations associated with accelerated vWF clearance are R1205H (vWD Vicenza), C1130G/F/R, W1144G, I1416N, and S1279F [5,86-88]. Research has shown that ABO blood types also influence vWF clearance and the severity of the phenotype in vWF gene mutation carriers. For example, Y1584C mutation was the most common type 1 vWD mutation in 3 type 1 studies with incomplete penetrance. Although this mutation causes intracellular retention, all symptomatic Y1584C carriers also had type O blood in the Canadian and UK type 1 vWD studies, and patients in the UK study had an elevated vWFpp to vWF:Ag ratio [89-92]. Similarly, C2362F carriers with type O blood had a more severe phenotype [93]. There are also some common type 1 vWD mutations for which the molecular pathogenesis has yet to be discerned. For example, R924Q is a recurrent mutation associated with a founder haplotype and marks a splicing defect that created a null allele in a Canadian patient that was a compound heterozygous for R816W type 2N mutation; however, other studies reported that R924Q variation is a polymorphism [94,95].

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Genetic testing in vWD Genetic testing of patients with inherited diseases has an important role in expanding our understanding of the molecular pathology of such diseases, and in decreasing disease-related morbidity and mortality. For some inherited complex disorders, including maturity onset diabetes of the young (MODY), genetic testing is important for differentiating disease subtypes and determining the optimal treatment method. Moreover, prenatal genetic diagnosis is extremely important for decreasing the frequency of inherited diseases as well as limiting the psychological and economic consequences for patients and their families. vWD is a complex inherited bleeding disorder with clinical and genetic heterogeneity. Incomplete penetrance and variable expression are the major roadblocks to clinical diagnosis. Clinical diagnosis of vWD is based on phenotypic data; however, high variation in assays or lower detection limit, particularly vWF:RCo, and unavailability of certain tests like vWF:FVIIIB (vWF:FVIII bonding assay) or multimer analysis would also lead to misdiagnosis or inefficient diagnosis of vWD. Genetic testing of patients with vWD is based on vWF gene analysis. The value of genetic testing in vWD depends on the subtype; it is useful for the differential diagnosis and determining the proper treatment in patients with type 2 vWD. Genetic testing could be helpful in differentiating type 2N vWD from hemophilia A, which is possible by analyzing the exons encoding the FVIII binding region (exons 17-25). Genetic testing could also be useful for differentiating type 2B vWD from platelet-type-vWD, which is based on analyzing just exon 28 in the vWF gene. In addition, genetic testing is important for the correct diagnosis of type 2A and type 2M vWD if multimer analysis cannot be performed. Genetic diagnosis of type 2A and type 2M vWD could also benefit the treatment of vWD, as type 2A patients respond to DDAVP, whereas type 2M patients do not. Clinical diagnosis of type 3 vWD is easily made based on phenotypic testing, as vWF is completely absent in the plasma. Nevertheless, genetic testing of type 3 vWD patients could be used for genetic counseling, prenatal diagnosis, and predicting inhibitor formation; however, the whole gene must be analyzed because mutations are scattered along the vWF gene. On the other hand, because correctly diagnosing type 1 vWD is clinically problematic and due to partial deficiency of vWF, molecular diagnosis is also problematic because of the complexity and mutational heterogeneity of the vWF gene. Many candidate mutations have been identified in type 1 vWD patients; in vitro expression studies are important for determining whether or not they are patho-

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genic variations. Hence, expression analysis of some candidate mutations showed they are just neutral polymorphisms. For some sequence variations, such as R924Q, the presence of a specific haplotype might be responsible for the disease phenotype. Moreover, it is likely that ≼35% of type 1 vWD patients do not have any vWF gene mutation. Finally, the presence of incomplete penetrance and the complex pathogenesis of vWD are major limitations to making a genotype-phenotype association in type 1 vWD patients. Consequently, although the use of genetic testing in type 1 vWD is of limited use, it could be used in patients with vWF:Ag <30% and in those with mutations that affect vWF clearance, such as R1205H mutation, for differentiating type 1 vWD from type 2 vWD [96-98]. Conclusion vWD is an inherited bleeding disorder with a complex molecular pathology. Although numerous studies in various geographic regions have considerably advanced our understanding of the molecular mechanism of vWD, cases of vWD not associated with vWF gene defects are still observed. Complete understanding of the molecular pathogenesis of vWD requires additional in vitro expression studies that observe the effects of the candidate vWF gene mutations. In addition, use of whole genome or exome (part of genome formed by exons) sequencing (novel technologies) might identify other genetic determinants of vWD and help to complete our understanding of vWD by demonstrating the genotype-phenotype relationship. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. References 1. Lillicrap D. Genotype/phenotype association in von Willebrand disease: Is the glass half ful lor empty?. J Thromb and Haemost 2009; 7:65-70. 2. Von Willebrand EA. Finska Lakarasallsk Hand 1926; 68:87. 3. Rodeghiero F, Castaman G, Dini E. Epidemiological investigation of the prevalence of von Willebrand’s disease. Blood 1987; 69:454-459. 4. Werner EJ, Broxson EH, Tucker EL, Giroux DS, Shults J, Abshire TC. Prevalence of von Willebrand disease in children: a multiethnic study. J Pediatr 1993; 123:893-898.

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Research Article

DOI: 10.5505/tjh.2012.13540

Upregulation of T-Cell-Specific Transcription Factor Expression in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL) Pediatrik T-Hücreli Akut Lenfoblastik Lösemide (T-ALL) T-Hücrelerine Özgü Transkripsiyon Faktörlerinin Artmış Gen Anlatımları Müge Sayitoğlu1, Yücel Erbilgin1, Özden Hatırnaz Ng1, İnci Yıldız2, Tiraje Celkan2, Sema Anak3, Ömer Devecioğlu3, Gönül Aydoğan4, Serap Karaman5, Nazan Sarper6, Çetin Timur7, Ümit Üre8, Uğur Özbek1 İstanbul University, Department of Genetics, Institute of Experimental Medicine, İstanbul, Turkey İstanbul University, Cerrahpaşa School of Medicine, Department of Pediatric Hematology, İstanbul, Turkey 3 İstanbul University, İstanbul School of Medicine, Department of Pediatric Hematology, İstanbul, Turkey 4 Bakırköy Maternity and Children’s Hospital, Department of Pediatrics, İstanbul, Turkey 5 Ministry of Health Şişli Etfal Teaching Hospital, Department of Pediatric Hematology, İstanbul, Turkey 6 Kocaeli School of Medicine, Department of Pediatric Hematology, Kocaeli, Turkey 7 Ministry of Health Göztepe Teaching Hospital, Department of Pediatric Hematology, İstanbul, Turkey 8 Ministry of Health Bakırköy Sadi Konuk Teaching Hospital, Department of Hematology, İstanbul, Turkey 1 2

Abstract Objective: T-cell acute lymphoblastic leukemia (T-ALL) is associated with recurrent chromosomal aberrations and abnormal ectopic gene expression during T-cell development. In order to gain insight into the pathogenesis of T-ALL this study aimed to measure the level of expression of 7 T-cell oncogenes (LMO2, LYL1, TAL1, TLX1, TLX3, BMI1, and CALM-AF10) in pediatric T-ALL patients.

Material and Methods: LMO2, LYL1, TLX1, TLX3, BMI1, TAL1, and CALM-AF10 expression was measured using quantitative real-time PCR in 43 pediatric T-ALL patients.

Results: A high level of expression of LMO2, LYL1, TAL1, and BMI1 genes was observed in a large group of T-ALL. Several gene expression signatures indicative of leukemic arrest at specific stages of normal thymocyte development (LYL1 and LMO2) were highly expressed during the cortical and mature stages of T-cell development. Furthermore, upregulated TAL1 and BMI1 expression was observed in all phenotypic subgroups. In all, 6 of the patients had TLX1 and TLX3 proto-oncogene expression, which does not occur in normal cells, and none of the patients had CALM-AF10 fusion gene transcription. Expression of LYL1 alone and LMO2-LYL1 co-expression were associated with mediastinal involvement; however, high-level oncogene expression was not predictive of outcome in the present pediatric T-ALL patient group, but there was a trend towards a poor prognostic impact of TAL1 and/or LMO2 and/or LYL1 protooncogene expression.

Address for Correspondence: Uğur ÖZBEK, M.D., İstanbul Universitesi, Deneysel Tıp Araştırma Enstitüsü, Genetik Anabilim Dalı, İstanbul, Turkey Phone: +90 212 414 22 00 E-mail: uozbek@istanbul.edu.tr Received/Geliş tarihi : October 26, 2010 Accepted/Kabul tarihi : February 21, 2012

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Conclusion: Poor prognostic impact of TAL1 and/or LMO2 and/or LYL1 proto-oncogene expression indicate the need for extensive study on oncogenic rearrangement and immunophenotypic markers in T-ALL, and their relationship to treatment outcome.

Key Words: T-ALL, Pediatric, Transcription factor, Expression, Prognosis

Özet Amaç: T-hücreli akut lenfoblastik lösemi (T-ALL), tekrarlı kromozom bozuklukları ve T-hücre gelişim basamaklarındaki anormal ektopik gen anlatım profili ile ilişkilidir. T-ALL patogenezine eğilebilmek için, çocukluk çağı T-ALL hastalarında, yedi farklı T-hücre onkogenlerinin anlatım düzeylerini belirlemeyi hedefledik (LMO2, LYL1, TAL1, TLX1, TLX3, BMI1 ve CALM-AF10).

Gereç ve Yöntemler: Çocukluk çağı T-ALL hastalarında (n=43) LMO2, LYL1, TLX1, TLX3, BMI1, TAL1, CALM-AF10 gen ekspresyonları kantitatif eş zamanlı PCR yöntemi ile tespit edildi. Bulgular: T-ALL hastalarının büyük çoğunluğunda, artmış LMO2, LYL1, TAL1 ve BMI1 gen anlatımları belirlendi. Normal timosit gelişiminin spesifik basamaklarındaki lösemik tutulumu gösteren genlerden olan LYL1 ve LMO2’ nin artmış ekspresyonu kortikal ve olgun timosit gelişim basamaklarında gözlendi. Bunun yanı sıra, artmış TAL1 ve BMI1 ekspresyonları bütün fenotipik alt gruplarda belirlendi. Altı hastada, normal koşullarda anlatımı görülmeyen TLX1 ve TLX2 gen anlatımları mevcut iken, hastalarımızın hiçbirinde CALM-AF10 füzyon gen transkripti belirlenmedi. Onkogen ekspresyonları ile klinik verileri karşılaştırdığımızda ise LYL1 ve LMO2-LYL1 genlerinin beraber anlatımları mediasten tutulumu ile ilişkili bulundu. Ancak, yüksek onkogen ekspresyonları pediatrik T-ALL grubumuzda hastaların son durumlarının önceden tahmin edilebilmesi açısından anlamlı değildir. Ama TAL1 ve/veya LMO2 ve/veya LYL1 gen ekspresyonlarının kötü prognozla ilişkili olabileceğine dair bir eğilimden bahsedebiliriz.

Sonuç: TAL1 ve/veya LMO2 ve/veya LYL1 gen ekspresyonlarının kötü prognozla olan ilişkisi, onkogenik yeniden düzenlenmeler ve immünfenotipik belirteçlerle beraber, daha büyük çalışma gruplarında çalışılması ve hastanın son durum ile olan ilişkisinin ortaya çıkarılması, gelecekteki tedavi protokolleri için önem arz etmektedir. Anahtar Sözcükler: T-ALL, Pediatrik, Yazılım faktörü, Gen anlatımı, Prognoz Introduction T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive malignancy of thymocytes and corresponds to a heterogeneous group of leukemia arrested at various stages of lymphoid development. T-ALL constitutes 15% of all childhood ALL and 25% of adult ALL; approximately 30% of patients relapse within the first year of treatment and the outcome is usually death [1]. T-ALL patients characteristically have recurrent and rare cytogenetic alterations that affect the gene expression profile of involved genes. These proto-oncogenes are associated with the pathways involved in T-cell development, such as differentiation, proliferation, survival, the cell cycle, and self-renewal. Most chromosomal translocations associated with T-ALL result in juxtaposition of T-cell antigen receptor (TCR) loci (α/δ or β) regulatory elements to proto-oncogenes, thus deregulating expression of the latter [2]. TCR promoters and enhancers are juxtaposed to a number of developmentally important transcription factor genes, including HOX11/TLX1, TLX3/HOX11L2, TAL1/SCL, TAL2, LYL1, bHLHB1, LMO1, and LMO2. Deregulation of these genes

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primarily arrest differentiation at specific stages of T-cell development [3-7]. TAL1, TAL2, LYL1, and bHLHB1 genes are part of the basic helix-loop-helix (bHLH) protein family and function as transcriptional co-factors that form complexes with E2A/ HEB. bHLH protein family members also bind to members of the LMO gene family. During normal human T-cell development, TAL2 and LMO1 are not expressed, whereas Lyl1, Tal1, and Lmo2 expression in mice are restricted to the earliest double-negative stages of T-cell maturation. TLX1 is a class II homeobox gene normally involved in spleen development that is not activated during normal T-cell development. TLX1-positive T-ALL cases share a similar gene expression profile characterized by arrest at the early cortical, CD1-positive thymocyte stage. Translocation/ectopic expression of these transcription factors have been reported with different percentage in pediatric T-ALL [8,9]. More recently, evidence of NOTCH1 mutations in 25%-50% of pediatric T-ALL patients has further enhanced the biologic heterogeneity of T-ALL [10,11]. An alternative mechanism of increased NOTCH1 activation

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via loss-of-function mutations of FBWX7 leads to inhibition of ubiquitin-mediated degradation of the activated form of NOTCH1 [12]. These transcription factors and their oncogenicity have been well documented in mouse models; however, few of these known oncogenetic markers have been shown to have prognostic significance in humans. Conflicting outcomes have been associated with TLX3 and TAL1 deregulation, and NOTCH1 mutations. TLX1-positive T-ALL patients frequently have activating NOTCH1 mutations, and TLX1 over expression and/or translocation confers a better prognosis, but this association varies by study [13-16]. In an earlier study we determined the NOTCH1 and FBXW7 mutation status in a pediatric T-ALL cohort [10]. In the present study we examined the transcription factor genes LMO2, LYL1, TAL1, TLX1, TLX3, and BMI1, and gene fusion of CALM-AF10 in an effort to identify T-cell-specific oncogenic transcription factors and their association with prognosis in pediatric T-ALL patients. Materials and Methods Participants At the time of diagnosis of T-ALL, bone marrow (BM) (n = 33) and peripheral blood (PB) (n = 10) samples were obtained from 43 pediatric patients. The study included pediatric T-ALL patients diagnosed at Istanbul University, Cerrahpaşa School of Medicine (n = 15) and Istanbul School of Medicine (n = 12), Bakirköy Maternity and Children’s Hospital (n = 5), Ministry of Health Şişli Etfal Teaching Hospital (n = 5), Kocaeli University, Kocaeli School of Medicine (n = 2), Ministry of Health Göztepe Teaching Hospital (n = 2), and Ministry of Health Haseki Teaching Hospital (n = 2). Patients were diagnosed based on French-American-British (FAB) Group criteria and their clinical characteristics are shown in Supplemental Table 1 [17]. Mean age of the 17 female and 26 male patients was 8.8 ± 4.1 years, and the median white blood cell (WBC) count was 68,400 x 109/L (range: 1300-580,000 x 109/L). In all, 20 of the patients were in complete remission (CR), 14 died, and 9 were lost to follow-up. T-ALL subgroups were classified according to European Group for the Immunological Characterization of Leukemias (EGIL) guidelines, as follows: immature: n = 16; cortical: n = 8; mature: n = 11 [18]. All T-ALL patients received chemotherapy, according to the Turkish ALL-Berlin-Frankfurt-Munster (Turkish BFM) protocol. Patients were already screened for NOTCH1 and FBXW7 mutations (Supplemental Table 2) [10]. Anonymous control thymocyte subsets were obtained

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from pediatric thymus tissues, using the same guidelines described for T-ALL subgroup classification [19]. Sorted CD34+ CD38– CD1a–, CD34+ CD38+ 1a–, CD34+ CD38+ 1a+, ISP (immature single positive), DP (double positive) CD3–, DP CD3+, SP (single positive), CD4+, and SP CD8+ cells were used as T-cell stage specific controls. Thymus cells were kindly provided by Dr. Frank J.T. Staal, Erasmus Medical Center, Department of Immunology, Rotterdam, The Netherlands. The Istanbul School of Medicine Ethics Committee (reference number and date: 2008/305 and 20.02.2008) approved the study protocol and informed consent was provided by all the patients. cDNA synthesis and quantitative real-time PCR (QRT-PCR) Total RNA was isolated using a Qiagen RNeasy Plus Mini Kit (Qiagen, GmbH, Germany), so as to eliminate the genomic DNA prior to RNA isolation. RNA quality and quantity were measured using a Nanodrop 1000 (Thermo Fisher Scientific, Germany), and cDNA was synthesized from 1 μg of total RNA using a random hexamer and MMLV reverse transcriptase, according to the enzyme manufacturer’s instructions (MBI Fermentase Life Sciences, Lithuania). Quantitative real-time PCR (QRT-PCR) was performed using an ABI 7700 (Applied Biosytems, Foster City, CA, USA) with specific primer-probes, as described by van Grotel et al. [20]. The level of expression of LMO2, LYL1, TLX1, TLX3, BMI1, TAL1, and CALM-AF10 was normalized to ABL gene expression. The threshold value, which is the maximum level of expression of each of the normal thymic subsets, was evaluated for each gene. Statistical analysis Relative gene expression was calculated according to the delta-delta Ct method-based mathematical model [21]. Analyzed gene expression was categorized as high and low, as compared to controls. Categorical variables were compared using Fisher’s exact test and comparison of medians was performed using the Mann-Whitney U test. Remission status was assessed after completion of induction chemotherapy. Treatment efficacy was analyzed according to in vivo response to induction therapy on d 33 (<5% BM blasts). Based on the 33-d response, patients were classified as good (<5% BM blasts) and poor (>5% BM blast) responders. CR was defined as the absence of leukemic blasts in the peripheral blood and cerebrospinal fluid, <5% lymphoblasts in BM aspiration smears, and no evidence of localized disease. Primary treatment failure was defined as persistence of PB blasts or ≥25% blasts in BM after induction therapy. Relapse was defined as the

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reappearance of PB blasts, >5% blasts in BM, or the appearance of extramedullary manifestations after CR was achieved. The Kaplan-Meier method was used to estimate survival rates. Median follow-up was 13.28 months (range: 1-130 months). Overall survival (OS) was defined as the interval from the date of diagnosis to the date of last follow-up or death. Relapse-free survival (RFS) was the time from the start of CR to the date of analysis or to the first event (failure to achieve remission - early death or resistant leukemia-, relapse or death in complete remission). Differences were compared using the 2-sided log-rank test. Mul-

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tivariate survival analysis was estimated according to the Cox regression model, and included the variables of gene expression, gender, age, WBC count, and immunophenotype. The level of statistical significance was set at P = 0.05. Statistical analysis was performed using SPSS v.12.0 for Windows (SPSS, Inc, Chicago, IL, USA) Results Up-regulated oncogene expression found in pediatric T-ALL patients The patients were grouped according to EGIL criteria (immature, cortical, and mature stages) and the levels of

Figure 1: QRT-PCR analysis of oncogenes in the pediatric T-ALL patient subgroups (according to EGIL criteria) and normal thymic control subsets. A. LYL1 expression was significantly higher in the cortical and mature stage subgroups, as compared to their specific controls (cortical versus control: P = 0.02; mature versus control: P = 0.007). B. TAL1 expression was significantly higher in all the phenotypic subgroups than in the controls (immature versus control: P = 0.01; cortical versus control: P = 0.01; mature versus control; P = 0.02 [Mann-Whitney U test]). C. LMO2 expression was significantly higher in the cortical and mature stage subgroups than in the controls (cortical versus control: P = 0.05; mature versus control: P = 0.007). D. BMI1 expression was higher in all the phenotypic subgroups than in their specific controls (immature versus control: P = 0.01; cortical versus control: P = 0.01; mature versus control: P = 0.01).

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gene expression were compared to normal thymic subsets. A group of patients had an up-regulated gene expression profile for LYL1, TAL1, and LMO2. In all, 25 of 41 patients (60.9%) had high-level TAL1 gene expression, 30% of the 43 T-ALL patients had LMO2 overexpression, and 10 patients (25.6%) had a high level of LYL1 gene expression, as compared to the controls. Individual gene expression levels did not significantly differ between the T-ALL phenotypic subgroups (immature, cortical, and mature). Comparison of the phenotypic subgroups and their stagespecific counterparts showed that LYL1 expression was significantly higher both in the cortical and mature stage subgroups than in their specific controls (P = 0.02 and P = 0.007, respectively), TAL1 expression was significantly higher in all the phenotypic subgroups (immature versus control: P = 0.01; cortical versus control: P = 0.01; mature versus control: P = 0.02 [Mann-Whitney U test]), and LMO2 expression was significantly higher in the cortical and mature stage subgroups (P = 0.05 and P = 0.007, respectively) (Figure 1A-C). LYL1 and TAL1 co-expression was significantly higher in the cortical (P = 0.008 [MannWhitney U test) and immature (P = 0.01) stage subgroups. Three of the patients had TLX1 expression and 3 others had TLX3 expression. BMI1 expression was observed in all of the patients and 53.4% of the 43 patients exhibited over-expression, as compared to the controls. BMI1 expression was higher in all the phenotypic subgroups, as compared to their specific counterparts (immature versus control: P = 0.01; cortical versus control: P = 0.01; mature versus control: P = 0.01 [Mann-Whitney U test]) (Figure 1D). One of the recurrent translocations in T-ALL patients is t(10;11)(p13;q14-21), which results in the CALM-AF10 fusion transcript; none of the pediatric patients were carrying CALM-AF10 fusion. Oncogene expression and outcome in the pediatric T-ALL patients To assess the prognostic significance of the above findings we compared the levels of oncogene expression and patient clinical characteristics, and then analyzed survival in the T-ALL patients. High-level LYL1 expression was correlated with mediastinal masses (P = 0.01) and other organ involvement (P = 0.04). LMO2-LYL1 co-expression was strongly associated with mediastinal involvement (P = 0.02 [Fisher’s exact test]). The patients with TLX1 expression had mediastinal masses (P = 0.003 [Fisher’s exact test]) and TLX1 expression was correlated with organ involvement, including the kidneys and heart P = 0.05 [Fisher’s exact test]). TLX3 expression was associated

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with central nervous system (CNS) involvement (P = 0.04 [Fisher’s exact test]). Preliminary comparison of the Kaplan-Meier plots showed that there wasn’t a significant difference between the individual oncogene expression groups, except for the TLX3 gene (patients with high-level TLX3 expression versus patients with no or low-level expression [Cox regression <0.0001; 95% CI: 1.6-18.6]). Additionally, we combined the oncogenes, such as TAL1 and/or LMO2 and/or LYL1, TLX1 and TLX3, TAL1 and/or LMO2 and/or LYL1 and/or TLX1, and TLX3 and/or BMI1. The outcome of these synergistic subgroups versus patients with normal expression of the oncogenes did not reach the level of statistical significance (Figure 2); however, the obtained data showed a trend toward longer RFS in the patients with normal TAL1 and/or LMO2 and/or LYL1 expression (P = 0.08 [Mantel-Cox test]). NOTCH1/FBXW7 mutations were observed in all the genetic subgroups (Supplemental Table 2). Although significant differences were not observed in RFS or OS between the NOTCH1/FBXW7 wild-type and mutated cases, as previously described, we noted a trend toward longer RFS in the mutant patients (Supplemental Figure 1) [10]. Hence, we considered NOTCH1/FBXW7 mutation to be a marker of good prognosis and re-analyzed the survival data for the oncogene expressions. As a result, we observed an independent correlation between a high oncogene expression profile and short OS and RFS both in NOTCH1/FBXW7 mutants and wild-type groups, even it did not show a significant difference. Discussion Deregulation of signaling pathways that control normal T-cell development in the thymus plays a crucial role in T-ALL leukemogenesis. These pathways—under normal circumstances—are strictly regulated by transcription factors, which are also proto-oncogenic proteins. A few molecular mechanisms suggested for T-ALL pathogenesis including the mutations in NOTCH1 and FBXW7 genes leading to NOTCH pathway activation and ectopic expressions of the specific transcription factors such as LYL1, TAL1, LMO2, TLX genes. Currently, there are no genetic markers that can be used to reliably predict treatment response and/or outcome in pediatric T-ALL patients [22]. LMO2, LYL1, and TAL1 genes are members of the bHLH protein family. Among the phenotypic subgroups in the present study (immature, cortical, and mature), LYL1 and LMO2 exhibited the highest level of expression in immature cases, as reported by Meijerink et al. [23].

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Figure 2: Outcome in pediatric T-ALL patients treated with the Turkish-BFM protocol. A. OS according to TAL1/LYL1/LMO2 expression. B. RFS according to TAL1/LYL1/LMO2 expression. C. OS according to TLX1/TLX3 expression. D. RFS according to TLX1/ TLX3 expression. E. OS according to TAL1/LYL1/LMO2/TLX1/TLX3/BMI1 expression. F. RFS according to TAL1/LYL1/LMO2/ TLX1/TLX3/BMI1 expression. P value ≤0.05 (two sided) was considered indicative of a statistically significant difference. None of the Kaplan-Meier analyses showed significant differences.

Recognition of specific T-cell developmental subgroups may have prognostic relevance in T-ALL. Among pediatric T-ALL patients, a pro-T immunophenotype was strongly correlated with poorer outcome than other T-cell phenotypes [24]. In contrast, comparison of LYL1 and LMO2 gene expression with their specific counterparts in the present study showed that the most significant differences occurred in mature stage cases. Moreover, LYL1 alone and LMO2-LYL1 co-expression were strongly associated with mediastinal involvement. We classified gene expression

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in the cases as TAL1/LYL1/LMO2 up-regulated or normal (Figure 2A and B), and analyzed the Kaplan-Meier estimates of OS and RFS. Although it was not significant but TAL1/LYL1/LMO2 up-regulated patients showed poor RFS rates. To date, it is unclear if TAL1/LYL1/LMO2 expression can be used to predict treatment outcome, although some studies suggest that it can [25]. The CALM-AF10 (MLLT10) fusion gene t(10;11) (p13;q21) is a common transcript in acute leukemia. It has been reported that 9% of adult T-ALL patients have

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CALM-AF10 fusion and it is restricted to immature lineage [26]. CALM-AF10-positive ALL is associated with TLX family members and their transcriptional regulator, BMI1 [26]. The BMI1 gene determines the proliferation capacity of normal and leukemic stem cells [27]. In the present study none of the T-ALL patients carried the CALM-AF10 fusion gene, whereas 53.4% of the cases had elevated BMI1 mRNA levels. Increased BMI1 expression in pediatric T-ALL cases is generally ectopic and independent of CALM-AF10 fusion. It was also suggested that up-regulated expression of BMI1 is responsible for the aggressive nature of T-ALL. Although a specific relationship between the present patient’s clinical features and high-level BMI1 expression was not observed, T-ALL is the most aggressive form of ALL. TLX genes are normally not expressed in adult tissues [28]. Approximately 7%-20% of childhood T-ALL patients have ectopic TLX1 and TLX3 expression [15,16]. In the present study 12% of the patients had ectopic expression of TLX1 and TLX3. Some studies indicate that ectopic TLX1 or TLX3 expression confers a poor response to treatment, whereas others report that they do not [5,13]. All the present study’s patients with TLX1 expression had mediastinal masses and those with TLX3 expression had significant CNS involvement, which may consider a possibility of poor prognosis. TLX gene expression may figure out TLX1 or TLX3 translocations but unfortunately these patients lack of cytogenetic data to validate. NOTCH1 activating mutations occur in 30%-60% of T-ALL patients [10,11]. Additionally, inactivating mutations in the E3-ubiquitin ligase gene FBXW7 contribute to aberrant expression of NOTCH1. TLX1 and NOTCH was reported to be synergistically activated to regulate transcription in T-ALL [29]. These mutations were observed in all the present study’s genetic subgroups, but weren’t correlated with oncogene expression or OS ans RFS, but the number of patients with TLX1 expression was insufficient for reaching any conclusion. The present findings offer some clues about the effects of activated transcription factors in pediatric T-cell leukemogenesis, prognostic parameters, and therapeutic applications in different thymic subsets. The most significant finding of the present study is that deregulation of multiple transcription factors (LMO2, LYL1, TAL1, TLX1, TLX3, and BMI1) was involved in the differentiation of Tcells, which is in agreement with other reports [1-5]. The present findings are relevant to 3 main topics. The first is the etiological point of view that the development of pediatric T-ALL is associated with up-regulation of several

Sayitoğlu M, et al : Transcription Factor Expressions in T-ALL

oncogenic transcription factors in a stage-specific manner. T-cell-specific oncogene expression has a greater impact in mature stage patients than in immature stage patients. The second is the prognostic implication of which identification and validation of oncogenic transcription factors in T-cell leukemia may lead to the development of new prognostic markers; these may then be useful for patient follow-up in the future. Lastly, identification of specific expression profiles in pediatric T-ALL subgroups may aid the development of new therapeutic applications and protocols. Acknowledgments This study was funded by the Scientific and Technological Research Council of Turkey (TÜBİTAK) (Project no: 106S112); Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations, relevant to the subject matter or materials included. References 1. Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med 2004; 350:1535-1548. 2. Cleary ML, Mellentin JD, Spies J, Smith SD. Chromosomal translocation involving the beta T cell receptor gene in acute leukemia. J Exp Med 1988;167:682-687. 3. Hatano M, Roberts CW, Minden M, Crist WM, Korsmeyer SJ. Deregulation of a homeobox gene, HOX11, by the t(10;14) in T cell leukemia. Science 1991;253:79-82. 4. Bach I. The LIM domain: Regulation by association. Mech Dev 2000; 91:5-17. 5. Ballerini P, Blaise A, Busson-Le Coniat M, Su XY, ZucmanRossi J, Adam M, van den Akker J, Perot C, Pellegrino B, Landman-Parker J, Douay L, Berger R, Bernard OA. HOX11L2 expression defines a clinical subtype of pediatric T-ALL associated with poor prognosis. Blood 2002; 100:991-997. 6. Berger R, Dastugue N, Busson M, Van Den Akker J, Pérot C, Ballerini P, Hagemeijer A, Michaux L, Charrin C, Pages MP, Mugneret F, Andrieux J, Talmant P, Hélias C, Mauvieux L, Lafage-Pochitaloff M, Mozziconacci MJ, Cornillet-Lefebvre P, Radford I, Asnafi V, Bilhou-Nabera C, Nguyen Khac F, Léonard C, Speleman F, Poppe B, Bastard C, Taviaux S, Quilichini B, Herens C, Grégoire MJ, Cavé H, Bernard OA. t(5;14)/HOX11L2-positive T-cell acute lymphoblastic leukemia. A collaborative study of the Groupe Francais de Cytogenetique Hematologique (GFCH). Leukemia 2003;17:1851-1857.

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27. Raaphorst FM. Self-renewal of hematopoietic and leukemic stem cells: A central role for the Polycomb-group gene Bmi1. Trends Immunol 2003;24:522-524. 28. De Keersmaecker K, Ferrando AA. TLX1 induced T-cell acute lymphoblastic leukemia. Clin Cancer Res 2011; 17:6381-6386.

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Supplemental Online Link: http://www.tjh.com.tr/file/Supplemental_ TurkJHematol_2012_29_325_333.pdf

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DOI: 10.5505/tjh.2012.03764

The Pattern of Hematological Malignancies at Al-Gamhouria Teaching Hospital, Aden, Yemen, from 2008 to 2010 Yemen, Aden’de Al-Gamhouria Eğitim Hastanesinde 2008 ile 2010 Arasında Hematolojik Malignansiler Paterni Gamal Abdul Hamid Al-Gamhouria Teaching Hospital, Department of Hematology-Oncology, Aden, Yemen

Abstract Objective: The aim of this study was to determine the pattern of hematological malignancies at Al-Gamhouria Teaching Hospital from 2008 to 2010.

Material and Methods: Hematological malignancies diagnosed at Al-Gamhouria Teaching Hospital, Department of Hematology-Oncology from January 2008 to December 2010 based on available hematological laboratory methods and histological examination were retrospectively analyzed. Clinical diagnoses were based on history and physical examination. Complete blood count was performed in all patients, bone marrow aspiration was performed in leukemia and multiple myeloma patients, and histopathological examination was performed in cases of malignant lymphoma. Results: Malignant hematological disorders were diagnosed in 300 patients aged 18-80 years (mean age: 43.6 years) with a male:female ratio of 1.23:1. Among the male patients the most common malignant disorder was non-Hodgkin’s lymphoma, followed by Hodgkin’s lymphoma and acute myeloid leukemia. Among the female patients the most common malignant disorder was non-Hodgkin’s lymphoma, followed by acute myeloid leukemia, and Hodgkin’s lymphoma. The study provides the frequencies for the main hematological disorders and present the different subtypes of the Hodgkin’s and non-Hodgkin’s lymphomas. Conclusion: The most common hematological malignancies in male patients were Non-Hodgkin’s lymphomas followed by Hodgkin’s lymphoma and acute myeloid leukemia. Gender distribution showed male predominance with striking predominance in Hodgkin’s lymphomas (M:F Ratio of 1.9:1). Key Words: Malignancies, Lymphoma, Leukemia, Multiple myeloma, Aden Yemen

Özet Amaç: Bu çalışmanın amacı Al-Gamhouria Eğitim Hastanesinde 2008 ile 2010 arasında hematolojik malignansiler paternini belirlemekti. Gereç ve Yöntemler: Al-Gamhouria Eğitim Hastanesinin Hematoloji-Onkoloji bölümünde Ocak 2008 ile Aralık 2010 yılları arasında mevcut hematolojik laboratuvar yöntemleri ve histolojik inceleme temelinde tanı konan hematolojik Address for Correspondence: Gamal Abdul HAMİD, M.D., Al-Gamhouria Teaching Hospital, Department of Hematology-Oncology, Aden, Yemen Phone: +00967-711323798 E-mail: drgamal2000@yahoo.com Received/Geliş tarihi : September 30, 2011 Accepted/Kabul tarihi : March 29, 2012

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malignansiler retrospektif olarak analiz edildi. Klinik tanılar öykü ve fiziksel incelemeyi temel aldı. Tüm hastalarda tam kan sayımı yapıldı, lösemi ve multipl myelom hastalarında kemik iliği aspirasyonu yapıldı ve malign lenfoma vakalarında histopatolojik inceleme yapıldı.

Bulgular: 18-80 yaşlar arasındaki (ortalama yaş: 43,6 yaş) ve 1,23:1 erkek:kadın oranına sahip 300 hastada malign hematolojik bozukluklar saptandı. Erkek hastalar arasında en sık görülen malign bozukluk non-Hodgkin lenfoma ve sonrasında Hodgkin lenfoma ve akut myeloid lösemiydi. Kadın hastalar arasında en sık görülen malign bozukluk nonHodgkin lenfoma ve sonrasında akut myeloid lösemi ve Hodgkin lenfomaydı. Bu çalışma ana hematolojik bozuklukların sıklıklarını vermekte ve Hodgkin ve non-Hodgkin lenfomaların farklı alt tiplerini sunmaktadır.

Sonuç: En sık görülen hematolojik malignansiler non-Hodgkin lenfoma ve sonrasında Hodgkin lenfoma ve akut myeloid lösemiydi. Cinsiyet dağılımı erkek predominansı gösterdi ve bu durum Hodgkin lenfomalarda özellikle belirgindi (E:K Oranı 1,9:1). Anahtar Sözcükler: Malignansiler, Lenfoma, Lösemi, Multipl myelom, Aden Yemen Introduction The pattern of hematological malignancies (HMs) in developing countries and developed countries differs. Among the different types of cancers, leukemia, lymphoma, and multiple myeloma appear to have greatly increased in frequency over the last 10 years. These malignancies are induced by genetic damage or mutation in somatic cells, which can result from environmental exposure to chemicals, ionizing radiation, and viral agents. Comparison of the incidence of HMs across geographic regions and time is complicated by the existence of different disease classification systems, and diagnostic criteria that vary by country, and even between hospitals and cancer registries (CRs) within a country[1]. Leukemia is a malignant neoplasm of hematopoietic stem cells characterized by diffuse replacement of bone marrow or peripheral blood by neoplastic cells; its etiology is obscure[2]. Leukemia typing is based on how quickly the disease develops and progresses. Leukemia is either chronic or acute. Furthermore, leukemia typing is also based on the type of white blood cell that is affected. There are 4 common types of leukemia: acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL)[3,4]. Most acute leukemias are classified as lymphoid or myeloid lineages via standard microscopic morphology, cytochemistry, and immunophenotyping[5]. Previous studies have shown that there are important differences in the incidence of the various leukemia subtypes according to geography, race/ethnicity, age, and trend pattern, indicating that the subtypes may have different etiological factors and that comprehensive global assessment of leukemia patterns is warranted[6,7]. Lymphomas are a heterogenous group of diseases with

differences in epidemiology, histology, and prognosis. The incidence of Hodgkin’s lymphoma (HL) and nonHodgkin’s lymphoma (NHL) is higher among males than females. Malignant lymphoma is the most common malignant tumor in southeastern Yemen, has a high incidence (12.1%) in both genders, and is the most common type of cancer in males (M:F 1.6:1)[8]. Multiple myeloma (MM) is included in the spectrum of diseases ranging from monoclonal gammopathy of unknown significance (MGUS) to plasma cell leukemia. Plasma cell proliferation usually results in extensive skeletal destruction, with osteolytic lesions, hypercalcemia, anemia, and occasionally plasma cell infiltration in different organs. Excessive production of a monoclonal (M) protein can lead to renal failure, hyperviscosity syndrome, or recurrent bacterial infections[9]. The annual incidence of MM in the US is 3-4/100,000 and accounts for 1% of all cancers diagnosed in the US and MM accounts for approximately 10% of all hematological malignancies and 20% of all hematological malignancies in African Americans. Median age at onset of MM in US is 71 years[10]. The aim of the present study was to determine the distribution and pattern of various HMs diagnosed at Al-Gamhouria Teaching Hospital, Aden, Yemen, and to compare them to those reported in other studies, so as to improve their diagnosis and treatment at our hospital. Patients and Methods All patients with HMs that were referred to, diagnosed, and managed at Al-Gamhouria Teaching Hospital (GTH), Department of Hematology-Oncology, Aden, Yemen, between 1 January 2008 and 30 December 2010 were included in the study. The Republic of Yemen has a population of approximately 22 million. GTH is one of the largest public hospitals in Yemen; it has 500 beds and pro-

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Turk J Hematol 2012; 29: 342-347

vides services to more than 60,000 hospitalized patients and more than 600,000 outpatients annually. Demographic data, including age and gender, and a detailed medical history were recorded for each patient. Each patient then underwent a detailed clinical examination, with particular emphasis on hematological parameters. Diagnoses were based on clinical features, blood counts, and peripheral blood films, and were confirmed via bone marrow cytology (Cytochemistry ;Myeloperoxidase and Periodic Acid Schiff) were used when indicated. Diagnosis of lymphoma was based primarily on documented histological reports. Other investigations performed for diagnosis or management included X-ray, creatinine, liver function, serum protein electrophoresis, serum total proteins, and albumin. Blood film examination for cell morphology was performed using Leishman’s stain. Bone marrow was examined following aspiration of the poste-

rior iliac spine, as described by Dacie and Lewis[11]. The study protocol was approved by the Al-Gamhouria Teaching Hospital Ethics Committee. Results In total, 300 patients aged between18-80 years (mean age: 43.6 years) with a male:female ratio of 1.23:1 were diagnosed with malignant hematological disorders. In table 1 NHL was the most common HM (37.66%), followed by HL (21.0%), AML (15.66 %), and ALL (7.66%). Among the chronic leukemia’s, CML was more common than CLL (9.33% and 4.66%, respectively). In all, 12 patients had MM (4.0%). The 1-year relative survival rate was 87% in the HL patients, 75% in the CML patients, 72% in the CLL patients, 71% in the NHL patients, 35% in the ALL patients, and 18% in the AML patients. Table 2 shows that most of the patients with AML (29.7%) were

Table 1: Distribution of HMs according to type of malignancy, gender, mean age, and survival.

Type of malignancy NHL HL AML ALL CML CLL MM Total

n Male 60 41 22 14 14 8 7 166

Total Female 53 22 25 9 14 6 5 134

n 113 63 47 23 28 14 12 300

% 37.66 21.00 15.66 7.66 9.33 4.66 4.00 100

Mean age (years) 50 34 35 24 45 57.5 60 43.6

1-Year Relative Survival 71% 87% 18% 35% 75% 72% 58% 59.4%

NHL: non-Hodgkin’s lymphoma HL: Hodgkin’s lymphoma AML: Acute myeloid leukemia ALL: Acute lymphoblastic leukemia CML: Chronic myeloid leukemia CLL: Chronic lymphatic leukemia MM: Multiple myeloma

Table 2: Distribution of HMs according to age.

Age groups (years) 18-30 31-40 41-50 51-60 61-70 71-80 Total

Total AML n/% 14/29.8 9/19.1 10/21.3 8/17.0 6/12.8 0 47/100

ALL n/% 17/73.9 1/4.35 2/8.7 2/8.7 1/4.35 0 23/100

CML n/% 7/25 6/21.4 7/25 5/17.9 3/10.7 0 28/100

CLL n/% 0 0 2/14.3 8/57.1 321.4 1/7.2 14/100

NHL n/% 22/19.5 13/11.5 18/15.9 28/24.8 23/20.3 9/8.0 113/100

HL n/% 37/58.75 6/9.5 8/12.7 3/4.75 914.3 0 63/100

MM n/% 0 0 1/8.3 5/41.6 4/33.3 2.16.6 12/100

n

%

97 35 48 59 49 12 300

32.33 11.66 16.00 19.66 16.33 4.00 100

AML: Acute myeloid leukemia ALL: Acute lymphoblastic leukemia CML: Chronic myeloid leukemia CLL: Chronic lymphatic leukemia MM: Multiple myeloma NHL: non-Hodgkin’s lymphoma HL: Hodgkin’s lymphoma

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aged 18-50 years and 73.9% of the ALL patients were aged 18-30 years. 21.4% of CML patients were aged 31-50 years and 57.1% of CLL patients were aged 51-60 years. NHL occurred in 24.8% patients aged 61-70 years, followed by 20.4% of those aged 51-60 years, whereas 58.7% of HL patients were aged 18-30 years. Table 3: Distribution of malignant lymphomas

HL according to Rye classification12 Mixed cellularity Nodular sclerosis Lymphocyte predominance Lymphocyte depletion Total

49.2 26.6 12.7 09.5 100

31 18 8 6 63

NHL according to working formulation NHL Low grade Intermediate grade High grade Miscellaneous Total

%

n 18 69 18 8 113

15.9 61.1 15.9 07.1 100

Table 4: Histological types of non-Hodgkin’s lymphoma.

Histological type

n (%)

Mean age

4 (3.5) 7 (6.2) 7 (6.2) 2 (1.8) 9 (8.0) 11 (9.7) 47 (41.6) 2 (1.8) 12 (10.6) 4 (3.5)

59 63 52 67 62 47 46 24 28 36

Others

8 (7.0)

47

Total

113 (100)

51.4

Small lymphocyte Follicular small cleaved cell Follicular mixed Follicular large Diffuse small cleaved cell Diffuse mixed Diffuse large cell Immunoblastic Lymphoblastic Burkitt’s?

According to Rye classification, [12] the most frequent type of HL was mixed cellularity (49.2%), followed by nodular sclerosis (26.6%), and lymphocyte depletion (9.5%) (Table 3). According to a working formulation for clinical usage, there are 3 major prognostic grades for HL: intermediate (n = 69 [61.1%]; low grade (n = 18 [15.9%]); high grade (n = 18 [15.9%]). The growth pattern was diffuse in 67 (59.3%) patients and follicular in 16 (14.16%) patients (Table 4). Among the various histological subtypes, 47 (41.6%) patients had diffuse large cell lymphoma and 12 (10.6%) patients had lymphoblastic lymphoma. Discussion Lymphoma and leukemia are common in Yemen (24.8%) [8,13,14]. Malignant lymphoma constituted 58.7% of all diagnosed HMs in the present study; this high prevalence rate is in agreement with earlier reports from Yemen and the US[13,15]. In the present study 23.3% of the patients had acute leukemias (AML and ALL), whereas CML was the fifth most common HM, accounted for 9.3% of patients and CLL accounted for 4.7%. In contrast, in Western countries CML accounts for 20% of leukemia subtypes and CLL accounts for 25%[16].Similar results have been reported by Al-Ghazaly from Sana’a, Yemen[14] and by Noor et al. from Kenya, [17] whereas ALL was reported to be the most common HM in Karachi,[18] which indicates the geographic variability of the incidence of acute leukemia. The incidence of lymphoma is increasing worldwide, largely due to NHL.[19-21] According to available data on cancer in southeastern Yemen (1983-1989), malignant lymphoma was the 2nd most common malignancy; among males it ranked 2nd and among females it ranked the 5th. [13] Data from developed countries indicate a male:female ratio of 1.4:1, whereas in developing countries the ratio ranges from 4.5:1 to 3.1:1. In general, many more HMs have been reported to occur in males than in females;[22] However, in the present study the number of males and females with CML and CLL was equal, and a slightly higher number of females than males had AML. In the present study the male:female ratio for HL and ALL was

Table 5: Lymphoma and leukemia age-standardized incidence rates in Yemen, Egypt, Jordan, and the US [25-26].

Yemen, 2002-2006

Egypt, 1999- 2001

Jordan, 1996-2001

Saudi Arabia, 2004

The US, 1999-2001

NHL

4.2

16.3

6.4

4.1

12.9

HL

2

2.1

2.5

2.1

2.4

Leukemia

5.6

6.0

6.3

2.7

8.8

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1.9:1 and 1.6:1, respectively, which is the opposite of that previously reported.[23-24] The age-standardized incidence rate for leukemia (Table 5) is 8.8% in the US, 6.3% in Jordan, 5.4% in Egypt, 4.2% in Yemen, and 3.9% in Saudi Arabia.[25-26] The agestandardized incidence rate for NHL in the US is among the highest in the world. Age-standardized incidence rate (ASR) in NHL have been reported to be low (6.5%) in East Asia, moderate (10.2%) in Africa and the Middle East, and high in Western Europe (17.9%), Australia( 25%) and Canada( 27.7%). International variation is due to differences in exposure to risk factors like the agricultural use of the pesticide 1,1,1-trichloro-2,2’bis (p-chlorophenyl)ethane (DDT) and increased risk of non-Hodgkin lymphoma. [27] The incidence rate of NHL increased in in Western Europe and North America during the 1990s and has leveled off in recent years due in part to AIDS; however, NHL as a complication of AIDS does not occur in a sufficient proportion of AIDS cases to fully account for the extent of the increase. Other possible explanations for the high NHL incidence rate in Egypt may be the high prevalence of hepatitis C virus HCV infection.[28] Human papilloma Virus 8 HHV8 infection, adverse environmental exposure and pollution in those countries. Mean survival in the present study was lower than that previously reported, [29,30] and may have been due to several factors, including late presentation to hospital or a shortage of chemotherapeutic drugs. Improve outcome was reported for patients with early presentation and early stages of disease. In the present study 45% of the patients with NHL and 53.7% of HL were aged 51-70 years and 18-30 years, respectively. In other studies the majority of NHL cases were aged 40-69 years—the peak age range being 50-59 years. In reports from Western countries HL was more prevalent among young adults (16 years) and the incidence of the disease makes pik late in the third decade of life (36 years), [31] which is similar to the present findings and indicates that there isn’t a significant difference in incidence rate of HL based on age. The MM patients in the present study had a median age of 60 years, which is younger than previously reported [32]. The mean age reported by Inamullah et al. also matches with our study, while the mean age reported by Dispenzieri et al. in the US was 71 years, which was higher than our results[32-33]. In our study the mean age in leukemia’s agrees with previous studies in Africa[34]. The difference in the age incidence observed in this study when compared to the western countries may be due to the interplay of both environmental and racial factors.[34]

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In general NHL with a follicular pattern of growth has a more favorable prognosis than NHL with a diffuse pattern—within the same cytological subtype. The growth pattern of NHL in the present study was diffuse in 59.3% of cases and follicular in only 14.2%, which is in sharp contrast to Western reports in which 30%-40% of all NHL cases are the follicular type[34]. In this study diffuse, mixed small and large cell lymphoma accounted for 9.7% of all the histological types, which is slightly higher than that reported from Western countries[33]. Moreover, 41.6% of NHL cases in the present study were diffuse large cell, without specification of the cell type. The frequency of Burkitt’s lymphoma in the present study was with the lower than that reported in Ilorin, Nigeria but was similar study reported from Sudan[35-36]. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. References 1. Carli PM, Delafosse P, Duchenet V, et al. Guide de recommandations pour l’enregistrement des he´mopathies malignes par les registres de cancer. Paris, France: INVS and FRANCIM; 2005. http://www.inversussante.fr/ publications/2005/guide_hemopathies_malignes/guide_ hemopathies_ malignes.pdf. Accessed March 28, 2010. 2. Dennis AC. and Mary CT. Acute leukemia . In manual of clinical oncology 6th edition London. Blackwell Science, 2009, 53-55. 3. Cassidy J, Bissett D, Roy AJ, Spence OBE. Acute leukemia. In oxford university press 2010; 528-539. 4. Thachuk DC, Hieschmann JV. Leukemia. In Wintrobes atlas of clinical hematology, 1st edition Lippincott Williams 2009; 48-136. 5. Bennett JM, Catovsky D,Daniel MT, Flandrin G, Galton DA, GralnickHR, Sultan C. Proposed revised criteria for the classification of acute myeloid leukemia: A report of the French-American-British Cooperative group. Ann Intern Med 1985; 103:620-625. 6. Kulshrestha R, Sah SP. Pattern of occurrence of leukemia at a teaching hospital in eastern region of Nepal – A six year study. J Nepal Med Assoc 2009; 48(173);35-40. 7. Estey EH, Faderl SH, Kantarjian HM. Hematology malignancies: Acute leukemia. Springer 1st edition. 2008 8. Hamid GA, Bawazir A, Tayeb MS. Malignant lymphoma in southeastern governorates of Yemen. University of Aden Journal of Natural and Applied Science, 2000; 4, 203-210.

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9. Kumar V, Abbas AK, Fausto N, Aster JC, editors . Pathologic Basis of Disease. 8th ed. Philadelphia:Saunders Elsevier 2010; 589-638. 10. Rajkumar SV, Kyle RA. Symposium on Oncology Practice: Multiple Myeloma: Diagnosis and Treatment. Mayo Clin Proc 2005; 80: 1371-1382. 11. Dacie JV and Lewis SM. Practical Haematology; Churchill Livingstone, 10 edition 2006. 12. Harris NL. Hodgkin’s disease: classification and differential diagnosis. Mod Pathol 1999;12:159–175. 13. Bawazir AA, Hamid GA, Morales E. Available data on cancer in southeastern of Yemen. Eastern Mediterranean Health Journal 1998; vol 4 No 3. 14. Al-Ghazaly J. Pattern of adult leukemia at Al-Jomhori educational hospital, Sanaa, Yemen. Turk J Haematol 2005; 22: 31-35. 15. Hermandez JA, Land KJ, McKenna RW. Leukemias, myeloma and other lymphoreticular neoplasms. Cancer 1995; 75: 381-394 16. Kurt Possinger , Anne C. Regierer. Leukemia: In Haematologie Onkologie 1st ed. ELSEVIER 2007; 331-58. 17. Noor NA, Masood M. Clinico-epidemiological study of Leukemia in Multan. Pak J MedResearch 1998; 28: 232243. 18. Bashir M, Zaman S, Rafatullah, Wazir F, Shoaib M, Biland B. Hematological and clinical presentation of acute leukemias at Khyber Pukhtoonkhwa; Gomal Journal of Medical Sciences July-December 2010, Vol. 8, No. 2: 130-140 19. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108. 20. Plesko I, Kramarova E, Vlasak V, Obstinikova A. Development of registration and cancer incidence rates and trends in Slovakia. Eur J Cancer 1991;27:1049-1052. 21. Rabkin CS, Biggar RJ, Horn JW. Increasing incidence of cancers associated with the human immunodeficiency virus epidemic. Int J Cancer 1991; 47:692-696. 22. Aftab K, Bhurgri Y, Pervez S. Small B cell Non-Hodgkins Lymphoma in Pakistan. J Pak Med Assoc 2006; 56:22-25. 23. Babatunde AS, Amiwero CE, Olantunji PO, Durotuve IA. Pattern of hematological malignancies in Ilorin, Nigeria: A ten year review. The internet Journal of Hematology. 2009:5 24. Omoti CE and Awoduo O. Adult leukemia in Niger delta region of Nigeria Omoti. PAk J Med Sci 2005 Vol. 21 No 3;253-257.

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25. Ba Saleem HO, Bawazir AA, Moore M, Al-Sakkaf KA.Five years cancer incidence in Aden Cancer Registry, Yemen (2002-2006). Asian Pac J Cancer Prev 2010;11:507-511. 26. Salim EI, Moore MA, Al-Lawati JA, Al-Sayyad J, Bazawir A, Bener A, Corbex M, El-Saghir N, Habib OS, Maziak W, Mokhtar HC, Seif-Eldrin IA, Sobue T. Cancer epidemiology and control in the arab world - past, present and future. Asian Pac J Cancer Prev 2009;10:3-16. 27. Parkin DM, Ferlay M, Hamdi-Cherif M, Sitas F, Thomas J, Wabinga H et al. Cancer in Africa: epidemiology and prevention. IARC scientific publication no. 153. Lyon (France): International Agency for Research on Cancer; 2003 28. Mueller NE, Mohar A, Evans A. Viruses other than HIV and non-Hodgkin’s lymphoma. Cancer Res 1992; 52:54795481. 29. Colby TV, Hoppe RT, Warnke RA. Hodgkins disease: A clinicopathological study of 659 cases. Cancer 1982; 49: 1848-1858. 30. Ferrà C, Marcos P, Misis M, Morgades M, Bordejé ML, Oriol A, Lloveras N, Sancho JM, Xicoy B, Batlle M, Klamburg J, Feliu E, Ribera JM. Outcome and prognostic factors in patients with hematologic malignancies admitted to the intensive care unit: A single-centre experience. Int J Hematol 2007; 85: 195-202. 31. Kumar V, Abbas AI Fausto N. Robbins and Cotran .Pathologic basis of disease.7th ed. Philadelphia, Pa.: Elsevier Saunders 2005 32. Inamullah, Raziq F, Tahir M, Wazir R, Rafiq A. Hmatological presentation of multiple myeloma in Khber Pakhtunkhwa. Gomal Journal of Medical Sciences . 2010, Vol. 8, No. 2, 130-133 33. Dispenzieri A, Lacy MQ, Greipp PR . Multiple Myeloma. In: Greer JP, Forester J, Lukens JN,Rodgers GM, Paraskevas F, Glader B. editors.Wintrobe’s Clinical Hematology. 11th ed. Philadelphia: Lippincot Williams & Wilkins 2004; 2583636 34. Harrington DS, Ye YL, Weisenburger DD, Armitage JO, Pierson J, Bast M, Purtilo DT. Malignant lymphoma in Nebraska and Guangzhou, China: a comparative study. Hum Pathol 1987;18: 924-928. 35. Oladipupo Williams CK, Bamgboye EA. Estimation of incidence of human leukemia subtypes in an urban African population. Oncology 1983; 40: 381-386. 36. Ahmed MA, Omer A, el Hassn AM. Malignant lymphomas at the Pathology Department, University of Khartoum, Sudan. East Afr Med J 1984; 61: 627-631.

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Research Article

DOI: 10.5152/tjh.2011.76

An In-House Method for Molecular Monitoring of BCR-ABL BCR-ABL Moleküler Takibi İçin Optimize Edilmiş Manuel Bir Yöntem Hakkı Ogün Sercan, Ilgın Öztürk, Ceyda Çalışkan, Melek Pehlivan, Zeynep Sercan Dokuz Eylül University, School of Medicine, Department of Medical Biology and Genetics, İzmir, Turkey

Abstract Objective: At present, there are a limited number of facilities in Turkey that can provide reliable real-time quantitative (RQ)-PCR BCR-ABL results. The present study aimed to test a cost-effective, in-house method of BCR-ABL quantification, including verification of the method by RQ-PCR validation tests.

Material and Methods: BCR-ABL and ABL target sequences were cloned into pJET1.2 vectors, from which calibrators were prepared and used as templates in RQ-PCR reactions to generate standard curves. Dilutions of K562 cells (representing an in vitro simulation of BCR-ABL transcript reduction) were analyzed.

Results: Standard curves were generated from calibrators. These curves were then used to calculate the BCR-ABL and ABL copy numbers; in which linear BCR-ABL results were obtained. Repetitive experiments showed that our methodology was able to detect 1 BCR-ABL positive cell from amnong 1x105 cells. Conclusion: The method described herein is suitable for implementation with any RQ-PCR instrument and/or kit for quantify BCR-ABL transcripts. Key Words: Chronic myeloid leukemia, BCR-ABL, Real-time PCR, Molecular response

Özet Amaç: Türkiye’de BCR-ABL gerçek zamanlı kantitatif polimeraz zincir tepkimesi ile BCR-ABL düzey tayini yapabilen merkezlerin sayısı, ihtiyacın altında kalmaktadır. BCR-ABL kantitasyonu için laboratuarımızda geliştirmiş olduğumuz yöntem temel alınarak, (PZT) ile kantitasyonun basamakları, iç kontrolleri ve geçerlilik testleri anlatılmıştır.

Gereç ve Yöntemler: BCR-ABL ve ABL hedef dizileri pJET1.2 vektörlerine klonlandıktan sonra kalibratörler hazırlanmış ve gerçek zamanlı PZT tepkimelerinde kalıp olarak kullanılarak standart eğriler çizilmiştir. Bu standart eğriler kullanılarak K562 hücre dilüsyonlarının analizi yapılmıştır. Bulgular: Önceden elde edilen standart eğriler kullanılarak BCR-ABL ve ABL kopya sayıları hesaplanmış; lineer sonuçlar elde edilmiştir. Tekrarlayan deneylerle yöntem duyarlılığının 1x105 hücrede bir BCR-ABL pozitif hücreyi saptayacak düzeyde olduğu gösterilmiştir.

Sonuç: Bu makalede tarif elden protokollerin geçerliliği/güvenirliliği gösterilmiştir. Kullanılan yaklaşım, BCR-ABL kantitasyonunun hedefleyen diğer gerçek zamanlı PZT kit ve cıhazlarına da uyarlanabilmektedir. Anahtar Sözcükler: Kronik miyeloid lösemi, BCR-ABL, Gerçek zamanlı PCR, Moleküler yanıt

Address for Correspondence: Hakkı Ogun SERCAN, M.D., Dokuz Eylül Üniversitesi Tıp Fakültesi, Tıbbi Biyoloji ve Genetik Anabilim Dalı İzmir, Turkey Phone: +90 232 412 46 23 E-mail: ogun.sercan@deu.edu.tr Received/Geliş tarihi : March 7, 2011 Accepted/Kabul tarihi : July 12, 2011

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Introduction Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cell origin. The Philadelphia chromosome (Ph)—der(22)t(9;22) (q34;q11)—is the hallmark of CML, and is formed via reciprocal translocation between the long arms of chromosomes 9 and 22. The translocation results in the juxtaposition of the 5’ end of the BCR gene and the 3’ end of the ABL gene, generating a BCR-ABL chimeric oncogene with aberrant kinase activity. The introduction of imatinib mesylate—a small-molecule competitive inhibitor of the BCR-ABL kinase—in 1998 substantially changed the management of CML [1]. Decades of research on CML has led to very effective molecularly targeted therapies. Since the late 1980’s the percentage of Ph-positive cells in bone marrow based on cytogenetic analysis has been the gold standard for monitoring the response to therapy [1,2,6]. Prior to the use of tyrosine kinase inhibitors for the treatment of CML a molecular response was rarely achieved and it was therefore not feasible to routinely monitor BCR-ABL transcript levels, with the exception of patients that underwent bone marrow transplantation [1-6]. Currently, tyrosine kinase inhibitors are the preferred first-line therapy for CML and based on IRIS trial data a 3-log reduction in BCR-ABL transcript levels after 12 months correlates with progression-free survival [1-6]. Molecular monitoring of CML patients has become clinically very important. Developments in real-time quantitative (RQ)-PCR technology as well as real-time chemistry have enabled routine, high throughput detection of BCR-ABL transcript levels. In turn, there has been an expected increase in the number of centers that provide RQ-PCR results for BCR-ABL mRNA. The rapid increase of these centers revealed that there were significant variations between individual laboratories in reporting RQ-PCR data [5-12]. The International Scale (IS) has been adopted for reporting BCR-ABL values based on the application of laboratory-specific conversion factors that are derived using patient samples [6-12]. Presently, few hospitals in Turkey can provide reliable RQ-PCR BCR-ABL results. Effective implementation of RQ-PCR requires knowledge of reaction kinetics and instrumentation, as well as extensive experience. Lack of experienced personnel could lead to over-dependence on the technical assistance of RQ-PCR kit manufacturers, which in turn may prove to be less than satisfactory. Herein we describe a cost-effective, in-house method developed in our laboratory, and detailed descriptions of validation

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tests and an in vitro simulation of transcript reduction, all which could be adapted to any RQ-PCR methodology chosen for detecting BCR-ABL transcript levels. Materials and Methods The study protocol was approved by the Dokuz Eylül University Clinic & Laboratory Reseach Ethics Committee. Primers and probes RQ-PCR results for BCR-ABL are usually presented as a ratio of the BCR-ABL transcript level and the reference control gene transcript level. Normalization to a reference gene compensates for variation in the efficiency of the reaction and RNA quality between samples. The most widely used reference control gene in BCR-ABL monitoring is the wild-type ABL gene [9,13]. PCR primers used for BCR-ABL facilitate amplification of b2a2 and b3a2 transcripts, which comprise the BCR exon b2/b3 and ABL exon 2 (primers synthesized by Metabion GmbH, Martinsried, Germany). A dual-labeled (5’ 6-FAM and 3’ TAMRA as quencher) hydrolysis probe (TaqMan Probe, Roche Diagnostics, Mannheim, Germany) was used for real-time detection of product accumulation during each cycle. The primer and probe sequences are shown in Figure 1. We used the wild-type ABL gene as the reference gene. The primers used most widely for quantification of ABL also amplify the BCR-ABL fusion transcript [9,12,13]. It has been reported, and also observed by us, that this may cause a bias in results, especially when a large

Figure 1: BCR-ABL fusion gene primer/probe sequences and locations (*sequences were obtained from reference 18).

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quantity of BCR-ABL is present in the sample [9,12,13]. The primer and hydrolysis probe sequences we used for ABL (Figure 1) were determined to hybridize only to wildtype ABL and not BCR-ABL. Cell lines K562 is a BCR-ABL-positive cell line derived from a CML patient in erythroid blast crisis. RS4;11 is a BCRABL-negative cell line derived from an acute myelocytic leukemia (AML) patient and was used as a BCR-ABL-negative cell line control. Both cell lines were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany) and grown in RPMI-1640 medium supplemented with 15% FBS and 1% L-glutamine in a 5% CO2-saturated incubator at 37 °C. Standard curves and copy numbers Calibrators necessary to create the standard curves for product quantity were generated by cloning the target DNA into plasmids, followed by copy number determination and preparation of serial dilutions. RNA or DNA standards may be used for RQ-PCR, but DNA standards are reported to have superior stability. The Europe Against Cancer (EAC) Programme established standardized protocols for fusion transcript quantification and concluded that DNA plasmids were appropriate for constructing standard curves, whereas RNA and cDNA did not provide the stability of plasmids [10,13]. Total RNA was extracted from K562 cells (Macherey Nagel-Nucleospin RNAII, Düren, Germany). To avoid genomic DNA contamination RNA samples were treated with RNase-free DNase I, according to the manufacturer’s instructions. cDNA was synthesized using a First Strand cDNA Synthesis Kit (MBI-Fermentas K1611, St. LeonRot, Germany), 2 μg of total RNA, and random primers, according to the manufacturer’s instructions. cDNA (5 µL) was used as the template in a conventional PCR reaction performed with primers ENF501 and ENR561 (Figure 1) to amplify the target BCR-ABL region. The PCR product was cloned into a pJET1.2 vector, in accordance with the manufacturer’s instructions (CloneJET PCR Cloning Kit, MBI Fermentas, St. Leon-Rot, Germany). Following colony selection, plasmid DNA was isolated (NucleoBond® PC20 Macherey Nagel, Düren, Germany) and analyzed spectrophotometrically. Similar procedures were performed for the ABL gene. Primers AblF and ENR561 (Figure 1) were used to amplify the ABL sequence in a conventional PCR reaction, in which cDNA from K562 cells were used as template. The amplification

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product was cloned into a pJET1.2 vector, followed by colony selection and plasmid analysis. To prepare the calibrators the plasmid copy number μL–1 volume was determined using the following equation (in brief, the molecular weight of the DNA template can be determined by multiplying the number of base pairs (bp) by the weight of 1 mol of a bp estimated to be 650 g; using 6.022 x 1023 [Avogadro’s number] molecules mol–1, the number of molecules of the template can be calculated by first converting to ng [multiplying 1 x 109] and then multiplying by the quantity of template) [14]: copy number μL–1 = [quantity (ng μL–1) x (6.022 x 10 )]/[plasmid length (bp) x 1.109x 650] 23

Calibrators containing 102, 103, 104, 105, and 106 gene copies were prepared for both the BCR-ABL and ABL genes. These calibrators were used as templates in RQ-PCR reactions to generate standard curves. RQ-PCR was carried out using a LightCycler 2.0 (Roche Diagnostics, Mannheim, Germany) instrument and LightCycler TaqMan Master Kit (Roche Diagnostics, 0453528001, Mannheim, Germany). Reactions were performed in a 20-μL volume with 10 pmol of each primer and probe. The same thermal profile was optimized for BCR-ABL and ABL: pre-incubation for 10 min at 95 °C, followed by 45 amplification cycles of denaturation at 95 °C for 10 s, primer annealing at 58 °C for 40 s, and primer extension at 72 °C for 2 s. dH2O was included as a no template control. Fluorescence was measured during the 72-°C segment in each cycle. Data were analyzed using LightCycler v.4.0.0.23 software (Roche Diagnostics, Mannheim, Germany). Standard curves for both BCR-ABL and ABL were generated and saved for further use (Figure 2). All samples in glass capillaries were subsequently run in 2% agarose gel electrophoresis to check for size and non-specific amplifications. All data were derived from independent experiments performed in triplicate. Serial dilutions of K562 cells Dilutions of K562 cells were prepared in the background of the BCR-ABL-negative RS4;11 cell line. Mixtures of BCR-ABL-positive K562 and BCR-ABL-negative RS4;11 cells were prepared, and the total cell number was always 105. The sample mixtures of K562/ RS4;11 cells were prepared so that they contained 10,000, 1000, 100, 10, 1, or 0 K562 cells. These samples represent in vitro simulation of BCR-ABL transcript reduction in a CML patient undergoing therapy. Total RNA isolation and cDNA preparation were performed as described in the previous section. The cDNA was used as a template for determining the

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A

B

BCR-ABL transcript levels in quantitative PCR reactions. RQ-PCR was performed in independent triplicate sets, as described in the previous section. Results and Interpretation RQ-PCR results for the K562/RS4;11 sample mixtures were analyzed using the previously determined standard

Figure 2: A. Standard curve, Cp values, and amplification curves for BCR-ABL during RQ-PCR. B. Standard curve, Cp values, and amplification curves for ABL during RQ-PCR

curves. These standard curves were used to calculate the quantity of BCR-ABL and ABL transcript (copy numbers) in the samples. Change/reduction in the BCR-ABL transcript level was expressed as the ratio of BCR-ABL:ABL (Figure 3). As expected, amplification product was not observed in the dH2O-negative controls, whereas amplification was evident in the 106 copies ΟL–1 BCR-ABL-pos-

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Figure 3: Dilutions of K562 cells were prepared in the background of the BCR-ABL-negative RS4;11 cell line and were subjected to RQ-PCR. Previously determined standard curves were used to calculate the BCR-ABL and ABL transcript copy numbers, and the results are plotted on a logarithmic scale (Y-axis). Change/reduction in the BCR-ABL transcript level is expressed as the ratio of BCR-ABL / ABL.

itive control sample. From the sample mixtures of K562/ RS4;11 cells only 1 sample that contained 105 RS4;11 cells (no K562 cells) was observed to be negative for the BCRABL transcript in repetitive experiments. The Cp value for ABL in this sample was very similar to other K562/RS4;11 mixtures, each of which contained 105 cells, indicating that the result for BCR-ABL was indeed a true negative. All other sample mixtures were positive for the BCR-ABL transcript, albeit in decreasing order. Repeated experimentation showed that our methodology was able to detect 1 BCR-ABL-positive cell from among 105 cells, which is the level of sensitivity generally accepted for BCR-ABL monitoring [15,16]. Analysis of the samples that contained varying quantities of BCR-ABL-positive cells is shown in Figure 3. Discussion RQ-PCR is a technically demanding, yet very powerful tool. The exquisite sensitivity of the assay is also its weakness. It is essential that extreme care be taken to avoid false-positive results due to cross-contamination and carry-over of RNA/DNA from a previous amplification. The calibrators themselves are the greatest threat of contamination, as they are cloned DNA containing DNA target regions. Their preparation, storage, and handling should not be performed in the same laboratory in which patient samples are processed. All equipment, including pipette sets, kits, reagents, paper, pens, workbooks, and lab coats should be dedicated for use only in that particular laboratory. It is of great importance that the rules of good labo-

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ratory practice be followed when setting up PCR assays and during post-PCR processing of samples. Though not addressed herein, there are excellent reviews of recommended pre-PCR procedures in the literature, including handling of blood samples, RNA extraction, cDNA synthesis, and reverse transcription [8-10,17]. A dH2O-negative control (BCR-ABL negative cells), along with low and high positive controls should be included in each RQ-PCR run to monitor assay performance. Any changes in technique, protocol, or instrument should be accompanied by a thorough evaluation. Standard curves should be generated after opening a new real-time kit or when a new batch of primer/probe is diluted. Over the past five years significant advances leading to more wide-spread adaptation of RQ-PCR for BCR-ABL have occurred. Implementation of the IS has improved the comparability of results between laboratories, and recently accredited reference reagents for BCR-ABL quantification have been developed [7]. It is essential that more diagnostic centers in Turkey qualify to report BCR-ABL results according to the IS. In essence, the procedure involves assignment of a laboratory-specific conversion factor (CF) to convert BCR-ABL measurements to the IS [15]. Log BCR-ABL values of the same sample set are compared to reference and local laboratories via linear regression [17]. The results are considered linear when the correlation coefficient of any 2 laboratories is >0.98 [17]. The prerequisite for the procedure is that all in-house validation tests be performed and confirmed prior to application. In this context, the present study aimed to describe a costeffective, in house method for BCR-ABL quantification and to illustrate an example for RQ-PCR validation testing, as well as to provide a description of DNA plasmids that may be implemented into any RQ-PCR methodology to quantify BCR-ABL transcripts. The primary advantage of the presented methodology over widely used commercial kits—in addition to being cost effective—is that once optimized and validated, both absolute and relative quantification can be performed, whereas most commercial kits are restricted to providing relative quantification results. In conclusion, the methodology described herein is suitable for implementation into any RQ-PCR instrument and/ or kit for quantifying BCR-ABL transcripts. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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References 1. Goldman JM. Chronic myeloid leukemia: A historical perspective. Semin Hematol 2010;47: 302-311. 2. Hughes T, Branford S. Molecular monitoring of BCR-ABL as a guide to clinical management in chronic myeloid leukaemia. Blood Rev 2006;20: 29-41. 3. Goldman J. Monitoring minimal residual disease in BCRABL-positive chronic myeloid leukemia in the imatinib era. Curr Opin Hematol 2005;12: 33-39. 4. Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML, Gathmann I, Bolton AE, van Hoomissen IC, Goldman JM, Radich JP; International Randomised Study of Interferon versus STI571 (IRIS) Study Group. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003;349: 1423-1432. 5. Hughes TP, Branford S. Monitoring disease response to tyrosine kinase inhibitor therapy in CML. Hematology Am Soc Hematol Educ Program 2009;477-487. 6. Hughes T. ABL kinase inhibitor therapy for CML: Baseline assessments and response monitoring. Hematology Am Soc Hematol Educ Program 2006;211-218. 7. White HE, Matejtschuk P, Rigsby P, Gabert J, Lin F, Lynn Wang Y, Branford S, Müller MC, Beaufils N, Beillard E, Colomer D, Dvorakova D, Ehrencrona H, Goh HG, El Housni H, Jones D, Kairisto V, Kamel-Reid S, Kim DW, Langabeer S, Ma ES, Press RD, Romeo G, Wang L, Zoi K, Hughes T, Saglio G, Hochhaus A, Goldman JM, Metcalfe P, Cross NC. Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA. Blood 2010;116: e111117. 8. Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J, Baccarani M, Cortes J, Cross NC, Druker BJ, Gabert J, Grimwade D, Hehlmann R, Kamel-Reid S, Lipton JH, Longtine J, Martinelli G, Saglio G, Soverini S, Stock W, Goldman JM. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006;108: 28-37 9. Cross NC. Standardisation of molecular monitoring for chronic myeloid leukaemia. Best Pract Res Clin Haematol 2009;22: 355-365. 10. Branford S, Cross NC, Hochhaus A, Radich J, Saglio G, Kaeda J, Goldman J, Hughes T. Rationale for the recommendations for harmonizing current methodology for detecting BCRABL transcripts in patients with chronic myeloid leukaemia. Leukemia 2006;20:1925-1930.

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11. Branford S, Fletcher L, Cross NC, Müller MC, Hochhaus A, Kim DW, Radich JP, Saglio G, Pane F, Kamel-Reid S, Wang YL, Press RD, Lynch K, Rudzki Z, Goldman JM, Hughes T. Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials. Blood 2008;112: 3330-3338. 12. Cross NC, Hughes TP, Hochhaus A, Goldman JM. International standardisation of quantitative real-time RT-PCR for BCR-ABL. Leuk Res 2008;32: 505-506. 13. Beillard E, Pallisgaard N, van der Velden VH, Bi W, Dee R, van der Schoot E, Delabesse E, Macintyre E, Gottardi E, Saglio G, Watzinger F, Lion T, van Dongen JJ, Hokland P, Gabert J. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program. Leukemia 2003;17: 2474-2486. 14. URI Genomics & Sequencing Center, Calculator for determining the number of copies of a template http://www. uri.edu/research/gsc/resources/cndna.html. 15. Cortes J, Talpaz M, O’Brien S, Jones D, Luthra R, Shan J, Giles F, Faderl S, Verstovsek S, Garcia-Manero G, Rios MB, Kantarjian H. Molecular responses in patients with chronic myelogenous leukemia in chronic phase treated with imatinib mesylate. Clin Cancer Res 2005;11: 3425-3432. 16. Luthra R, Sanchez-Vega B, Medeiros LJ. TaqMan RT-PCR assay coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type. Mod Pathol 2004;17: 96-103. 17. Müller MC, Cross NC, Erben P, Schenk T, Hanfstein B, Ernst T, Hehlmann R, Branford S, Saglio G, Hochhaus A. Harmonization of molecular monitoring of CML therapy in Europe. Leukemia 2009;23: 1957-1963 18. Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cavé H, Pane F, Aerts JL, De Micheli D, Thirion X, Pradel V, González M, Viehmann S, Malec M, Saglio G, van Dongen JJ. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program. Leukemia 2003;17: 2318-2357

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Research Article

DOI: 10.5152/tjh.2012.03

The Prognostic Value of CD49d Expression in Turkish Patients with Chronic Lymphocytic Leukemia Türk Kronik Lenfositik Lösemi Hastalarında CD49d Ekspresyonunun Hastalık Seyri Üzerindeki Etkisi Ant Uzay1, Tayfur Toptaş2, Işık Kaygusuz2, Emel Ekşioğlu Demiralp2, Tülin Fıratlı Tuğlular2, Mahmut Bayık2 Erzurum Regional Training and Research Hospital, Hematology Department, Erzurum, Turkey Marmara University, Pendik Training and Research Hospital, Hematology Department, İstanbul, Turkey

1 2

Abstract Objective: The aim of this study was to assess the prognostic value of CD49d expression in Turkish chronic lymphocytic leukemia patients.

Material and Methods: Data for 118 patients from a single center were evaluated. In all, the study included 73 patients for whom complete clinical follow-up data, and flow cytometry test results for CD5/19, CD23/43, CD38, Zap70, Kappa, and Lambda light chains, and CD49d were available. The effect of the level of CD49d expression on overall survival (OS) and time to treatment (TTT) was investigated retrospectively. Results: Patients with high CD49d expression (≥30%) had more advanced disease at the time of diagnosis (median Rai stage 3 vs. Rai stage 1, P = 0.03). Patients resistant to treatment had higher CD49d expression than patients that responded to treatment (mean CD49d expression of 58% vs. 46%, P = 0.08). The level of CD49d expression was not associated with OS or TTT. Conclusion: The study’s findings show that the patients with high CD49d expression at the time of diagnosis had more advanced disease and poorer response to therapy; however, their overall survival did not differ from that of the patients with advanced disease stage, but lower levels of CD49d expression. Key Words: Chronic lymphocytic leukemia, CD49d, Prognosis

Özet Amaç: Bu çalışmada, kronik lenfositik lösemi hastalarında, CD49d ekspresyonun hastalık seyri üzerindeki etkisi araştırılmıştır.

Gereç ve Yöntemler: Merkezimizde kayıtlı 118 hastanın laboratuvar ve tıbbi kayıtları değerlendirmiştir. CD5/CD19, CD23/CD43, CD38, Zap-70, kappa, lambda ve CD49d ekspresyonuna bakılmış olan toplam 73 hasta çalışmaya dahil edilmiştir. Bunlardan, CD49d’nin hastalık seyri üzerindeki etkileri geriye dönük olarak araştırılmıştır. Sonuç: CD49d’yi yüksek oranda (≥%30) eksprese eden hastaların daha ileri evrede oldukları görülmüştür (ortanca Rai evre 3’e karşı Rai evre 1, p= 0.03). İki ve üç basamak tedavi alan hastaların, sadece bir basamak tedavi alanlara göre

Address for Correspondence: Ant UZAY, M.D., Erzurum Bölge Eğitim ve Araştırma Hastanesi, Hematoloji Bölümü Erzurum, Turkey Phone: +90 442 232 52 90 E-mail: teteantuzay@yahoo.com Received/Geliş tarihi : September 19, 2011 Accepted/Kabul tarihi : October 25, 2011

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daha yüksek oranda CD49d eksprese ettikleri görülmüştür (%58’e karşı %46, p= 0.08). İleri evre hastalarda, CD49d ekspresyonun, tedaviye kadar geçen zaman ve toplam sağ kalım üzerinde etkisi bulunmamıştır.

Bulgular: Çalışmamızda, yüksek CD49d ekspresyonu ileri evre ve kötü tedavi yanıtı ile ilişkili bulunmasına rağmen, düşük CD49d ekspresyonu gösteren diğer ileri evre hastalarla karşılaştırıldığında, tedaviye kadar geçen zaman ve toplam sağ kalım açısından belirleyici olmadığı görülmüştür.

Anahtar Sözcükler: Kronik lenfositik lösemi, CD49d, Prognoz Introduction Chronic lymphocytic leukemia (CLL) is the most prevalent malignancy in the elderly. As populations in developed countries continue to age, the disease is becoming a greater burden for national health care systems. Even though CLL is classified as an indolent lymphoma, for practical purposes it is a unique clinical entity [1]. The disease’s clinical presentation is diverse. There are welldefined CLL classification systems (i.e. Rai and Binet), but some patients with the same stage of disease have different clinical outcomes. This highlights the need for a better understanding of the histological and genetic properties of CLL cells. Determination of prognostic parameters during the early stages of the disease provides valuable clues concerning the disease course and aids in establishing an effective treatment strategy. The most commonly used prognostic parameters at present are ZAP-70, IGHV mutation status, CD38 expression, and cytogenetic abnormalities, all of which are associated with early disease progression [2-6]. The role of these surface antigens in cell survival is not fully known. Nevertheless, many studies have shown that these antigens play an important role in cell-to-cell signaling, interaction with the microenvironment, and cytokinemediated effects [7].

functions, it also serves as a signaling receptor that up regulates Bcl-2 transcription, which in turn inhibits apoptosis [13,14]. Many researchers have investigated CD49d expression in CLL, and its impact on prognosis and overall survival (OS). Expression of CD49d in CLL differs from that of other B-cell malignancies, and is generally lower than that of normal B-cells [15]. Some studies report that high CD49d expression is associated with advanced-stage disease and the presence of lymphadenopathy [16-18]. Based on these findings, other researchers studied the effect of CD49d expression on OS and its potential as a prognostic parameter. Shanafelt et al. reported that high CD49d expression (>45%) is strongly correlated with poor prognosis and shorter time to treatment (TTT) in previously untreated CLL patients [19]. CD49d is increasing in popularity as a molecule in research on CLL prognostic markers to such an extent that some researchers think it is a major factor to which a prognostic antigen profiling score should be assigned [20]. The aim of the present study was to examine the prognostic value of CD49d expression in Turkish CLL patients. Materials and Methods Patients

It is interesting that CLL cells can proliferate steadily and become resistant to apoptosis in vivo, but become susceptible when exposed to an in vitro environment [8]. This suggests that there are various factors that promote cell-microenvironment interaction, enhancing cell proliferation and survival [9]. Some of the most important factors responsible for survival of CLL cells are integrins (alfa4, beta1 integrin, and CD49d), and ligands (VCAM-1, fibronectin) [10,11].

This study included CLL patients that were followedup at Marmara University Hospital, Istanbul, Turkey, between 1998 and 2008. Clinical and laboratory data for 118 patients were obtained, of which 73 had sufficient data, including immunophenotyping and pathology findings, as well as well-documented clinical follow-up results. Each participant provided written informed consent before undergoing any procedure. The study protocol was in compliance with the principles of Good Clinical Practice and was approved by the Marmara University Hospital Institutional Review Board.

CD49d is a surface antigen of the integrin family (integrin-α 4) that plays an important role in cell survival, activation, and migration. Interacting with fibronectin and VCAM-1, CD49d bonds B-cells to the marrow microenvironment and stromal cells found in the germinal centers of lymphoid follicles [12]. Except for the anchorage

Diagnostic criteria for CLL was as follows: an absolute lymphocyte count >5000 mm–3 in peripheral blood; a monoclonal lymphocytic population in peripheral blood, lymph node, or bone marrow (defined as CD5+/CD10–/ CD19+/CD22+/CD23+) in >30% of lymphocytes. Patients were classified according to the Rai staging system for CLL,

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as follows: stage 0: only peripheral blood lymphocytosis >15000 mm–3 or lymphocytes >40% in bone marrow; stage 1: stage 0 and lymphadenopathy; stage 2: stage 0 or 1 and splenomegaly, hepatomegaly, or both; stage 3: stage 0-2 and Hb <11 g dL–1; stage 4: stage 0-3 and a platelet level <100,000 109/L. Only patients in whom treatment was indicated according to National Cancer Institute-Working Group (NCI-WG) criteria received treatment [21]. Immunophenotypic analysis Prognostic evaluation was based on Rai stage and flow cytometric parameters, including CD38, Zap-70, and CD49d expression. CD49d, Zap-70, and CD38 were analyzed via flow cytometry (FACS CANTO®-Becton Dickinson, Mountain View, CA, USA) using direct immunofluorescence labeling. After gating CD11a (+) cells (total lymphocytes), the CD5+/CD19+ population as a percentage (CLL cells) was calculated and CD49d+ cells were gated in that population, providing the actual percentage of CD49d expression among the CLL cells. Similarly, Zap-70 expression was analyzed on CD20+ cells and CD38 expression was analyzed on CD22+ lymphocytes. High-level expression cut-off values for CD49d, Zap-70, and CD38 were set at ≥30%, ≥20%, and ≥20%, respectively.Cytogenetic data were inconsistent and scarce, and were not included.

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dows (SPSS Inc. Chicago IL, USA) and Stata/SE v.10.0 for Macintosh (Stata Corporation, TX, USA). Results Among the 73 patients, 25 were female (34.3%) and 48 were male (65.7%). Median age was 64 years (range: 38-83 years). Median follow-up was 36 months (range: 2-240 months). In all, 22% (n = 16) of the patients had advanced-stage disease (Rai stage 3 and 4). Patient characteristics are shown in Table 1. There weren’t any differences in age or gender between the patients with CD49d expression ≥30% and <30% (median age: 64 years vs. 63.5 years, P = 0.67, respectively; male/female 23/12 vs. 25/13, P = 0.99, respectively). The distribution of high CD49d expression according to Rai stage showed that patients with CD49d expression ≥30% had a significantly higher median Rai stage at the time of diagnosis than those with CD49d expression <30% (median Rai stage 3 vs. 1, P = 0.034). Analysis of the correlation between high CD49d expression and TTT showed that as CD49d expression increased TTT decreased, but the difference between patients with high and low CD49d

None of the patients was treated with autologous or allogeneic stem cell transplantation. Treatments given to patients with progressive disease were highly heterogeneous. Whereas some patients received fludarabine-based treatments—with or without rituximab—others had chlorambucil- or CHOP-based regimens. Considering the small study population, we had to exclude the effects of treatment on prognosis. Statistical analysis Normality of the distribution of data was evaluated using the binominal and Ryan Joiner’s tests. Data are presented as mean, median, standard error of mean, standard deviation, range, and 95% confidence interval. Two-tailed P values <0.05 were considered statistically significant. Parametric and non-parametric variables with abnormal distribution were evaluated using the Mann-Whitney χ2, Yates corrected χ2, Fisher’s exact, and KolmogorovSmirnov’s tests. TTT and OS were estimated according to the Kaplan-Meier method, and analyzed via univariate and multivariate Cox proportional hazards models. TTT was calculated as the time from diagnosis to the start of firstline treatment or last contact. OS was estimated as the time from diagnosis to death from any cause or last contact. All analyses were performed using SPSS v.15.0 for Win-

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A

B Figure 1: The effect of CD49d expression level on (A) TTT and (B) OS.

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expression was not statistically significant (Figure 1a, Table 1). Patients with higher CD49d expression had longer OS, but the difference between patients with high and low CD49d expression was not statistically significant (Figure 1b, Table 1). Patients those were resistant to treatment (patients those received >1 line treatment) had higher CD49d expression than patients that received only 1 line treatment, but the difference in CD49d expression was not statistically significant (median CD49d expression: 44.5% vs. 25%, respectively, P = 0.58). The difference in TTT and OS between the patients with high and low CD49d expression that were in the same disease stage was not statistically significant (Figure 2a and b). The present study also investigated the effect of CD38+/CD49d+ co-expression on TTT and OS. Patients with CD38+/CD49d+ coexpression had shorter TTT and OS, but only the difference in TTT was statistically significant (Figure 3a and b). Multivariate analysis showed that only advanced Rai stage was associated with shorter OS and TTT (Table 2).

Uzay A, et al : CD49d Expression in CLL

Discussion The aim of the present study was to determine the prognostic value of CD49d expression in Turkish CLL patients, . Rai disease stage was inversely correlated with OS and TTT. Three of the most commonly used flow cytometric parameters—CD38, Zap-70, and CD49—were included as parameters indicative of poor prognosis, and their effect on TTT and OS was investigated. Lymphocytosis at the time of diagnosis and high Zap-70 expression were not associated with shorter OS or shorter TTT.High expression of Zap-70 is a well-known parameter indicating poor prognosis [6]; however, an arbitrary cut-off value of 20% was not associated with a negative effect on the disease course in the present study. Had we used a Zap-70 positivity cut-off value ≥30%, the results would probably revealed the negative prognostic value of the marker more clearly CD49d was investigated alone and together with CD38 expression in order to understand its effect on the disease course. Some researchers used >45% as a cut-off value [19], whereas many others used >30%; as such, the latter cut-off value was used in the present study [20,22]. High

A

A

B

B

Figure 2: The effect of CD49d expression level on (A) TTT [*p=0.36, **p=0.66 (log-rank)] and (B) OS [*p=0.33, **p=0.18 (log-rank)] in different RAI stages.

Figure 3: The effect of CD38/CD49d co-expression status on (A) TTT [*p=0.33, **p=0.004 (log-rank)] and (B) OS [*p=1.0, **p=0.13 (log-rank)].

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Turk J Hematol 2012; 29: 354-360

Table 1: Clinical characteristics of 73 CLL patients

Variable Follow-up Median Range Age Median Range <65 years ≥65 years Gender Female Male RAI 0-2 3-4 Initial lymphocytosis ≥25x103/µL <25x103/µL ZAP-70 <20% ≥20% CD38 <30% ≥30% CD49d <30% ≥30%

Number (%)

3-y TTT (%)

P (log-rank)

3-y OS (%)

P (log-rank)

64 years 38-83 years 39 (53.4) 34 (46.6)

41.8 42.0

.37

100 88.6

.39

25 (34.3) 48 (65.7)

35.1 45.4

.31

95.2 93.3

.66

57 (78.1) 16 (21.9)

54.1 0

<.001

97.0 85.1

.036

38 (52.1) 35 (47.9)

33.7 52.2

.21

88.6 100

.055

15 (20.6) 58 (79.4)

46.7 39.0

.91

92.3 93.9

1.0

31 (42.5) 42 (57.5)

61.0 20.5

.031

100 86.4

.022

35 (48.0) 38 (52.0)

48.4 35.7

.21

100 89.1

.069

36 months 2-240 months

TTT and OS denote time-to-treatment and overall survival, respectively.

Table 2: Cox proportional hazard model for OS and TTT

Variable TTT RAI CD38 OS RAI

HR

95% CI

p

5.10 1.84

2.51-10.39 .91-3.59

<.001 .093

4.33

.45-42.02

.21

TTT and OS denote time-to-treatment and overall survival, respectively.

CD49d expression was associated with more advanced disease stages, as previously reported [16]. Even though high CD49d expression was associated with shorter TTT and OS, the results were not statistically significant, which is contrast to previous published data by other investigators [19,22]. Shanafelt and Gattei studied CD49d as an independent prognostic factor in untreated CLL patients. They

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reported that high CD49d expression was associated with shorter OS and treatment-free survival (TFS) [19,22]. In our study, the patients who were in early disease stage (Rai 0-2) had significantly better OS and TTT than those in adveanced disease stage (Rai 3-4), regardless of the level of CD49d expression. As the advanced disease patients had higher CD49d expression at the time of diagnosis, it is logical to conclude that the negative effect of high CD49d expression on OS and TTT may not have been due to CD49d per se, but to more advanced disease stage. Shanafelt and Gattei investigated CD49d expression by assigning patients into risk groups , but their results do not reveal clearly the clinical course in low-risk patients with high CD49d expression [19,22] The present study also investigated the effect of CD49d expression in highrisk patients and observed that there wasn’t a difference in OS or TTT between the high-risk patients with high and

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low CD49d expression, which supports our hypothesis that disease stage, not CD49d expression, is a predictive prognostic factor. Advanced-stage disease is associated with high tumor mass, which in turn is associated with extensive organ and tissue invasion by CLL cells. Thus, in patients with advanced-stage disease there should be more production of integrins and ligands, as expected. This might explain why CD49d expression is associated with advanced stage disease. The actual prognostic value of high CD49d expression would become more definitive if investigated in earlystage CLL patients. As low-risk (Rai 0-2) CLL patients have long-term OS and TFS, it is extremely challenging to perform a prospective study on the effect of CD49d expression on disease course. It is known that high CD49d expression is associated with advanced-stage disease and shorter OS and TTT; however, most of these patients are diagnosed with advanced CLL and are expected to have limited survival anyway. It would be interesting to examine how the disease progresses in low-risk CLL patients that have high CD49d expression at the time of diagnosis. If early-stage patients with high CD49d expression are shown to have more rapid disease progression, then the prognostic value of CD49d expression would be firmly established. CD38 expression in CLL patients is associated with shorter OS and poor response to treatment [3-5]. Some studies reported that CD49d expression is correlated with CD38 expression, [23,24]. The present study analyzed the prognostic effect of CD38+/CD49d+ co-expression on TTT and OS. only patients with high levels of coexpression (CD38 ≥30% and CD49d ≥30%) had shortOS with no statistical significance, these patients had also significantly short TTT. These findings show that CD38+/CD49d+ coexpression had a negative effect on disease course, as previously reported [23, 24]. The small cohort included in the present study might have prevented observation of the probable prognostic importance of CD49d. Another important limitation of the present study is the heterogeneity of the treatment modalities provided to the patients with progressive disease, which is why it was not possible to investigate the heterogeneous treatment effect on prognosis. In conclusion, the present findings indicate that CD49d expression may not be a completely independent prognostic factor, but probably should be evaluated in conjunction with CD38 expression. Currently, there is a lack of solid statistical evidence to prove that and as such, additional prospective research is required in order to determine if CD38+/CD49d+ co-expression is a stronger prognostic factor than CD49d expression alone.

Uzay A, et al : CD49d Expression in CLL

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. References 1. Caligaris-Cappio F, Hamblin TJ. B-cell chronic lymphocytic leukemia: A bird of a different feather. J Clin OncoI 1999;17:399-408. 2. Molica S, Levato D, Dattilo A, Mannella A. Clinicoprognostic relevance of quantitative immunophenotyping in B-cell chronic lymphocytic leukemia with emphasis on the expression of CD20 antigen and surface immunoglobulins. Eur J Haematol 1998;60:47-52. 3. Del Poeta G, Maurillo L, Venditti A, Buccisano F, Epiceno AM, Capelli G, Tamburini A, Suppo G, Battaglia A, Del Principe MI, Del Moro B, Masi M, Amadori S. Clinical significance of CD38 expression in chronic lymphocytic leukemia. Blood 2001;98:2633-2639. 4. Ibrahim S, Keating M, Do KA, O’Brien S, Huh YO, Jilani I, Lerner S, Kantarjian HM, Albitar M. CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 2001;98:181-186. 5. Chevallier P, Penther D, Avet-Loiseau H, Robillard N, Ifrah N, Mahé B, Hamidou M, Maisonneuve H, Moreau P, Jardel H, Harousseau JL, Bataille R, Garand R. CD38 expression and secondary 17p deletion are important prognostic factors in chronic lymphocytic leukaemia. Br J HaematoI 2002;116:142-150. 6. Dürig J, Nückel H, Cremer M, Führer A, Halfmeyer K, Fandrey J, Möröy T, Klein-Hitpass L, Dührsen U. ZAP-70 expression is a prognostic factor in chronic lymphocytic leukemia. Leukemia 2003;17:2426-2434. 7. Calgaris-Cappio F. Role of the microenvironment in chronic lymphocytic leukemia. Br J Haematol 2003;123:380-388. 8. Kay NE, Shanafelt TD, Strege AK, Lee YK, Bone ND, Raza A. Bone biopsy derived marrow stromal elements rescue chronic lymphocytic leukemia B-cells from spontaneous and drug induced cell death and facilitates an ‘’angiogenic switch’’. Leuk Res 2007;31:899-906. 9. Ghia P, Granziero L, Chilosi M, Caligaris-Cappio F. Chronic B cell malignancies and bone marrow microenvironment. Semin Cancer Biol 2002;12:149-155. 10. Lagenaux L, Delforge A, De Bruyn C, Bernier M, Bron D. Adhesion to bone marrow stroma inhibits apoptosis of chronic lymphocytic leukemia cells. Leuk Lymphoma 1999;35:445-453.

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11. De la Fuente MT, Casanova B, Garcia-Gila M, Silva A, GarciaPardo A. Fibronectin interaction with alpha4beta1 integrin prevents apoptosis in B cell chronic lymphocytic leukemia: Correlation with Bcl-2 and Bax. Leukemia 1999;13:266274. 12. Rose DM, Han J, Ginsberg MH. Alpha4 integrins and the immune response. Immunol Rev 2002;186:118-124. 13. Koopman G, Keehnen RM, Lindhout E, Newman W, Shimizu Y, van Seventer GA, de Groot C, Pals ST. Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells. J Immunol 1994;152:3760-3767. 14. Hayashida K, Shimaoka Y, Ochi T, Lipsky PE. Rheumatoid arthritis synovial stromal cells inhibit apoptosis and up-regulate Bcl-xL expression by B cells in a CD49/CD29CD106-dependent mechanism. J Immunol 2000;164:11101116. 15. Baldini L, Cro L, Calori R, Nobili L, Silvestris I, Maiolo AT. Differential expression of very late activation antigen-3 (VLA-3)/VLA-4 in B-cell non-Hodgkin lymphoma and B-cell chronic lymphocytic leukemia. Blood 1992;79:26882693. 16. Eksioğlu-Demiralp E, Alpdoğan O, Aktan M, Firatli T, Oztürk A, Budak T, Bayik M, Akoğlu T. Variable expression of CD49d antigen in B cell chronic lymphocytic leukemia is related to disease stages. Leukemia 1996;10:1331-1339. 17. Behr SI, Korinth D, Schriever F. Differential adhesion pattern of B cell chronic lymphocytic leukemia cells. Leukemia 1998;12:71-77. 18. Lúcio PJ, Faria MT, Pinto AM, da Silva MR, Correia Júnior ME, da Costa RJ, Parreira AB. Expression of adhesion molecules in chronic B-cell lymphoproliferative disorders. Haematologica 1998;83:104-111.

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19. Shanafelt TD, Geyer SM, Bone ND, Tschumper RC, Witzig TE, Nowakowski GS, Zent CS, Call TG, Laplant B, Dewald GW, Jelinek DF, Kay NE. CD49d expression is an independent predictor of overall survival in patients with chronic lymphocytic leukaemia: A prognostic parameter with therapeutic potential. Br J Haematol 2008;140:537546. 20. Zucchetto A, Bomben R, Dal Bo M, Sonego P, Nanni P, Rupolo M, Bulian P, Dal Maso L, Del Poeta G, Del Principe MI, Degan M, Gattei V. A scoring system based on the expression of six surface molecules allows the identification of three prognostic risk groups in B-cell chronic lymphocytic leukemia. J Cell Physiol 2006;207:354-363. 21. Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O’Brien S, Rai KR. National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: re- vised guidelines for diagnosis and treatment. Blood 1996;87:4990-4997. 22. Gattei V, Bulian P, Del Principe MI, Zucchetto A, Maurillo L, Buccisano F, Bomben R, Dal-Bo M, Luciano F, Rossi FM, Degan M, Amadori S, Del Poeta G. Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia. Blood 2008;111:865-873. 23. Dürig J, Nückel H, Hüttmann A, Kruse E, Hölter T, Halfmeyer K, Führer A, Rudolph R, Kalhori N, Nusch A, Deaglio S, Malavasi F, Möröy T, Klein-Hitpass L, Dührsen U. Expression of ribosomal and translation-associated genes is correlated with a favorable clinical course in chronic lymphocytic leukemia. Blood 2003;101:2748-2755. 24. Pittner BT, Shanafelt TD, Kay NE, Jelinek DF. CD38 expression levels in chronic lymphocytic leukemia B cells are associated with activation marker expression and differential responses to interferon stimulation. Leukemia 2005;19:2264-2272.

Research Article

DOI: 10.5152/tjh.2011.41

The Dual Diverse Dynamic Reversible Effects of Ankaferd Blood Stopper on EPCR and PAI-1 Inside Vascular Endothelial Cells With and Without LPS Challenge Lipopolisakkarit Varlığı ve Yokluğunda Vasküler Endotelyal Hücrelerde Endotelyal Protein C Reseptörü ve Plazminojen Aktivatör İnhibitör-1 Üzerinde Ankaferd’in Geri Dönüşümlü İkili Karşıt Etkileri Afife Karabıyık1, Erkan Yılmaz1, Şükrü Güleç1, İbrahim Haznedaroğlu2, Nejat Akar1 Ankara University, Department of Pediatric Molecular Genetics, Ankara, Turkey Hacettepe University, Department of Hematology, Ankara, Turkey

1 2

Abstract Objective: Ankaferd blood stopper (ABS) is comprised of a mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. ABS is used as a topical hemostatic agent due to its antihemorrhagic effect, yet its hemostatic mechanism of action remains to be investigated. ABS does not affect the levels of coagulation factors II, V, VII, VIII, IX, X, XI and XII. The aim of this study was to investigate the effects of ABS on endothelium and immune response. As such, we evaluated changes in endothelial cell protein C receptor (EPCR) and plasminogen activator inhibitor type-1 (PAI-1) expression inside human umbilical vein endothelial cells (HUVECs) in the presence and absence of lipopolysaccharides (LPSs).

Material and Methods: We exposed HUVECs to 10 μL and 100 μL of ABS for 5 min, 25 min, 50 min, 6 h, and 24 h. Additionally, 10 μg mL–1 of LPS was administered for 1 h to observe the effects of LPS challenge on HUVECs, and then the cells were treated with ABS for 5 min, 25 min, 50 min, and 6 h to observe the effects of ABS on HUVECs. Total RNA was isolated from HUVECs and then the level of expression of EPCR and PAI-1 mRNA was measured.

Results: Cells were microscopically observed to arise from the surface and adhere to each other following the administration of ABS to HUVECs. Additionally, after 24 h the cells had normal growth and physiology, which suggests that the adhesive cellular effects of ABS might be reversible. ABS had a negative effect on EPCR and PAI-1 expression; the effect in response to 100 µL was greater than that to 10 µL. EPCR and PAI-1 expression increased over time in response to LPS and 10 µL of ABS. EPCR and PAI-1 expression was very low during the first hour of exposure to LPS and 100 µL of ABS, but after 6 h increased to levels similar to those observed in response to LPS and 10 µL of ABS. Conclusion: It was observed that ABS had dual diverse dynamic reversible effects on EPCR and PAI-1 expression in HUVECs, which were dependent on dose and concentration. ABS might play a role in numerous cellular mechanisms, in addition to having hemostatic effects.

Key Words: Ankaferd, EPCR, PAI-1, LPS, HUVEC Address for Correspondence: Afife KARABIYIK, M.S., Ankara Üniversitesi, Pediatrik Moleküler Genetik Anabilim Dalı, Ankara, Turkey Phone: +90 312 595 63 48 E-mail: afifekrbyk@gmail.com Received/Geliş tarihi : October 3, 2009 Accepted/Kabul tarihi : April 30, 2010

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Karabıyık A, et al : Ankaferd’s Effects on EPCR and PAI-1

Turk J Hematol 2012; 29: 361-366

Özet Amaç: Ankaferd, Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum ve Urtica dioica bitkilerinden oluşan bir karışımdır. Kanamayı durdurucu etkisinden dolayı topikal hemostatik ajan olarak kullanılmaktadır. Hemostatik çalışma mekanizması araştırılmaktadır. Koagülasyon faktörleri II, V, VII, VIII, IX, X, XI ve XII seviyeleri Ankaferd’den etkilenmemektedir. Çalışmamızda Ankaferd’in endotel üzerindeki ve immün yanıttaki etkisini araştırmak amacıyla, İnsan Umbilical Ven Endotel Hücreleri (HUVEC) kullanılarak Endotelyal Protein C Reseptörü (EPCR) ve Plazminojen Aktivatör İnhibitör (PAI-1) gen ekspresyonları üzerindeki etkisi araştırılmış ve farklı doz ve zamanlarda oluşturduğu değişikliklerin incelenmesi, lipopolisakkarit (LPS) muamelesinden sonra EPCR ve PAI-1 ekspresyonları üzerindeki olası etkisinin gösterilmesi amaçlanmıştır.

Gereç ve Yöntemler: HUVEC’lere 10 µl ve 100 µl konsantrasyonlarda Ankaferd 5 dk, 25 dk, 50 dk, 6 saat ve 24 saat süreyle uygulanmıştır. Ayrıca immün yanıt üzerindeki etkisinin incelenebilmesi için 10 µg/ml LPS ile 1 saatlik muamelenin ardından hücreler 10 µl ve 100 µl Ankaferd’le birlikte 5 dk, 25 dk, 50 dk ve 6 saatlik sürelerde maruz bırakılmıştır. HUVEC hücrelerinden RNA izolasyonu yapılmış; EPCR ve PAI-1 gen ekspresyonları incelenmiştir. Bulgular: Yapılan mikroskobik incelemede Ankaferd uygulamasıyla hücrelerin yüzeyden kalkıp toplanarak birbirlerine yapıştıkları ve 24 saatin içerisinde büyüme ve gelişmelerinin normale döndüğü gözlenmiştir. Bu, Ankaferd’in etkisinin zamanla azaldığı anlamına gelebilir. Ankaferd muamelesiyle başlangıçta gözlenen EPCR ve PAI-1 ekspresyonundaki değişim zamanla azalmıştır. Ayrıca ekspresyonlardaki hızlı değişim göz önüne alındığında Ankaferd’in artan konsantrasyonlarda etkisini daha da fazla gösterdiği söylenebilir. Böylece Ankaferd’in EPCR ve PAI-1 üzerinde hem doz, hem de zamana bağlı etkisi belirlenmiştir. LPS muamelesi sonrasında da hücreler üzerinde Ankaferd benzer bir etki göstermekte, doz arttıkça daha da etkili hale gelmekte ve zamanla etkisini yitirmeye başlamaktadır.

Sonuç: Ankaferd’in HUVEC’lerde EPCR ve PAI-1 ekspresyonu üzerinde doz ve konsantrasyona bağımlı etkisinin varlığı saptanmış olup, hemostatik etkileri yanısıra hücresel birçok mekanizmayı da etkileyebileceği düşünülmektedir. Anahtar Sözcükler: Ankaferd, EPCR, PAI-1, LPS, HUVEC Introduction Ankaferd blood stopper (ABS) is a unique medicinal plant extract mixture, which has been historically used as a hemostatic agent in Turkish folk medicine [1,2]. ABS is comprised of a standardized mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. Following numerous preclinical experiments [3-7] and a clinical phase I study [8], ABS was approved for use in Turkey as a medicinal product for the management of external hemorrhage, the post-dental surgery period, and bleeding refractory to conventional anti-hemorrhagic agents [9-16]. The basic mechanism of action of ABS is formation of an encapsulated protein network that provides focal attachment points for very rapid (<1 s) vital erythrocyte aggregation—known as the hemostatic ABS-web [1,17]. ABS-induced protein network formation with blood cells—particularly erythrocytes—covers the primary and secondary hemostatic system without disturbing individual coagulation factors [1,5,7,17]. ABS also has antiinfection and anti-neoplastic effects [18-20]. The distinct important molecular components of the ABS-induced hemostatic network involve vascular endothelium, pro-

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teins, and blood cells [17,21-24]. Endothelial protein C receptor (EPCR) plays a role in numerous hemostatic, vascular, and immunological actions [25-33]. Likewise, plasminogen activator inhibitor type-1 (PAI-1) is a very important biological mediator of fibrinolysis, infection, neoplasia, obesity, and wound healing [34-36]. The aim of the present study was to examine the intracellular effects of ABS on EPCR and PAI-1 expression in human umbilical vein endothelial cells (HUVECs), as these molecules may be novel administrators at the center of ABS-induced pleiotropic effects. Lipopolysaccharides (LPSs) are large molecules that act as endotoxins and elicit strong immune responses within the vascular system [37]. LPS challenge is the process of exposing a biological environment to an LPS that may behave as a toxin in order to observe immunological and hemostatic responses. Hence, the present study aimed to investigate the effects of ABS— with and without LPS challenge—on HUVECs, based on changes in EPCR and PAI-1 expression. Materials and Methods ABS (10 μL and 100 μL) was administered to HUVECs (75 cm2 and ~75% fullness) for 5 min, 25 min, 50 min, 6 h, and 24 h. Nuclei were isolated from HUVECs, and

Turk J Hematol 2012; 29: 361-366

the level of expression of EPCR and PAI-1 was determined using a Roche LightCycler 1.5 (Basel, Switzerland). Fluorescence-marked primers were used to analyze EPCR and PAI-1 expression. Water with a pH of 2 (likely to be similar to the pH of ABS) was used as a control. In addition, to observe the effects of LPS challenge on HUVECs and those of ABS on HUVECs 10 μg mL–1 of LPS (Sigma, Germany) was administered for 1 h to the test platform. Then, the cells were exposed to ABS for 5 min, 25 min, 50 min, and 6 h, so as to measure ABS-induced changes in EPCR and PAI-1 expression in relation to LPS. All experiments were repeated at least 2 times. Statistical analysis was based on two-way ANOVA with Bonferroni post test using GraphPad Prism v.5.0 (GraphPad Software, San Diego California, USA, http://www.graphpad.com). Results Cells were microscopically observed to arise from the plastic surface and adhere to each other in response to ABS administration to HUVECs. Additionally, after 24 h the cells had normal growth and physiology, which suggests that the adhesive cellular effects of ABS might be reversible. PAI-1 and EPCR expression was negatively affected by 24 h of exposure to 100 µL of ABS (Figures 1 and 2), but not by exposure to 10 µL of ABS. These findings indicate that the dose-dependent effects of ABS rely on PAI-1 and EPCR gene expression. When LPS only was administered to HUVECs, EPCR and PAI-1 expression was higher than in HUVECs not exposed to LPS (data not shown). When LPS, and 10 µL and 100 µL of ABS were administered, the level of PAI-1 expression was stable and similar to that in the control after 6 h (Figure 3), whereas EPCR expression was very

Figure 1: The effect of 10 μL and 100 μL of ABS on PAI-1 mRNA expression (*P < 0.05 and **P < 0.01). Error bars represent Means ± SD.

Karabıyık A, et al : Ankaferd’s Effects on EPCR and PAI-1

low during the first h of exposure, but increased after 6 h (Figure 4).

Figure 2: The effect of 10 μL and 100 μL of ABS on EPCR mRNA expression (**P < 0.01 and ***P < 0.001).

Figure 3: The effect of LPS, and 10 µL and 100 µL of ABS on PAI-1 mRNA expression. Error bars represent Means ± SD.

Figure 4: The effect of LPS, and 10 µL and 100 µL of ABS on EPCR mRNA expression (*P < 0.05 and ***P < 0.001). Error bars represent Means ± SD.

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Discussion ABS exhibited dual diverse dynamic reversible effects on EPCR and PAI-1 expression in HUVECs. The observed immediate increase in the level of expression of pro-hemostatic PAI-1 and down-regulation of anti-coagulant EPCR following exposure to ABS are compatible with previous reports of ABS’s sudden anti-hemorrhagic effect [3-7] and clinical backgrounds [8-10,13-15,20]. The topical hemostatic efficacy of ABS has been previously tested in animals with normal [4,6] and dysfunctional hemostasis [5,7]. Experimental studies have led to the preclinical stage of this hemostatic product’s development. Short-term oral systemic administration of ABS in rabbits was reported to be hematologically and biochemically safe, as acute mucosal toxicity, hematotoxicity, hepatotoxicity, nephrotoxicity, and biochemical toxicity were not observed during the short-term follow-up [3]. Those preclinical results reflect a starting point to search any possible systemic confounding effect of ABS when applied to internal topical surfaces. The use of ABS as a hemostatic agent for treating external hemorrhages and for dental treatment in humans is thie first indication the ABS is safe and efficacious in humans [8]. A phase I double-blind, randomized crossover placebo-controlled clinical study with a 5-d washout period between cross-over periods that included healthy volunteers reported that ABS was safe. Physiological cellbased coagulation was clinically obtained in response to topical ABS administered for the prevention and treatment of bleeding associated with many distinct clinicopathological states [8-10,13-15,20]. The ABS-induced hemostatic network consists of distinct important molecular components. Vital erythroid aggregation occurs in the spectrin, ankyrin, and actin proteins in red blood cell membranes. Essential erythroid proteins (ankyrin recurrent and FYVE bundle containing protein 1, spectrin alpha, actin-depolymerization factor, actin-depolymerizing factor, LIM bundle and actin-binding subunit 1 isoform a, LIM bundle and actin-binding subunit 1 isoform b, NADP-dependent malic enzyme, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, mitochondrial NADP [+]-dependent malic enzyme 3, ribulose bisphosphate carboxylase large chain, and maturase K) and the required ATP bioenergy (ATP synthase, ATP synthase beta subunit, ATP synthase alpha subunit, ATP-binding protein C12, TP synthase H+ transporter protein, ADF, and alpha-1,2-glycosyltransferase ALG10-A) are included in the ABS protein library. ABS also upregulates the GATA/ FOG transcription system, affecting erythroid functions and urotensin II [21,23,24].

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Turk J Hematol 2012; 29: 361-366

The initial vascular dynamic response to ABS is vasoconstriction, whereas the late response is vasodilatation [22]. The clinical control of critical bleeding states associated with deficiencies of either primary or secondary hemostasis has been previously examined [13, 38]. Patients with bleeding diathesis that could not be controlled with standard anti-hemorrhagic methods were successfully treated with topical ABS [9,13,16,38]. The present study’s results provide additional evidence that ABS might also affect vascular anticoagulant (namely EPCR) and antifibrinolytic (namely PAI-1) pathways in a balanced way to regulate hemostasis, even in individuals with clotting defects. In addition to its hemostatic activity, ABS might also inhibit the growth of bacteria [19]. The anti-infection activity of ABS may bolster its current clinical use, as it inhibits the growth of bacteria in the region in which it is applied primarily for its hemostatic activity, such as infected wounds. The antimicrobial activity of ABS was tested against many pathogens, including A. baumannii, E. coli, K. pneumonia, P. aeruginosa, Enterobacter spp., Stenotrophomonas maltophilia, methicillin-resistant coagulase-negative Staphylococcus, vancomycin-susceptible Enterococcus, and VRE. ABS was reported to exhibit antibacterial activity against several gram-positive and gramnegative food and human pathogens [18]; however, the mechanism of action of ABS’s anti-infection effect is currently unknown. EPCR is an important molecule involved in the regulation of biological responses to severe infection [25,39]. LPS-induced endotoxemia requires the enzymatic active site of EPCR and PAR-1 [39]. In the present study ABS up-regulated the expression of EPCR and PAI-1 in the presence of LPS. The anti-infection effect of ABS might be related to its hemostatic effects on distinct steps of coagulation and vascular endothelium. Preliminary observations indicated that ABS might have wound healing effects in different clinical states, such as oral infections, rectal ulcers, and neoplastic lesions [12,14,17]. PAI-1 is involved in tissue preservation and wound repair [34,35]. The overexpression of PAI-1 in response to ABS observed in the present study is an initial clue to set further experiments to search the importance of fibrinolysis regulators in the biological effects of ABS. PAI-1 is also involved in tumor responses [39-41]. In a case series topical ABS inhibited tumor angiogenesis [20], prothrombotic affect of PAI-1 in this respect shall also be further searched based on the observation in this study. In conclusion, ABS exhibited dual de novo affects on EPCR and PAI-1 in HUVECs. HUVECs exposed to LPS challenge caused ABS-induced up-regulation of the expres-

Turk J Hematol 2012; 29: 361-366

sion of EPCR and PAI-1, indicating that ABS might act as a topical biological response modifier. As ABS is currently being developed in basic and clinical grounds, these novel observations indicate that additional research on the pleiotropic effects of this unique hemostatic agent is warranted. Acknowledgement None of the authors have any conflicts of interest related to the materials used or data presented herein. ABS was obtained from Ankaferd Drug Inc., Istanbul, Turkey. References 1. Goker H, Haznedaroglu IC, Ercetin S, Kirazli S, Akman U, Ozturk Y, Firat HC. Haemostatic actions of the folkloric medicinal plant extract ankaferd blood stopper. J Int Med Res 2008;36:163-170. 2. Haznedaroglu IC. Time to take a healthier view of history. Nature 1998;396:108. 3. Bilgili H, Captug O, Kosar A, Kurt M, Kekilli M, Shorbagi A, Kurt OK, Ozdemir O, Goker H, Haznedaroglu I. Oral systemic administration of ankaferd blood stopper has no short-term toxicity in an in vivo rabbit experimental model. Clin Appl Thromb Hemost. 2010; 16: 533-536. 4. Bilgili H, Kosar A, Kurt M, Onal IK, Goker H, Captug O, Shorbagi A, Turgut M, Kekilli M, Kurt OK, Kirazli S, Aksu S, Haznedaroglu IC. Hemostatic efficacy of ankaferd blood stopper in a swine bleeding model. Med Principl Pract 2009;18:165-169. 5. Cipil H, Kosar A, Kaya A, Uz B, Haznedaroglu IC, Goker H, Ozdemir O, Koroglu M, Kirazli S, Firat H. In vivo hemostatic effect of the medicinal plant extract ankaferd blood stopper in rats pretreated with warfarin. Clin Appl Thromb Hemost 2009;15:270-276 6. Huri E, Akgul T, Ayyildiz A, Ustun H, Germiyanoglu C. Hemostatic role of a folkloric medicinal plant extract in a rat partial nephrectomy model: Controlled experimental trial. J Urol 2009;181:2349-2354. 7. Kosar A, Cipil HS, Kaya A, Uz B, Haznedaroglu IC, Goker H, Ozdemir O, Ercetin S, Kirazli S, Firat HC. The efficacy of ankaferd blood stopper in antithrombotic drug-induced primary and secondary hemostatic abnormalities of a ratbleeding model. Blood Coagul Fibrinolysis 2009;20:185190. 8. Firat H, Ozdemir O, Kosar A, Goker H, Haznedaroglu I. Annual review of ankaferd 08-09. Istanbul, Naviga Scientific Publications, 2009: pp. 1-175. 9. Arslan S, Haznedaroglu IC, Oz B, Goker H. Endobronchial application of ankaferd blood stopper to control profuse lung bleeding leading to hypoxemia and hemodynamic instability. Resp Med 2009;2:144-146

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10. Dogan OF, Ozyurda U, Uymaz OK, Ercetin S, Haznedaroglu I. New anticoagulant agent for CABG surgery. Eur J Clin Invest 2008;38:341. 11. Huri E, Akgul T, Ayyildiz A, Germiyanoglu C. Hemostasis in retropubic radical prostatectomy with ankaferd bloodstopper: A case report. Kaohsiung J Med Sci 2009;25:445-447. 12. Ibis M, Kurt M, Onal IK, Haznedaroglu IC. Successful management of bleeding due to solitary rectal ulcer via topical application of ankaferd blood stopper. J Altern Complement Med 2008;14:1073-1074. 13. Kurt M, Akdogan M, Onal IK, Kekilli M, Arhan M, Shorbagi A, Aksu S, Kurt OK, Haznedaroglu IC. Endoscopic topical application of ankaferd blood stopper for neoplastic gastrointestinal bleeding: A retrospective analysis. Dig Liver Dis 2010;42:196-199. 14. Kurt M, Disibeyaz S, Akdogan M, Sasmaz N, Aksu S, Haznedaroglu IC. Endoscopic application of ankaferd blood stopper as a novel experimental treatment modality for upper gastrointestinal bleeding: A case report. Am J Gastroenterol 2008;103:2156-2158. 15. Kurt M, Kacar S, Onal IK, Akdogan M, Haznedaroglu IC. Ankaferd blood stopper as an effective adjunctive hemostatic agent for the management of life-threatening arterial bleeding of the digestive tract. Endoscopy 2008;40 Suppl 2:E262. 16. Öner AF, Doğan M, Kaya A, Sal E, Bektaş MS, Yesilmen O, Ayhan H, Acikgoz M. New coagulant agent (ankaferd blood stopper) for open hemorrhages in hemophilia with inhibitor. Clin Appl Thromb Hemost 2010;16:705-707. 17. Haznedaroglu BZ, Haznedaroglu IC, Walker SL, Bilgili H, Goker H, Kosar A, Aktas A, Captug O, Kurt M, Ozdemir O, Kirazli S, Firat HC. Ultrastructural and morphological analyses of the in vitro and in vivo hemostatic effects of ankaferd blood stopper. Clin Appl Thromb Hemost 2010; 16:446-453. 18. Akkoc N, Akceik M, Haznedaroglu I, Goker H, Aksu S, Kirazli S, Firat H. In vitro anti-bacterial activities of ankaferd blood stopper. Int J Lab Hematol 2008;30:95. 19. Tasdelen Fisgin N, Tanriverdi Cayci Y, Coban AY, Ozatli D, Tanyel E, Durupinar B, Tulek N. Antimicrobial activity of plant extract ankaferd blood stopper. Fitoterapia 2009;80:48-50. 20. Turhan N, Kurt M, Akdogan M, Haznedaroglu I. Topical ankaferd blood stopper administration to bleeding gastrointestinal carcinomas can decrease tumor vascularization. Am J Gastroenterol 2009; 104:2874-2877. 21. Demiralp DO, Haznedaroglu IC, Akar N: Functional proteomics of ankaferd blood stopper. Turkish Journal of Hematology 2010;27: 70-77.

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22. Aktas A, Er N, Onur MA: Effects of ankaferd blood stopper on vascular response in rat carotid artery. UHOD Int J Hematol Oncol. 2010; 20: 156-162. 23. Haznedaroglu IC. Molecular basis of the pleiotropic effects of ankaferd blood stopper. IUBMB Life 2009;61:290. 24. Yılmaz E, Gulec S, Haznedaroglu IC, Akar N: Effects of ankaferd on huvec transcription factors and erythrocyte protein profile: Turkish Journal of Hematology 2011;28: 276-285 25. Esmon CT. The endothelial protein C receptor. Curr Opin Hematol 2006; 13(5): 382-385. 26. Akar N, Gokdemir R, Ozel D, Akar E. Endothelial cell protein c receptor (EPCR) gene exon III, 23 bp insertion mutation in the turkish pediatric thrombotic patients. Thromb Haemost 2002; 88:1068-1069. 27. Eroglu A, Ulu A, Kurtman C, Cam R, Akar N. 23-bp endothelial protein c receptor (EPCR) gene insertion mutation in cancer patients with and without thrombosis. Am J Hematol 2006;81:220. 28. Kendirli T, Ince E, Ciftci E, Dogru U, Egin Y, Akar N. Soluble endothelial protein c receptor level in children with sepsis. Pediatr Hematol Oncol 2009;26:432-438. 29. Orhon FS, Ergun H, Egin Y, Ulukol B, Baskan S, Akar N. Soluble endothelial protein C receptor levels in healthy population. J Thromb Thrombolysis 2010;29:46-51. 30. Ulu A, Gunal D, Tiras S, Egin Y, Deda G, Akar N. EPCR gene a3 haplotype and elevated soluble endothelial protein c receptor (sEPCR) levels in Turkish pediatric stroke patients. Thromb Res 2007;120:47-52. 31. Yalcindag FN, Batioglu F, Ozdemir O, Cansizoglu E, Egin Y, Akar N. Soluble endothelial protein c receptor levels in behcet patients with and without ocular involvement. Graefes Arch Clin Exp Ophthalmol 2008;246:1603-1608. 32. Sipahi T, Kara A, Kuybulu A, Egin Y, Akar N. Congenital Thrombotic Risk Factors in Beta-Thalassemia. Clin Appl Thromb Hemost 2009;15:581-584.

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33. Yürürer D, Teber S, Deda G, Egin Y, Akar N. The Relation Between Cytokines, Soluble Endothelial Protein C Receptor, and Factor VIII Levels in Turkish Pediatric Stroke Patients. Clin Appl Thromb Hemost 2009;15:545-551. 34. Ozturk MA, Ertenli I, Kiraz S, Haznedaroglu, IC, Celik I, Kirazli S, Calguneri M. Plasminogen activator inhibitor-1 as a link between pathological fibrinolysis and arthritis of Behcet’s disease. Rheumatol Int 2004;24:98-102. 35. Providence KM, Higgins PJ. PAI-1 expression is required for epithelial cell migration in two distinct phases of in vitro wound repair. J Cell Physiol 2004;200:297-308. 36. Oguzulgen IK, Yilmaz E, Demirtas S, Erkekol FO, Ekim N, Demir N, Numanoglu N, Ozel D, Ulu A, Akar N. The role of plasminogen activator inhibitor-1 polymorphism, factor-V-Leiden, and prothrombin-20210 mutations in pulmonary thromboembolism. Clin Appl Thromb Hemost. 2009;15:73-77. 37. Stewart I, Schluter PJ, Shaw GR. Cyanobacterial lipopolysaccharides and human health - a review. Environ Health 2006; 5:7. 38. Turgut M, Tutkun F, Çelebi N, Muğlalı M, Goker H, Haznedaroglu IC: Topical ankaferd bloodstopper in the management of critical bleedings due to hemorrhagic diathesis. UHOD Int J Hematol Oncol.2011;21: 160-165. 39. Kerschen EJ, Fernandez JA, Cooley BC, Yang XV, Sood R, Mosnier LO, Castellino FJ, Mackman N, Griffin JH, Weiler H. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exp Med 2007;204:2439-2448. 40. Angenete E, Langenskiold M, Palmgren I, Falk P, Oresland T, Ivarsson ML. Upa and PAI-1 in rectal cancer-relationship to radiotherapy and clinical outcome. J Surg Res 2009;153:4653. 41. Fabre-Guillevin E, Malo M, Cartier-Michaud A, Peinado H, Moreno-Bueno G, Vallee B, Lawrence DA, Palacios J, Cano A, Barlovatz-Meimon G, Charriere-Bertrand C. PAI-1 and functional blockade of snai1 in breast cancer cell migration. Breast Cancer Res 2008;10:100.

Research Article

DOI: 10.5505/tjh.2012.59254

Influence of Blood Collection Systems on Coagulation Tests Kan Alma Sistemlerinin Koagülasyon Testleri Üzerinde Etkileri Soner Yavaş1, Selime Ayaz2, S.Kenan Köse3, Fatma Ulus4, A.Tulga Ulus1 Türkiye Yüksek İhtisas Education and Research Hospital, Cardiovascular Surgery Clinic, Ankara, Turkey Türkiye Yüksek İhtisas Education and Research Hospital, Hematology Laboratory, Ankara, Turkey 3 Ankara University Faculty of Medicine, Department of Biostatistics, Ankara, Turkey 4 Atatürk Chest Disease and Thoracic Surgery Education and Research Hospital, Anesthesiology and Reanimation Clinic, Ankara, Turkey 1 2

Abstract Objective: Coagulation tests are influenced by pre-analytic conditions such as blood collection systems. Change of glass collection tubes with plastic ones will cause alteration of the test results. The aim of this study was to compare three plastic blood collection tubes with a standard glass blood collection tube and each plastic collection tube with the other two for possible additional tube-to- tube differences.

Material and Methods: A total of 284 blood samples were obtained from 42 patients receiving warfarin during their routine controls, besides 29 healthy volunteers. Subgroup analyses were done according to health status. Results: Our study demonstrated that different blood collection tubes have a statistically significant influence on coagulation tests. The magnitude of the effect depends on the tube used. However most of the tests performed on samples obtained from any tube correlated significantly with results obtained from other tube samples. Conclusion: Although blood collection tubes with different brands or properties will have distinct effects on coagulation tests, the influence of these blood collection tubes may be relatively small to interfere with decision-making on dose prescription, therefore lack clinical importance. Correlations between the results showed that, one of these plastic blood collection tubes tested in our study, can be used interchangably for a wide variety of coagulation assays. Key Words: Coagulation testing, Glass tubes, Plastic tubes

Özet Amaç: Koagülasyon testleri, farklı kan alma sistemleri gibi preanalitik durumlardan etkilenirler. Kullanılan cam tüplerin, plastik olanlarla değiştirilmesi, test sonuçlarında farklılık yaratabilir. Çalışmanın amacı 3 farklı plastik kan alma tüpünün, standart cam kan alma tüpü ile karşılaştırılmasıdır. Tüpler arası olası ek bir farklılığın saptanması açısından, plastik tüpler de ayrıca kendi aralarında karşılaştırıldı.

Gereç ve Yöntemler: Rutin kontrolleri sırasında varfarin tedavisi alan 42 hastadan ve 29 sağlıklı gönüllüden toplam 284 kan örneği alındı. Subgrup analizleri deneklerin sağlık durumlarına göre yapıldı.

Bulgular: Her ne kadar farklı marka veya özellikteki kan alma tüpleri koagülasyon testleri üzerinde belirgin etki yaratabilse de, bu etki doz ayarlamasını etkilemeyecek ve klinik önem arz etmeyecek kadar küçük olabilir. Sonuçlar Address for Correspondence: Soner YAVAŞ, M.D., Yüksek İhtisas Eğitim ve Araştırma Hastanesi, Kalp ve Damar Cerrahisi Kliniği, Ankara, Turkey Phone: +90 312 306 17 84 E-mail: drsoneryavas@gmail.com Received/Geliş tarihi : August 31, 2010 Accepted/Kabul tarihi : June 01, 2011

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Turk J Hematol 2012; 29: 367-375

arasındaki korelasyon, çalışmamızda kullanılan plastik kan alma tüplerinden herhangi birinin, bir çok koagülasyon testi için, diğerlerinin yerine kullanılabileceğini göstermektedir.

Sonuç: Çalışmamız farklı kan alma tüplerinin koagülasyon testleri üzerinde istatistiksel olarak anlamlı etkisi olduğunu gösterdi. Bu etkinin derecesi kullanılan tüpe göre değişmektedir. Fakat, herhangi bir tüpten alınan örneklerle gerçekleştirilen testlerin çoğunun, diğer tüplerden alınan örnekler ile belirgin korelasyon gösterdiği gözlendi. Anahtar Sözcükler: Koagülasyon testleri, Cam tüpler, Plastik tüpler Introduction Siliconized glass collection tubes have traditionally been used in the coagulation tests for the determination of International Normalized Ratio (INR), prothrombin time (PT) and coagulation factor levels.[1-5] However, the potential risk of sharp injury and biohazardous exposure due to broken glass during handling or centrifugation, rendered the need of newer plastic collection tubes and clinical laboratories gradually replaced glass collection tubes with plastic ones.[1-4] Since coagulation tests are influenced by pre-analytic conditions such as the blood collection systems, change of glass collection tubes with plastic ones raised concerns about the potential for in vitro activation of the clotting cascade, hence alteration of test results.[1-13] Therefore, we planned to compare three different plastic blood collection tubes with a standard glass blood collection tube which is used worldwide as in our hospital, to find out whether glass and plastic blood collection tubes have significantly different influence on coagulation tests, both in patients under oral anticoagulant therapy (OAT) and in healthy volunteers. Each plastic collection tube was also compared with each other for possible additional tube-to-tube differences. To the best of our knowledge, no study in current literature has also compared the plastic collection tubes at the same time. Patients and Methods A total of 284 blood samples were obtained from 42 patients receiving warfarin during their routine controls, as well as 29 healthy volunteers, upon approval from Education, Planning and Coordination Committee (Ethical Committee) in our hospital. Our study was performed according to the principles outlined in the appropriate version of 1964 Declaration of Helsinki and informed consent was obtained from each subject. Blood samples were taken using a 21G needle and BD Vacutainer reuseable standard size tube holder (Becton Dickinson, USA), allowing natural vacuum of tube to withdraw specimen into tube by clean venopuncture after 8-12

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hours of fasting and prior to the daily dose of warfarin in the patient group. The test tubes contained sodium citrate 3.2%, with a ratio of one part anticoagulant to nine parts whole blood. The precedure was completed when vacuum no longer continued to withdraw. All samples were obtained from peripheral arm veins. For each case, 1 tube of blood sample was collected into a glass collection tube and 3 additional samples were collected into 3 different plastic collection tubes, in a random order. Tubes were delivered in the laboratory, checked for adequate tube filling, and centrifuged for 10 minutes at 2,000 G to prepare plateletpoor plasma (<10,000 platelets per microliter). In keeping accordance with the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) guidelines, collection tubes were kept unopened at 18°C to 24°C before separation of cells from plasma. Hemolytic and/or lipemic samples were excluded. All samples were tested as fresh plasma and processed within 2 hours of collection to avoid the loss of activity of coagulation factors. All four samples from the same person were processed and analyzed at the same time. Patient demographics, including sex, age, primary diseases, and medication history were recorded. Blood collection tubes were grouped as; Tube Group I: BD Vacutainer® Citrate Tubes - Glass (BD Vacutainer®, 9NC Sodium Citrate, 3.2%, 4.5 mL, Becton, Dickinson and Company, UK.), Tube Group II: VACUETTE® Blood Collection Tubes (Vacuette® Coagulation Tubes, 9NC Sodium citrate, 3.2%, 4 mL, Greiner Bio-One GmbH, Germany.), Tube Group III: BD Vacutainer® Plus Plastic Citrate Tube (BD Vacutainer® 9NC Sodium Citrate, 3.2%, 2.7 mL, Becton, Dickinson and Company, UK.), Tube Group IV: BD Vacutainer® Plus Plastic Citrate Tube (BD Vacutainer® 9NC Sodium Citrate, 3.2%, 1.8 mL, Becton, Dickinson and Company, UK.). Subgroup analyses were done according to health status as demonstrated below: Group P: Patients receiving warfarin therapy (n= 42, 59.2%),

Turk J Hematol 2012; 29: 367-375

Yavaş S, et al : Blood Collection Tubes and Coagulation Tests

Group H: Healthy Volunteers (n= 29, 40.8%). Mean INR, PT%, PT, activated partial thromboplastin time (APTT), activated partial thromboplastin time ratio (APTT R) and fibrinogen levels were obtained both in healthy volunteers (Group H) and patients under OAT (Group P). D-Dimer, protein C, protein S, antithrombin, thrombin time, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, plasminogen, Alpha 2 antiplasmin, activated protein C resistance test (APCR), Lupus antibodies (Lupus Ab) and von Willebrand factor antigen (vWF Ag) were available only in healthy volunteers. Statistical Analyses The data were presented as mean and standard deviations, and percentage, where appropriate. Chi square test and Paired-Samples T test were used to compare patient characteristics and test values. Pearson correlation test was

utilized to determine the correlations between groups. Probability (p) values below 0.05 were considered significant. Confidence intervals (CI) were calculated at the 95% level. Correspondences of results between Tube Groups were undertaken using the Bland-Altman procedure, and BlandAltman plots were performed to assess the magnitude of disagreement between the results, plotting the mean of the results for the two methods on the x axis against the arithmetic or percentage difference on the y axis [14]. Results A total of 284 samples were obtained from 71 cases (29 men (40.8%), 42 women (59.2%)). Mean age of the patients was 44.4±14.6 years. Without any subgrouping; When all mean values were compared, INR, PT, APTT and fibrinogen results obtained in Tube Group II were different but not significantly from the other groups (Table 1).

Table 1: Comparison of tube groups without any subgrouping. Mean Values

p Values

I

II

III

IV

I vs II

I vs III

I vs IV II vs III II vs IV III vs IV

2.0±0.9

1.8±0.9

2.0±1.0

2.0±0.9

0.0001

0.594

0.297

0.0001

0.0001

0.194

PT% (%)

55.0±33.7

58.9±36.2

55.5±34.3

54.6±34.8

0.0001

0.060

0.612

0.0001

0.005

0.995

PT (Sec)

23.7±12.0

22.4±11.3

23.7±12.1

23.9±12.0

0.0001

0.372

0.344

0.0001

0.0001

0.511

APTT (Sec)

45.8±13.4

49.3±17.3

44.7±12.7

45.5±12.7

0.0001

0.002

0.176

0.0001

0.0001

0.038

APTT R (R)

1.4±0.3

1.6±0.5

1.4±0.3

1.4±0.3

0.0001

0.002

0.051

0.0001

0.0001

0.113

Fibrinogen (g/L)

3.1±0.8

3.1±0.9

3.1±0.8

3.1±0.8

0.010

0.556

1.000

0.004

0.002

0.432

0.3±0.3

0.7±1.8

0.2±0.1

0.2±0.1

0.207

0.367

0.407

0.169

0.175

0.695

0.730

0.356

0.067

0.310

0.087

0.019

INR (INR)

D Dimer (mg/L) # Protein C (%) #

127.4±29.0 125.9±30.3 132.7±34.8 115.4±39.9

Protein S (%) #

96.8±18.4

94.6±13.0

96.4±19.3

95.8±18.9

0.540

0.926

0.821

0.576

0.739

0.906

AntiThrombin (%) #

94.5±10.7

109.1±10.7

96.1±10.6

96.0±9.0

0.0001

0.373

0.309

0.0001

0.0001

0.939

18.0±1.5

19.7±2.2

18.0±4.0

20.1±5.0

0.0001

0.997

0.043

0.027

0.763

0.071

Factor V (%) #

Thrombin Time (Sec) #

115.5±27.8 135.4±29.3 118.6±29.1 114.7±26.3

0.002

0.640

0.788

0.008

0.003

0.441

Factor VII (%) #

104.8±26.0 121.7±42.6 107.4±28.3 107.3±32.5

0.001

0.489

0.422

0.022

0.005

0.984

Factor VIII (%) #

137.7±57.7 128.0±53.0 124.6±43.9 109.2±27.8

Factor IX (%) #

88.5±20.1

110.2±29.6

89.1±16.3

0.232

0.042

0.001

0.558

0.007

0.002

88.2±26.0

0.0001

0.843

0.961

0.0001

0.010

0.865

Factor X (%) #

100.6±16.7 115.9±23.2 102.5±17.0

95.4±20.3

0.0001

0.252

0.181

0.0001

0.0001

0.086

Factor XI (%) #

89.5±27.8

92.5±22.2

0.001

0.053

0.499

0.121

0.013

0.115

Factor XII (%) #

124.5±47.2 122.4±55.6 124.5±49.8 110.5±47.4

Plasminogen (%) #

103.8±18.4 112.6±17.6 104.8±20.4

104.3±28.5

Alpha 2 antiplasmin (%) # 106.1±19.4 107.7±17.8 APCR/ FVL (NR) #

1.2±0.1

1.0±0.3

98.2±15.6

0.898

0.106

0.281

0.983

0.162

0.134

94.5±14.8

0.0001

0.220

0.334

0.0001

0.0001

0.011

100.4±6.8

106.0±13.6

0.113

0.455

0.143

0.129

0.714

0.173

1.2±0.3

1.1±0.3

0.203

0.855

0.322

0.274

0.441

0.411

Lupus Ab (Sec) #

37.1±2.0

38.7±2.3

38.3±3.2

37.8±2.9

0.140

0.401

0.546

0.694

0.289

0.411

vWF Ag (%) #

93.1±21.7

104.7±25.3

94.4±23.6

91.9±22.3

0.0001

0.285

0.100

0.0001

0.0001

0.095

# Only in healthy volunteers not receiving warfarin. INR: International Normalized Ratio, PT: Prothrombin time, APTT: Activated Partial Thromboplastin Time, APTT R: Activated Partial Thromboplastin Time Ratio, APCR: Activated protein C resistance test, Lupus Ab: Lupus antibodies, vWF Ag: von Willebrand factor antigen.

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Table 2: Correlations and correspondences between tube groups without any subgrouping (Correlations/Correspondences).

INR (INR) PT% (%) PT (Sec) APTT (Sec) APTT R (R) Fibrinogen (g/L) D Dimer (mg/L) # Protein C (%) # Protein S (%) # AntiThrombin (%) # Thrombin Time (Sec) # Factor V (%) # Factor VII (%) # Factor VIII (%) # Factor IX (%) # Factor X (%) # Factor XI (%) # Factor XII (%) # Plasminogen (%) # Alpha 2 antiplasmin (%) # APCR # Lupus Ab (Sec) # vWF Ag (%) #

I vs II +/+/+/+/+/+/-/+/+ -/+/+ +/+ +/+ +/+/+ +/+ +/+/+ +/+ +/+ +/+ -/-/+/-

Pearson Correlations / Correspondences Between Groups I vs III I vs IV II vs III II vs IV III vs IV +/+ +/+ +/+/+/+ +/+/+ +/+/+ +/+ +/+ +/+ +/+/+/+ +/+/+ +/+/+/+ +/+/+ +/+/+/+/+ +/+ +/+/+ +/+ +/+ +/+ -/-/+/+ +/+ +/+ +/+ +/+ +/+ -/-/+/-/-/+/+ +/+ +/+ +/+ +/+ -/-/-/-/-/-/-/+/+ -/+/+ +/+ +/+ +/+/+/+ +/+/+/+ +/+/+/+ -/+/-/-/+/+ +/+ +/-/-/+/+/+ +/+/+ +/+ +/+ +/+ +/+ -/+/+ +/+ +/+ +/+ +/+ +/+ -/-/-/-/+/+ -/-/-/-/-/-/-/-/-/+/+ +/+ +/+ +/+ +/+/-

INR: International Normalized Ratio, PT: Prothrombin time, APTT: Activated Partial Thromboplastin Time, APTT R: Activated Partial Thromboplastin Time Ratio, APCR: activated protein C resistance test, Lupus Ab: Lupus antibodies, vWF Ag: von Willebrand factor antigen.

Pearson correlation test showed significant correlations between total mean values. Using Bland-Altman procedure, significant correspondences were also valid between Tube Group I, III and IV. (Table 2) Correspondences between mean INR values were shown in Figure 1 as BlandAltman plots. When mean D-Dimer, protein C, protein S, antithrombin, thrombin time, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, plasminogen, Alpha 2 antiplasmin, activated protein C resistance test (APCR), Lupus antibodies (Lupus Ab) and von Willebrand factor antigen (vWF Ag) values were compared, significant differences were observed between Tube Group II and the others, although some comparisons had significant correlations and correspondences (Table 1 and 2).

370

According to Group P and Group H; Demographic data of the patients were listed in Table 3. Since Group H consisted of young healthy volunteers, the only statistically significant difference was between mean ages (p= 0.0001). When mean values were compared, statistically significant differences existed especially between Tube Group II and the others (Table 4). When Pearson correlation test was performed, significant correlations were observed between mean values. But, Bland-Altman procedure showed poor correspondences between Tube Group II and the others (Table 5). Coagulation Factors were studied in Group H and variable results were obtained when mean values were com-

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YavaĹ&#x; S, et al : Blood Collection Tubes and Coagulation Tests

Figure 1: Correspondences between mean INR values according to Tube Groups without any subgrouping (Bland-Altman plots). Upper-Left: Tube Group I vs Tube Group II, Upper-Middle: Tube Group I vs Tube Group III, Upper-Right: Tube Group I vs Tube Group IV, Lower-Left: Tube Group II vs Tube Group III, Lower-Middle: Tube Group II vs Tube Group IV, Lower-Right: Tube Group III vs Tube Group IV. p< 0.05: No correspondence.

pared (Table 1 and 2). While some of the tests were affected by plastic blood collection tubes, others were not.

in turn may cause bleeding or thromboembolic complications.[15]

Discussion

Glass collection tubes are unblocked and have siliconized interior. On the other hand, plastic collection tubes have a double-wall technology (sandwich tubes – plastic within plastic) for reliable analysis results. The outer tube is made of polyethylene terephthalate and ensures a long shelf-life for the vacuum, while the inner tube is made out

Different blood collection tubes will have an influence on laboratory tests and may result in changes in therapeutic dosage adjustment in patients receiving warfarin therapy. This may result in overdosing or underdosing which

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Table 3: Demografic data according to Group P and H.

Group P 59.2% (n= 42) Group H 40.8% (n= 29) Gender Male Female Age (years) Warfarin dose (mg/week) # Duration of warfarin use (months) # DM # HT # HL # CRF # COPD # Valve Replacement # Atrial Fibrilation # DVT # Drugs # Warfarin Cardiac glycosides β Blockers ACEI ARB CCB ASA Insulin Oral antidiabetics H2 receptor antagonists Proton-pump inhibitors Diuretics Antihyperlipidemics Bronchodilators

p

37.5 (15) 64.3 (27) 52.5±12.9 32.8±16.8 57.2±59.7 9.5 (4) 31.0 (13) 7.1 (3) 2.4 (1) 4.8 (2) 88.1 (37) 2.4 (1) 9.5 (4)

48.3 (14) 51.7 (15) 32.7±7.2 -

0.290 0.0001 -

100.0 (42) 31.0 (13) 33.3 (14) 19.0 (8) 4.8 (2) 7.1 (3) 26.2 (11) 2.4 (1) 2.4 (1) 11.9 (5) 4.8 (2) 38.1 (16) 2.4 (1) 4.8 (2)

-

-

INR: International Normalized Ratio, DM: Diabetes mellitus, HT: Hypertension, HL: Hyperlipidemia, CRF: Chronic renal failure, COPD: Chronic obstructive pulmonary disease, DVT: Deep venous thrombosis, ACEI: Angiotensin converting enzyme inhibitors, ARB: Angiotensin receptor blockers, CCB: Calcium channel blockers, ASA: Acetyl salicylic acid.

of polypropylene and prevents the citrate solution from evaporating. Polypropylene is ideal for sensitive coagulation parameters, due to its inert characteristics.[1]

port has compared plastic collection tubes at the same time, we also focused on comparing each plastic collection tube with each other.

There are conflicting reports about the effects of plastic blood collection tubes on coagulation testing and most are limited to PT analyses.[1,5,12,16-23] Previous studies reported significant differences in thrombin time and PT test results.[1,5,12]

Our study demonstrated that different blood collection tubes had a statistically significant influence on coagulation tests. The magnitude of the effect depended on the tube used.

The aim of our study was to determine whether conversion to plastic tubes from glass tubes would result in significant differences in laboratory results. Since no re-

372

It has been suggested that INR differences below 10% do not seriously interfere with oral anticoagulant dosage regulation.[6,23] Our study showed that the mean INR values increased or decreased by the influence of differ-

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Yavaş S, et al : Blood Collection Tubes and Coagulation Tests

Table 4: Comparison of tube groups according to Group P and H.

Mean Values INR (INR) PT% (%) PT (Sec) APTT (Sec) APTT R (R) Fibrinogen (g/L)

Group P Group H Group P Group H Group P Group H Group P Group H Group P Group H Group P Group H

I 2.6±0.7*£ 1.0±0.1* 28.7±10.2*£ 93.0±12.2* 31.8±9.2*£ 12.0±0.8* 54.0±11.4*# 33.9±4.1 1.6±0.3*# 1.0±0.1 3.5±0.6* 2.4±0.4

II 2.5±0.7*$€ 0.9±0.1*$ 30.5±10.8*$€ 99.0±14.9*$€ 30.0±8.8*$€ 11.5±1.2*$e 59.8±15.2*$e 34.5±4.7 1.7±0.4*€ 1.0±0.1 3.7±0.7*€ 2.3±0.4

III 2.6±0.7$j 1.0±0.1$ 28.7±10.2$j 94.3±12.6$ 31.8±9.2$j 11.9±0.9$ 52.4±10.7#$j 33.5±3.6 1.5±0.3#j 1.0±0.1 3.6±0.6 2.3±0.4

IV 2.6±0.7£€j 1.0±0.1 29.0±10.3£€j 94.5±17.2€ 31.5±9.2£ej 12.1±1.7€ 53.0±10.7€j 34.0±3.3 1.5±0.3€j 0.9±0.1 3.6±0.7€ 2.3±0.4

INR: International Normalized Ratio, PT: Prothrombin time, APTT: Activated Partial Thromboplastin Time, APTT R: Activated Partial Thromboplastin Time Ratio. (P<0.05; *I vs II, #I vs III, £I vs IV, $II vs III, €II vs IV, jIII vs IV )

Table 5: Correlations and Correspondences Between Tube Groups according to Group P and H (Correlations/Correspondences).

INR (INR) PT% (%) PT (Sec) APTT (Sec) APTT R (R) Fibrinogen (g/L)

Group P Group H Group P Group H Group P Group H Group P Group H Group P Group H Group P Group H

Pearson Correlations+ and Correspondences Between Tube Groups I vs II I vs III I vs IV II vs III II vs IV III vs IV +/+/+ +/+ +/+/+/+ +/+/+ +/+/+ +/+ +/+ +/+/+ +/+ +/+/+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+/+ +/+ +/+/+/+ +/+/+ +/+ +/+ +/+ +/+ +/+/+ +/+ +/+/+/+ -/+/+ +/-/-/+/+ +/+/+ +/+ +/+/+/+ -/+/+ +/+ -/-/+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+

INR: International Normalized Ratio, PT: Prothrombin time, APTT: Activated Partial Thromboplastin Time, APTT R: Activated Partial Thromboplastin Time Ratio

ent blood collection systems. Mean INR value was lowest in Tube Group II. Values obtained in Tube Group II and IV were 4.1% and 0.3% lower, respectively, and in Tube Group III was 0.02% higher than Tube Group I. Although statistical differences not existed in INR Group I, III and IV, values were nearly equal in practice. Although, some of the statistical analyses showed no differences for some of the assays like Alpha 2 antiplasmin, APCR, protein S or Lupus Ab between different tubes,

poor correlations were observed. It is unclear why some of the tests would be affected by plastic blood collection methods while others would not. Type or International Sensitivity Index (ISI) of the reagent used, blood-tube surface interaction, the dynamic properties of the coagulation factors, use of individual vacuum tubes or clinically insignificant differences in tube blood volume (although all samples were checked for adequate filling) are some of the factors affecting the results.[1,2,5,7,8,10,11,12,24-26]

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YavaĹ&#x; S, et al : Blood Collection Tubes and Coagulation Tests

Besides, as seen in Tube Group II, different brands may also have distinct effects on coagulation tests. Tube Group II was the only group with a different brand, but differences related to Tube Group II did not reach clinical significance because the difference below 10% does not seriously interfere with oral anticoagulant dosage regulation. [6,23] Correlations between the results showed that, plastic blood collection tubes can be used in place of glass tubes or instead of the other plastic tubes for a wide variety of coagulation assays but the clinicians should be aware of the fact. The influence of blood collection tubes on a single coagulation analyzer using a single thromboplastin reagent with a constant ISI, may be insufficiently small to interfere with decision-making on dose prescription. On the other hand it must be kept in mind that probably the combination of multiple systematic variables such as different brands, reagents or analyzers may cumulatively lead to important INR differences. To reduce the resultant total error from system combinations, the influence of the blood collection systems may have to be eliminated. Detection of inter-laboratory multicenter calibration standards for the establishment of an international reference will also be helpful. It should be a note of caution that, when any laboratory plans to change their blood collection method, clinicians must be alerted about the new method. Actually, the best way to gain experience with a new blood collection method may be the use of both the old and new tubes simultaneously for an adaptation period to avoid unmeant trouble. Since analytical or statistical significance is only numerical data, the most important judgment is clinical experience based on patient-dose-response triangle. Conflict of Interest Statement None of the authors have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included. References 1. Gosselin RC, Janatpour K, Larkin EC, Lee YP, Owings JT. Comparison of samples obtained from 3.2% sodium citrate glass and two 3.2% sodium citrate plastic blood collection tubes used in coagulation testing. Am J Clin Pathol 2004;122:843-848.

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2. Fiebig EW, Etzell JE, Ng VL. Clinically relevant differences in prothrombin time and INR values related to blood sample collection in plastic vs glass tubes. Am J Clin Pathol 2005;124:902-909. 3. Toulon P, Ajzenberg N, Smahi M, Guillin MC. A new plastic collection tube made of polyethylene terephtalate is suitable for monitoring traditional anticoagulant therapy (oral anticoagulant, unfractionated heparin, and low molecular weight heparin). Thromb Res 2007;119:135-143. 4. Kratz A, Stanganelli N, Van Cott EM. A comparison of glass and plastic blood collection tubes for routine and specialized coagulation assays: A comprehensive study. Arch Pathol Lab Med 2006;130:39-44. 5. Flanders M, Crist R, Rodgers G. A comparison of blood collection in glass versus plastic vacutainers on results of esoteric coagulation assays. Lab Med 2003;34:732-735. 6. van den Besselaar AM, Hoekstra MM, Witteveen E, Didden JH, van der Meer FJ. Influence of blood collection systems on the prothrombin time and international sensitivity index determined with human and rabbit thromboplastin reagents. Am J Clin Pathol 2007;127:724-729. 7. Duncan EM, Casey CR, Duncan BM, Lloyd JV. Effect of concentration of trisodium citrate anticoagulant on calculation of the International Normalised Ratio and the International Sensitivity Index of thromboplastin. Thromb Haemost 1994;72:84-88. 8. Adcock DM, Kressin DC, Marlar RA. Effect of 3.2% vs 3.8% sodium citrate concentration on routine coagulation testing. Am J Clin Pathol 1997;107:105-110. 9. Danielson CF, Davis K, Jones G, Benson J, Arney K, Martin J. Effect of citrate concentration in specimen collection tubes on the International Normalized Ratio. Arch Pathol Lab Med 1997;121:956-959. 10. Chantarangkul V, Tripodi A, Clerici M, Negri B, Mannucci PM. Assessment of the influence of citrate concentration on the International Normalized Ratio (INR) determined with twelve reagent-instrument combinations.Thromb Haemost 1998; 80:258-262. 11. Palmer RN, Gralnick HR. Cold-induced contact surface activation of the prothrombin time in whole blood. Blood 1982;59:38-42. 12. Tripodi A, Chantarangkul V, Bressi C, Mannucci PM. How to evaluate the influence of blood collection systems on the international sensitivity index. Protocol applied to two new evacuated tubes and eight coagulometer/thromboplastin combinations. Thromb Res 2002;108:85-89. 13. Rapaport SI, Vermylen J, Hoylaerts M, Saito H, Hirsh J, Bates S, Dahlbäck B, Poller L. The multiple faces of the partial thromboplastin time APTT. J Thromb Haemost 2004;2:2250-2259.

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14. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307-10. 15. Schulman S. Clinical practice. Care of patients receiving longterm anticoagulant therapy. N Engl J Med 2003;349:675683. 16. van den Besselaar AM, Meeuwisse-Braun J, Witteveen E, van Meegen E. Effect of evacuated blood collection tubes on thromboplastin calibration. Thromb Haemost 1998;79:1062-1063. 17. van den Besselaar AM, Bertina RM, van der Meer FJ, den Hartigh J. Different sensitivities of various thromboplastins to two blood collection systems for monitoring oral anticoagulant therapy. Thromb Haemost 1999;82:153-154. 18. van den Besselaar AM, Chantarangkul V, Tripodi A. A comparison of two sodium citrate concentrations in two evacuated blood collection systems for prothrombin time and ISI determination. Thromb Haemost 2000;84:664-667. 19. D’Angelo G, Villa C. Measurement of prothrombin time in patients on oral anticoagulant therapy: Effect of two different evacuated tubes. Haematologica 1999;84:656-657. 20. Biron-Andréani C, Mallol C, Séguret F, Schved JF. Plastic versus siliconized glass tubes: Evaluation in current laboratory practice. Thromb Haemost 2000;83:800-801. 21. Foucher K, Toulon P, Schved JF, Polack B. Suggestions for an appropriate methodological approach for the validation of an evacuated blood collection tube made in plastic material including the effect of age. Thromb Haemost 2004;91:845847.

Yavaş S, et al : Blood Collection Tubes and Coagulation Tests

22. Ridyard J, Bhavnani M, Seal LH. Laboratory control of oral anticoagulant therapy: Preservation of prothrombin time specimens using a polypropylene collection system. Clin Lab Haematol 1998;20:369-372. 23. Poller L, Keown M, Chauhan N, van den Besselaar AM, Tripodi A, Shiach C, Jespersen J. European Concerted Action on Anticoagulation: A multicentre calibration study of WHO international reference preparations for thromboplastin, rabbit (RBT/90) and human (rTF/95). J Clin Pathol 2005;58:667-669. 24. Adcock DM, Kressin DC, Marlar RA. Minimum specimen volume requirements for routine coagulation testing: dependence on citrate concentration. Am J Clin Pathol 1998;109:595-599. 25. Peterson P, Gottfried EL. The effects of inaccurate blood sample volume on prothrombin time (PT) and activated partial thromboplastin time (aPTT). Thromb Haemost 1982;47:101-103. 26. Reneke J, Etzell J, Leslie S, Ng VL, Gottfried EL. Prolonged prothrombin time and activated partial thromboplastin time due to underfilled specimen tubes with 109 mmol/L (3.2%) citrate anticoagulant. Am J Clin Pathol 1998;109:754-757.

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Research Article

DOI: 10.5505/tjh.2012.57338

The Prognostic Significance of the Serum p53 Protein Concentration in Chinese Patients with Non-Hodgkin Lymphoma Non-Hodgkin Lenfomalı Çinli Hastalarda Serum p53 Proteini Konsantrasyonunun Prognostik Önemi Min Zhou, Ling Cen, Tao Chen, Rong Xiao, Jianhe Yang, Nai-ke Giang, Yan Zhang Changzhou Second People’s Hospital Affiliated Nanjing Medical University, Department of Hematology, China

Abstract Objective: To investigate the prognostic significance of cytogenetic abnormalities, staging, patient factors, and the serum p53 protein concentration in Chinese non-Hodgkin lymphoma (NHL) patients.

Material and Methods: The study included 43 patients with NHL that were identified between August 2003 and December 2008. Patient clinical characteristics patients were determined based on morphological, immunohistochemical, and cytogenetic analysis, and the serum p53 protein concentration was measured quantitatively. Results: Following conventional chemotherapy, the complete/partial remission (CR/PR) rate was significantly higher and overall survival (OS) was significantly longer in the patients with early-stage (stage I-II) lymphoma, normal karyotype, and a low serum p53 protein concentration than in those with advanced-stage (stage III-IV) lymphoma, cytogenetic abnormalities, and a high serum p53 protein concentration (≥0.35 U/mL). Bone marrow infiltration was also a predictor of poor response and OS. There weren’t any significant differences in disease remission between the male and female patients, older and younger patients (aged <70 years vs. ≥70 years), or B-cell lymphoma and T-cell lymphoma patients. Conclusion: Staging is an effective means of assessing the severity of NHL. Cytogenetic examination can provide useful information for diagnosis, staging, and prognostication. The serum p53 protein level may be a potential prognostic marker in patients with NHL. Key Words: Non-Hodgkin lymphoma, p53 protein, Karyotype

Özet Amaç: Çinli non-Hodgkin lenfoma (NHL) hastalarında sitogenetik anormallikler, evrelendirme, hasta faktörleri ve serum p53 proteini konsantrasyonunun prognostik önemini araştırmak. Gereç ve Yöntemler: Çalışmaya Ağustos 2003 ile Aralık 2008 arasında tanımlanan 43 NHL hastası dahil edildi. Hasta klinik özellikleri morfolojik, immünohistokimyasal ve sitogenetik analiz temelinde belirlendi ve serum p53 proteini konsantrasyonu kantitatif olarak ölçüldü. Bulgular: Geleneksel kemoterapi sonrasında erken dönem (evre I-II) lenfoma, normal karyotip ve düşük serum p53 protein konsantrasyonu olan hastalarda tam/kısmi remisyon oranı ileri dönem (evre III-IV) lenfoma, sitogenetik Address for Correspondence: Min ZHOU, M.D., Changzhou Second People’s Hospital Affiliated Nanjing Medical University, Department of Hematology, Changzhou 21300, China Phone: 0086-519-88119221 E-mail: zhoumin@medmail.com.cn Received/Geliş tarihi : April 1, 2011 Accepted/Kabul tarihi : March 29, 2012

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Zhou M, et al : Prognosis in Chinese Non-Hodgkin Lymphoma Patients

bozukluklar ve yüksek serum p53 proteini konsantrasyonu (≥0,35 U/mL) olan hastalara göreönemli ölçüde daha yüksek ve genel sağkalım önemli ölçüde daha uzundu. Kemik iliği infiltrasyonu da kötü cevap ve genel sağkalımın bir prediktörüydü. Erkek ve kadın hastalar, yaşlı ve genç hastalar (<70 yaş ve ≥70 yaş) veya B-hücreli lenfoma ve T-hücreli lenfoma hastaları arasında hastalık remisyonu açısından önemli farklar yoktu.

Sonuç: Evrelendirme NHL şiddetini değerlendirmenin etkin bir yoludur. Sitogenetik inceleme tanı, evrelendirme ve prognoz belirleme için faydalı bilgiler sunabilir. Serum p53 proteini düzeyi NHL hastalarında olası bir prognostik işaret olabilir. Anahtar Sözcükler: Non-Hodgkin lenfoma, p53 proteini, Karyotip Introduction Non-Hodgkin lymphoma (NHL) is a heterogeneous disease that is generally categorized as indolent or aggressive, based on the morphology and proliferation of cancerous cells [1,2]. Differences in clinical features and treatment responses are evaluated based on the morphological, histological, immunophenotypic, cytogenetic, genetic, and molecular features that affect and indicate the aggressiveness of the disease. Nonetheless, the critical events associated with disease progression remain poorly understood, and their prognostic relevance has yet to be fully validated. In addition, p53 protein is known to be a tumor suppressor that maintains genomic stability—either by inducing cell cycle arrest or apoptosis. Although some studies have proposed that p53 protein expression is involved in lymphomagenesis, no study has analyzed the serum p53 protein level in Chinese NHL patients. As such, the present study aimed to evaluate the prognostic significance of the karyotype detected at the time of diagnosis, staging, patient factors, and the serum p53 protein concentration, according to the complete/partial remission (CR/PR) rate and overall survival (OS) in a cohort of Chinese NHL patients. In addition, the potential relationships between these factors were also evaluated. Materials and Methods Patients Of the 43 Chinese NHL patients included in this study, 37 had B-cell neoplasm and 6 had T-cell neoplasm. All the patients were admitted to our hospital between August 2003 and December 2008. At the time of diagnosis the median age of the 30 male and 13 female patients was 51 years (range: 10-81 years). NHL was diagnosed according to the World Health Organization (WHO) classification system, based on morphological, immunophenotypical, and clinical features [3]. Initial clinical data for all patients were recorded, including age, sex, disease classification and stage, bone marrow infiltration, karyotype, serum p53 protein concentration, mortality, and follow-up. The

study protocol was approved by the Ethics Committee and was performed in accordance with the Declaration of Helsinki. All the patients provided written informed consent to participate in the study. Enzyme-linked immunosorbent assay and research into NHL Blood samples were collected from each patient. Peripheral blood and/or bone marrow aspirate smears stained with Wright-Giemsa were analyzed and classified according to the WHO classification for NHL [3]. Suspensions were collected from bone marrow aspirate material and cultured in RPMI 1640 medium supplemented with 20% fetal calf serum, penicillin-streptomycin, minimum essential medium vitamins, and glutamine at 37 °C for 24 h. Cells were exposed to colcemid overnight, followed by hypotonic treatment (KCL 0.075 M) for 30 min, and then fixation in ethanol and acetic acid (3:1). Chromosome analysis was based on reverse heat Giemsa (RHG)-banded metaphases. Chromosomal abnormalities were defined according to the 2005 International System for Human Cytogenetic Nomenclature [4]. The immunophenotype of each patient was confirmed via paraffin section immunoperoxidase, as previously described [5]. Serum samples were collected upon admission—prior to the administration of any treatment. Enzyme-linked immunosorbent assay (ELISA) for quantitative detection of human serum p53 protein was performed, according to the manufacturer’s instructions (Bender MedSystems, Germany). Stage-modified international prognostic index (IPI) The stage-modified IPI was designed according to the IPI for which the original Ann Arbor stage II was substituted by the Lugano staging system for NHL. Response criteria Response to treatment was evaluated based on imaging studies. Response criteria were defined according to International Working Group recommendations. CR was

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defined as the complete disappearance of all physical and radiological evidence of disease for ≼4 weeks. Biopsies of lymphomas were performed to confirm CR. Patients without CR at the end of treatment were considered treatment failures. OS was defined as the time from the date of diagnosis to the date of the final follow-up or time of death due to any cause. Statistical analysis Fisher’s exact test and logistic regression models were used to determine if any of the study parameters (age, sex,

Turk J Hematol 2012; 29: 376-384

disease classification and stage, bone marrow infiltration, karyotype, and serum p53 protein) were independent prognostic factors of the overall response rate (CR + PR). Survival data were analyzed according to the Kaplan-Meier method, and were compared using the log-rank test. Multivariate regression analysis using the Cox proportional hazard model was used to identify risk factors on OS. Data were analyzed using SPSS v.12.0. A P value less than 0.05 was considered statistically significant.

Table 1: Cytogenetic data.

Classification DLBCL FL SLL MZBL MCL BL AIL Ki-1 ALCL Adult T-cell lymphoma

Patients (n)

Chromosomal Abnormalities

15 7 6 5 3 1 3 2 1

6 (40%) t(14;18)(q32;q2l) 3 (42.9%) t(14;18)(q32;q21) 1 (16.7%) de1 13q14 2 (40%) t(11;18)(q21;q21) 2 (66.7%) t(11;14)(q13;q32) 1 (100%) t(8;14)(q24;q32) 2 (100%) +3, +5, and +X 1 (50%) t(2;5)(p23;q35) 1 (100%) inv(14)(q11;q32)

Table 2: Characteristics in nodal NHL patients, according to p53 protein expression and response to chemotherapy.

p53 Expression <0.35 U/mL (n = 20) n (%) Age (years) <70 >70 Sex Male Female Stage I-II III-IV T/B-cell T B p53 <5% >5%

378

Complete

>0.35 U/mL (n = 23) n (%)

CR + PR (n = 24) n

(%)

No CR + PR (n = 14) n (%)

10/29 3/14

(34.5) (21.4)

19/29 11/14

(64.5) (78.6)

15/25 9/13

(60.0) (69.2)

10/25 4/13

(40.0) (30.8)

9/30 5/13

(30.0) (38.5)

21/30 8/13

(70.0) (61.5)

17/26 7/12

(65.4) (58.3)

9/26 5/12

(34.6) (41.7)

6/18 8/25

(33.3) (32.0)

12/18 17/25

(66.7) (68.0)

11/16 13/22

(68.8) (59.1)

5/16 9/22

(31.2) (40.9)

1/6 14/37

16.7 37.8

5/6 23/37

(83.3) (62.2)

2/5 22/33

(40.0) (66.7)

3/5 11/33

(60.0) (33.3)

20/32 4/16

(62.5) (25.0)

12/32 12/16

(37.5) (75.0)

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Figure 1: DLBCL karyotypes: 47; XY; del(1)(q41); l(2;18)(p13;q23); t(3;13)(q27;q14); +5[3]/46; XY[47].

Results Cytogenetic findings Chromosomal abnormalities were observed in 19 patients and are summarized in Table 1. Abnormalities were taken into consideration if they were observed in a majority of abnormal cells or were known to be associated with a particular subtype of NHL. The main abnormalities showed a degree of association with the underlying diagnosis, although a few exceptions were apparent. The following translocations were observed: t(14;18)(q32;q2l) in 6 of 15 patients with diffuse large B-cell lymphoma (DLBCL); t(14; 18) in 3 of 7 patients with follicular lymphoma (FL); (q32;q21)t(11;14) in 2 of 3 patients with mantle cell lymphoma (MCL); t(11;18)(q21;q21) in 2 of 5 patients with Marginal zone b-cell lymphoma (MZBL); de1(13)(q14q31) in 1 of 6 patients with small lymphocytic lymphoma (SLL); t(8;14)(q24;q32) in the 1 patient

with Burkitt’s lymphoma (BL); +3, +5, and +X in 2 of 3 patients with angioimmunoblastic lymphoma (AIL); t(2;5) (p23;q35) in 1 of 2 patients with Ki-l+ anaplastic large-cell lymphoma (Ki-1 ALCL); inv(14)(q11;q32) in the 1 patient with adult T-cell lymphoma. Figure 1 shows the DLBCL karyotypes. In this case, the histology of the lymph node predominantly showed the presence of small-cleaved cells with sparsely spaced large cells (14;18) was detectable cytogenetically. p53 Expression and NHL In total, p53 protein expression <0.35 U/mL was observed in 1 of the 6 cases (16.7%) of T-cell NHL and in 14 of the 37 cases (37.8%) of B-cell NHL. According to REAL classification, p53 protein expression <0.35 U/mL was observed in 6 of the 15 DLBL patients, 3 of the 6 (50%) SLL patients, the 1 (100%) BL patient, 2 of the 6 (33.3%) peripheral T-cell lymphoma (PTCL) patients, 1 of the 3 (33.3%) MCL patients, 3 of the 7 (42.9%) FL patients, and

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Turk J Hematol 2012; 29: 376-384

Table 3: Fisher’s P values for risk factors associated with the overall response rate.

Factors Female vs. male Male Female Age (years) <70 ≥70 Classification B-cell T-cell Disease stage I-II III-IV BMI No Yes p53 protein level <0.35 U/mL >0.35 U/mL Karyotype Normal Abnormal

Patients (n) 30 13 29 14 37 6 18 25 25 18 20 23 24 19

CR + PR (%) 46.67 69.23 55.17 50.00 56.76 33.33 88.89 28.00 80.00 16.67 90.00 21.74 79.17 21.05

P 0.2025 1.0000 0.3929 0.0001 0.0001 0.0000 0.0002

BMI: Bone marrow infiltration; CR: complete remission; PR: partial remission.

Table 4: Results of multivariate analysis using a logistic regression model.

Factors Female vs. male Age Classification Disease stage BMI p53 protein level Karyotype

RC 1.5904 –0.0198 –1.2152 0.6504 –0.1346 –24.4950 –0.0383

OR 4.9055 0.9804 0.2967 1.9163 0.8741 0.0000 0.9624

SE 1.43217 0.03129 1.66742 0.94326 1.99277 11.06465 1.86852

P 0.950 0.548 0.285 0.650 0.468 0.017 0.505

BMI: Bone marrow infiltration; OR: odds ratio; RC: regression coefficients; SE: standard error.

1 of the 3 AIL patients (Table 2). There wasn’t a correlation between p53 protein expression status, and age, sex, disease stage, T/B-cell type, or IPI score. Analysis of short-term curative effect Following conventional chemotherapy, 9 patients achieved CR and 14 patients achieved PR. The overall response rate (CR + PR) was 53.49%. In all, 20 patients had disease progression during treatment. Significantly more patients with early-stage (stage I-II) lymphoma, normal karyotype, and a low p53 protein level had CR/PR than did those with advanced-stage (stage III-IV) lymphoma, cytogenetic abnormalities, and a high serum p53 protein level. Bone marrow infiltration was also a predictor of poor response, as indicated by Fisher’s exact test, although there wasn’t a significant difference in the rate of disease remission between male and female patients, younger and

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older patients (aged <70 vs. ≥70 years), or B-cell lymphoma and T-cell lymphoma patients. Table 3 summarizes patient characteristics and Fisher P values associated with the overall response rate. Advanced-stage, bone marrow infiltration, and cytogenetic abnormalities were observed to be adverse prognostic factors based on univariate analysis, but not according to multivariate analysis; however, the serum p53 protein level was an independent prognostic factor for disease remission, based on the logistic regression model. The most likely cause of this is the existence of multicollinear relationships (high degree of correlation [correlation coefficient >0.7] between these independent variables) between the 4 variables mentioned above. As such, tumor stage, karyotype, and bone marrow infiltration might be considered prognostic factors that affect the overall response

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Zhou M, et al : Prognosis in Chinese Non-Hodgkin Lymphoma Patients

rate and the p53 protein level. The results of multivariate analysis using the logistic regression model are summarized in Table 4. Analysis of long-term curative effect

Figure 2: OS curve for bone marrow infiltration and noninfiltration.

OS was defined as the time from the date of diagnosis to the last follow-up evaluation or time of death due to any cause. Median survival time (MST) was 33 months. Bone marrow infiltration, cytogenetic abnormalities, and a low serum p53 protein level were all strongly associated with OS (Figures 2-4). The log-rank P values for OS are shown in Table 5. Log-rank testing showed that being female had a positive effect on prognosis (P = 0.0107). OS was significantly longer in patients with early-stage (stage I-II) lymphoma than in those with advanced-stage (stage III-IV) lymphoma; however, there wasn’t a significant difference in OS between younger (<70 years) and older (≼70 years) patients, or between patients with T-cell and B-cell lymphoma. Multivariate analysis using the Cox proportional hazard regression model was performed to assess the effect of the factors mentioned above on OS (Table 6). The data showed that sex and the p53 protein concentration affected OS, whereas advanced-stage lymphoma, bone marrow infiltration, and cytogenetic abnormalities were not significant prognostic factors. Multicollinearity may be the most important reason, and therefore the 3 variables were still significantly associated with tumor-related OS. Discussion

Figure 3: OS curve for low p53 protein level and high p53 Protein level.

Figure 4: OS curve for normal and abnormal karyotypes.

A number of recurrent chromosomal abnormalities associated with histopathological subtypes and clinical outcomes have been identified in NHL [6]. Translocation t(14;18) and t(8;14) are strongly associated with FL and BL, t(11;14) is associated with MCL, and t(2;5) is associated with anaplastic large-cell lymphoma, and 3q27 abnormalities are associated with DLBCL [7]. Some chromosomal aberrations are associated with significantly poor prognosis; for example, rearrangement of 8q24, +7q, and del (13q) are independently associated with significantly shorter event-free survival in DLBCL, whereas del (13q) and +7q have a similar effect in DLBCL and BL [8]. In the present study 19 of the 43 NHL patients had cytogenetic abnormalities. It is difficult to assess the prognostic significance of individual cytogenetic alterations in a small patient cohort with a short follow-up period; however, the present findings strongly support the concept that chromosomal aberrations noted at the time of diagnosis are useful for predicting the overall response rate and/or OS using univariate and multivariable analysis.

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Table 5: Prognostic factors and log-rank P values associated with OS.

Factors Female vs. male Age (years) Classification Disease stage BMI p53 protein level Karyotype

Male Female <70 ≥70 B-cell T-cell I-II III-IV No Yes <0.35 ≥0.35 Normal Abnormal

Patients (n) 30 13 29 14 37 6 18 25 25 18 20 23 24 19

MST (months) 19.3 >33.0 33.0 12.9 33.0 18.0 >19.3 18 >33.0 12.9 >19.3 18.0 >33.0 18.0

P 0.0107 0.2150 0.5170 0.0000 0.0000 0.0001 0.0006

BMI: Bone marrow infiltration; MST: median survival time.

Table 6: Multivariate analysis of OS based on Cox regression model means.

Factors Female vs. male Age Classification Disease stage BMI p53 protein level Karyotype

RC

RR

SE

P

–3.423 0.979 –0.713 –1.868 1.831 6.557 –3.268

30.3 0.4 2.0 6.5 0.1 0.0 26.2

0.8 1.5 1.2 0.4 0.6 0.3 0.6

0.037 0.368 0.245 0.184 0.452 0.001 0.063

BMI: Bone marrow infiltration; RR: relative risk; RC: regression coefficients; SE: standard error.

Chromosomal abnormalities were also observed in some patients with normal morphological characteristics, suggesting that bone marrow aspirate smears from a single puncture site may be of limited value in determining if the bone marrow is involved. Chromosomal analysis could be used as a supplementary technique. Another advantage of cytogenetic analysis is its ability to detect balanced translocations that would otherwise be missed by some DNAbased techniques [9]. Thus, it is necessary to perform cytogenetic examination to obtain useful information for diagnosis, staging, and prognostication. Molecular analysis of chromosome rearrangements has resulted in identification of several genes directly implicated in the biology of NHL; however, only with cytogenetic analysis will it become possible to identify additional events that could lead to further characterization of these

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genetic transpositions. Studies based on chromosome painting and in situ hybridization with breakpoint-specific probes may help elucidate the nature of these complex translocations and the genes involved in the secondary events. A sequential study may also establish the relative importance of these rearrangements to different stages of the lymphoma-leukemia development process and define the roles of the additional abnormalities described herein. The p53 gene is a cancer suppressor gene located at chromosome 17p13.1. It encodes a 53-kDa nuclear phosphoprotein (p53 protein). It acts as a negative regulator of the cell cycle [10]. It has been shown that p53 protein is activated in response to DNA damage and oncogenic stress via 2 distinct signaling pathways involving kinase-mediated phosphorylation of p53 protein by ATM/CHK2/1 cascade and inhibition of MDM2 via p19Arf, which results in

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p53 protein stabilization [11-14]. Solid tumors, including non-small-cell lung cancer, breast cancer, colorectal cancer, osteosarcoma, bladder cancer, and prostate cancer, exhibit increased apoptosis in response to Advexin [15]. Because serum p53 protein can be found in a small percentage of normal controls, its presence is not considered a diagnostic marker of cancer. Whether its serum concentration should be used as a screening marker or a predictive factor for the development of cancer remains to be determined. It has been reported to be variably present in the sera of patients with various malignancies. Several investigators have reported that the mean preoperative serum concentration of p53 protein in patients with head and neck squamous cell carcinoma was significantly higher than that in healthy controls. Higher serum p53 protein levels were also observed in lung cancer, pancreatic carcinoma, and colorectal cancer patients than in normal controls [16-19]. In the present study p53 protein was observed in the sera of NHL patients, which confirmed its value as a marker of the p53 concentration in NHL. The present data show that over-expression of p53 protein (≥0.35 U/ mL) was associated with a lower CR/PR rate and poor OS. Although the following conclusion cannot be considered definitive because of the small sample size, the findings suggest that serum p53 protein may be a potential prognostic marker for NHL. Unlike staging, sex is not a conventional means of assessing disease severity, and there is no consistent concept of its role in the prognosis of NHL. The present data show that female gender was positively correlated with longer OS than male gender, but this gender difference must be investigated further by studies with larger samples. Conflict of Interest Statement None of the authors have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included. References 1. Fisher RI. Overview of non-Hodgkin’s lymphoma: Biology, staging, and treatment. Semin Oncol 2003; 30:3-9. 2. Evans LS, Hancock BW. Non-Hodgkin lymphoma. Lancet 2003; 362:139-146. 3. Diebold J, Jaffe ES, Raphael M, Warnke RA. Burkitt lymphoma. In: Jaffe ES, Harris NL, Stein H, Wardiman JW, eds. World Health Organization Classification of tumours of Haematopoietic and Lymphoid Tissues,Lyon, France:IARC Press 2001.

Zhou M, et al : Prognosis in Chinese Non-Hodgkin Lymphoma Patients

4. Brothman AR, Persons DL, Shaffer LG. Nomenclature evolution: Changes in the ISCN from the 2005 to the 2009 edition. Cytogenet Genome Res 2009; 127:1-4. 5. Sukpanichnant S, Sonakul D, Piankijagum A, Wanachiwanawin W, Veerakul G, Mahasandana C, Tanphaichitr VS, Suvatte V. Malignant lymphoma in Thailand. Changes in the frequency of malignant lymphoma determined from a histopathologic and immunophenotypic analysis of 425 cases at Siriraj Hospital. Cancer 1998; 83:1197-1204. 6. Donner LR. Cytogenetics of lymphoma: A brief review of its theoretical and practical significance. Cancer Genet Cytogenet 1997;94:20-26. 7. Ong ST, Le Beau MM. Chromosomal abnormalities and molecular genetics of non-Hodgkin’s lymphoma. Semin Oncol 1998;25: 447–460. 8. Poirel HA, Cairo MS, Heerema NA, Swansbury J, Aupérin A, Launay E, Sanger WG, Talley P, Perkins SL, Raphaël M, McCarthy K, Sposto R, Gerrard M, Bernheim A, Patte C. Specific cytogenetic abnormalities are associated with a significantly inferior outcome in children and adolescents with mature B-cell non-Hodgkin’s lymphoma: Results of the FAB/LMB 96 international study. Leukemia 2009; 23:323331. 9. Johnson NA, Al-Tourah A, Brown CJ, Connors JM, Gascoyne RD, Horsman DE. Pognostic Significance of Secondary Cytogenetic Alterations in Follicular Lymphomas. Genes Chromosomes Cancer 2008; 47(12):1038-1048. 10. Milner BJ, Allan LA, Eccles DM, Kitchener HC, Leonard RC, Kelly KF, Parkin DE, Haites NE. p53 mutation is a common genetic event in ovarian carcinoma. Cancer Res 1993;53:2128–2132. 11. Zhang Y, Xiong Y, Yarbrough WG. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 1998; 92:725–734. 12. Winrow CJ, Pankratz DG, Vibat CR, Bowen TJ, Callahan MA, Warren AJ, Hilbush BS, Wynshaw-Boris A, Hasel KW, Weaver Z, Lockhart DJ, Barlow C. Aberrant recombination involving the granzyme locus occurs in Atm-/- T-cell lymphomas. Hum Mol Genet 2005; 14:2671-2684. 13. Sanchez E, Chacon I, Plaza MM, Muñoz E, Cruz MA, Martinez B, Lopez L, Martinez-Montero JC, Orradre JL, Saez AI, Garcia JF, Piris MA. Clinical outcome in diffuse large B-cell lymphoma is dependent on the relationship between different cell-cycle regulator proteins. J Clin Oncol 1998; 16:1931-1939. 14. Xu Y, Baltimore D. Dual roles of ATM in the cellular response to radiation and in cell growth control. Genes Dev 1996; 10:2401–2410.

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15. Hawley RS, Friend SH. Strange bedfellows in even stranger places: The role of ATM in meiotic cells, lymphocytes, tumors and its functional links to p53. Genes Dev 1996; 10:2383–2388. 16. Sherr CJ. Tumor surveillance via the ARF-p53 pathway. Genes Dev 1998; 12:2984–2991. 17. Chada S, Menander KB, Bocangel D, Roth JA, Ramesh R. Cancer targeting using tumor suppressor genes. Front Biosci 2008; 13:1959–1967.

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18. Chow V, Yuen AP, Lam KY, Ho WK, Wei WI. Prognostic significance of serum p53 protein and p53 antibody in patients with surgical treatment for head and neck squamous cell carcinoma. Head Neck 2001; 23:286–291. 19. Luo JC, Zehab R, Anttila S, Ridanpaa M, HusgafvelPursiainen K, Vainio H, Carney W, De Vivo I, Milling C, Brandt-Rauf PW. Detection of serum p53 protein in lung cancer patients. J Occup Med 1994; 36:155-160.

Research Article

DOI: 10.5505/tjh.2012.48640

The Value of Serum Immunoglobulin Free Light Chain Assessment in Patients with Monoclonal Gammopathies and Acute Renal Failure Akut Böbrek Yetmezlikli Monoklonal Gammopatili Hastalarda Serum İmmunglobulin Hafif Serbest Zincirlerinin Değerlendirilmesinin Önemi Mustafa Cirit1, Atilla Üzüm1, Pınar Özen1, Banu A. Şentürk2, Giray Bozkaya3, Bahriye Payzin4, Orçun Ural5 İzmir Atatürk Training and Research Hospital, Department of Nephrology, İzmir, Turkey İzmir Atatürk Training and Research Hospital, Department of Biochemistry, İzmir, Turkey 3 İzmir Training and Research Hospital, Department of Biochemistry, İzmir, Turkey 4 İzmir Ataturk Training and Research Hospital, Department of Hematology, İzmir, Turkey 5 Manisa State Hospital, Department of Internal Medicine, Manisa, Turkey 1 2

Abstract Objective: Immunoglobulin free light chain (FLC) abnormalities are common in patients with monoclonal gammopathies and the kidneys are the most affected organs. Immunoassays that provide quantitative measurement of FLC in serum indicate monoclonal FLC production based on the presence of an abnormal FLC kappa:lambda (κ:λ) ratio. The aim of this study was to assess the utility of serum FLC measurement as a diagnostic tool for detecting plasma cell dyscrasias in comparison to standard assays, and to ascertain its sensitivity and specificity in patients with acute renal failure (ARF).

Material and Methods: Sera from 82 patients with ARF were assessed using serum protein electrophoresis (SPE), serum immunofixation electrophoresis (SIFE), and FLC measurement. The sensitivity and specificity of the FLC ratio in identifying which ARF patients had multiple myeloma (MM) was compared to those of SPE and SIFE. Results: Among the 82 patients with ARF, 7 were diagnosed as MM using SPE, SIFE, and bone marrow biopsy techniques. In total, 8 patients did not have a FLC κ:λ ratio that was within the published reference range (0:26-1:65); the FLC κ:λ ratio based on FLC measurement had a specificity of 96% and sensitivity of 71%, and positive and negative predictive values of 62.9% and 97.3%, respectively, for the diagnosis of MM. Conclusion: The sensitivity and specificity of the FLC κ:λ ratio for diagnosing MM in patients that presented with ARF were lower than those of SPE and SIFE. To further delineate the utility of the FLC κ:λ ratio additional prospective, well-designed large-scale studies are needed.

Key Words: Multiple myeloma, Acute renal failure, Immunoglobulin free light chain

Address for Correspondence: Mustafa CİRİT, M.D., İzmir Atatürk Eğitim ve Araştırma Hastanesi, Nefroloji Bölümü, İzmir, Turkey Phone: +90 232 244 44 44 E-mail: mustafacirit@yahoo.com Received/Geliş tarihi : March 14, 2011 Accepted/Kabul tarihi : August 10, 2011

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Özet Amaç: Monoklonal gammopatili hastalarda immunglobulin hafif serbest zincir anormalliklerine sık rastlanır ve bu hastalarda böbrekler en sık etkilenen organlardan biridir. Serum immunglobulin hafif serbest zincir düzeylerinin sayısal ölçümünü sağlayan yöntemlerle, monoklonal immunglobulin hafif serbest zincir üretiminin varlığı anormal κ/λ oranlarına dayanmaktadır. Bu çalışmada akut böbrek yetmezliği ile başvuran hastalarda serum hafif serbest zincir analizinin plazma hücre diskrazilerinin saptanmasında tanısal değerinin standart yöntemlerle karşılaştırılarak, spesifite ve sensitivitesinin değerlendirilmesi amaçlanmıştır. Gereç ve Yöntemler: Akut böbrek yetmezliği ile başvuran 82 hasta serum protein elektroforezi, serum immunfiksasyon elektroforezi ve hafif serbest zincir ölçüm yöntemleriyle değerlendirildi. Akut böbrek yetmezlikli multipl myelom tanılı hastalarda hafif serbest zincir oranının sensitivite ve spesifitesi serum protein elektroforezi, serum immunfiksasyon elektroforez yöntemleriyle kıyaslandı. Bulgular: Akut böbrek yetmezliği ile başvuran 82 hastanın 7’sinde serum protein elektroforezi, immunfiksasyon elektroforezi ve kemik biopsisi ile multipl myelom tanısı kondu. Hastaların 8’inde κ/λ oranı yayınlarda bildirilen referans değerlerinin (0.26-1.65) dışında saptandı; serbest hafif zincir ölçümlerine dayanarak κ/λ oranı myelom tanısı için %96 spesifite,%71 sensitivite, %62.9 pozitif prediktif değer ve%97.3 negatif prediktif değere sahipti.

Sonuç: Akut böbrek yetmezlikli multipl myelomlu hastalarda serum protein elektroforezi, serum immunfiksasyon elektroforez yöntemleriyle kıyaslandığında κ/λ hafif serbest zincir oranları düşük sensitivite ve spesifite değerine sahipti. Bu yöntem için daha doğru yorum yapabilmek için daha iyi dizayn edilmiş, prospektif ve çok sayıda hasta içeren çalışmalara ihtiyaç vardır. Anahtar Sözcükler: Multipl myelom, Akut böbrek yetmezliği, İmmunglobulin serbest hafif zinciri Introduction Multiple myeloma (MM) is a malign disease characterized by abnormal proliferation of plasma cells. Monoclonal immunoglobulin secreted by these cells can be quantitatively measured to monitor the disease course [1,2]. The kidneys are the most commonly affected organs in patients with MM. Impaired renal function is present in 22%-43% of MM patients and is associated with survival. The most common pathogenetic lesion that causes renal failure is the so-called myeloma kidney, which is the result of light chain cast nephropathy, and occurs in approximately 65% of patients with myeloma. Among patients diagnosed as myeloma, 25% present with acute renal failure (ARF) [36]. Because renal dysfunction has an impact on survival, early diagnosis is critically important in patients with MM, as it can help prevent renal damage. Until recently, serum free light chain (FLC) measurement has been used for the diagnosis and screening of monoclonal gammopathy, light chain amyloidosis (AL), and MM [7-10]. Determination of monoclonal FLC production is based on the presence of an abnormal FLC kappa:lambda (κ:λ) ratio (reference range: 0:26-1:65) [11]. The aim of the present study was to determine the utility of serum FLC measurement as a diagnostic tool for detecting plasma cell dyscrasias, as compared with standard assays, and to determine its sensitivity and specificity in patients with ARF.

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Materials and Methods Study design and participants The study protocol was approved by the Izmir Ataturk Training Hospital Ethics Committee. The study included 82 patients with ARF that were admitted to hospital between May 2008 and December 2008. Exclusion criteria were age <50 years, renal ultrasonography findings indicative of obstruction and chronic kidney disease, diabetic nephropathy, polycystic kidney disease, pregnancy, malignancy, and collagen tissue diseases. Renal ultrasonography, renal function testing, serum protein electrophoresis (SPE), serum immunofixation electrophoresis (SIFE), and FLC measurement were performed in all the patients. If indicated, bone marrow aspiration and biopsy were performed. We estimated the glomerular filtration rate (GFR) using the Modified Diet in Renal Disease (MDRD) equation. The decision to initiate dialysis was made by a consultant nephrologist based on the following indications: uremia, hyperkalemia, metabolic acidosis, and fluid overload (defined as an estimated GFR [eGFR] <15 mL·min–1·1.73 m–2, according to the MDRD equation). Diagnosis of MM was made by a consultant hematologist in accordance with international diagnostic criteria. ARF was diagnosed based on medical history, physical examination, biochemical parameters, AKIN criteria, and ultrasound findings. Detailed ARF causes cannot be stated because of inconvenience of renal biopsy application.

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Laboratory assessment After a 12-h fasting period, venous blood samples were obtained from the participants and serum was separated via centrifugation at 3000 rpm for 15 min. Samples were stored in small aliquots at –200 °C until analyzed. The serum free light chain concentration was measured using a commercially available latex-enhanced immunoassay kit (Freelite® Human Lambda/Kappa Free Kit, The Binding Site Ltd., Birmingham, UK) and an Olympus AU640 autoanalyzer (Olympus, Tokyo, Japan). The assay consists of 2 separate measurements: one quantifies λκ FLC and to the other quantifies κ FLC. In addition to measuring λ and κ FLC, the assay determines the FLC κ:λ ratio (the reference ranges used [0:26-1:65] were supplied by the manufacturer). Patients with an FLC κ:λ ratio >1:65 have excess κ FLC and are presumed to be producing clonal λ FLC. Patients with a FLC κ:λ ratio <0:26 have excess λ FLC and are presumed to be producing clonal λ FLC. SIFE was performed using a Hydrasys Automate, according to the manufacturer’s instructions, and Hydragel 15 and 30 protein(e), and Hydragel 2 and 4 immunofixation gels (Sebia, Lisses, France). Serum BUN and creatinine levels were determined using photometric methods, an Olympus AU 640 autoanalyzer (Olympus, Tokyo, Japan), and commercial kits. Table 1: Baseline patient demographic and biochemical data.

Mean

Std

Deviation

Min-Max

Age (years)

69.9

9.35

47–87

BUN (mg dL–1)

67.9

28.61

22-162

4.6

2.63

1.8-12.8

16.3

9.61

3-39

Creatinine (mg dL–1) eGFR

A Toshiba Famia S ultrasound machine was used to assess the urinary system in all patients. Renal parenchymal echogenicity and urinary tractus were assessed. If indicated, bone marrow aspiration and biopsy were performed; samples were fixed with 10% formalin and stained with Hematoxylin-Eosin, and then histopathological evaluation was performed via light microscopy. Statistical analysis Statistical analysis was performed using SPSS v.15.0. Patient groups were compared using the Mann-Whitney U test, Student’s t test, and chi-square test. Pearson’s correlation test was used for correlation analysis. Statistical significance was set at a P value of less than 0.05. Results Patient baseline demographic and biochemical data are shown in Table 1. Mean age of the patients was 69 ± 9 years; 53.7% were male. Renal ultrasonography findings were normal in all the patients. Among the 82 patients that presented with ARF—confirmed based on medical history, and laboratory and ultrasonographic findings—7 were diagnosed as MM via SPE, SIFE, and bone marrow biopsy findings. Among the 8 patients that did not have an FLC κ:λ ratio within the published reference range (0:261:65), 5 had an FLC κ:λ ratio >1:65 and 3 had an FLC κ:λ ratio <0:26 (Figure). The FLC κ:λ ratio based on FLC measurement had a specificity of 96% and sensitivity of 71%, and positive and negative predictive values of 62.9% and 97.3%, respectively, for diagnosing MM. Characteristics of the patients with an abnormal FLC κ:λ ratio are shown in Table 2. Among the 5 patients that had an FLC κ:λ ratio >1:65, 2 were diagnosed as MM, whereas 3 of the patients with an FLC κ:λ ratio <0:26 were diagnosed as MM. Characteristics of patients that had a normal FLC κ:λ ratio and were diagnosed as MM (false

Table 2: Characteristics of patients with an abnormal FLC k:λ ratio.

Patients

Diagnosis

Age (years)

Gender

eGFR

US

κ

λ

κ:λ

SPE

SIFE

1 2 3 4 5 6 7 8

MM MM Other Other Other MM MM MM

75 80 80 61 77 69 74 78

M M F F M M F M

9 6 5 15 8 15 12 13

Normal Normal Normal Normal Normal Normal Normal Normal

24,3 12.5 17.2 18.5 25.7 8.9 5.1 1.8

11,3 6.6 8.7 10.9 15.4 52.5 45.2 418

2:3 1:9 2:0 1:7 1.7 0:2 0:1 0:01

+ + + + +

IgG κ IgG κ Normal Normal Normal IgG λ FLC λ IgG λ

US: Ultrasonography.

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Figure: Flow diagram of FLC measurement results and MM diagnoses.

negative) are shown in Table 3. In all, 1 of 2 patients with normal SPE and SIFE findings was diagnosed as MM based on bone marrow biopsy findings and had normal FLC measurements indicating the diagnosis of non-secretory myeloma, whereas the other patient had a normal FLC κ:λ ratio and was as diagnosed as MM based on SPE, SIFE, and bone marrow biopsy findings. Table 4 shows the comparison between the patients with and without MM. Age, BUN, creatinine, λ, and the FLC κ:λ ratio did not differ between the 2 groups, but the κ value was significantly different. When considering SPE and SIFE findings together, the specificity, sensitivity,

and positive and negative predictive values were higher than those for the FLC κ:λ ratio and SPE+FLC κ:λ ratio co-assessment (Table 5). Correlation analysis between age, eGFR, FLC κ, and FLC λ values showed that there was a positive correlation between FLC κ and λ values (P = 0.027, r = 0.87) and a negative correlation between eGFR and FLC κ values (P = 0.027, r = –0.25). The 2 patient groups assessed according to eGFR as 15ml/min after exclusion of MM patients. There wasn’t a difference between FLC λ and the FLC κ:λ ratio, but BUN, creatinine, and FLC κ values significantly differed between the 2 patient groups, with respect to eGFR (Table 6). Pearson’s correlation analysis showed that creatinine was positively correlated with FLC κ (P = 0.019, r = 0.27) and FLC λ (P = 0.018, r = 0.272) levels, and eGFR was inversely correlated with FLC κ (P = 0.023, r = –0.262) and FLC λ (P = 0.034, r = –0.246) levels. Furthermore, the FLC κ level was positively correlated with FLC λ (p<0.001, r = 0.908) and the FLC κ:λ ratio (p<0.001, r = 0.543). Discussion Recently, an assay for serum FLC became commercially available for use as a marker for diagnosing and screening monoclonal gammopathies [7-10]. There are 3 indications for using serum FLC to diagnose and screen MM and related diseases. First, screening serum using SPE and SIFE with FLC levels is highly sensitive and eliminates the necessity of using 24-h urine collection for the diagnosis

Table 3: Patients with a normal FLC k:λ ratio diagnosed as MM (false negative cases).

Patients 1* 2

Diagnosis MM MM

Age (years) 69 57

Gender M F

eGFR 22 4

USG Normal Normal

κ 5.6 6.5

λ 4.9 6.4

κ:λ 1:1 1:0

SPE – +

SIFE Normal IgG κ

*Patient 1 was diagnosed as having non-secretory myeloma

Table 4: Comparison of the patients with and without MM.

Age (years) BUN (mg dL–1) Creatinine (mg dL–1) eGFR κ λ κ/λ

388

MM (+) mean 71.7 61.6 5.8 11.6 9.2 77.8 0.9

n=7 SD 7.7 20.8 2.9 6.0 7.4 151.3 0.9

MM (–) mean 69.7 68.5 4.5 16.8 15.6 12.4 1.2

n = 75 SD 9.5 29.3 2.6 9.8 6.7 4.7 0.2

P 0.67 0.85 0.12 0.22 *0.024 0.84 0.21

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Table 5: The sensitivity, specificity, and positive and negative predictive values for SPE+SIFE, the FLC k:λ ratio, and the FLC k:λ ratio+SPE.

Positive Predictive Value Negative Predictive Value Specificity Sensitivity

FLC κ:λ Ratio 62.9% 97.3% 96% 71%

SPE+SIFE 100% 98.7% 100% 86%

SPE+FLC κ:λ Ratio 100% 97.4% 100% 71%

Table 6: Patients assessed according to eGFR, after exclusion of MM patients.

Age (years) BUN (mg dL–1) Creatinine (mg dL–1) κ λ Κ:λ

Group 1 (eGFR < 15 mL min–1) n = 39 Mean SD 68.8 8.8 75.5 25.1 6.2 2.4 17.1 7.1 13.4 5.1 1:3 0:3

of related diseases, except for light chain amyloidosis. Second, FLC assessment has prognostic importance for all plasma cell dyscrasias. Third, FLC assay provides insight into the screening of oligo-secretory plasma cell dyscrasias, quantitatively [9,12,13]. Most kidney disease associated with monoclonal gammopathies is caused by monoclonal FLCs, which is a consequence of the kinetics of FLC clearance from the serum by the kidneys. As such, any structural and functional abnormalities in the kidneys and/or excess production of FLCs can lead to deposition and precipitation in situ [8]. The mechanisms involved in the nephrotoxicity of monoclonal FLCs are activation of inflammatory mediators in proximal tubules, proximal tubule necrosis, acquired Fanconi syndrome due to FLC deposition, cast nephropathy, light chain amyloidosis, and light chain deposition disease [14,15]. There are limited data concerning the use FLC assays in patients that present with renal disease. In general, as renal clearance decreases, FLC κ and λ increase, but no change occurs in the FLC κ:λ ratio; however, different reference ranges must be evaluated in patients with renal disease in order to increase the specificity and sensitivity. Some

Group 2 (eGFR > 15 mL min–1) n = 36 Mean 70.7 60.9 2.5 13.8 11.3 1:2

P SD 10.2 31.8 0.5 5.8 4.0 0.2

0.38 *0.031 <0.001 *0.032 0.05 0.37

patients with plasma cell dyscrasias may present with renal pathologies, and delayed identification and treatment leads to irreversible renal failure [8,10]. The present study aimed to evaluate the sensitivity and specificity of the FLC κ:λ ratio for detecting paraproteinemias in patients that presented with ARF. Paraproteinemias are more prevalent in older populations; therefore, we included patients aged >50 years, and because of elevated FLC levels in patients with diabetes, liver diseases, and collagen tissue diseases, such patients were excluded. It has been reported that 24-h urine specimens can be obtained in 50% of patients [16,17]; however, in the present study urine specimens could not be evaluated because the patients presented with oligo and anuria. Studies have reported that cast nephropathy is the predominant cause of renal failure in patients with paraproteinemia that present with ARF [14,15]. In the present study, however, histologic types of ARF in the patients with and without MM could not be determined due to hemorrhagic diathesis and other medical conditions that contraindicated renal biopsy. Based on the published reference range for the FLC κ:λ ratio (0:26-1:65), it had a sensitivity of 71% and specificity of 96%, and a positive and negative predictive value of

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62.9% and 97.3%, respectively, for the diagnosis of MM. In the present study there weren’t any significant differences in the sensitivity, specificity, or positive and negative predictive values between the FLC κ:λ ratio and SPE. Hutchison et al. reported that based on the published reference range, the sensitivity of the FLC κ:λ ratio was 100% and its specificity was 93% [17]. The specificity of the FLC κ:λ ratio increased to 99% with no change in sensitivity when the renal reference range used was 0:373:1. Moreover, based on the published reference range Katzmann et al. reported that the FLC κ:λ ratio has a sensitivity of 97%, specificity of 100%, and positive and negative predictive values of 100% and 99%, respectively [11]. The authors used nephelometric methods in these reports. Yet, Jaslowski et al. [18] reported that the FLC κ:λ ratio has lower sensitivity than SIFE (91.4% versus 72%) when turbidimetric methods are used, as in the present study. They examined 483 consecutive serum samples with abnormal SIFE findings and reported some of the samples had a normal FLC κ:λ ratio. As such, they suggest that in routine examination the FLC κ:λ ratio has lower diagnostic value. In the present study we also observed low specificity and sensitivity with turbidimetric methods; however, the present study population was small and this method was used firstly in our hospital. FLC measurement is accepted as sufficiently sensitive for diagnosing non-secretory myeloma [19]; however, in the present study patients with MM confirmed via bone marrow biopsy findings that had normal SPE and SIFE findings also had a normal FLC κ:λ ratio (1:14). Despite the advantages of FLC assays, they do have some limitations, including reagent lot variability, low ratios due to high antigen levels, innumerable epitopes, and excess polymerization [20,21]. Elevated FLC κ and λ levels correlate strongly with declining kidney function [10]. To address the effect of renal function on FLC levels, patients assessed according to GFR as below and above to eGFR as 15 mL·min–1·1.73 m–2. κ levels had a statistical significance between two groups but λ and κ/λ values had no significance. Based on Pearson’s correlation analysis in the present study, creatinine was positively correlated with FLC κ (P = 0.019, r = 0.27) and λ (P = 0.018, r = 0.272) levels, and eGFR was inversely correlated with FLC κ (P = 0.023, r = –0.262) and λ (P = 0.034, r = –0.246) levels. These results are consistent with other reports that emphasize the correlation between FLC levels and renal function [10]. The present study included only 82 patients and FLC levels were assessed using turbidimetric methods. The FLC κ:λ

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ratio had lower sensitivity and specificity than SPE and SIFE in the patients with MM that presented with ARF. In conclusion, additional, well-designed large-scale prospective studies are required to further delineate the diagnostic utility of the FLC κ:λ ratio. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. References 1. Kyle RA. Sequence of testing for monoclonal gammopathies: Serum and urine assays. Arch Pathol Lab Med 1999;123:114118. 2. Smith A, Wisloff F, Samson D. Guidelines on the diagnosis and management of multiple myeloma 2005. Br J Haematol 2005;132:410-451 3. Kyle RA. Multiple myeloma: Review of 869 cases.Mayo Clin Proc 1975;50:29-40. 4. Bladé J, Fernández-Llama P, Bosch F, Montolíu J, Lens XM, Montoto S, Cases A, Darnell A, Rozman C, Montserrat E. Renal failure in multiple myeloma: presenting features and predictors of outcome in 94 patients from a single institution. Arch Intern Med 1998;158:1889-1893. 5. Knudsen LM, Hjorth M, Hippe E. Renal failure in multiple myeloma: reversibility and impact on the prognosis. Nordic Myeloma Study Group. Eur J Haematol 2000;65:175-181 6. Dimopoulos MA, Kastritis E, Rosinol L, Blade J, Ludwig H. Pathogenesis and treatment of renal failure in multiple myeloma. Leukemia 2008;22:1485-1493. 7. Pratt G. The evolving use of serum-free light chain assays in haematology. Br J Haematol 2008;141:413-422. 8. Hutchison CA, Basnayake K, Cockwell P. Serum free light chain assessment in monoclonal gammopathy and kidney disease. Nat Rev Nephrol 2009;5:621-628. 9. Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, Hajek R, Palumbo A, Jagannath S, Blade J, Lonial S, Dimopoulos M, Comenzo R, Einsele H, Barlogie B, Anderson K, Gertz M, Harousseau JL, Attal M, Tosi P, Sonneveld P, Boccadoro M, Morgan G, Richardson P, Sezer O, Mateos MV, Cavo M, Joshua D, Turesson I, Chen W, Shimizu K, Powles R, Rajkumar SV, Durie BG. International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia 2009;23:215-224.

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10. Hutchison CA, Harding S, Hewins P, Mead GP, Townsend J, Bradwell AR, Cockwell P. Quantitative assessment of serum and urinary polyclonal free light chains in patients with chronic kidney disease. Clin J Am Nephrol 2008;3:16841690. 11. Katzmann JA, Clark RJ, Abraham RS, Bryant S, Lymp JF, Bradwell AR, Kyle RA. Serum reference intervals and diagnostic ranges for free Îş and free Îť immunoglobulin light chains: Relative sensitivity for detection of monoclonal light chains. Clin Chem 2002;48:1437-1444. 12. Katzmann JA, Dispenzieri A, Kyle RA, Snyder MR, Plevak MF, Larson DR, Abraham RS, Lust JA, Melton LJ 3rd, Rajkumar SV. Elimination of the need for urine studies in the screening algorithm for monoclonal gammopathies by using serum immunofixation and free light chain assays. Mayo Clin Proc 2006;81:1575-1578. 13. Tate JR, Gill D, Cobcroft R, Hickman PE. Practical consideration for the measurement of free light chains in serum. Clin Chem 2003;49:1252-1257 14. Herrera GA. Renal Lesions Associated With Plasma Cell Dyscrasias: Practical Approach to Diagnosis, New Concepts, and Challenges. Arch Pathol Lab Med 2009;133:249-267 15. Gu X, Herrara GA. Light-chain-mediated acute tubular interstitial nephritis: A poorly recognised pattern of renal disease in patients with plasma cell dyscrasia. Arch Pathol Lab Med 2006;160:165-169.

Cirit M, et al : Free Light Chains Acute Renal Failure

16. Hill PG, Forsyth JM, Rai B, Mayne S. Serum free light chains: An alternative to the urine Bence Jones proteins screening test for monoclonal gammopathies. Clin Chem 2006;52:1743-1748. 17. Hutchison CA, Plant T, Drayson M, Cockwell P, Kountouri M, Basnayake K, Harding S, Bradwell AR, Mead GP. Serum free light chain measurement aids the diagnosis of myeloma in patients with severe renal failure. BMC Nephrol 2008;9:11. 18. Jaskowski TD, Litwin CM, Hill HR. Detection of kappa and lambda light chain monoclonal proteins in human serum: automated immunoassay versus immunofixation electrophoresis. Clin Vaccine Immunol 2006; 13:277-280. 19. Drayson M, Tang LX, Drew R, Mead GP, Carr-Smith H, Bradwell AR. Serum free lightchain measurements for identifying and monitoring patients with nonsecretory multiple myeloma. Blood 2001;97:2900-2902. 20. Tate JR, Mollee P, Dimeski G, Carter AC, Gill D. Analytical performance of serum free light-chain assay during monitoring of patients with monoclonal light-chain diseases. Clin Chim Acta 2007 376:30-36. 21. Daval S, Tridon A, Mazeron N, Ristori JM, Evrard B. Risk of antigen excess in serum free light chain measurements. Clin Chem 2007;53:1985-1986.

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Case Report

DOI: 10.5505/tjh.2012.50490

Corticosteroid-Responsive Pulmonary Toxicity Associated with Fludarabine Monophosphate: A Case Report Fludarabin Monofosfat ile İlişkili Kortikosteroide Cevap Veren Pulmoner Toksisite: Bir Olgu Sunumu Milda Rudzianskiene, Rasa Griniute, Elona Juozaityte, Arturas Inciura, Viktoras Rudzianskas, Greta Emilia Kiavialaitis Lithuanian University of Health Sciences Hospital, Kauno Klinikos, Oncology and Hematology Department, Kaunas, Lithuania

Abstract Fludarabine monophosphate is an effective drug for the treatment of lymphoid malignancies. Myelosuppression, opportunistic infections, and autoimmune hemolytic anemia are the most common side effects of fludarabine. Herein we report a 55-year-old female that presented with fever and dyspnea after completing her third cycle of FMD (fludarabine, mitoxantrone, and dexamethasone) chemotherapy for stage IV non-Hodgkin follicular lymphoma. Chest X-ray revealed bilateral pneumofibrotic changes and chest CT showed bilateral diffuse interstitial changes with fibrotic alterations. No evidence of infectious agents was noted. The patient had a reduced carbon monoxide transfer factor (45%). Her symptoms and radiographic findings resolved following treatment with prednisolone. The literature contains several cases of fludarabine-associated interstitial pulmonary toxicity that responded to steroid therapy. Fludarabine-induced pulmonary toxicity is reversible with cessation of the drug and administration of glucocorticosteroids.

Key Words: Fludarabine monophosphate, non-Hodgkin lymphoma, Pulmonary toxicity

Özet Fludarabin monofosfat lenfoid malignansilerin tedavisinde etkin bir ilaçtır. Fludarabinin en sık görülen yan etkileri myelosupresyon, fırsatçı enfeksiyonlar ve otoimmün hemolitik anemidir. Burada evre IV non-Hodgkin foliküler lenfoma için FMD (fludarabin, mitoksantron ve deksametazon) kemoterapisinin üçüncü kürünü tamamladıktan sonra ateş ve dispneyle gelen 55 yaşında bir kadını bildiriyoruz. Akciğer röntgeninde bilateral pnömofibrotik değişiklikler varken göğüs BT’de fibrotik değişikliklerle bilateral difüz interstisyel değişiklikler görüldü. Herhangi bir enfeksiyöz ajan bulgusu saptanmadı. Hastanın karbonmonoksit transfer faktörü azalmıştı (%45). Belirtileri ve radyografik bulguları prednizolon tedavisiyle geçti. Literatürde steroid tedavisine cevap veren, fludarabinle ilişkili birkaç interstisyel pulmoner toksisite olgusu vardır. Fludarabin tarafından indüklenen pulmoner toksisite ilacın kesilmesi ve glukokortikosteroidlerin uygulanmasıyla geri çevrilebilir.

Anahtar Sözcükler: Fludarabin monofosfat, non-Hodgkin lenfoma, Pulmoner toksisite

Address for Correspondence: Milda RUDZIANSKIENE, M.D., Lithuanian University of Health Sciences Hospital, Kauno Klinikos, Oncology and Hematology Department, Kaunas, Lithuania Phone: 00370 37 326954 E-mail: mildalietuva@yahoo.com Received/Geliş tarihi : July 11, 2011 Accepted/Kabul tarihi : September 27, 2011

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Introduction Fludarabine monophosphate—a purine analogue—is an effective drug used as a single agent or in combination with other drugs for the treatment of patients with previously treated or newly diagnosed chronic lymphocytic leukemia, as well as those with low-grade non-Hodgkin lymphoma [1-3]. Myelosuppression, opportunistic infections caused by Pneumocystis jiroveci, mycobacteria, Candida spp., and other agents, neurotoxicity, and autoimmune hemolytic anemia are the most common side effects of fludarabine. The literature includes several reports of fludarabineassociated interstitial pulmonary toxicity. Pulmonary toxicity due to fludarabine responds well to steroid therapy [4-8]. In some reported cases other drugs were administered concurrently with or prior to fludarabine, and pathological confirmation of pneumonitis was not available [4,5,8]. Herein we describe a case of pulmonary toxicity following fludarabine therapy that responded to steroid treatment. Case A 55-year-old female with a history of stage IV nonHodgkin follicular lymphoma was treated with the FMD (fludarabine, mitoxantrone, and dexamethasone) chemotherapy regimen. She was receiving IV fludarabine 25 mg m–2 for 3 d every 28 d. Then, 2 weeks after the last cycle of chemotherapy she was admitted to the hematology depart-

Rudzianskiene M, et al : Pneumonitis Caused by Fludarabine

ment with fever and dyspnea. Her history of lung disease, exposure to toxins, tuberculosis, and smoking was negative. Her blood pressure was 136/85 mmHg, pulse rate was 116/min , respiratory rate was 36/min, temperature was 38.9 °C, and pulse oximetry was 96% on room air. Examination of the cardiovascular and respiratory systems was normal. Lymphadenopathy and hepatosplenomegaly were not observed. Chest X-ray showed bilateral pneumofibrotic changes (Figure 1); previous chest X-rays were normal. The patient’s laboratory findings were as follows: white blood cell count: 4.7 x 109 L–1, with 71% neutrophils, 17% lymphocytes, 11% monocytes, and 1% eosinophils; hemoglobin: 10.7 g dL–1; platelet count: 301 x 109 L–1; CRP: 47 mg L–1. Blood and urine microbiological cultures were negative. Chest CT showed bilateral diffuse interstitial changes with fibrotic alterations in the lower parts of the lungs (Figure 2). Atypical pneumonia or Mycoplasma pneumoniae infection was considered, and empirical treatment with cefuroxime (1.5 g t.i.d. IV) and clarithromycin (500 mg b.i.d. p.o.) was administered for 7 d, but the patient did not respond. Clarithromycin was withdrawn after laboratory findings verified that IgM antibodies against M. pneumoniae were negative. Antibodies against cytomegalovirus were also negative. Pneumonitis caused by P. jiroveci was then a consideration and bronchoscopy was performed. The patient was given antibiotic therapy, together with oral trimethoprim/sulfamethoxazole 960 mg t.i.d. for 4 d, but she did not respond. Microbiological cultures of secretions obtained during bronchoscopy were negative for evidence of P. jiroveci. Radiological investigation and bronchoscopy with bronchoalveolar lavage were performed, but no evidence of sarcoidosis was observed. Laboratory data, including ANA, ANCA, anti-DNR, and Scl-70, did not support the existence of systemic rheumatic disease. The reduced carbon monoxide transfer factor (TLCO) was 45%. The findings, including fever, moderate CRP level, diffuse interstitial changes with bilateral fibrotic alterations in the lungs, and a reduced TLCO led to the diagnosis of pneumonitis, and the suspected cause was fludarabine. Antibiotic therapy was withdrawn and the patient was started on prednisolone (1 mg·kg–1·d–1). After 2 d of prednisolone therapy dyspnea and fever disappeared, and chest X-ray showed complete resolution of diffuse interstitial changes (Figure 3). Prednisolone was tapered over the course of 1 month. Post treatment TLCO was 75%. Fludarabine treatment was not reinstated.

Figure 1: Bilateral pneumofibrotic changes in the chest X ray.

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Figure 2: Chest CT: bilateral diffuse interstitial changes with fibrotic alterations in the lower parts of the lungs.

Figure 3: Chest X-ray showed complete resolution of diffuse interstitial changes after prednisolone therapy.

Discussion Development of diffuse interstitial changes in the lungs can occur due to various reasons, such as connective tissue diseases, drugs, exposure to occupational and environmental toxins, and inherited conditions [9]. Many diverse etiologies of diffuse interstitial changes in the lungs may produce difficulties to establish the cause of pneumonitis. Fludarabine monophosphate depletes CD4 cells, altering the CD4:CD8 ratio and producing a syndrome that is clinically and immunologically similar to acquired immunodeficiency [10-11]. The most common infections seen in patients treated with fludarabine are opportunistic infections caused by P. jiroveci, mycobacteria, cytomegalovirus, and Candida spp. [7]. Interstitial pneumonitis induced by P. jiroveci presents with acute hypoxia, fever, and non-productive cough.

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Chest X-ray in such cases shows pulmonary infiltrates and a very low arterial oxygen level. Chest X-ray in the presented patient showed diffuse interstitial changes with fibrotic alterations in the lungs, which are not characteristic of P. jioveci infection, and microbiological cultures of secretions obtained from bronchoscopy didn’t show evidence of P. jiroveci, the arterial oxygen level was 96%, and there was no response to trimethoprim/sulfamethoxazole treatment. The observed diffuse interstitial changes strongly suggested atypical pneumonia caused by M. pneumoniae or viral infection; however, viral infection in the lungs was excluded because it does not cause TLCO reduction. Antibodies against cytomegalovirus were negative. Pneumonitis due to M. pneumoniae was excluded because IgM antibodies were negative and there was no response to clarithromycin therapy. Laboratory data did not support the existence of connective tissue diseases. The patient’s anamnesis was negative for exposure to toxins, such as asbestosis, silica dust, and chest radiotherapy. The incidence of fludarabine-associated pulmonary toxicity is not known. Helman et al. conducted a retrospective analysis of 105 patients with chronic lymphoproliferative diseases that were treated with fludarabine or fludarabine-containing regimens, and reported an 8.6% incidence of fludarabine-associated pulmonary toxicity [12]. The literature includes several case reports of interstitial pneumonitis related to fludarabine therapy [4-8,13]. In all cases the patients had non-productive cough, dyspnea, and fever, which typically began 1-2 weeks after the last course of chemotherapy. A relationship between the number of chemotherapy cycles and the development of pneumonitis was not established. In the four previous cases patients presented with the certain degree of hypoxia [4,5, 8, 13]. Garg et al. reported a patient with no signs of hypoxia [7]. In the presented patient pulse oximetry was 96% on room air. Kane et al. reported pneumonitis with severe respiratory failure [5] and Disel et al. reported a

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case of pneumonitis with rapidly progressing severe dyspnea, cyanosis, and massive pulmonary bleeding [8]. No deaths have been reported. Radiographic changes consist mostly of diffuse reticular infiltrates, with or without nodularity, usually in the middle or lower zones of the lungs [7,14]. Chest X-ray in the presented patient showed bilateral pneumofibrotic changes and chest CT showed bilateral diffuse interstitial changes with the fibrotic alterations in the lower parts of the lungs. In all reported cases, as in the presented patient, the causes of infection (viral, bacterial, P. jiroveci, fungal) were excluded and antibiotic therapy was ineffective. In the presented patient a lung function test was performed (TLCO) and the result was 45%. The TLCO is a test used to diagnose, grade, and monitor diseases that affect gas transfer at the alveolar-capillary surface area. The test is useful for identification of disorders that affect lung parenchyma, interstitial lung diseases, and anemia that result from pulmonary hemorrhage, as is seen in chronic thromboembolic disease or pulmonary hypertension [15]. In previously reported cases this test was not performed and interstitial pneumonitis was pathologically confirmed via open lung biopsy in only in 1 published case [5]. In previously reported cases, as in the presented patient, corticosteroids were administrated after excluding causes of infection that could have led to the pneumonitis. All reported patients had clinical response and resolution of radiographic findings. Tapering corticosteroid therapy for pneumonitis resulted in relapse in 1 patient, but after steroids were again administered a secondary response was induced [4]. Following reinstatement of fludarabine therapy recurrence of interstitial pneumonitis was observed in some patients [4,6,16]. The mechanism of lung injury due to fludarabine is unknown. Response to steroids suggests an immunological mechanism, although direct toxicity cannot be ruled out [7]. Fludarabine therapy can induce pulmonary toxicity, ranging from mild to severe respiratory failure. If symptoms and radiographic findings indicate development of interstitial pneumonitis and possible etiological infections are excluded, fludarabine therapy should be terminated and steroid therapy should be initiated. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

Rudzianskiene M, et al : Pneumonitis Caused by Fludarabine

References 1. Adkins JC, Peters DH, Markham A. Fludarabine. An update of its pharmacology and use in the treatment of hematological malignancies. Drugs 1997;53:1005-1037. 2. Chun HG, Leyland Jones B, Cheson BD. Fludarabine phosphate: A synthetic purine antimetabolite with significant activity against lymphoid malignancies. J Clin Oncol 1991;9:175-188. 3. Hallek M, Fischer K, Fingerle-Rowson G, Fink AM, Busch R, Mayer J, Hensel M, Hopfinger G, Hess G, von Grunhagen U, Bergmann M, Catalano J, Zinzani PL, Caligaris-Cappio F, Seymour JF, Berrebi A, Jager U, Cazin B, Trneny M, Westermann A, Wendtner CM, Eichhorst BF, Staib P, Buhler A, Winkler D, Zenz T, Bottcher S, Ritgen M, Mendila M, Kneba M, Dohner H, Stilgenbauer S; International Group of Investigators; German Chronic Lymphocytic Leukaemia Study group. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: A randomised, open-label, phase 3 trial. Lancet 2010;376:1164–1174. 4. Hurst PG, Habib MP, Garewal H, Blustain M, Paquin M, Greenberg BR. Pulmonary toxicity associated with fludarabine monophosphate. Invest New Drugs 1987;5:207210. 5. Kane GC, McMichael AJ, Patrick H, Erslev AJ. Pulmonary toxicity and acute respiratory failure associated with fludarabine monophosphate. Respir Med 1992;86:261-263. 6. Levin M, Aziz M, Opitz L. Steroids responsive interstitial pneumonitis after fludarabine therapy. Chest 1997;111:1472-1473. 7. Garg S, Garg MS, Basmaji N. Multiple pulmonary nodules: An unusual presentation of fludarabine pulmonary toxicity: Case report and review of literature. Am J Hematol 2002;70:241-245. 8. Disel U, Paydas S, Yavuz S, Karakoc E. Severe pulmonary toxicity associated with fludarabine and possible contribution of rituximab. Chemotherapy 2010;56:89-93. 9. King TE Jr. Clinical advances in the diagnosis and therapy of the interstitial lung diseases. Am J Respir Crit Care Med 2005;172: 268–279. 10. Schilling PJ, Vadhan-Raj S. Concurrent cytomegalovirus and pneumocystis pneumonia after fludarabine therapy for chronic lymphocytic leukemia (Letter). N Engl J Med 1990;323:833-834. 11. Ravandi F, O’Brien S. Immune defects in patients with chronic lymphocytic leukemia. Cancer Immunol Immunother 2006;55:197-209. 12. Helman DL, Byrd JC, Ales NC, Shorr AF. Fludarabine-related pulmonary toxicity. A distinct clinical entity in chronic lymphoproliferative disorders. Chest 2002;122:785-790.

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13. Cervantes F, Salgado C, Montserrat E, Rozman C. Fludarabine for prolymphocytic leukemia and risk of interstitial pneumonitis. Lancet 1990;336:1130. 14. Rossi SE, Erasmus JJ, McAdams HP, Sporn TA, Goodman PC. Pulmonary drug toxicity: Radiologic and pathologic manifestations. Radiographics 2000;20:1245-1259. 15. Peter G. Gibson,Michael Abramson,Richard Wood-Baker. Evidence-based respiratory medicine. BMJ Books, 2005.

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16. Trojan A, Meier R, Licht A, Taverna C. Eosinophilic pneumonia after administration of fludarabine for the treatment of non-Hodgkin’s lymphoma. Ann Hematol 2002;81:535-537.

Case Report

DOI: 10.5505/tjh.2012.23600

Metastatic Pulmonary Calcinosis and Leukocytoclastic Vasculitis in a Patient with Multiple Myeloma Multipl Myelomlu Bir Hastada Lökositoklastik Vaskulit ve Metastatik Pulmoner Kalsifikasyon Seçkin Çağırgan1, Nur Soyer1, Filiz Vural1, Güray Saydam1, Ilgın Yıldırım Şimşir1, Ayhan Dönmez1, Taner Akalın2, Selen Biçeroğlu3, Murat Tombuloğlu1 Ege University, School of Medicine, Department of Hematology, İzmir, Turkey Ege University, School of Medicine, Department of Pathology, İzmir, Turkey 3 Ege University, School of Medicine, Department of Radiology, İzmir, Turkey 1 2

Abstract Both leukocytoclastic vasculitis and metastatic pulmonary calcification are conditions that rarely occur during the course of multiple myeloma. We present a multiple myeloma patient that had severe dyspnea due to metastatic pulmonary calcinosis, and ulceronecrotic skin lesions caused by leukocytoclastic vasculitis. After 3 courses of standarddose chemotherapy all skin and pulmonary lesions disappeared. Autologous peripheral stem cell transplantation was performed and during 1 year of follow-up the patient was in complete remission; after 1 year, laboratory test results indicated disease relapse. Although the patient was treated with bortezomib and dexamethasone, the disease progressed. Non-myeloablative allogeneic stem cell transplantation was performed, but despite of all treatment the patient died due to disease progression.

Key Words: Multiple myeloma, Metastatic calcification, Leukocytoclastic vasculitis

Özet Hem lökositoklastik vaskülit hemde metastatik pulmoner kalsifikasyon, multipl myelomda nadir olarak görülmektedir. Biz, lökositoklastik vaskulit nedeniyle gelişen ülseronekrotik cilt lezyonları ve ciddi dispneye yol açan metastatik pulmoner kalsifikasyonla tanısı konan bir multipl myelom hastasını sunmak istedik. Üç kür standart doz kemoterapi sonrası hastanın hem cilt lezyonları hemde pulmoner lezyonları düzeldi. Otolog kök hücre nakli yapılan hasta 1 yıl tam remisyonda izlendi. Daha sonra laboratuvar testlerinde relaps saptandı. Hastaya bortezomib ve deksametazon başlandı. Bu tedaviye rağmen progresyonu olan hastaya myeloablatif olmayan rejimle allojeneik kök hücre nakli yapıldı. Bütün tedavilere rağmen hasta progresif hastalıkla kaybedildi.

Anahtar Sözcükler: Multipl myelom, Metastatik kalsifikasyon, Lökositoklastik vaskülit

Address for Correspondence: Nur SOYER, M.D., Ege Üniversitesi, Tıp Fakültesi Hastanesi, İç Hastalıkları, Hematoloji Bilim Dalı İzmir, Turkey Phone: +90 232 390 42 87 E-mail: drakadnur@yahoo.com Received/Geliş tarihi : September 17, 2010 Accepted/Kabul tarihi : March 05, 2011

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Çağırgan S, et al : Metastatic Pulmonary Calcinosis Leukocytoclastic Vasculitis Myeloma

Introduction Multiple myeloma is a clonal malignant plasma cell disease. Manifestations of the disease develop due to bone marrow and skeletal infiltration, and immunoglobulin production by malignant plasma cells [1]. Leukocytoclastic vasculitis is a systemic vasculitis characterized by involvement of small vessels in the skin that present as palpable purpura. It may present with systemic manifestations, such as fever, arthralgia as a finding of arthritis, and less commonly renal, neurological, and gastrointestinal functional compromise [2-4]. This disorder is related to hypersensitivity mechanisms caused by various antigens, primarily infections, drugs, or autoantigens in connective tissue diseases and malignant neoplasms [3]. Most reports of cutaneous leukocytoclastic vasculitis occurred in patients with hematological malignancies and presented as paraneoplastic vasculitis [3-6]. The incidence of paraneoplastic vasculitis in patients with multiple myeloma is approximately 0.8% [3]. Metastatic pulmonary calcification is the deposition of calcium in pulmonary interstitium and parenchyma. Predisposing factors include chronic renal failure, hypercalcemia, and elevated tissue alkalinity [7,8]. Hypercalcemia is a common manifestation of multiple myeloma and, as such, metastatic calcification is an expected consequence; however, to the best of our knowledge an association between metastatic calcification and cutaneous leukocytoclastic vasculitis in patients with multiple myeloma has not been reported. Herein we present a multiple myeloma patient that presented with severe dyspnea due to metastatic pulmonary calcinosis, and ulceronecrotic skin lesions. Case Report Written informed consent was obtained from the patient. A 35-year-old female presented with severe dyspnea, back pain, and multiple painful purpuric ulceronecrotic skin lesions on her face and extremities in November 2005. She was hospitalized with the presumed diagnosis of respiratory failure based on findings of hypoxemia and scattered density changes on chest X-ray. Physical examination showed widespread fine rales in chest auscultation and diffuse palpable purpura of variously sized patches, some of which were necrotic, that formed well-delimited ulcers (Figure 1). The patient was afebrile and there were no signs of an infectious focus. Initial laboratory analysis results were as follows: hemoglobin: 7 g dL–1; hematocrit: 20.3%; leukocyte count: 12.1

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x 109 L–1; platelet count: 123 x 109 L–1; serum urea 76 mg dL–1 (normal range: 10-50 mg dL–1); creatinine: 1.66 mg dL–1 (normal range: 0.6-1.1 mg dL–1); creatinine clearance: 26 mL s–1 (normal range: 70-145 mL s–1); calcium: 13.8 mg dL–1 (normal range: 8.2-10.4 mg dL–1); globulin: 5 g dL–1 (normal range: 2.5-3.5 g dL–1). Serum protein electrophoresis showed a monoclonal peak in the gammaglobulin zone. Serum immunoelectrophoretic analysis showed there was immunoglobulin G (IgG) and kappa chain fixation. Serum IgG, IgA, and IgM levels were 3349 mg dL–1 (normal range: 650-1600 mg dL–1), <25 mg dL–1 (normal range: 40-350 mg dL–1), and 27 mg dL–1 (normal range: 50-300 mg dL–1), respectively. Urinary kappa and lambda levels were 847 mg dL–1 (normal range: 138375 mg dL–1) and 16 mg dL–1 (normal range: 92-242 mg dL–1), respectively. Daily urinary protein excretion was 1 g d–1 and Bence-Jones proteinuria was negative. Analysis of a bone marrow biopsy specimen showed that there was 90% immature plasma cell infiltration. Multiple osteolytic lesions were observed in the skull, scapula, clavicle, humerus, femurs, and tibias. The patient was diagnosed as stage III-A IgG-kappa multiple myeloma, according to Durie-Salmon classification. Thoracic high-resolution computed tomography (HRCT) showed bilateral widespread centrilobular acinar opacities and a ground-glass appearance, which was consistent with metastatic calcinosis of the lungs (Figure 2). Whole-body scintigraphy also showed metastatic calcium depositions in soft tissues. Biopsy specimens obtained from the skin lesions showed findings of leukocytoclastic vasculitis, as well as diffuse fibrin thromboses in small vessels and necrosis on the epidermis. Immunofluorescence examination showed the presence of fibrinogen in superficial dermis and vessel walls, as well as granular C3 deposition on the vessel walls (Figure 3). Calcium and amyloidal deposition was not detected based on histological examination with special Congo red and von Kossa dyes. The patient was treated with standard-dose VAD (vincristine, adriamycin, and dexamethasone) chemotherapy and zoledronic acid. All skin and pulmonary lesions disappeared after the third course of chemotherapy, and serum calcium and creatinine levels returned to normal. The patient achieved partial remission. Following the fourth course of chemotherapy, the patient was treated with high-dose melphalan (200 mg m–2) and autologous peripheral stem cell transplantation. During 1 year of post transplantation follow-up the patient was in complete remission. One year after the transplantation laboratory test results showed relapse of multiple myeloma and that

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Çağırgan S, et al : Metastatic Pulmonary Calcinosis Leukocytoclastic Vasculitis Myeloma

Figure 1: Well-delimited necrotic skin ulcers.

Figure 3: Leukocytoclastic vasculitis with widespread fibrin thromboses in small vessels (H&E, 200x).

Discussion The presented patient presented with severe dyspnea and ulceronecrotic skin lesions, and was diagnosed as multiple myeloma. A skin biopsy specimen obtained from ulceronecrotic lesions showed leukocytoclastic vasculitis and HRCT of the lungs showed metastatic pulmonary calcification. To the best of our knowledge this is the first case reported with these 3 findings. After 3 courses of standard first-line multiple myeloma chemotherapy, the patient achieved partial remission, and all skin and pulmonary lesions disappeared.

Figure 2: HRCT at the level of the upper lobes shows nodular ground glass opacities with a predominately centrilobular distribution.

the patient’s IgG level was 3344 mg dL–1. She was treated with 3 courses of bortezomib and dexamethasone, after which time her serum IgG level increased to 4199 mg dL–1. Because of disease progression she underwent non-myeloablative allogeneic stem cell transplantation (busulphan 3.2 mg·kg·d–1 for 2 d, cyclophosphamide 350 mg m–2 for 3 d, and fludarabine 30 mg m–2 for 3 d) from an HLA fullymatched sibling donor. Following the transplantation acute graft versus host disease was not observed; however, disease response was not achieved. Three months after the transplantation donor lymphocyte infusion was administered in order to control the progression of disease, but despite all treatment the patient died at home due to disease progression in October 2007.

Leukocytoclastic vasculitis is a paraneoplastic syndrome, which is observed most commonly in cases of hematological malignancy, such as lymphoid neoplasm or myelodysplastic syndrome [4,6]. Leukocytoclastic vasculitis rarely occurs during the course of multiple myeloma [2]. This vasculitis is an inflammatory necrotizing condition of the superficial dermal vessels, and is characterized by neutrophilic, angiocentric, segmental inflammation with endothelial cell injury and fibrinoid necrosis of the blood vessel walls [9]. Clinical symptoms are variable and include palpable purpura, and hemorrhagic-necrotizing, bullous, nodular, and urticarial lesions [10]. Sanchez et al. [2] reported a case of multiple myeloma associated with paraneoplastic vasculitis and reviewed 8 other reported patients. In all, 6 of the 9 patients were diagnosed as type IgA myeloma. All the patients had palpable purpura involving the legs and/or trunk, and 2 patients had ulceronecrotic lesions, as in the presented case. Skin lesions in 6 of the patients improved after treatment of multiple myeloma. Bayer-Garner et al. reported 8 patients with multiple myeloma that developed leukocyto-

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clastic vasculitis [11]. They reported that 4 of the patients were diagnosed as IgG myeloma with diffuse skin lesions, as was the presented case. Metastatic pulmonary calcification generally occurs in patients with hypercalcemia [12]; multiple myeloma is less commonly a cause of metastatic pulmonary calcification. Generally, patients with metastatic pulmonary calcification are asymptomatic, but restrictive lung function, decreased diffusing capacity, hypoxemia, and respiratory failure may occur [12]. Plain radiography of the chest is usually normal. HRCT generally shows centrilobular ground glass nodular opacities. In cases of metastatic calcification calcium deposits may accumulate to such tissues as lung, stomach, skin, and kidney [13,14]. Weber et al. reported a multiple myeloma patient with metastatic pulmonary calcification. After treatment of multiple myeloma, pulmonary infiltration decreased, as in the presented case [15]. Marchiori et al. [12] reported 3 patients who presented with metastatic pulmonary calcification with unusual HRCT findings, 1 of which died due to diffuse metastatic calcification in the parenchyma of major organs, including the heart, lungs, kidneys, meninges, and skin, and was diagnosed as multiple myeloma during autopsy. Another study reported 4 cases diagnosed as paraneoplastic hypercalcemia with metastatic calcification, based on autopsy, 1 of which presented with paraneoplastic hypercalcemia and was diagnosed as multiple myeloma [16]. Most of the reported cases of metastatic calcinosis died early because of acute pulmonary failure or because the underlying disease was refractory to treatment; even though symptoms resolved following treatment, they died due to disease progression. In conclusion, both leukocytoclastic vasculitis and metastatic pulmonary calcification are conditions that rarely occur during the course of multiple myeloma. Treatment of multiple myeloma may improve skin lesions and metastatic calcification, but the prognosis appears to be poor despite administration of the most effective treatments. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. References 1. Davies FE, Jack As, Morgan GJ. The use of biological variables to predict outcome in multiple myeloma. Br J Haematol 1997; 99: 719-725.

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2. Sanchez NB, Canedo IF, Garcia-Patos PE, de Unamuno Pérez P, Benito AV, Pascual AM. Paraneoplastic vasculitis associated with multiple myeloma. J Eur Acad Dermatol Venereol 2004;18: 731-735. 3. Kurzrock R, Cohen PR. Vasculitis and cancer. Clin Dermatol 1993;11: 175-187. 4. Kurzrock R, Cohen PR, Markowitz A. Clinical manifestations of vasculitis in patients with solid tumors. Arch Intern Medical 1994; 154: 334-340. 5. Garcia-Porrua C, Gonzalez-Gay MA. Cutaneous vasculitis as paraneoplatic syndrome in adults. Arthritis Rheum 1998; 41:1133-1136. 6. Hayem G, Gomez MJ, Grossin M, Meyer O, Kahn MF. Systemic vasculitis and epithelioma. Rev Rhum Engl ED 1997; 64:816-824. 7. Bendayan D, Barziv Y, Kramer MR. Pulmonary calcifications: A review. Respir Med 2000; 94:190-193. 8. Lingam RK, the J, Sharma A, Friedman E. Metastatic pulmonary calcification in renal failure: A new HRCT pattern. Br J Radiol 2002; 75:74-77. 9. Cem Ar M, Soysal T, Hatemi G, Salihoglu A, Yazici H, Ulku B. Successful management of cryoglobulinemia-induced leukocytoclastic vasculitis with thalidomide in a patient with multiple myeloma. Ann Hematol 2005; 84: 609-613. 10. Marini A, Fenk R, Plettenberg H, Ruzicka T, Haas R, Hengge UR. Rare types of vasculitis as markers of plasmocytoma. Hautarzt 2006; 57: 137-143. 11. Bayer-Garner IB, Smoller BR. Leukocytoclastic (small vessel) vasculitis in multiple myeloma. Clin Exp Dermatol 2003; 28:521-524. 12. Marchiori E, Muller NL, Souza AS Jr, Escuissato DL, Gasparetto EL, de Cerqueira EMl. Unusual manifestations of metastatic pulmonary calcification: high-resolution CT and pathological findings. J Thorac Imaging 2005; 20: 66-70. 13. Hirose Y, Tachibana J, Sugai S, Konda S, Hayabe T, Konishi F. Metastatic calcification in the stomach demonstrated by a bone scan in Bence Jones lambda myeloma. Jpn J Med 1987; 26: 72-75. 14. Chaves Alverez AJ, Herrera Saval A, Merquez Enriquez J, Camacho Martinez F. Metastatic calcinosis cutis in multiple myeloma. Br J Dermatol 2000;142:820-822. 15. Weber CK, Friedrich JM, Merkle E, Prümmer O, Hoffmeister A, Mattfeldt T, Frickhofen N. Reversible metastatic pulmonary calcification in a patient with multiple myeloma. Ann Hematol 1996; 72: 329-332. 16. Liou JH, Cho LC, Hsu YH. Paraneoplastic hypercalcemia with metastatic calcification--clinicopathologic studies. Kaohsiung J Med Sci 2006; 22:85-88.

Case Report

DOI: 10.5505/tjh.2012.65148

An Unusual Presentation of Galactosemia: Hemophagocytic Lymphohistiocytosis Galaktozeminin Sıradışı Prezentasyonu: Hemofagositik Lenfohistiyositozis Ahmet Afşin Kundak1, Ayşegül Zenciroğlu1, Neşe Yaralı2, Belma Saygılı Karagöl1, Arzu Dursun1, Selim Gökçe3, Nilgün Karadağ1, Nurullah Okumuş1 Dr.Sami Ulus Hospital, Neonatology Department, Ankara, Turkey Ankara Pediatric Hematology Oncology Training and Research Hospital, Ankara, Turkey 3 Dr.Sami Ulus Hospital, Gastroenterology Department, Ankara, Turkey 1 2

Abstract Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening condition. Uncontrolled proliferation of activated lymphocytes secreting high amounts of inflammatory cytokines seems to be the main pathogenesis. The diagnosis of HLH can often be difficult. It may presents in many forms such as fever of unknown origin, hepatitis, acute liver failure, and sepsis-like illness. Here we present a newborn galactosemia case presented with HLH. Close monitoring of the diagnostic criteria of HLH during the course of galactosemia-associated hemophagocytosis, both before and after dietary treatment, should be performed in order to fully determine if the triggering mechanism is infection or accumulation of metabolites.

Key Words: Hemophagocytic lymphohistiocytosis, Galactosemia, Newborn

Özet Hemofagositik lenfohistiyositozis (HLH), nadir görülen, yaşamı tehdit eden bir durumdur. Patogenezinde; yüksek miktarda sitokin üreten aktive lenfositlerin kontrolsüz proliferasyonunun olduğu düşünülmektedir. HLH tanısını koymak genellikle güçtür. Hastalar nedeni bilinmeyen ateş, hepatit, akut karaciğer yetmezliği, sepsis gibi çok farklı klinik bulgularla başvurabilir. Burada HLH ile başvuran galaktozemili bir yenidoğan vakasını sunduk. Galaktozemi ile ilişkili HLH vakalarında tanı kriterlerinin diyet tedavisi öncesi ve sonrasında yakın takip edilmesi hastalığı tetikleyen mekanizmanın enfeksiyon mu yoksa metabolit birikimi mi olduğunu daha iyi açıklayabilmek için gereklidir.

Anahtar Sözcükler: Hemofagositik lenfohistiyositozis, Galaktozemi, Yenidoğan

Address for Correspondence: Ahmet Afşin KUNDAK, M.D., Dr. Sami Ulus Hastanesi, Neonatoloji Bölümü, Ankara, Turkey Phone: +90 312 305 62 84 E-mail: drafsin@hotmail.com Received/Geliş tarihi : May 21, 2012 Accepted/Kabul tarihi : August 22, 2012

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Introduction Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening condition characterized by severe multisystem hyperinflammation [1]. The term HLH does not refer to a specific disease, as it has many genetic and acquired causes. Primary HLH, also known as familial HLH (FHL), is an inherited autosomal recessive disorder that occurs more commonly in cases of parental consanguinity. Secondary or acquired HLH occurs following strong immunologic activation, such as immunodeficiency, systemic infection, or malignancy. Both forms are characterized by abnormal activation of T-lymphocytes and macrophages. Uncontrolled proliferation of activated lymphocytes that secrete large quantities of inflammatory cytokines is thought to be the primary pathogenesis [2]. FHL is diagnosed either via mutation analysis of genes, including perforin, UNC 13D, syntaxin 11, and syntaxinbinding protein 2, or via family history. HLH can be diagnosed based on fulfillment of 5 of the following 8 clinical/biochemical changes: fever, splenomegaly, cytopenias, hypertriglyceridemia/hypofibrinogenemia, hemophagocytosis in bone marrow, abnormal natural killer cell function assay, elevated soluble IL-2R alpha level, and elevated ferritin level. These 8 HLH criteria can also be observed in both FHL and secondary HLH [3]. Acquired HLH is primarily thought to be associated with several infectious agents, including viral, bacterial, fungal, and parasitic pathogens [4-7], and is also associated with autoimmune diseases, malignancies, and metabolic diseases [8]. Herein we present a full-term newborn with galactosemia that presented with HLH. HLH associated with galactosemia is rarely reported. Case Report A 3-d-old full-term girl born to consanguineous (firstdegree cousins) parents was admitted to our neonatal intensive care unit with jaundice and fever (37.6 °C). Her birth weight was normal (3300 g) and had jaundice and poor feeding 1 day prior to admission. She was the offspring of the mother’s second gravida. The first gravida was terminated via abortion. Prenatal history, mother’s laboratory findings, and family history were unremarkable. The patient was delivered via Caesarian section and had normal APGAR scores. On physical examination she weighed 3000 g and her vital signs were stable. She had icterus, lethargy, and weak sucking. The remainder of the physical examination was unremarkable. Initial laboratory findings were as follows: white blood cell (WBC) count: 10.3 x 109/L; hemoglobin

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(Hb): 15.7 g/dL; red blood cell (RBC) count: 4.68 x 1012/L; platelet (Plt) count: 233x109/L; C-reactive protein (CRP): 3.34 mg/dL; total bilirubin: 15.6 mg/dL: direct bilirubin: 1 mg/dL; aspartate aminotransferase (AST): 1367 U/L; alanine aminotransferase (ALT): 504 U/L; prothrombin time (PT): >60 s; international normalized ratio (INR): >6; activated partial thromboplastin time (aPTT): 88 s. The blood of the patient and her mother was O Rh +; Coombs test was negative. Serum sodium, potassium, and creatinine were 147 mEq/L, 4.5 mEq/L, and 0.46 mg/dL, respectively. Microbiological work-up, including blood culture, was performed and ampicillin-gentamicin was empirically started. The patient received supportive treatment with vitamin K and fresh frozen plasma due to acute liver failure (ALF). The patient’s clinical condition deteriorated on d 2 of hospitalization and her body temperature increased to 39 °C. Due to upper gastrointestinal bleeding oral feeding was stopped. The following screening tests performed due to elevated liver enzymes and coagulopathy were negative: Epstein-Barr virus, toxoplasma, rubella, cytomegalovirus, herpes simplex virus, human immunodeficiency virus, human parvovirus B19, treponema pallidum, and hepatitis A, B, and C. The patient’s ferritin level was 5644 ng/ mL, and her fibrinogen level was 147 mg/dL, despite fresh frozen plasma treatment. During this period the patient was given an anti-oxidant cocktail that included selenium, n- acetylcysteine, vitamin E, and desferrioxamine due to the possibility of hemochromatosis, but there was no improvement in clinical or laboratory parameters. For the differential diagnosis of neonatal hemochromatosis buccal biopsy was performed, but no evidence of hemochromatosis was observed. On d 12 of hospitalization hepatosplenomegaly was noted on physical examination and the patient’s WBC count, Hb, and Plt count decreased to 3.8 x 109/L, 6.5 g/ dL, and 48 x 109/L, respectively. Hemophagocytosis was observed in her bone marrow aspirate (Figure 1) and CRP increased to 19 mg/dL. The patient was diagnosed as HLH based on fulfillment of 6 diagnostic criteria (fever, hemophagocytosis, hypofibrinogenemia, pancytopenia, splenomegaly, and hyperferritinemia). Antibiotic treatment was changed to imipenem due to suspected sepsis, and then vancomycin was added. Blood cultures were negative and the patient was given high-dose (1 mg/kg) intravenous immunoglobulin (IVIG) to stop the inflammatory cascade, because diverse bacterial infections were suspected to have caused HLH. Her work-up for inborn errors of metabolism showed non-specific changes, except for urine,

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Kundak AA, et al : A Galactosemia Case With HLH

The mechanism of HLH is not clear in metabolic disorders; it could be associated with tissue damage, and impaired lymphocyte and histiocyte functions, or activation of macrophages via accumulation of metabolites in some way [8,12]. In the presented case the underlying endogenous mechanism may have been tissue damage caused by galactose or its metabolites; exogenous agents such as gram-negative microorganisms that could have had an additive effect were not detected in blood culture. In galactosemic neonates the results of tests for evaluating neutrophil function, including chemiluminescence, chemotaxis, and adherence, were significantly low due to galactosemiainduced immune deficiency [13,14].

Figure 1: Hemophagocytosis in bone marrow aspirate.

which was positive for reducing substance; however, thin layer sugar chromatography was negative on 3 separate occasions. Liver histology showed focal hepatic necrosis, macrovesicular steatosis, vacuolar degeneration, and canalicular cholestasis. Due to the possibility of galactosemia, lactose-free formula was commenced together with the second dose of IVIG, after which time the patient’s clinical and laboratory findings improved. As galactose-1-phosphate uridyl transferase (GALT) enzyme analysis would have been noninformative due to previous erythrocyte transfusion during hospitalization, her GALT enzyme level was evaluated at age 3 months (72 U/L [n > 262]) and the diagnosis of galactosemia was confirmed. At age 5 months the patient’s physical examination and laboratory findings were normal. Discussion We described a neonatal case of galactosemia with secondary HLH and ALF. The patient recovered following IVIG treatment and a galactose-free diet. HLH is a rare and fatal disorder resulting from uncontrolled proliferation of activated lymphocytes and histiocytes that secrete large quantities of inflammatory cytokines. HLH is a clinical syndrome, rather than a single disease, associated with a variety of underlying conditions [9]. The underlying mechanism of secondary HLH is not fully understood and it can manifest at any age. It is observed in the context of infection, underlying autoimmune disorders, and some metabolic disorders [7,9]. To the best of our knowledge the presented case is only the third report of HLH secondary to galactosemia [10,11]. Previously described cases also had sepsis (confirmed via blood culture).

The diagnosis of HLH can often be difficult, as it may present in many forms, including fever of unknown origin, hepatitis, acute liver failure, and sepsis-like illness [15]. HLH diagnostic criteria may not be observed initially, as in the present case and, as such, it is important to carefully follow the clinical signs and laboratory markers of HLH during the course of illness. Acute systemic immune activation and specific findings, such as cytopenias, decreased fibrinogen or increased triglycerides, hemophagocytosis, and elevated ferritin, are criteria that differentiate HLH from other inflammatory disorders [3]. The presented case was difficult to diagnose; hemochromatosis was an initial consideration due to ALF with hyperferritinemia, and both buccal biopsy and liver biopsy were performed to confirm the diagnosis. Moreover, the presented case gradually developed the diagnostic criteria of HLH (cytopenia and splenomegaly) during the course of the disease, which were not initially observed. A lactose-free diet and high-dose IVIG stopped the hyperinflammation cascade in the presented case; the elevated liver enzymes may have been due to galactosemia or HLH. The principle challenge in treating patients with HLH is to diagnose as early as possible. It is also critical to search for and treat underlying triggers of HLH, and to begin specific therapy [9]. Marcoux et al. reported a neonate with a favorable outcome following antibiotic and corticosteroid treatment [10]. Treatment with IVIG, as in the presented case, or corticosteroids, as in the case reported by Marcoux et al. [10] might have an effect on satisfactory outcome and stop the hyperinflammatory process. Additionally, it is important to block triggering of the hyperinflammatory process via a lactose-free diet. As the presented case was clinically unstable, the response specific to the lactose-free diet could not be determined. In secondary HLH cases conditions leading to the hyperinflammatory process are generally exogenous agents,

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such as infections, endogenous products causing tissue damage, rheumatic diseases, and malignant diseases [9]. Sepsis-associated secondary HLH should be considered in the differential diagnosis of HLH, especially in neonates [16]. In cases of galactosemia, overlap of tissue damage and the potential risk of gram-negative infection might predispose patients to HLH. In cases of severe galactosemia with sepsis that do not respond to antibiotic treatment in addition to standard therapy the presence of HLH should be investigated. Diagnosis of FHL was not possible in the presented case. HLH improves following treatment of galactosemia and administration of IVIG, which indicates the presence of secondary HLH. In conclusion, it is difficult to differentiate neonatal HLH from metabolic disease and sepsis; however, differentiation of these entities should be performed. In neonates with an acute presentation, with sepsis and ALF, metabolic diseases should also be investigated, especially in regions in which consanguineous marriage is common, as in Turkey. In the literature three reports of HLH cases with galactosemia are remarkable. Close monitoring of the diagnostic criteria of HLH during the course of galactosemia-associated hemophagocytosis, both before and after dietary treatment, should be performed in order to fully determine if the triggering mechanism is infection or accumulation of metabolites. References 1. Freeman HR, Ramanan AV. Review of haemophagocytic lymphohistiocytosis. Arch Dis Child 2011; 96:688-693. 2. Filipovich AH. Hemophagocytic lymphohistiocytosis (HLH) and related disorders. Hematology Am Soc Hematol Educ Program 2009:127-131. 3. Jordan MB, Allen CE, Weitzman S, Filipovich AH, McClain KL. How I treat hemophagocytic lymphohistiocytosis. Blood 2011; 118:4041-4052. 4. Henter JI, Horne A, Aricó M, Egeler RM, Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D, Winiarski J, Janka G. HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007;48:124-131.

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5. Imashuku S, Hlbi S, Todo S. Hemophagocytic lymphohistiocytosis in infancy and childhood. J Pediatr 1997; 130:352-357. 6. Fisman DN. Hemophagocytic syndromes and infection. Emerg Infect Dis 2000; 6:601-608. 7. Gurgey A, Secmeer G, Tavil B, Ceyhan M, Kuskonmaz B, Cengiz B, Ozen H, Kara A, Cetin M, Gumruk F. Secondary hemophagocytic lymphohistiocytosis in Turkish children. Pediatr Infect Dis J 2005; 24:1116-1117. 8. Gokce M, Unal O, Hismi B, Gumruk F, Coskun T, Balta G, Unal S, Cetin M, Kalkanoglu-Sivri HS, Dursun A, Tokatlı A. Secondary hemophagocytosis in 3 patients with organic acidemia involving propionate metabolism. Pediatr Hematol Oncol 2012; 29:92-98. 9. Janka GE. Familial and acquired hemophagocytic lymphohistiocytosis. Eur J Pediatr 2007; 166:95-109. 10. Marcoux MO, Laporte-Turpin E, Alberge C, Fournie-Gardini E, Castex MP, Rolland M, Brivet M, Broue P. Congenital galactosaemia: An unusual presentation. Arch Pediatr. 2005 Feb;12:160-162 11. Yaralı N, Yıldırım I, Arık E, Zorlu P, Tanır G. Hemophagocytic Syndrome Associated with Bacterial Infections. J Pediatr Inf 2010; 4: 162-164 12. Malaguarnera L. Chitotriosidase: the yin and yang. Cell Mol Life Sci 2006; 63:3018-3029. 13. Kobayashi RH, Kettelhut BV, Kobayashi AL. Galactose inhibition of neonatal neutrophil function. Pediatr Infect Dis1983; 2: 442-445. 14. Waggoner DD, Buist NR, Donnell GN. Long-term prognosis in galactosaemia: Results of a survey of 350 cases. J Inherit Metab Dis 1990; 13: 802-818. 15. Bas AY, Demirel N, Zenciroglu A, Yarali N, Metin A. Familial haemophagocytic lymphohistiocytosis in two newborn siblings: A good mimicker of newborn sepsis. Ann Trop Paediatr 2007; 27:231-235. 16. Aygun C, Tekinalp G, Gurgey A. Infection- associated Hemophagocytic syndrome due to Pseudomonas aeruginosa in preterm infants. J Pediatr Hematol Oncol 2003; 25:665667

Case Report

DOI: 10.5505/tjh.2012.82542

Imatinib Use in Pregnancy Hamilelikte İmatinib Kullanımı Michael J Webb1, Debbie Jafta2 Division of Clinical Haematology, Department of Internal Medicine, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa 2 Department of Haematology and Cell Biology, National Health Laboratory Service, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa 1

Abstract The outcome in patients with chronic myeloid leukemia (CML) has dramatically improved over the last decade due to the widespread use of novel tyrosine kinase inhibitors such as imatinib. As overall survival has improved, the number of women with CML that wish to become pregnant has increased. As such, attending physicians are faced with a dilemma⎯continue life-prolonging medication to treat the cancer, or interrupt its use due to its potential teratogenicity. Herein we describe 2 CML patients that gave birth. Case 1 was managed via substitution of imatinib with interferon. The patient’s child underwent genetic evaluation at age 3 years, achieved normal developmental milestones, and despite being shorter than his peers was proportional. In terms of morphology, the child had clinodactyly, short fifth fingers, and slightly downward slanting palpebral fissures, but otherwise appeared normal. In case 2 imatinib was continued throughout the pregnancy. This patient’s child underwent postpartum evaluation by a geneticist and was observed to be morphologically normal, except for clinodactyly and low-set ears.

Key Words: Imatinib, Tyrosine kinase inhibitors, Chronic myeloid leukemia, Pregnancy

Özet Kronik myeloid lösemili (KML) hastaların sonuçları son on yıl içinde yeni tirozin kinaz inhibitörlerinin yaygın olarak kullanılmasıyla dramatik bir düzelme göstermiştir. Daha iyi genel sağkalımla hamile kalan KML hastası kadınların sayısında bir artış görülmüştür. İlgili doktor bir ikilemle karşı karşıyadır: kanseri tedavi etmek için yaşamı uzatıcı ilaçları devam ettirme veya olası teratojenisite nedeniyle kullanımlarını kesme. KML’li kadınlarda iki başarılı hamileliği sunuyoruz . Birinci vaka imatinib yerine interferon kullanılmasıyla takip edildi. Bu vakada çocuğa üç yaşındayken genetik değerlendirme yapıldı. Normal gelişimsel referans noktalarına ulaşmıştı ama yaşıtlarından fiziksel olarak daha küçüktü. Ancak orantılar normaldi. Morfolojik olarak klinodaktili, kısa 5. parmaklar ve palpebral fissürlerde hafif aşağıya doğru eğim olması dışında normal görünüyordu. İkinci vakada imatinib hamilelik boyunca devam ettirildi. Çocuğa postpartum bir genetik uzmanı tarafından değerlendirme yapıldı ve klinodaktili ve alçak yerleşimli tırnaklar dışında morfolojik olarak normal bulundu.

Anahtar Sözcükler: İmatinib, Tirozin kinaz inhibitörleri, Kronik myeloid lösemi, Hamilelik

Address for Correspondence: Mike WEBB, M.D., Department of Internal Medicine (G73), Faculty of Health Sciences, University of the Free State, PO Box 339, Bloemfontein 9300, South Africa Phone: + 27 51 405 3154 E-mail: webbmj@ufs.ac.za Received/Geliş tarihi : May 23, 2012 Accepted/Kabul tarihi : June 12, 2012

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Turk J Hematol 2012; 29: 405-408

Introduction

Clinical examination showed that her spleen was enlarged to 6 cm below the costal margin. She was initially started on hydroxyurea 2 g/d p.o. until imatinib could be procured via the Glivec® International Patient Assistance Program (GIPAP). She was started on imatinib 400 mg/d in June 2005, and achieved complete hematological response in September 2005 and complete cytogenetic remission (CCyR) in June 2006. In February 2007 she wanted to become pregnant. Qualitative PCR was performed in March 2007, which was negative for the BCRABL gene rearrangement. Following patient consultation, imatinib treatment was withdrawn in April 2007 and subcutaneous (SQ) interferon-α was initiated at a dose of 3 MU/m2 5 times/week.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm associated with the Philadelphia chromosome. The Philadelphia chromosome results from the reciprocal translocation of chromosomes 9 and 22, leading to production of the BCR-ABL fusion protein. This fusion protein is thought to be the initiating event in the pathogenesis of CML. Inhibition of this protein was a logical therapeutic target and led to the development of imatinib mesylate (Gleevec®, Novartis, Basel, Switzerland), a firstgeneration tyrosine kinase inhibitor. The International Randomized Study of Interferon Versus STI571 (IRIS) showed conclusively that imatinib 400 mg was superior to the standard care offered at the time of the study [1]. To date, imatinib remains the optimal firstline therapy for patients with CML. In terms of monitoring patients treated with imatinib, time-related improvement in hematological, cytogenetic, and molecular markers of disease is important [2]. To achieve optimal outcome, imatinib should be administered at 400 mg po /day [3]. Treatment compliance is important and dose interruptions have a negative impact on outcome [4]. With the advent of imatinib, CML has been transformed from a universally fatal disease to a disease with an estimated 7-year overall survival of 86% [5]. Along with the increase in the number of CML patients experiencing long-term survival come additional challenges, such as an increase in the number of female patients that want to become pregnant. This poses a potential predicament for attending physicians, as based on limited data available in human and animal models, it is recommended that imatinib (a life-saving therapy) not be administered to pregnant women [6]. Herein we describe 2 female CML patients treated with imatinib therapy that subsequently gave birth. Case 1 A newly married 26-year-old female presented to her general physician with headache, depression, and splenomegaly. She was subsequently diagnosed with Philadelphia chromosome-positive chronic-phase CML in November 2004. Her full blood count findings were as follows: WBC count: 300 x 109/L (normal range: 4.0-11.0 x 109/L); Hb: 10.0 g/dL (normal range: 11.5-16.0 g/dL), Plt count: 249 x 109/L (normal range: 150-450 x 109/L). The differential count included a neutrophil count of 90 x 109/L (normal range: 2.0-7.5 x 109/L) and an increase in immature granulocytes. Her peripheral blast count was 3% (normal value: 0%). The patient’s bone marrow was hypercellular without an increase in the blast count.

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Contraception was then stopped and 2 months later she was pregnant. The patient remained on interferon-α through out her pregnancy, without the need for dose reduction due to side effects. The pregnancy was carefully monitored in conjunction with the patient’s obstetricians and she delivered a 1540-g boy in November 2007 via a Caesarian section at 33 weeks of gestation. The premature birth was due to pre-eclampsia. As a neonate the child was diagnosed with a patent foramen ovale and patent ductus arteriosus, which were considered complications of the premature delivery. The child then developed grade 3 hyaline membrane disease and hospital-acquired pneumonia. The congenital heart lesions resolved without intervention and the child fully recovered from his pulmonary complications. The patient remained in CCyR for the duration of her pregnancy. Imatinib was reinstituted post delivery and the patient was advised not to breastfeed. The patient maintained CCyR and exhibited a continuous decrease in her BCR-ABL transcript level, although she still did not achieve a major molecular response (not adjusted to the international scale). The patient’s child was evaluated by a geneticist at age 3 years. The boy achieved normal developmental milestones, but was just below the third percentile for height and weighthe was, however, proportional. In terms of morphology, the child had clinodactyly, short fifth fingers, and slightly downward slanting palpebral fissures, but otherwise appeared normal. Case 2 A 15-year-old female was diagnosed with Philadelphia chromosome-positive chronic-phase CML in 2004. Her symptoms included fatigue, peripheral edema, and bilateral hip pain. She presented with a WBC count of 347 x 109/L, Hb of 6.0 g/dL, and a Plt count of 16 x 109/L. Her

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differential count included 27% neutrophils, 11% metamyelocytes, 45% myelocytes, and 3% blasts. Her bone marrow was hypercellular without an increase in the blast count. Clinical examination showed that her spleen was enlarged to 4 cm below the costal margin. She was initially started on hydroxyurea 1.5 g/d p.o. until imatinib could be procured via the GIPAP program. She achieved complete hematological remission after 3 months of treatment and cytogenetic remission after 12 months. After 18 months of treatment she had not achieved major molecular response. The imatinib dose was not increased because the patient developed neutropenia, which required a dose interruption. In October 2009 (at age 21 years), having subsequently attained major molecular response, the patient discovered that she was 8 weeks pregnant. The treatment options were discussed with the patient and she decided to continue taking imatinib 400 mg/d during the pregnancy. The pregnancy was carefully monitored in conjunction with her obstetricians and she delivered a boy in March 2010 via Caesarian section, which was indicated due to intrauterine growth restriction. At birth the infant weighed 1980 g and had APGAR scores of 9 and 10. The child was evaluated by a geneticist and except for clinodactyly and low-set ears the child was morphologically normal. The mother was advised against breastfeeding. Discussion Treating patients with CML that wish to become pregnant is challenging for a number of reasons. There is no therapy that can be offered to a pregnant woman that is both completely safe and effective, and clinicians are therefore faced with the challenge of balancing the safety of the mother and treating her malignancy against the safety of the unborn child. Data on pregnancy outcomes in patients treated with imatinib are limited. One of the largest collections of data on the effect of imatinib on pregnancy outcome was compiled by Pye et al. [7]. They obtained data on 180 pregnancies in patients receiving imatinib from attending doctors that reported to the Novartis Pharmaceutical Company in Switzerland, the Hammersmith Hospital in London, or the M. D. Anderson Cancer Center in Huston. Fetal abnormalities were observed in 12 cases. A matter of great concern highlighted by the researchers was the increased preponderance of bony abnormalities, which indicated a potential correlation with imatinib treatment. Both of the presented patients’ children had clinodactyly, which can be considered a normal variant or a minor malformation, as its incidence varies from 1% to 19% [8]. Clinodactyly was not considered to be causally

Webb MJ and Jafta D : Imatinib in Pregnancy

linked to the use of imatinib in the presented cases. Data from the Stop Imatinib (STIM) Trial indicate that imatinib can be discontinued in a select subgroup of patients that achieve complete molecular response and maintain it for 2 years [9]. This may well be the ideal method to manage a pregnancy, but may not be applicable to all patients. Patients with disease that is not optimally controlled, as per the European LeukemiaNet (ELN) guidelines [2], may be even more difficult to manage. In such cases continuation of therapy with close monitoring of the pregnancy may facilitate continuation of effective therapy, but pose the risk of adverse fetal outcome. Other modalities of therapy that have been used include hydroxyurea, interferon-ι, and leukapheresis [10-12], each of which is potentially problematic during pregnancy, although interferon may be a safer option though less efficacious than imatinib [13,14]. We described 2 CML patients that gave birth following different approaches to the management of their pregnancies. Case 1 was managed in what may be considered a more classical approachinterruption of imatinib and the use of interferon. This option resulted in a successful pregnancy without the loss of disease control. Case 2 was more complex because the patient failed to achieve an optimal response, according to published guidelines [2]. Consequently, there was concern about disease progression and resistance should therapy be interrupted. In the light of normal fetal ultrasonographic findings at the end of the first trimester, and consultation with colleagues and the patient, imatinib was continued during the pregnancy with close monitoring, which resulted in a favorable outcome. According to Pye et al. [7] and preclinical animal models [6], it is important that women be made aware of the potential complications of therapy for CML during pregnancy; however, in selected cases continuation of imatinib with close monitoring of the pregnancy may facilitate control of the disease and delivery of a normal infant. Acknowledgement The authors thank Dr. Daleen Struwig, University of the Free State, Faculty of Health Sciences, for technical and editorial preparation of the manuscript for publication. Conflict of Interest Statement None of the authors have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

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References 1. O’Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F, Cornelissen JJ, Fischer T, Hochhaus A, Hughes T, Lechner K, Nielsen JL, Rousselot P, Reiffers J, Saglio G, Shepherd J, Simonsson B, Gratwohl A, Goldman JM, Kantarjian H, Taylor K, Verhoef G, Bolton AE, Capdeville R, Druker BJ. Imatinib compared with interferon and lowdose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348: 994–1004. 2. Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio G, Apperley J, Cervantes F, Deininger M, Gratwohl A, Guilhot F, Hochhaus A, Horowitz M, Hughes T, Kantarjian H, Larson R, Radich J, Simonsson B, Silver RT, Goldman J, Hehlmann R. Chronic myeloid leukemia: An update of concepts and management recommendations of European LeukemiaNet. J Clin Oncol 2009; 27: 6041–6051. 3. Deininger MWN, O’Brien SG, Ford JM, Druker BJ. Practical management of patients with chronic myeloid leukemia receiving imatinib. J Clin Oncol 2003; 21(8): 1637–1647. 4. Marin D, Bazeos A, Mahon FX, Eliasson L, Milojkovic D, Bua M, Apperley JF, Szydlo R, Desai R, Kozlowski K, Paliompeis C, Latham V, Foroni L, Molimard M, Reid A, Rezvani K, de Lavallade H, Guallar C, Goldman J, Khorashad JS. Adherence is the critical factor for achieving molecular responses in patients with chronic myeloid leukemia who achieve complete cytogenetic responses on imatinib. J Clin Oncol 2010; 28: 2381–2388. 5. O’Brien SG, Guilhot F, Goldman JM, Hochhaus A, Hughes TP, Radich JP, Rudoltz M, Filian J, Gathmann I, Druker BJ, Larson RA. International Randomized Study of Interferon Versus STI571 (IRIS) 7-year follow-up: Sustained survival, low rate of transformation and increased rate of major molecular response in patients with newly diagnosed chronic myeloid leukemia in chronic phase treated with imatinib. Blood 2008; 112: Abstract 186.

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6 Novartis Pharmaceutical Company. Gleevec® (imatinib) prescribing information. Available at www.pharma. us.novartis.com/product/pi/pdf/gleevec_tabs.pdf (Accessed 3 December 2012). 7. Pye SM, Cortes J, Ault P, Hatfield A, Kantarjian H, Pilot R, Rosti G, Apperley JF. The effects of imatinib on pregnancy outcome. Blood 2008; 111: 5505–5508. 8. Flatt AE. The troubles with pinkies. BUMC Proceedings 2005; 18: 341–344. 9. Mahon FX, Réa D, Guilhot J, Guilhot F, Huguet F, Nicolini F, Legros L, Charbonnier A, Guerci A, Varet B, Etienne G, Reiffers J, Rousselot P. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: The prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol 2010; 11: 1029–1035. 10. Lipton JH, Derzko CM, Curtis J. Alpha-interferon and pregnancy in a patient with CML. Hematol Oncol 1996; 14: 119–122. 11. Patel M, Dukes IA, Hull JC. Use of hydroxyurea in chronic myeloid leukemia during pregnancy: A case report. Am J Obstet Gynecol 1991;165: 565–566. 12. Ali R, Ozkalemkaş F, Ozkocaman V, Ozçeklic T, Ozan U, Kimya Y, Tunali A. Successful pregnancy and delivery in a patient with chronic myelogenous leukemia (CML), and management of CML with leukapheresis during pregnancy: a case report and review of the literature. Jpn J Clin Oncol 2004; 34: 215–217. 13. Pons JC, Lebon P, Frydman R, Delfraissy JF. Pharmacokinetics of interferon-alpha in pregnant women and fetoplacental passage. Fetal Diagn Ther 1995; 10: 7–10. 14. Bristol-Myers Squibb Company. Hydrea® (hydroxyurea) prescribing information. Available at http://packageinserts. bms.com/pi/pi_hydrea.pdf (Accessed 3 December 2012).

Case Report

DOI: 10.5505/tjh.2012.57855

Intramesenteric Steroid Treatment for SteroidRefractory Gastrointestinal Graft Versus Host Disease Steroid Dirençli Gastrointestinal Graft Versus Host Hastalığında İntramezenterik Steroid Uygulaması Aynur Uğur Bilgin1, Pervin Topcuoğlu2, Tanzer Sancak3, Nahide Konuk2, Mutlu Arat2 Necmettin Erbakan University, Meram Faculty of Medicine, Division of Hematology, Department of Internal Medicine, Konya, Turkey Ankara University, School of Medicine, Division of Hematology, Department of Internal Medicine, Ankara, Turkey 3 Ankara University, School of Medicine, Division of Radiology, Ankara, Turkey 1 2

Abstract Currently, steroid-refractory severe gastrointestinal (GI) graft versus host disease (GVHD) is among the most important complications of allogeneic transplantation, and as yet there is no standard approach to its treatment. Herein we report two cases with steroid-refractory GI GVHD that received intramesenteric steroid treatment. In both cases the frequency and volume of diarrhea resolved completely following intramesenteric methylprednisolone (MP) injection. In conclusion, intra-arterial steroid injection might be an alternative treatment approach for steroid-refractory GI GVHD.

Key Words: Steroid-refractory GVHD, Intramesenteric steroid

Özet Günümüzde, steroid dirençli gastrointestinal (Gİ) graft versus host hastalığı (GvHH) allogeneik transplantasyon yapılan hastalarda izlenen en sık komplikasyonlardan biridir ve henüz standart bir tedavi yaklaşımı yoktur. Burada, steroid refrakter GvHH olan iki vakanın intramezenterik steroid uygulamasına dair sonuçları bildirilmiştir. Her iki hastada intramezenterik metilprednizolon (MP) enjeksiyonunu takiben diarenin sıklığı ve miktarı tamamen düzelmiştir. Sonuç olarak, intra-arteriel steroid verilimi bu tip olgularda alternative bir tedavi yaklaşımı olabilir.

Anahtar Sözcükler: Steroid dirençli GvHH, İntramezenterik steroid Introduction Steroid-refractory severe gastrointestinal (GI) graft versus host disease (GVHD) is one of the most challenging complications of allogeneic hematopoietic cell transplantation (allo-HCT), and is associated with very high morbidity and mortality rates. Treatment with various salvage regimens has been used, but the results have not been satisfactory. In recent years, regional administration of steroids directly through the arteries that supply blood to

the liver and intestines has resulted in varying degrees of effectiveness [1-3]. Herein we present 2 cases with steroidrefractory severe GI GVHD in which significantly stable recovery followed a single intra-arterial injection of methylprednisolone (MP) through the superior and inferior mesenteric arteries. Case 1 A 44-year-old male patient with AML underwent alloHCT from an HLA-identical sister following a myeloabla-

Address for Correspondence: Aynur UĞUR BİLGİN, M.D., Necmettin Erbakan Üniversitesi, Meram Tıp Fakültesi, İç Hastalıkları Anabilim Dalı, Hematoloji Bilim Dalı, Konya, Turkey Phone: +90 312 595 73 49 E-mail: aynurugurbilgin@yahoo.com Received/Geliş tarihi : January 14, 2011 Accepted/Kabul tarihi : June 13, 2011

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tive conditioning regimen. Symptoms of grade III (skin, liver, and GI) acute GVHD emerged on d 30 of hematological recovery, which were confirmed based on pathological examination. Skin symptoms resolved following IV MP 2 mg·kg–1·d–1, but frequent diarrhea (maximum volume: 6 L d–1) persisted. Due to exacerbation of GI symptoms, i.e. severe abdominal pain and hematochezia, antithymocyte globulin was initiated on d 35 of hematological recovery. As the patient’s diarrhea did not abate and he had an elevated bilirubin level and metabolic disorders, 4 weeks of infliximab 10 mg kg–1 QWK was started on d 41 of hematological recovery. Despite of all the immunosuppressive treatments, the patient’s diarrhea continued at the rate of 3 L d–1; therefore, intramesenteric arterial MP infusion was administered on d 54 of hematological recovery. Briefly, under digital subtraction angiography (DSA) guidance (Multistar Plus/T.O.P., Siemens AG, Forchheim, Germany), via the right femoral artery No. 5 French catheters (Contra 2, Imager II, Boston Scientific/Medi-Tech, Annacotty, Limerick, Ireland) were placed with their mesenteric tips. Selective views were obtained via injection of 6-8 mL of non-ionic contrast agent (Iomeron 400 mg/dl, Bracco SpA, Milan, Italy) in order to precisely visualize the vascular supply of the superior and inferior mesenteric arteries. Additionally, selective celiac angiography was performed in order to determine if there was anatomical variation. Selectively, 1 mg kg–1 of MP in 10 mL of sterile saline was infused over the course of 3 min into the superior and inferior mesenteric arteries (Figures 1 and 2).

Figure 1: Digital subtraction angiography of the hepatic artery.

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The patient’s diarrhea decreased in volume and frequency (1 L d–1 2 d) after administration of intra-arterial steroid. The patient’s metabolic and clinical status began to recover on the fourth day. The symptoms of GI GVHD were completely resolved 90 d post treatment (Figure 3) and he was discharged with CsA alone as immunosuppressive therapy. Unfortunately, the patient died due to systemic infection 224 d post treatment. Case 2 A 56-year-old male with idiopathic myelofibrosis underwent allo-HCT from an HLA identical sibling donor and non-myeloablative conditioning in November 2005. Donor lymphocyte infusion was administered on 10 May 2006 because of increasing-donor type chimerism. The patient developed grade II acute GVHD in the skin and liver 1 month after donor lymphocyte infusion (DLI). Steroid plus CsA was administered, but acute GVHD progressed to extensive chronic GVHD, involving the eyes, the GI system, and bone marrow. The results of skin and colon biopsies confirmed grade III skin and GI GVHD. Oral budesonide, mycophenolate mofetil, sirolimus, and extracorporeal photopheresis (ECP) were added for the treatment of GVHD. Despite all treatment, GI GVHD could not be controlled. We administered intramesenteric steroid infusion, as described in case 1. Marked improvement in the patient’s symptoms and a decrease in the volume of diarrhea was noted 72 h following the procedure. Complete resolution of diarrhea occurred 15 d after steroid infusion. As of 3 years following the procedure (at

Figure 2: Digital subtraction angiography of the superior mesenteric artery.

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UÄ&#x;ur Bilgin A, et al : Steroid-Refractory GI GVHD and Intramesenteric Steroid

enteric steroid infusion are not exactly known. Rogler and Schottelius reported a decrease in the number and affinity of steroid receptors in GI tract mucosa in patients with inflammatory bowel disease [8,9]. Administration of regional steroid infusion versus systemic may cause higher concentration of steroids in the mesenteric circulation and inflammatory mucosa in patients with GI GVHD. Hence, regional steroid application may overcome the refractoriness caused by a decrease in the number and affinity of steroid receptors. Figure 3: Volume of diarrhea and the bilirubin level prior to and after regional steroid injection in case 1. IMAMP: Intramesenteric artery MP.

the time this manuscript was prepared) the patient did not have recurrence of GI GVHD. Discussion Corticosteroids are currently used as front-line therapy for acute and chronic GVHD; however, less than 50% of patients, especially those with grade II-IV acute GVHD, achieve durable response following initial treatment. Mortality is very high in patients with steroid-refractory disease and therapeutic options are limited. Several immunosuppressive drugs, including mycophenolate mofetil, sirolimus, pentostatin, rituximab, and ECP, have been used as salvage regimens [4]. To date, however, the most effective strategy remains unknown. GI GVHD has similar features as severe ulcerative colitis (UC). Steroid administration via the superior and inferior mesenteric arteries is very effective in UC patients that are unresponsive to conventional therapy [5,6]. An extensive review of literature showed that there are several publications on the use of regional steroids in GVHD. Sato et al. [1] were the first to report the beneficial role of this approach and subsequently many other case studies reported that similar patients were also successfully treated with the same approach [2,3,7]. Encouraged by these reports we used the same approach to treat steroid-refractory severe GI GVHD and observed complete resolution of the symptoms of GI GVHD within a few days of administration of the treatment. Unlike previously reported patients, the 2 presented cases were not only refractory to high-dose systemic steroid treatment, but were also refractory to secondary salvage therapy. In the patients with refractory to systemic steroids, the mechanisms underlying regional and low-dose intrames-

In conclusion, the two presented cases with severe GI GVHD, and refractoriness to steroid and secondary salvage therapies responded dramatically to regional administration of steroids via the mesenteric artery. A single dose of intramesenteric artery steroid infusion appears to be effective, even in patients that have not responded to multiple treatment regimens, and is not associated with extensive systemic immunosuppression. Nonetheless, we cannot exclude that the treatment responses observed in the two presented cases were not due to the delayed therapeutic effect of their initial steroid treatment or other immunosuppressive agents. However, the resolution of GI symptoms within 72 h of intra-arterial administration demonstrate the efficiency of this novel treatment. Based on the present findings, intra-arterial administration of steroids is easy, reliable, and safe, and can be considered a first-line treatment, especially for avoiding severe systemic immunosuppression in patients with severe GI GVHD. On the other hand, controlled studies are required to exclude the role of previous immunosuppressive treatments. Informed consent was obtained. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. References 1. Sato T, Sakamaki S, Nagaoka Y, Kuribayashi K, Nagamachi Y, Morii K, Honma H, Kogawa K, Kato J, Niitsu Y. IntraMesenteric Artery Steroid Administration Relieved Severe Refarctory Gastro-Ä°ntestinal Graft-Vs.-Host Disease in an Allogeneic Bone Marrow Transplantation Patient. Am J Hematol 1997; 56:277-280. 2. Tfayli A, Selby G, Maqbool F, Bierbaum W, Hamadani M. Role of Intra-arterial Steroid Administration in the Management of Steroid Refractory Acute Gastrointestinal Graft-Versus-Host Disease. Am J Hematol 2006; 81:959962.

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3. Nakai K, Tajima K, Tanigawa N, Matsumoto N, Zen K, Nomura S, Fujimoto M, Kishimoto Y, Amakawa R, Sawada S, Fukuhara S. Intra-arterial Steroid-injection therapy for Steroid Refractory Acute Graft-Versus-Host Disease with the evaluation of angiography. Bone Marrow Transplant 2004; 33, 1231–1233. 4. Bolaños-Meade J, Vogelsang GB. Novel strategies for steroid-refractory acute graft-versus-host disease. Curr Opin Hematol 2005; 12:40-44 5. Ishii N, Chiba M, Suzuki T, Iizuka M, Masamune O, Kubo N. A successful case of intraarterial prednisolone injection therapy in severe ulserative colitis unresponsive to an intensive intravenous regimen. Gastroenterol Jpn 1993; 28:725-729 6. Momoshima S, Kohda E, Hiramatsu K, Asakura H. Intraarterial Prednisolone Infusion Therapy in Ulserative Colitis. AJR Am J Roentgenol 1985; 145:1057-1060

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7. Shapira MY, Bloom AI, Or R, Sasson T, Nagler A, Resnick IB, Aker M, Zilberman I, Slavin S, Verstanding A. Intraarterial catheter directed therapy for severe graft-versushost disease. Br J Haematol 2002; 119:760-764 8. Rogler G, Meinel A, Lingauer A, Michl J, Zietz B, Gross V, Lang B, Andus T, Schölmerich J, Palitzsch KD. Glucocorticoid receptors are down-regulated in inflamed colonic mucosa but not in peripheral blood mononuklear cells from patients with inflammatory bowel disease. Eur J Clin Invest. 1999 Apr;29:330-336. 9. Schottelius A, Wedel S, Weltrich R, Rohde W, Buttgereit F, Schreiber S, Lochs H. Higher expression of glucocorticoid receptor in peripheral mononuklear cells in inflammatory bowel disease. Am J Gastroenterol 2000; 95:1945-1999

Case Report

DOI: 10.5505/tjh.2012.03360

Nasal Natural Killer/T-cell Lymphoma with Skin, Eye, and Peroneal Nerve Involvement Cilt, Göz ve Peroneal Sinir Tutulumu ile Seyreden Nazal Tip NK/T-Hücreli Lenfoma Burcu Türker1, Burak Uz2, Metin Işık3, Özlen Bektaş2, Haluk Demiroğlu2, Nilgün Sayınalp2, Aysegül Üner4, Osman İlhami Özcebe2 Hacettepe University, Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey Hacettepe University, Faculty of Medicine, Department of Hematology, Ankara, Turkey 3 Hacettepe University, Faculty of Medicine, Department of Rheumatology, Ankara, Turkey 4 Hacettepe University, Faculty of Medicine, Department of Pathology, Ankara, Turkey 1 2

Abstract Nasal-type natural killer (NK)/T-cell lymphoma (NKTL) is a rare disease strongly associated with Epstein-Barr virus and is often localized to the upper aerodigestive tract at presentation. Extranodal NKTL may involve any extranodal site and disease beyond the nasal cavity is highly aggressive, with short survival time and poor response to therapy. Herein we present a 57-year-old male that had been treated with systemic chemotherapy and cranial radiotherapy for nasaltype NKTL in the palate with skin, right eye, and right peroneal nerve involvement. He was given salvage chemotherapy consisting of 3 cycles of ICE and his response to the therapy was satisfactory, except for persistent right drop foot. About 6 weeks later, the patient presented with bilateral total loss of vision and proptosis; therefore, DHAP chemotherapy was started. Unfortunately, after 1 cycle of the second salvage chemotherapy, he died due to severe fungal infection of the hard palate. Despite the fact that involvement of any extranodal site is possible, concurrent involvement of many systems in NKTL patients is unusual. Nasal-type NKTL has a poor prognosis, despite local radiotherapy and systemic chemotherapy. Physicians should be aware of this rare disorder than can only be diagnosed after extensive immunohistochemical studies.

Key Words: Nasal-type NK/T-cell lymphoma, Radiotherapy, Peroneal nerve, Drop foot

Özet Nazal tip NK/T-hücreli lenfoma, Epstein-Barr virüs ile yüksek oranda ilişkili ve başvuru anında sıklıkla üst solunum/ sindirim sistemine yerleşen nadir bir hastalıktır. Nazal bölge dışındaki tutulumlar sıklıkla agresif seyirli olup, tedaviye yanıtları kötüdür ve yaşam süreleri oldukça kısadır. Damakta nazal tip NK/T-hücre lenfoma tanısı olup sistemik kemoterapi ve kranial radyoterapi tedavileri uygulanmış bir erkek hasta, cilt, sağ göz ve sağ peroneal sinir tutulumları ile karşımıza çıktı. 3 kür ICE kurtarma kemoterapisi ile başarılı bir yanıt elde edildi, fakat sağda düşük ayak deformitesi devam etti. Yaklaşık 6 hafta sonra her iki gözde tam görme kaybı ve proptozis ile başvurdu. Bunun üzerine DHAP kemoterapisine geçildi. 1 kür sonrası sert damakta meydana gelen fungal enfeksiyon neticesinde hasta ex oldu. Bu Address for Correspondence: Burak UZ, M.D., Hacettepe Üniversitesi Tıp Fakültesi Hematoloji Bilim Dalı Samanpazarı, Sıhhıye, 06100 Ankara, Turkey Phone: +90 312 305 15 36 E-mail: burakuz78@gmail.com Received/Geliş tarihi : January 29, 2011 Accepted/Kabul tarihi : October 6, 2011

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tür lenfomalarda herhangi bir bölgenin tutulumu olabilmekle birlikte, aynı hastada birçok bölgenin etkilenmesi sık görülen bir durum değildir. Nazal tip NK/T-hücreli lenfomalarda lokal radyoterapi ve sistemik kemoterapiye rağmen prognoz kötüdür. Klinisyenler ileri immünhistokimyasal çalışmalarla tanı konabilen bu nadir hastalığa karşı uyanık olmalıdırlar.

Anahtar Sözcükler: Nazal tip NK/T-hücreli lenfoma, Radyoterapi, Peroneal sinir, Düşük ayak Introduction Nasal-type NK/T-cell lymphoma (NKTL) is a rare entity strongly associated with Epstein-Barr virus (EBV). The neoplastic cells derived from NK cells and/or cytotoxic T-lymphocytes involve the nasal cavity and paranasal sinuses. NKTL is most prevalent in Asians, and the Native American population of Mexico, Central America, and South America. Irrespective of ethnic origin, EBV is the most probable pathogenetic agent [1]. Extranodal NKTL almost always exhibits extranodal presentation. The disease is often localized to the upper aerodigestive tract at presentation and bone marrow involvement is uncommon. Patients with nasal involvement present with symptoms of nasal obstruction and epistaxis. Lymphoma may extend to adjacent tissues and/or may disseminate rapidly to various sites (e.g. skin, gastrointestinal tract, testis, and cervical lymph nodes). Extranodal NKTL occurring beyond the nasal cavity is highly aggressive, and is associated with a short survival time and poor response to therapy [1]. Although any extranodal site involvement may be seen in this disease, concurrent involvement of many systems is unusual. Herein, we present the clinical, morphological, and immunohistochemical features, and clinical findings in a case of relapsed nasal-type NKTL with skin, eye, and peroneal nerve involvement. It is noteworthy that colon adenocarcinoma and NKTL occured in the presented patient, which is unusual and probably had a negative effect on prognosis. Case A 57-year-old male presented to our outpatient unit in March 2009 with abdominal distention, abdominal pain, and constipation of 15-d duration. To evaluate the onset of constipation colonoscopic evaluation was performed and a tumoral lesion on the ascending colon was noted. Pathological evaluation was compatible with adenocarcinoma, which was extending to the serosa, and right hemicolectomy was performed. Further treatment was not planned, because the surgical margins were clear of tumoral tissue and there was no lymph node metastasis. Additionally, the tumor was moderately differentiated. Four months later the patient was re-evaluated due

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to a new lesion on the palate. The white, painful lesion was rapidly enlarging and had a tendency to bleed. In a short time, a hard immobile 3-cm mass on the right cheek appeared and the patient began to complain of nasal congestion, dyspnea, and fever, which were especially severe at night. He also lost 5 kg of body weight, and was diagnosed as sinusitis at a different hospital and was treated with antibiotics. Because there was no clinical improvement, incisional biopsy was performed and nasal-type NKTL was diagnosed (Figures 1-3); the initial stage was III ESB, according to the Ann-Arbor staging system. International Prognostic Index (IPI) score at presentation was 0 (low risk). Serum ELISA tests were positive for EBV EBNA IgG (47.5 RU mL–1) (normal range: 0-20 RU mL–1) and EBV VCA IgG (147.7 RU mL–1) (normal range: 0-20 RU mL–1). The presence of EBV small ribonucleic acids (RNAs) in the neoplastic cells was observed via in situ hybridization using EBV-encoded small RNA (EBER) oligonucleotides (Figure 4). Neoplastic cells stained positive for CD3, CD56, and granzyme B, and negative for CD20. The Ki-67 proliferation index was 80%-90%. Nasopharyngeal MRI showed a giant lesion measuring 6.1x4.9x7.1cm located in the nasal cavity and the palate that extended to the pterygopalatine fossa, filling the nasopharynx and the oropharynx, and narrowing the airway. Furthermore, bilateral cervical lymphadenopathies, which increased in size and in metastatic nature, were observed. Abdominal CT showed a splenic hypodense mass measuring 17 mm, but thoracic CT and bone marrow biopsy were normal. Colon biopsy specimens that were obtained at the time adenocarcinoma was diagnosed were re-evaluated and determined to be negative for lymphoma involvement. As such, chemotherapy and local radiotherapy were scheduled. On 1 September 2009 we initiated chemotherapy (CHOP regimen: cyclophosphamide, vincristine, adriamycin, and prednisolone) in 21-d cycles plus central nervous system (CNS) prophylaxis with methotrexate 15 mg t.w. for 3 weeks. Following 3 cycles of CHOP, 3 cycles of cranial radiotherapy were administered. Cervical and thoracic CT post treatment were normal. Follow-up abdominal CT showed that the splenic mass had decreased in size

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Figures 1-3: H&E staining of the lesion located on the palate shows abundant lymphocytes.

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(1 cm). Colonoscopic evaluation was performed again and the findings were normal. On 11 June 2010 the patient developed erythematous lesions on his arms and legs, which increased in diameter and ulcerated during the days that followed (Figures 5 and 6). E. Coli and S. Haemolyticus were noted in culture specimens and IV antibiotic therapy was started. A skin biopsy specimen was obtained and reported as leukemic infiltration. Immunohistochemical examination of neoplastic cells showed diffuse positivity with CD56 staining. On 21 June 2010 the patient developed right drop foot. MRI of the right knee showed increased contrast involvement in the peroneal nerve trace, indicative of a malignant

Figure 4: The presence of EBV small RNAs in the neoplastic cells was observed via in situ hybridization using EBV-encoded small RNA (EBER) oligonucleotides.

infiltrate. Electromyographic findings were compatible with an axonal lesion in the proximal region of the peroneal nerve. On 6 July 2010 vision in his right eye deteriorated; orbital MRI showed subretinal hemorrhage, edema, and thickening of the eyelids, which were more prominent in the right eye. He was thought to have exudative retinal detachment as a result of tumor infiltration (Figure

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Figure 5: Lesion on the right knee.

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Figure 8: Paraneoplastic lesions on the legs that ocurred during the course of chemotherapy.

7). The 4x4 cm lesion in his right knee was debrided and improved with therapy. He was then given 3 cycles of ICE (iphosfamide, carboplatin, and etoposide) chemotherapy and 6 cycles of intrathecal chemotherapy. During the first cycle of chemotherapy skin lesions regressed within a few days, but new erythematosus lesions appeared, resembling a drug eruption. Although biopsy specimens from these lesions were not obtained, they were thought to be paraneoplastic and regressed during the course of chemotherapy (Figure 8). In addition, conjunctival hyperemia and corneal epithelial defects regressed.

Figure 6: Lesion on the right arm.

Figure 7: The patient’s eyes, hyperemia, and edema.

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Unfortunately, the patient’s general condition deteriorated rapidly during a 2-week period and he developed bilateral total loss of vision, conjunctival chemosis, and proptosis (Figure 9). In addition, he had swelling on his left testis (Figure 10). Scrotal ultrasound showed bilateral thickening of the tunica layers and a right-sided varicocele. Orbital MRI showed a mass lesion surrounding the bilateral bulbus oculi that extended throughout the optic nerves to the retrobulbar region, infiltrating the medial and superior rectus muscles. This lesion showed significant progression, as compared to previous MRI findings. Conjunctival biopsy showed atypical lymphoid cells with significant nucleoli. Some of these cells stained positive for CD3 and CD56, and a small number of scattered cells stained positive for granzyme B. Salvage chemotherapy with DHAP (dexamethasone, cytarabine, and cisplatin) was then initiated. After 1 cycle of chemotherapy a crusty necrotic lesion appeared on the hard palate. A biopsy specimen taken from this lesion showed fungal infection, probably associated with rhinocerebral mucormycosis;

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the presented patient was based on a combination of morphology, positive granzyme B and CD56 expression, and negative staining for B-cell markers (such as CD20 and CD79a). The presence of EBV small RNAs was observed via in situ hybridization using EBER oligonucleotides; however, the cells expressed both CD56 and granzyme B. An unusual feature of the presented case was the expression of CD3, which might have been due to the presence of CD3 molecule zeta chain cytoplasmically in NK cells, that would not be detected on the cell surface by flow-cytometry, but can occasionally reach the detection threshold for immunohistochemical analysis, which can detect surface and cytoplasmic expression simultaneously.

Figure 9: Photograph taken a few days before the patient died shows bilateral proptosis and conjunctival chemosis.

Figure 10: Swelling of the left testis.

therefore, posaconazole treatment was scheduled, but the patient died within a few days. Discussion NKTL is a predominantly extranodal malignancy that is most commonly positive for EBV and CD56, and is usually located in the nasal cavity or paranasal sinuses [1]. Nasaltype NKTL is an aggressive disorder with a tendency to invade local tissues and metastasize to the CNS. The presented patient had nasal congestion, dyspnea, and fever, and subsequently developed chronic nasal obstruction and bleeding. These non-specific symptoms may cause a delay in diagnosis, as in the presented patient. Diagnosis of

Rodriquez et al. reported the involvement of unusual extranodal sites, such as the testes, lungs, liver, CNS, bone marrow, and peripheral blood in 6 of 10 patients that presented with local disease [2]. Apart from the nasal cavity, the skin is the most common site of involvement, and may be a primary or secondary manifestation of the disease; nearly 10%-20% of patients with nasal lymphomas also have skin involvement [1]. Our patient presented to hospital with skin, eye, and peroneal nerve involvement 3 months after the last cycle of CHOP chemotherapy. Skin biopsy findings were compatible with leukemic involvement; therefore, salvage chemotherapy with ICE was initiated a second time. The patient’s skin lesions improved rapidly in response to this therapy. The close proximity of the nasal cavity, paranasal sinuses, and eyes may by why the presented patient had ocular metastasis. Primary intraocular T-cell and NKTL are extremely rare and primarily represent as a secondary manifestation of either a cutaneous or systemic lymphoma [3]. They accounted for <3% of all primary ocular lymphoproliferative lesions in reported series [4-6]. Hon et al. reported that 6 of 24 primary nasal and nasopharyngeal NKTL patients suffered vision-threatening complications [7]; therefore, examination of the eyes is very important in the diagnosis and management of this type of lymphomatous disease. Neurologic deficits are rare in patients with NKTL. Nasal-type NKTL presents with transverse myelopathy and third cranial nerve palsy [8,9]. More recently, nasaltype NKTL of the cauda equina has been reported [10]. In the presented patient right-sided peroneal nerve involvement with drop foot was diagnosed on the basis of clinical, MRI, and electromyographic findings. The pathogenesis of this involvement might have been due to ischemia secondary to angiocentric and angiodestructive infiltration by lymphoma cells [1].

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The prognosis of nasal NKTL is variable. Unfavorable prognostic factors include advanced-stage disease (stage III or IV), unfavorable IPI, bone or skin invasion, an elevated circulating EBV DNA level, and the presence of EBVpositive cells in bone marrow [1]. The presented patient had a severe clinical course, despite having a low-risk IPI. Although patients with low IPI scores (≤1) have better survival rates, the prognostic significance of the IPI score is not as consistent in NKTL patients as in those with other types of aggressive NHL [11]. A new prognostic model for NKTL defined by Na et al. was constructed using the LDH level, performance status, B symptom, and stage [12]. This patient was allocated to group 4 according to his weightage scores, and his 1-year survival and median survival time were 0% and 3 months, respectively. Measurement of the circulating viral DNA load in peripheral blood is useful for diagnosis, monitoring, and prognostication of the disease [13]. EBV-DNA can therefore be used as a marker to predict tumor burden [14,15], but prediction can potentially be affected by the presence of EBV unrelated to lymphoma [13]. Extranodal NKTL occurring beyond the nasal cavity is highly aggressive and is associated with short survival time and poor response to therapy. Historically, the survival rate is poor (30%-40%), but survival has improved in recent years due to the use of more intensive therapy, including upfront radiotherapy. Local radiotherapy is the mainstay of treatment, whereas additional multi-agent chemotherapy improves local disease control and survival [16]. Despite intensive radiotherapy, local failure accounts for 50% of NKTL relapses [17]. Nasal NKTL located in the palate is a rare entity, which is diagnosed based on extensive immunohistochemical studies. Nonetheless, it should be kept in mind that if a lesion on the palate or in the nasal cavity is resistant to treatment, malignancy should be an alternative diagnosis. In addition, baseline and regular ophthalmic, neurologic, and testicular assessment is warranted for this type of lymphoma, particularly in patients with NKTL. Unfortunately, nasal-type NKTL has a poor prognosis, despite local radiotherapy and systemic chemotherapy. Conflict of Interest Statement None of the authors has any conflicts of interest, including specific financial interests, relationships, and/ or affiliations, relevant to the subject matter or materials included in this manuscript. Written informed consent was obtained from the patients’ wife.

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References 1. WHO classification of tumours of haematopoietic and lymphoid tissues. Eds: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. Extranodal NK/T-cell lymphoma, nasal type. Chan JKC, Quintanilla-Martinez L, Ferry JA, Peh SC. 285-8. Lyon 2008. 4th Edition. International Agency for Research on Cancer. 2. Rodriquez J, Romaguera JE, Manning J, Ordonez N, Ha C, Ravandi F, Cabanillas F. Nasal-type T/NK lymphomas: A clinicopathologic study of 13 cases. Leuk Lymp 2000;39: 139-144. 3. Coupland SE, Joussen A, Anastassiou G, Stein H. Diagnosis of a primary uveal extranodal marginal zone B-cell lymphoma by chorioretinal biopsy: Case report. Graefes Arch Clin Exp Ophthalmol. 2005;243:482-486. 4. Cheung MM, Chan JK, Lau WH, Foo W, Chan PT, Ng CS, Ngan RK. Primary non-Hodgkin’s lymphoma of the nose and nasopharynx: Clinical features, tumor immunophenotype, and treatment outcome in 113 patients. J Clin Oncol 1998; 16:70-77. 5. Liang R, Todd D, Chan TK, Chiu E, Lie A, Kwong YL, Choy D, Ho FC. Treatment outcome and prognostic factors for primary nasal lymphoma. J Clin Oncol 1995; 13:666-670. 6. Liang R, Chiu E, Todd D, Chan TK, Choy D, Loke SL. Combined chemotherapy and radiotherapy for lymphomas of Waldeyer’s ring. Oncology 1991;48:362-364. 7. Hon C, Chim JC, Au WY. Vision threatening complications of nasal T/NK lymphoma. Am J Ophthalmol 2002;134:406410. 8. Sadahira Y, Wada H, Nakamura E, Terayama K, Sugihara T, Yamada O, Mikami Y, Shirabe T. Nasal NK/T cell lymphoma presenting as transverse myelopathy. Wirchows Arch 2000; 436:393-397. 9. Chen CS, Miller NR, Lane A, Eberhart C. Third canial nerve palsy caused by intracranial extension of a sino-orbital matural killer T-cell lymphoma. J Neuro Ophthalmol 2008; 28:31-35. 10. Morita M, Osawa M, Naruse H, Nakamura H. Primary NK/T-cell lymphoma of the cauda equina: A case report and literature review. Spine 2009; 34:882-885. 11. Chim CS, Ma SY, Au WY, Choy C, Lie AK, Liang R, Yau CC, Kwong YL. Primary nasal natural killer cell lymphoma: long-term treatment outcome and relationship with the Internal Prognostic Index. Blood 2004; 103:216-221. 12. Na II, Kang HJ, Park YH, Lee SS, Yoo HJ, Choe DH, Ryoo BY, Yang SH. Prognostic factors for classifying extranodal NK/T cell lymphoma, nasal type, as lymphoid neoplasia. Eur J Haematol 2007; 79:1-7.

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13. Suzuki R, Takeuchi K, Ohshima K, Nakamura S. Extranodal NK/T-cell lymphoma: Diagnosis and treatment cues. Hematol Oncol 2008; 26:66-72. 14. Lei KIK, Chan LYS, Chan W-Y, Johnson PJ, Dennis Lo YM. Diagnostic and prognostic implications of circulating cell-free Epstein–Barr virus DNA in natural killer/T-cell lymphoma. Clin Cancer Res 2002; 8:29-34. 15. Au WY, Pang A, Choy C, Chim CS, Kwong YL. Quantification of circulating Epstein–Barr virus (EBV) DNA in the diagnosis and monitoring of natural killer cell and EBV-positive lymphomas in immunocompetent patients. Blood 2004;104: 243-249.

16. Kwong YL, Chan AC, Liang R, Chiang AK, Chim CS, Chan TK, Todd D, Ho FC. CD56+ NK lymphomas: clinicopathological features and prognosis. Br J Haematol 1997; 97:821-829. 17. Kim GE, Cho JH, Yang WI, Yang WI, Chung EJ, Suh CO, Park KR, Hong WP, Park IY, Hahn JS, Roh JK, Kim BS. Angiocentric lymphoma of the head and neck: Patterns of systemic failure after radition treatment. J Clin Oncol 2000; 18:54-63.

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Letter to the Editor

DOI: 10.5505/tjh.2012.71473

TNF-α-308 G/A Polymorphism in Egyptian Budd-Chiari Syndrome Patients Mısır Kökenli Budd-Chiari Sendromlu Hastalarda TNF-α -308 G/A Polimorfizmi Yonca Eğin1, Solaf Elsayed2, Mohamed Sakr3, Nejat Akar4 Ankara University, Department of Pediatric Genetics, Ankara, Turkey Ain Shams University, Pediatrics Department, Genetics Unit, Cairo, Egypt 3 Ain Shams University, Cairo, Egypt 4 TOBB Economy and Technology University Hospital, Ankara, Turkey 1 2

To the Editor, Budd-Chiari syndrome (BCS) is an uncommon condition induced by thrombotic or non-thrombotic obstruction of hepatic venous outflow. BCS most often occurs in patients with underlying thrombotic diathesis, including such myeloproliferative disorders (MPDs) as polycythemia vera and paroxysmal nocturnal hemoglobinuria, and pregnancy, oral contraceptives, tumors, chronic inflammatory diseases, clotting disorders, and infections [1]. Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine produced primarily by macrophages and T-cells that has a range of inflammatory and immunomodulatory activity [2]. TNF-α is a pro-inflammatory cytokine with -308 promoter region G/A polymorphism. This polymorphism has been shown to affect the level of expression of the gene in vitro. Population-based studies on the effect of TNF-α -308 G/A on the occurrence of thromboembolic disease have reported inconsistent findings [3-5].

Recently, Fleischman et al. reported that Jak-2 V617F activity is positively correlated with TNF-α mRNA expression, suggesting that Jak2 V617F directly up regulates TNF-α mRNA in myeloproliferative neoplasm (MPN) patients [4]. As such, we aimed to study a polymorphic site located in the promotor region of the TNF-α gene (TNF- α -308 G/A) in Egyptian BCS patients and compared to an Egyptian control group. DNA was obtained from peripheral blood samples of BCS patients (n = 84) and healthy controls (n = 101). DNA was isolated using a MagnaPure LC 2.0 automatic isolation system (Roche Diagnostics, Rotkreuz, Switzerland). The TNF-α -308 promoter region was then amplified using specific primers. A 194-bp product was amplified using the primers, F: 5’-ATTGGAAATAGGTTTTGAGGGTCAT-3’ and R:5’TCTCGGTTTCTTCTCCATCGC-3’ (MWG, Germany). These PCR products were digested using PagI (Fermantas, Lithuania) restriction enzyme. Re-

Table: Distribution of TNF-α -308 G/A polymorphisms in the BCS patients and controls.

TNF-α -308 G/A G/G G/A A/A

Control n­= 101 93 8 -

Patients n = 84 77 7 -

Address for Correspondence: Yonca EĞİN, M.D., Ankara Üniversitesi, Pediatrik Genetik Bilim Dalı, Ankara, Turkey Phone: +90 312 595 63 48 E-mail: yonca_egin@yahoo.com Received/Geliş tarihi : June 28, 2012 Accepted/Kabul tarihi : September 19, 2012

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OR

95% CI

P

1.05 (0.3-3.0) -

0.36-3.04 -

0.86 -

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stricted fragments were run on 3% agarose gel and viewed under UV light [3]. Among the 84 BCS patients, TNF-α -308 G/A polymorphism was present in 8.3% (n = 7), whereas the frequency of TNF-α -308 G/A polymorphism in the healthy Egyptian controls was 7.9%. The distribution of TNF-α -308 G/A polymorphisms in the BCS patients and controls is shown in the Table. Fleischman et al. reported that TNF-α plays a central role in promoting clonal dominance of Jak2 V617Fexpressing cells in MPN. They showed that Jak2 V617F kinase regulates TNF-α expression in cell lines and primary MPN cells, and that TNF-α expression is correlated with Jak2 V617F allele burden [4]. Ghaffar et al. recently reported that factor V Leiden (FVL) was a major etiological factor associated with thrombosis in Egyptian BCS patients, as compared to the frequency of FVL in the general Egyptian population, [6,7]. Elevated TNF-α might be associated with an increase in the risk of thrombotic complications due to the effect of this cytokine on the endothelium. The frequency of TNF-α -308 G/A polymorphism did not differ between Egyptian BCS patients and healthy controls in the present study. Conflict of Interest Statement None of the authors have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

References 1. Shetty S, Ghosh K. Thrombophilic dimension of Budd chiari syndrome and portal venous thrombosis-a concise review. Thromb Res 2011; 127:505-512. 2. Allen RD. Polymorphism of the human TNF-alpha promoter-random variation or functional diversity? Mol Immunol 1999; 36:1017-1027. 3. Akar N, Hasipek M. Tumour Necrosis Factor-Alpha Gene Polymorphism (-308 G-A) in Turkish Pediatric Thrombosis Patients. Turk J Hematol 2002; 19:39-41. 4. Fleischman AG, Aichberger KJ, Luty SB, Bumm TG, Petersen CL, Doratotaj S, Vasudevan KB, Latocha DH, Yang F, Press RD, Loriaux MM, Pahl HL, Silver RT, Agarwal A, O’Hare T, Druker BJ, Bagby GC, Deininger MW. TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms. Blood 2011; 118:6392-6398. 5. Elahi MM, Asotra K, Matata BM, Mastana SS. Tumor necrosis factor alpha -308 gene locus promoter polymorphism: An analysis of association with health and disease. Biochim Biophys Acta 2009; 1792:163-172. 6. Abdel Ghaffar TY, Elsayed SM, Sakr MA, Elsobky ES, Abdelhakam SM Yousuf S, Eğin Y, Akar N. Factor V G1691A (Leiden) is a major etiological factor in Egyptian BuddChiari syndrome patients. Turk J Hematol 2011; 28:299305 7. Ulu A, Elsobky E, Elsayed M, Yıldız Z, Tekin M, Akar N. Frequency of five thrombophilic polymorphisms in the Egyptian population. Turk J Hematol 2006; 23:100-103

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Letter to the Editor

DOI: 10.5505/tjh.2012.98853

Rituximab-Related Reversible Hepatocellular Damage Ritüksimab İlişkili Geridönüşlü Hepatosellüler Hasar Selami K. Toprak, Sema Karakuş Başkent University, School of Medicine, Department of Hematology, Ankara, Turkey

To the Editor, Since its approval in 1997 by the US Food and Drug Administration, use of rituximab (MabThera®, Roche, Switzerland) has become widespread, especially for the treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) [1]. Rituximab is a monoclonal chimeric antibody that targets the CD20 antigen on the surface of normal B-cells and malignant cells in patients with CD20 B-cell lymphoproliferative disorders. The toxic effects of rituximab are mild and usually limited to its initial administration [2]. Reactions to its infusion include hypotension, fever, chills, rigors, urticaria, bronchospasm, angioedema, nausea, fatigue, headache, pruritus, dyspnea, rhinitis, vomiting, flushing, and pain at the disease site [3]. Such reactions usually occur at the beginning of the initial infusion within 30 min-2 h. Other possible and more serious adverse reactions are tumor lysis syndrome, mucocutaneous reaction, progressive multifocal leukoencephalopathy, hepatitis B reactivation with fulminant hepatitis, infection, cardiac arrhythmias, renal toxicity, and bowel obstruction and perforation [3,4]. A moderate increase in liver function test findings—depending on monoclonal anti-CD20 treatment—have been reported; however, no cases of a 15-20-fold increase in transaminases have been reported. Herein we present a case of rituximab treatmentrelated deterioration in liver function test results in a patient with CLL. Case Report A 50-year-old female was given 2 cycles of fludarabine/ cyclophosphamide (FC) combination therapy as a first line treatment for Rai stage II CLL. She was then given 2 cycles

of rituximab/fludarabine/cyclophosphamide (R-FC) treatment, and was admitted to our clinic for preparation and evaluation before her fifth cycle of treatment. Her workup was within normal limits and, as such, on d 1 she was given rituximab 375 mg/m2 (total dose: 600 mg/d) during monitorization and for a time period in accordance with its prospectus information. Although the results of all tests performed before administration of the drug were normal, the patient’s alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and gamma glutamyl transferase (GGT) levels increased sharply the day following administration (Table). The patient had no clinical complaints and her physical examination did not show any associated pathology. In addition, the D-dimer level, prothrombin time, bilirubin level, and hemolysis test results were within normal limits, as were blood electrolytes, kidney function test results, and the whole blood count. The patient’s history of other medical conditions was negative and she did not report regular use of any medication. Administration of the scheduled subsequent chemotherapeutics (FC) according to the treatment protocol was delayed. Hepatobiliary ultrasonography was performed and the results were normal. Screening for hepatitis A, B, and C, and other infectious serologies, including EpsteinBarr virus, cytomegalovirus, human immunodeficiency virus, toxoplasma gondii, rubella, herpes zoster, and herpes simplex, was performed, all of which were negative for acute infection. Autoimmune serologies, including antiliver kidney microsome, anti-smooth muscle antibody, and anti-nuclear antibody, were also negative. The patient was referred to the gastroenterology department for consulta-

Address for Correspondence: Selami K. TOPRAK, M.D., Başkent University School of Medicine Department of Hematology 5. Sokak No:48 Bahçelievler, Ankara 06490, Turkey Phone: +90 532 656 02 06 E-mail: sktoprak@yahoo.com Received/Geliş tarihi : May 16, 2012 Accepted/Kabul tarihi : June 19, 2012

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Table: Liver function biomarker levels.

Parameter ALT AST LDH ALP GGT Total Bilirubin Direct Bilirubin

Normal <55 U/L 5-34 U/L 125-243 U/L 40-150 U/L 5-36 U/L 0.2-1.2 mg/dL 0-0.5 mg/dL

Pre-treatment 29 34 458 134 13 0.2 0.1

1st Post-treatment Day 611 509 704 169 49 0.3 0.2

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; LDH: Lactate dehydrogenase; ALP: Alkaline phosphatase; GGT: Gamma glutamyl transferase.

tion and was followed-up with daily liver function testing and intravenous hydration. She required no additional treatment and on d 7 of the above-described treatment, as her laboratory parameters regressed to basal values, the FC combination therapy was successfully completed. No clinical or laboratory problems were encountered and the patient was discharged with scheduled close-monitoring follow-up. Discussion Based on the presented patient’s negative viral and autoimmune serologies, and rapid recovery after cessation of rituximab therapy, and the fact that rituximab was the only drug the patient received immediately preceding liver function deterioration, we think that this was a case of monoclonal anti-CD20 drug-induced impairment. The half-life of rituximab is approximately 22 d and it is detectable months after administration; however, the presented patient developed abnormal liver function 1 d after administration of the drug, when the concentration is expected to be high [5]. Several viral infections associated with rituximab, particularly viral hepatitis, have been reported [6]. Viral reactivation is a well-known side effect of the drug; however, the presented case highlights the possibility that direct hepatotoxicity may result from rituximab therapy. The question that remains is why rituximab caused no side effects the first 2 times it was given to this patient, but did result in toxicity when administered the third time. Winkler et al. studied 11 patients that underwent rituximab treatment for fludarabine-resistant recurrent CLL/ NHL and reported that serum concentrations of liver enzymes, including ALT, AST, and GGT, increased to levels that exceeded the normal range by a factor greater than 5, whereas the concentration of ALP, and direct and indirect bilirubin remained stable throughout antibody treatment

[7]. Additionally, the LDH level increased in 9 of the patients during treatment, peaking in 2 of the patients at values >2,000 U/mL [7]. In the presented patient the marked increase in peripheral lymphocytes was associated with elevated LDH and liver transaminases; however, the lymphocyte count remained in the normal range and not differ from previously obtained values. Researchers have suggested that in order to prevent rituximab-related toxicity it is reasonable to lower the number of circulating tumor cells to <50.0 x 109/L using chemotherapeutic regimens prior to administering rituximab. In contrast to our expectations based on the literature, in the presented case limited and reversible hepatocellular damage due to rituximab was thought to occur directly, instead of triggering viral reactivation, and presented with laboratory findings only—there were no associated clinical signs. As such, we recommend close follow-up of transaminases following rituximab treatment. Ethical Consideration Written informed consent was obtained from the patient. Conflict of Interest Statement None of the authors have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included. References 1. Toma MB, Medina PJ. Update on Targeted Therapy-Focus on Monoclonal Antibodies. J Pharm Pract 2008;21:4-16. 2. Murawski N, Pfreundschuh M. New drugs for aggressive B-cell and T-cell lymphomas. Lancet Oncol 2010;11:10741085.

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3. Lang DS, Hagger C, Pearson A. Safety of rapid rituximab infusion in adult cancer patients: A systematic review. Int J Nurs Pract 2011;17:357-69. 4. Vogel WH. Infusion reactions: Diagnosis, assessment, and management. Clin J Oncol Nurs 2010;14:E10–21. 5. Del Prete CJ, Cohen NS. A case of rituximab-induced hepatitis. Cancer Biother Radiopharm 2010;25:747-748.

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6. Aksoy S, Harputluoglu H, Kilickap S, Dede DS, Dizdar O, Altundag K, Barista I. Rituximab-related viral infections in lymphoma patients. Leuk Lymphoma 2007; 48:1307-1312. 7. Winkler U, Jensen M, Manzke O, Schulz H, Diehl V, Engert A. Cytokine-release syndrome in patients with B-cell chronic lymphocytic leukemia and high lymphocyte counts after treatment with an anti-CD20 monoclonal antibody (rituximab, IDEC-C2B8). Blood 1999; 94:2217-2224.

Letter to the Editor

DOI: 10.5505/tjh.2012.96977

Hemolytic Crisis as the Initial Presentation of Hereditary Spherocytosis Induced by Parvovirus B19 and Herpes Virus Infection in a Patient with the Thalassemia Trait: A Case Report Talasemi Taşıyıcılığı Olan Bir Hastada Parvovirüs B19 ve Herpes Virüs Enfeksiyonu ile Tetiklenen Herediter Sferositozun Başlangıç Tablosu Olarak Hemolitik Kriz: Olgu Sunumu Meriç Kaymak Cihan1, Hafize Gökçe2, Meral Oruç1, Lale Olcay1 Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Department of Pediatric Hematology, Ankara, Turkey Ankara University, School of Medicine, Department of Pediatric Hematology, Ankara, Turkey

1 2

To the Editor, Human parvovirus B19 (PV-B19) causes erythema infectiosum, hydrops fetalis, and transient aplastic crisis in immunocompromised patients with chronic hemolytic anemia, arthralgia, and chronic pure red cell aplasia [1]. It may also cause autoimmune hemolytic anemia [2] (which presents as aplastic crisis with reticulocytopenia or increased erythropoiesis with reticulocytosis) [3], autoimmune thrombocytopenia/neutropenia, myelodysplastic syndrome, leukoerythroblastosis, hemophagocytic lymphohistiocytosis, and leukemia [1]. Transient aplastic crisis manifests as sudden exacerbation of anemia in patients with chronic hemolytic anemia, with severe reticulocytopenia lasting 7-10 days in the absence of erythroid precursors due to lysis of the precursors by PV-B19 [4]—the hallmark of which is giant pronormoblasts in the bone marrow [5]. Hemolytic crisis in hereditary spherocytosis (HS) is characterized by more pronounced jaundice than that in stable state of HS due to accelerated hemolysis and probable viral infection [6]. Herein we describe a patient that presented with hemolytic crisis as the initial manifestation

of HS during PV-B19 infection—in contrast to the expectation of transient aplastic crisis [7]. This condition was attributed to coexistent herpes virus infection. The patient had a low mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) due to the thalassemia trait, which masked spherocytosis. A 15-year-old female was presented for evaluation of anemia. She had a 4-month history of progressive pallor, fatigue, scleral icterus, and dark urine. She has had intractable pallor since nearly 3 years of age Icterus was not noted until four months prior to her presentation to our hospital. Her brother also had pallor without icterus. Physical examination showed pallor, scleral icterus, and hepatosplenomegaly (spleen and liver extended the costal margins by 4 cm and 3 cm, respectively). Laboratory findings were as follows: hemoglobin level: 75 g/L; hematocrit: 24.1%; red blood cell count (RBC):4.11x1012/dL; platelet count: 279 x 109/L; white blood cell count: 7.3 x 109/L: mean corpuscular volume (MCV): 59.5 fL: mean corpuscular hemoglobin concentration MCHC 31.5 g/dl (N: 32.7-35.6 g/dl); mean corpuscular hemoglobin (MCH): 18.5 pg (normal range: 27.2-33.5

Address for Correspondence: Lale OLCAY, M.D., Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Department of Pediatric Hematology, Ankara, Turkey Phone: +90 312 336 09 09 E-mail: laleolcay@hotmail.com.tr Received/Geliş tarihi : March 24, 2012 Accepted/Kabul tarihi : March 29, 2012

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pg); red blood cell distribution width: 21% (normal range: 11.8%-14.3%); reticulocyte count: 2.2% (normal range: 0.6%-2.6%). Peripheral blood smear showed anisocytosis, poikilocytosis, hypochromia, and spherocytes. Direct and indirect Coombs tests were negative, and total/indirect bilirubin was 1.68/1.33 mg/dL, haptoglobin was <5.83 mg/dL (normal range: 36-195mg/dL), plasma hemoglobin was 7% (normal: <3), serum lactate dehydrogenase was 254 IU/L, and Hb A2 was 4.37%. Bone marrow aspiration showed erythroid hyperplasia, glucose-6-phosphate dehydrogenase, pyrimidine 5’ nucleotidase, pyruvate kinase, CD 55, and CD 59 evaluation of CD 55, and CD 59 on granulocytes by flow cytometry were normal. Collagen tissue disease markers were negative. Abdominal ultrasonography showed splenomegaly and elevated osmotic fragility. Herpes simplex virus HSV type 1-2 IgM was positive. PV-B19 PCR was 75.3 copies/mL in plasma, based on real-time PCR (normal: negative). The patient was diagnosed as hereditary spherocytosis and thalassemia trait, with coexistent PV-B19 and herpes virus infections.The hemoglobin level was found to have increased spontaneously to 88 g/L and her pallor and scleral icterus were found resolved at the end of in her evaluation made on 46 th day of admission At this time blood smear findings and mild indirect hyperbilirubinemia persisted and PV-B19 DNA showed 13 copies and HSV type 1-2 IgM was still positive. Viral serological tests may cross react with each other and we couldn’t perform HSV PCR. But HSV IgG was found to have increased from 6.15 to 113.2 RU/mL on the 46 th day of admission and this confirmed that PV-B19 and HSV infected the patient coexistently., The presented patient had mild HS, which manifested first as hemolytic crisis due to herpes virus infection, although infection by other viruses we didn’t test for could not be excluded. Her RBC count was within the lower limits of normal, which is unexpected in a patient with hemolytic anemia and the thalassemia trait. We think the normal RBC and reticulocyte counts, despite erythroid hyperplasia of the bone marrow, were indicative of repressed erythropoiesis due to PV-B19 infection.

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The presented case highlights the fact that, in the setting of chronic mild hemolytic anemia, patients in hemolytic crisis that have normal reticulocyte and RBC counts should be tested for PV-B19 infection and accompanying other viral infections . Clinicians should be aware that MCH and MCHC are not elevated in HS patients with thalassemia trait. Conflict of Interest Statement None of the authors have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included. References 1. Yetgin S, Elmas SA. Parvovirus–B19 and hematologic disorders. Turk J Hematol 2010; 27:224-233 2. Lehmann HW, Landenberg P, Modrow S. Parvovirus B19 infection and autoimmune disease. Autoimmunity Rev 2003; 2:218-223 3. de la Rubia J, Moscardó F, Arriaga F, Monteagudo E, Carreras C, Marty ML. Acute parvovirus B19 infection as a cause of autoimmune hemolytic anemia. Hematologica 2000; 85:995-997 4. Carzavec D, Gacina P, Vasilj A, Katovic SK. Aplastic crisis induced by Human Parvovirus B19 as an initial presentation of hereditary spherocytosis. Coll Antropol 2010; 34:619621 5. Gallagher PG, Forget BG. Hereditary spherocytosis, elliptocytosis, and related disorders. In: Beutler E, Coller BS, Lıchtman AM, Kıpps TJ, Selıghsohn U, eds. Williams Hematology. 6th ed. New York: McGraw-Hill, 2001: 508 6. Lanskowsky P. Red cell membrane and enzyme defects. Manual of Pediatric Hematology and Oncology. 5th ed. San Diego, CA: Elsevier, 2011:175-179 7. Tavil B, Özdel S, Özkasap S, Yaralı N, Tunç B. Aplastic crisis induced by human parvovirus B19 infection as an initial presentation of HS. Indian J Pediatr 2010; 77:1191-1192

Letter to the Editor

DOI: 10.5505/tjh.2012.21703

A Search for Beta Thalassemia Trait in India Hindistan’da Beta Thalassemia Taşıyıcı Araştırması Veda Parthasarathy Department of Pathology, ESI PGIMSR, Bangalore, India

To the Editor, The beta-thalassemia trait (BTT)—or beta-thalassemia minor—is a heterozygous condition in which only a single beta-globin gene is affected. The estimated prevalence of BTT in different regions of India is reported to vary between 2.7% and 14.9% (mean: 4.5%) [1,2]. Most individuals with BTT are asymptomatic and are identified incidentally when their complete blood count (CBC) shows microcytosis [3]. Red blood cells are considered to be microcytic when the mean corpuscular volume (MCV) is <80 fL [4]. Common causes of microcytosis are iron deficiency anemia (IDA), BTT, anemia of chronic disease (ACD), lead poisoning, and sideroblastic anemia [5]; BTT must be differentiated from IDA and other causes of microcytosis. Automated red cell parameters, such as MCV, RBC count, and red cell distribution width (RDW), have been used to identify patients with a high probability of BTT [5]. The aim of the present study was to determine which routine CBC parameters would best differentiate BTT from other microcytic anemias. The study included 200 adult patients with microcytosis (MCV <80 fL). Detailed clinical history, CBC, blood smear, quantitative assessment of hemoglobin A2 (HbA2), and serum ferritin were evaluated in all cases. An HbA2 concentration >3.5% was considered diagnostic of BTT [1,6]. Iron deficiency was diagnosed based on a ferritin level <15 ng/mL [7]. Serum ferritin is the best parameter to use for IDA screening and in the absence of inflammation, a normal ferritin level generally excludes iron deficiency [3]. Of the 200 cases evaluated, the 39 that had an HbA2 level >3.5% constituted the BTT group (figure 1); the

remaining 161 cases with an HbA2 level ≤3.5% constituted the non-BTT group. In the BTT group 3 cases had concomitant iron deficiency. In the non-BTT group 120 cases had a serum ferritin level <15 ng/mL and were diagnosed as IDA; of the remaining 41cases, 12 had associated chronic illness indicative of ACD (anemia of chronic disease) and in the other 29 the cause of microcytosis could not be identified. IDA was the most frequent cause of microcytosis in the present study, followed by BTT. The various red cell parameters examined in the 36 BTT cases, 120 IDA cases, and 12 ACD cases are shown in Table 1; the 3 BTT cases with concomitant iron deficiency are excluded. Mean Hb was highest in the BTT cases (10.9 g/dL) and lowest in the IDA cases (8.7 g/dL). The degree of microcytosis was most severe (62.9 fL) in the BTT cases, followed by the IDA cases (63.4 fL) and ACD cases (70 fL). The mean RBC count was highest in the BTT cases (5.7 x 1012/L), and slightly lower in those with IDA and ACD (4.8 x 1012/L). Anisocytosis (mean RDW-CV) was most common in the IDA cases (21.7%), followed by ACD (19.1%) and BTT cases (17.6%). The BTT cases had the lowest mean MCV (62.9 fL) and highest mean RBC count (5.7 x 1012/L). While evaluating microcytosis it is essential to differentiate IDA and BTT. In the present study patients with IDA had the most severe anemia and most severe degree of anisocytosis, as they had the lowest mean Hb (8.7 g/ dL) and highest mean RDW (21.7%). In contrast, the BTT group had the highest mean Hb (10.9 g/dL) and lowest mean RDW (17.6%), indicating less severe anemia and less severe anisocytosis. Shalev et al. [8] reported that the

Address for Correspondence: Veda Parthasarathy, M.D., 10, Sri Devi Krupa, 1st Main, 1st Block, RT Nagar, Bangalore- 560032. India Phone: 09945841386 E-mail: vedalallu@yahoo.com Received/Geliş tarihi : July 14, 2012 Accepted/Kabul tarihi : July 26, 2012

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Turk J Hematol 2012; 29: 427-429

Figure 1: HPLC Chromatograms displaying the hemoglobin fractions obtained in BTT and non-BTT cases. A and B belong to betathalassemia carriers having HbA2 concentration of 4.5% and 6.8% respectively. C represents a normal control with HbA2 fraction of 2.3%. Table 1: Comparison of various red cell parameters in BTT, IDA and ACD. Values within parenthesis indicate the mean.

Indices Hb (g/dL) MCV (fL) MCH (pg) MCHC (%) RBC (1012/L) RDW-CV (%)

BTT n = 36

IDA n = 120

ACD n = 12

7.3-15.6 (10.9) 54.5-78 (62.9) 15.2-29.1 (19.4) 25.8-35.1 (30.3) 3.95-8.5 (5.70) 12.3-23.2 (17.6)

3.4-11.8 (8.7) 56.9-77.6 (63.4) 13.9-23 (17.9) 24.3-32.9 (27.9) 3.56-4.91 (4.80) 18.5-31.2 (21.7)

8.9-11.2 (10.2) 68.3-79.2 (70.0) 18.5-24.2 (19.7) 25.3-33 (29.2) 3.90-5.82 (4.80) 14.2-22.9 (19.1)

combination of a high RBC count and low MCV is characteristic of BTT. It has been suggested that the RBC count is the most efficient single test for differentiating BTT and IDA [5,9,10]. Eldibany et al. [11] reported that the RBC count, MCV, and RDW are the most useful indices for differentiating BTT and IDA.

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Etiology unknown n = 29 8.7-13.6 (10.9) 71-76.6 (70.3) 17.4-24.0 (20.0) 24.9-33.9 (30.6) 3.78-5.3 (4.9) 17.6-21.3 (17.5)

Controversy continues regarding the ideal red cell indices and their cut-off values for differentiating BTT and IDA. Kotwal et al. [5] conducted a study with 640 adult patients with microcytosis (MCV <80 fL), plotting receiver operator characteristic (ROC) curves and recalculating the cut-off values for the Indian setting. The cutoff values of

Turk J Hematol 2012; 29: 427-429

Parthasarathy V, : Search for Beta Thalassemia Trait

Table 2: The sensitivity, specificity, PPV, NPV and Youden’s index obtained by using various parameters. Youden’s Index = sensitivity +specificity.

Cutoff values MCV <76 fL RBC count ≥4.9 x 1012/L RDW ≤18 %

Sensitivity %

Specificity %

PPV %

NPV %

Youden’s Index

94.87

65.21

73.1

92.7

1.6

76.92

81.37

80.50

77.90

1.58

58.97

90.06

85.58

68.70

1.49

PPV: Positive predictive value; NPV: negative predictive value.

MCV <76 fL, RBC count ≥4.9 x 1012/L, and RDW ≤18% were suggested to be associated with a high probability of BTT. In the present study the same cutoff values were applied to both the BTT and non-BTT groups in order to determine the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Youden’s index (Table 2). We attempted to determine the efficacy of these cutoff values for differentiating BTT from all other causes of microcytosis. In the present study an MCV <76 fL was observed in 94.9% (n = 34) of those in the BTT group and 34.8% (n = 56) of those in the non-BTT group. An RBC count >4.9 x 1012/L was observed in 76.9% (n = 27) of the BTT cases and 18.6% (n = 30) of the non-BTT cases. An RDW ≤18% was noted in 58.9% (n = 21) of the BTT cases and 9.9% (n = 16) of the non-BTT cases (Table 2). These findings indicate that MCV was the most sensitive parameter for identifying BTT; however, MCV lacks specificity when used as a single parameter [5]. Moreover, atypical carriers of BTT with a normal MCV will not be identified [9]. The RBC count and RDW have greater specificity than MCV. Youden’s index was highest for RDW, followed by the RBC count and MCV; therefore, we think a combination of MCV, RDW, and the RBC count is more effective for identifying BTT and differentiating it from other non-thalassemic microcytosis; however, it should be noted that patients with BTT and concomitant iron, vitamin B12, or folic acid deficiency, and double heterozygous δβ-thalassemics can have an elevated RDW [6,9,12]. Concomitant nutritional deficiency can also alter HbA2 levels in BTT. Microcytosis accompanied by a high RBC count and normal RDW is suggestive of BTT. These automated red cell parameters are routinely examined and offer a rapid and reliable method for BTT screening. Adequate utilization of these parameters can facilitate identification of the majority of BTT cases at no additional cost to the health care system. Identifying carriers and counseling them about the genetic implications of marrying another carrier is the most effective method for preventing beta-thalassemia major.

References 1. Madan N, Sikka M, Sharma S, Rusia U. Frequency of coincident iron deficiency and beta-thalssemia trait. J Clin Pathol 1996; 49:1021-1022. 2. Sachdev R, Dam AR, Tyagi G. Detection of Hb variants and hemoglobinopathies in Indian population using HPLC: Report of 2600 cases. Indian J Pathol Microbiol 2010; 53:57-62 3. Muncie LH, Campbell JS. Alpha and Beta Thalassemia. American Family Physician 2009; 80:339-344. 4. Tiwari AK, Chandola I. Comparing prevalence of Iron Deficiency Anemia and Beta Thalassemia Trait in microcytic and non-microcytic blood donors: Suggested algorithm for donor screening. Asian J Transfus Sci 2009; 3: 99-102. 5. Kotwal J, Saxena R, Choudhry VP, Dwivedi SN, Bhargava M. Erythrocyte indices for discriminating thalassaemic and non-thalassaemic microcytosis in Indians. Natl Med J India 1999; 12:266-267. 6. Clarke GM, Higgins TN. Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update. Clinical Chemistry. 2000;46:8(B) 1284–1290. 7. Clark SF. Iron Deficiency Anemia. Nutr Clin Pract 2008; 23:128-141. 8. Shalev O, Yehezkel E, Rachmilewitz EA. Inadequate Utilization of Routine Electronic RBC Counts to Identify Beta Thalassemia Carriers. AJPH 1988;78:1476-1477. 9. Rathod DA, Kaur A, Patel V, Patel K, Kabrawala R, Patel M, Shah P. Usefulness of Cell Counter–Based Parameters and Formulas in Detection of β-Thalassemia Trait in Areas of High Prevalence. Am J Clin Pathol 2007; 128:585-589 10. Demir A, Yarali N,Fisgin T, Duru F,Kara AR. Most reliable indices in differentiation between thalassemia trait and iron deficiency anemia. Pedia Int 2002 : 44: 612-616. 11. Eldibany MM, Totonchi KF, Joseph NJ, Rhone D. Usefulness of certain red blood cell indices in diagnosing and differentiating thalassemia trait from iron deficiency anemia. Am J Clin Pathol 1999; 111:676-682. 12. Niazi M, Tahir M, Raziq F, Hameed A. Usefulness of Red cell indices in differentiating Microcytic Hypochromic Anemias. Gomal Journal of Medical Sciences 2010; 8:125-129.

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Letter to the Editor

DOI: 10.5505/tjh.2012.92609

Severe Iron Deficiency Anemia Due to Late Presentation of Congenital Diaphragmatic Hernia in a Toddler Oyun Çocuğunda, Geç Bulgu Veren Doğumsal Diyafram Hernisine Bağlı Ağır Demir Eksikliği Anemisi Nazan Sarper1, Emine Zengin1, Suar Çakı Kılıç1, Melih Tugay2, Ayşen Aydoğan3, Özlem Kayabey1 Kocaeli University, Pediatric Hematology, Kocaeli, Turkey Kocaeli University, Pediatric Surgery, Kocaeli, Turkey 3 Kocaeli University, Pediatric Gastroenterology, Kocaeli, Turkey 1 2

To the Editor, A 3-year-old female toddler was referred to our pediatric emergency unit with a 2-week history of fatigue, anorexia, progressive pallor, and vomiting. Medical history showed that iron deficiency anemia was diagnosed one year before and oral iron-sulfate was given. She also had a one year history of intermittent vomiting. Her diet seemed adequate in iron-rich foods. Chest X-ray and abdominal ultrasonographic examination performed in a medical center were normal. Complete blood count findings were as follows: Hb: 1.7 g/dL; WBC count: 8.0 x 109/L; ANC: 4.6 x 109/L; Hct: 6.9%; RBC count: 1.19 x 1012/L; RDW: 20.5; MCV: 58 fL; Plt count: 485 x 109/L; absolute reticulocyte count: 45.5 x 109/L (normal: 50-150 x 109/L). Peripheral blood smear showed hypochromic microcytic red cells, polychromasia, and nucleated red cells. Serum iron was 5 µg/dL, total iron binding capacity was 450 µg/dL, and ferritin was <1 ng/mL. She was hospitalized and given packed red cell transfusion. The following day she suddenly developed respiratory distress after breakfast. Chest X-ray showed a radiolucent lesion in the right paracardiac area (Figure). Congenital diaphragmatic hernia (CDH) was confirmed via barium

gastrointestinal X-ray and computed tomography of the chest. During surgery the retrosternal diaphragmatic hernia sack was excised, and the stomach, intestines, and

Figure: X-ray of the thorax showing the radiolucent area with clear borders in the right paracardiac region.

Address for Correspondence: Nazan SARPER, M.D., Kocaeli Üniversitesi Tıp Fakültesi Hastanesi Umuttepe Yerleşkesi Kocaeli 41300, Turkey Phone: +90 262 303 72 16 E-mail: nazan_sarper@hotmail.com Received/Geliş tarihi : July 30, 2012 Accepted/Kabul tarihi : August 3, 2012

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transverse colon were reduced to the abdominal cavity. Nissen fundoplication was also performed in an effort to prevent postoperative gastroesophageal reflux; however, another defect was noted on the left posterolateral side of the diaphragm. The defect was repaired and Thal anterior fundoplication was performed. The patient had both Morgagni and Bochdalek defects. At 6 weeks post surgery the patient had no gastrointestinal symptoms, but her Hb dropped to 7.9 g/dL and fecal occult blood test findings were positive (Hct: 25.6%; RBC count: 3.63 x 1012/L; MCV: 70.5 fL; MCH: 21.9; MCHC: 31; RDW: 24; WBC count: 9.8 x 109/L; ANC: 4.3 x 109/L, Plt count: 453 x 109/L; absolute reticulocyte count: 11 x 109/L) [normal 50-150 x 109/L]). Peripheral blood smear showed hypochromic microcytic red cells. Esophagogastroduodenoscopy and esophageal biopsy showed erosions, gastroesophageal reflux, chronic inflammation, and hyperplasia of the epithelium. A proton pump inhibitor, domperidone, and anti-acid medications were started. Iron sucrose was administered twice weekly for 3 weeks and the Hb increased (Hb: 12.2 g/dL; Htc: 34.6%; RBC count: 4.76 x 109/L; MCV: 72fL; MCH: 25.6; MCHC: 35.1; RDW: 17.4; WBC count: 12.2 x 109/L; ANC: 8.2 x 109/L; Plt count: 348 x 109/L, absolute reticulocyte count: 85 x 109/L [normal: 50-150 x 109/L]; ferritin: 16 ng/mL). Blood smear showed normal red cells. At the 2-year follow-up the patient had no gastrointestinal symptoms or anemia. The coexistence of Morgagni and Bochdalek defects is rarely reported [1]. About 5%-30% of CDH cases present after the neonatal period, which poses a diagnostic challenge [2]. Such symptoms as vomiting and respiratory distress may be acute or intermittent—sometimes due to gastric volvulus or spontaneous reduction of the hernia to the abdomen. During asymptomatic periods imaging findings may be normal [3]. The presented patient had

Sarper N, et al: Severe Iron Deficiency Anemia due to Diaphragma Hernia

normal abdominal ultrasound and chest X-ray findings before referral to our department. The presented patient most probably had chronic occult blood loss from the gastrointestinal tract due to reflux esophagitis and mechanical trauma to the diaphragm. Sinaki et al. reported 2 patients—a 19 month-old and 6-year-old—that presented with gastrointestinal symptoms and an Hb of 5.8 g/dL and 6 g/dL, respectively [4]. Zaki et al. reported a 5-year-old patient with persistent anemia that did not respond to adequate hematinics and blood transfusion. The patient had abdominal pain, melena, an Hb of 4.8 g/dL, and multiple linear gastric erosions on the mucosal folds of the lesser curve of the stomach [5]. CDH must be included in the differential diagnosis of severe iron deficiency anemia in the absence of such obvious causes as nutritional deficiency, melena, hematochezia, and malabsorption. Physicians must be aware that a history of intermittent vomiting and/or sudden onset respiratory distress in young children are indications for imaging of the upper gastrointestinal tract and thorax. References 1. Jelin EB, Kim TN, Nathan N, Miniati D. Synchronous ipsilateral Bochdalek and Morgagni diaphragmatic hernias: A case report. J Pediatr Surg 2011;46: 2383-2386. 2. Sridhar AV, Nichani S. Late presenting congenital diaphragmatic hernia. Emerg Med J 2004; 21: 261-262. 3. Davenport M, Holmes K. Current management of congenital diaphragmatic hernia. Br J Hosp Med 1995; 53: 95-101. 4. Sinaki B, Jayabose S, Sandoval C. Iron-deficiency anemia associated with hiatal hernia: Case reports and literature review. Clin Pediatr (Phila) 2010;49: 984-985. 5. Zaki SA, Dadge D, Shanbag P. Diaphragmatic hernia presenting as gastrointestinal bleeding. Indian Pediatr 2010; 47: 185-187.

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Letter to the Editor

DOI: 10.5505/tjh.2012.71324

Splenic Infarct in a Patient with Autoimmune Hemolytic Anemia Otoimmun Hemolitik Anemili Bir Hastada Splenik İnfarkt Hava Üsküdar Teke1, Samet Karahan2, Ümmügülsüm Gümüş3 Kayseri Education and Research Hospital, Department of Hematology, Kayseri, Turkey Kayseri Education and Research Hospital, Department of Internal Medicine, Kayseri, Turkey 3 Kayseri Education and Research Hospital, Department of Radiology, Kayseri, Turkey 1 2

To the Editor, Splenic infarct may be observed during the course of several diseases; however, the incidence is quite low. Splenic infarct is related to thromboembolic events; various diseases, including sickle cell anemia, chronic myeloproliferative diseases, lymphoma, and leukemia, can also cause splenic infarct [1]. In patients with autoimmune hemolytic anemia (AIHA) massive hemolysis causes activation of the immune response, destruction of erythrocytes, and splenomegaly [2]. Splenic infarct is rarely observed in AIHA patients and to the best of our knowledge the literature contains only 3 cases of splenic infarct due to AIHA [3-5]. Herein we describe a 67-year-old female that presented to the emergency department with fatigue, tachycardia, and weakness. The patient’s vital signs were as follows: temperature: 37 °C; pulse: 120/min; blood pressure: 120/70 mmHg. Physical examination showed pale conjunctivas, icteric sclerae, and a spleen that was palpable 3 cm below the costa. Lymphadenopathy was not observed. Electrocardiography (ECG) and lung radiography results were normal. Complete blood count findings were as follows: Hb: 5.7 g/dL; MCV: 115 fL, WBC count: 6.4 x 109/L; Plt count: 157 x 109/L. Biochemical examination findings were as follows: total bilirubin: 2.0 mg/dL; indirect bilirubin: 1.5 mg/dL; LDH: 466 U/L. Her reticulocyte count was 6.38%, direct anti-globulin test with IgG and C3d complement was 4 positive, and the indirect anti-globulin test was 4

positive. Erythrocyte agglutination, polychromasia, and spherocytes were observed in her peripheral blood smear. The patient was further examined to determine if there existed other secondary causes for her complaints; there were no signs of hepatitis, her IgG, IgA, and IgM levels were normal, and monoclonality was not observed on the serum protein electrophoresis. Mycoplasma pneumonia, ANA, anti-ds DNA, and HIV tests were negative. Lymphoma was eliminated via computed tomography (CT). Bone marrow aspiration showed the presence of erythroid hyperplasia. Based on these findings, the patient was diagnosed with AIHA. The patient was given erythrocyte suspension because of her symptomatic status. The patient was started on 1 mg/kg/d methylprednisolone and folic acid after the diagnosis of idiopathic mixed type AIHA. After 3 weeks of the steroid treatment, the patient developed left upper quadrant pain and and the abdominal ultrasonography revealed splenic infarct complicated by splenic hemorrhage. Upper abdominal MRI was performed to confirm the presence of splenic infarct. MRI showed that the spleen was 155 mm and that a wedge-shaped hemorrhagic infarct was present around the spleen (Figure). The patient was experiencing severe pain in the upper left quadrant and underwent splenectomy, a common treatment for AIHA. The patient’s pain disappeared following the splenectomy. To eliminate hypercoagulability causes protein C, protein S, antithrombin, factor VIII, factor V Leiden mutation, prothrombin mutation, lupus anticoagu-

Address for Correspondence: Samet KARAHAN, M.D., Kayseri Eğitim Araştırma Hastanesi, İç Hastalıkları Kliniği, Kayseri 38010, Turkey Phone: +90 352 336 88 84 E-mail: doktorsamet@yahoo.com Received/Geliş tarihi : June 5, 2012 Accepted/Kabul tarihi : September 10, 2012

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Üsküdar Teke H, et al: Splenic Infarct in a Patient with Autoimmune Hemolytic Anemia

Medical treatment can be administered for uncomplicated splenic infarcts. Splenectomy is preferred in cases with persistent symptoms or complicated cases with a splenic pseudocyst, abscess, or hemorrhage; however, early splenectomy is required to prevent mortality [7]. The presented case had pain that was unresponsive to analgesic treatment, and her splenic infarct was complicated by the presence of a hemorrhage; therefore, splenectomy was performed with early intervention. Following the splenectomy the patient’s complaints disappeared.

Figure: Axial T2-weighted fat-suppressed MRI shows a wedgeshaped hyperintense area of infarction in the spleen (arrow), as well as decreased signal intensity of the liver.

lant, anti-cardiolipin antibodies, and homocysteine were measured, all of which were normal. ECG showed a normal sinus rhythm. Transthoracic echocardiography did not show any cardiac valve pathology. Thromboemboli were not present during the observation. The patient was started on azathioprine for AIHA, as she was unresponsive to steroid treatment and splenectomy. The most common cause of splenic infarct is cardiovascular thromboembolism (22% of all cases), followed by thrombophilia[5]. In addition, the third most common cause of splenic infarct is hematological diseases, including sickle hemoglobinopathies, myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, leukemia, and lymphoma, which account for 10% of all cases [1]; however, only 3 cases of splenic infarct due to AIHA have been reported [3-5]. Thrombogenic events and splenic vasculitis are not the only causes of splenic infarct. Other immune-mediated mechanisms related to cryoglobulin synthesis can cause splenic infarct [6]. In cases of cardiovascular thromboembolism or thrombophilia-induced infarct, the lungs, kidneys, heart, and intestine, together with splenic infarct, are affected by multiple emboli; however, in hematological diseases infarct is usually limited to the spleen [5]. The presented patient was evaluated for thrombophilia, but there was neither a laboratory evidence for hypercoagulability nor any cardiac cause. Moreover, the presented case only had an infarct that was localized in the spleen. Splenic infarct may have arisen in the present case due to splenomegaly or immune-mediated mechanisms caused by cold antibodies; the patient had both warm and cold antibodies.

In conclusion, in patients with such hematological diseases as AIHA, hemoglobinopathies, polycythemia vera, myelofibrosis, leukemia, and lymphoma, and complaints of left upper quadrant pain, splenic infarct should be suspected and MRI or CT should be performed for diagnostic purposes. As thrombophilia can also cause infarct, patients should also be evaluated for thrombophilia. If a patient with splenic infarct complains of severe persistent pain and the case is complicated the patient should undergo splenectomy immediately to avoid mortality. Conflict of Interest Statement None of the authors have any conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included. References 1. Antopolsky M, Hiller N, Salameh S, Goldshtein B, Stalnikowicz R. Splenic infarction: 10 years of experience. Am J Emerg Med 2009; 27:262-265. 2. Eichner ER. Splenic function: Normal, too much and too little. Am J Med 1979; 66:311-320. 3. Horeau J, Robin C, Guenel J, Nicolas G. An unusal complication of aqcuired hemolytic anemia, splenic infarction. Concours Med 1963; 85:663-666. 4. Tzanck A, Andre R, Dreyfus B. Aqcuired hemolytic anemia infarct of the spleen; splenectomy; recovery. Bull Mem Soc Med Hop Paris 1951; 67:286-290. 5. Park MY, Kim JA, Yi SY, Chang SH, Um TH, Lee HR. Splenic infarction in a patient with autoimmune hemolytic anemia and protein C deficiency. Korean J Hematol 2011; 46:274278. 6. Giovanardi P, Mannucci C, Vandelli MR, Sansone R, Bondi M. Splenic infarcts in chronic hepatopathy accompanied by cryoglobulinemia: two case reports. Minerva Gastroenterol Dietol 2010; 56:81-85. 7. Jaroch MT, Broughan TA, Hermann RE. The natural history of splenic infarction. Surgery 1986; 100:743-750.

433

Letter to the Editor

DOI: 10.5505/tjh.2012.48642

Co-Existence of Hereditary Pyrimidine 5’-Nucleotidase Deficiency and Heterozygous α-Thalassemia: A Case Presentation Kalıtsal Pirimidin 5’- Nükleotidaz Eksikliği ve Alfa Talasemi Taşıyıcı Birlikteliği: Olgu Sunumu A. Agapidou1, S. Theodoridou1, K. Tegos3, E. Mandala4, E. Leukou1, O. Karakasidou1, B. Aletra1, A. Sevastidou1, M. Alemayehou1, E. Voskaridou2 Hippokration Hospital of Thessaloniki, Hemoglobinopathy Prevention Unit, Thessaloniki, Greece Laikon Hospital of Athens, Thalassemia Center, Athens, Greece 3 NIMITS, Military Hospital, Athens, Greece 4 Aristotelion University of Thessaloniki, 4th Unit of Internal Medicine, Thessaloniki, Greece 1 2

To the Editor, Pyrimidine 5’-nucleotidase (P5N) is an intra-erythrocytic isoenzyme that catalyzes the dephosphorylation of pyrimidine ribonucleotides, which are the product of DNA catabolism. A reduction in P5N reactivity leads to accumulation of pyrimidine products in erythrocytes, which increases its life span, resulting in hemolytic anemia and intense basophilic stippling [1]. P5N deficiency is inherited in an autosomal recessive fashion and is one of the most important causes of inherited non-spherocyte anemia, as well as G-6PD and pyruvate kinase deficiency. P5N deficiency is characterized by hemolytic anemia, splenomegaly, and intense basophilic stippling. There are several reports in the literature concerning reduced P5N reactivity in patients with intermediate and heterozygous β-thalassemia; however, the co-existence of P5N deficiency in a patient with α-thalassemia is a rare event [2,3,4]. We report the clinical and hematological findings in a 25-year-old female that was screened for hemoglobinopathies due to her pregnancy (6th week).Based on her medical

records, she was known to be a carrier of 5PN deficiency. The proposita’s hematological parameters were as follows: Hb: 10.7 g dL—1; Hct: 34%; RBC: 4.35 x 106; MCV: 77 fL; MCH: 24.7 pg; RDW: 16.2%; reticulocytes: 3.97%; ferritin: 30 (normal range=20-81ngr/Ml). Microscopic examination of a stained peripheral blood smear showed severe anisocytosis, microcytosis, and basophilic stippling. Biochemical analysis of hemoglobin using high-performance liquid chromatography (HPLC) showed normal values for hemoglobin A2 (HbA2: 2.4%) and hemoglobin F (HbF: 0.5%). No abnormal hemoglobins were detected. Due to an unclear hematological picture in the proposita’s husband, who was thought to be heterozygous for thalassemia, genetic analysis of the proposita’s DNA was performed. Molecular analysis showed that she was heterozygous for the mild mutation of α-thalassemia (-α3,7/ NI). In conclusion, mismatch between the hematological parameters of the presented pregnant patient and the usual hematological phenotype associated with α-thalassemia (-α 3,7/NI) were due to the lack of P5N (P5N: 1.88 U g–1 of Hb).The patient never required blood transfusion. The co-existence of hereditary P5N deficiency and heterozy-

Address for Correspondence: A. AGAPIDOU, M.D., Alexandra Agapidou, Hippokration Hospital of Thessaloniki, Hemoglobinopathy Prevention Unit, Thessaloniki, Greece Phone: 00302310892819 E-mail: alekagapidou@yahoo.gr Received/Geliş tarihi : January 26, 2012 Accepted/Kabul tarihi : January 30, 2012

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gous α-thalassemia is extremely interesting due to the fact that to the best of our knowledge this is the first report of such a case. Conflict of Interest Statement The authors have no conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials presented.

Agapidou A, et al: Pyrimidine 5’-Nucleotidase and α-Thalassemia

References 1. Evdokia Mandala, Study of Pyrimidine 5- Nucleotide(P5N) Activity In Hereditary Hemoglobinopathies. DoctorateThesis, Thessaloniki 1997. 2. Zerez CR, Lachant NA, Lent KM, Tanaka KR. Decreased pyrimidine nucleoside monophosphate kinase activity in sickle erythrocytes. Blood 1992; 80:512-516. 3. David O, Sacchetti L, Vota MG, Comino L, Perugini L, Pescarmona GP. Pyrimidine 5’-nucleotidase and oxidative damage in red blood cells transfused to beta-thalassemic children. Haematologica 1990; 75:313-318. 4. Dell’Edera D, Malvasi A, Tinelli A, Mazzone E, Leo M, Monti V, Epifania AA. Importance of the molecular diagnosis in the screening of alpha-thalassemia. Recenti Prog Med 2011; 102(7-8):302-306. doi: 10.1701/913.10050. Italian.

435

Letter to the Editor

DOI: 10.5505/tjh.2012.80106

Is a High Homocysteine Level Related to Thrombosis? Yüksek Homosistein Seviyesi Trombozla İlgili mi? Şinasi Özsoylu Retired Prof of Pediatrics, Hematology, Hepatology, Ankara, Turkey Honorary fellow of American Academy of Pediatrics Honorary member of American Pediatric Society

To the Editor, The study by Eğin and Akar [1] entitled, First observation of MTHFR 678C-A (Ala 222 Ala) single nucleotide polymorphism, in the recent issue of the Journal (2012; 29: 204-5) requires questioning its importance to thrombosis in the light of Dayal et al.’s findings in a mouse model [2]. I used to think that high homocysteine level important in thrombosis, whereas currently I question such knowledge. As such, I think the authors must show the functional effect of their observation in blood coagulation and its relationship to the thermolabile variant of MTHFR ?.

Address for Correspondence: Şinasi ÖZSOYLU, M.D., Beysukent Altınşehir Sitesi No:30, Ankara, Turkey Phone: +90 312 235 41 88 E-mail: sinasiozsoylu@hotmail.com Received/Geliş tarihi : June 29, 2012 Accepted/Kabul tarihi : June 29, 2012

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References 1. Eğin Y, Akar N. First Observation of MTH FR 678 C-A (Ala222Ala) Single Nucleotide Polymorphism. Turk J Hematol 2012; 29:204-205 2. Dayal S, Chauhan AK, Jensen M, Leo L, Lynch CM, Faraci FM, Kruger WD, Lentz SR. Paradoxical absence of a prothrombotic phenotype in a Mouse model of severe hyperhomocysteinemia. Blood 2012;119:3176-3183.

Images in Hematology

DOI: 10.5505/tjh.2012.05826

Intraleukocytic Candida is Diagnostic of Pathological Candidemia Lökosit İçi Kandida Patolojik Kandidemi İçin Tanı Koydurucudur Anil Handoo1, Sarada Nagoti1, Adarsh Choudhary2 Medanta The Medicity, Haematology, Gurgaon, India Medanta The Medicity, Surgical Gastroenterology, Gurgaon, India

1 2

Candidemia is the presence of Candida species in blood. It is the most frequently encountered invasive fungal infection (IFI) in hospitalized patients, and ranks as the fourth most common cause of nosocomial blood stream infection [1]. Despite its high incidence and the availability of a variety of techniques for its detection, the sensitivity of blood cultures for detecting candidemia remains only 50% [2]. As such, candidal bloodstream infections may be vastly under diagnosed and under reported. A number of risk factors associated with candidemia have been identified, including the presence of a central intravenous line, mechanical ventilation, dialysis dependence, major surgery, and use of broad-spectrum antimicrobial agents or total parenteral nutrition [3]. Timely diagnosis and initiation of proper antifungal therapy are critical in the management of candidemia and for the prevention of its complications. Most institutions rely on blood cultures alone to diagnose candidemia. Budding yeasts on peripheral blood film are considered a contaminant, whereas their intracytoplasmic presence— in neutrophils or monocytes—should be indicative of the pathological nature of the findings. A 16-year-old male undergoing follow-up for polyarteritis nodosa (PAN) was admitted to our hospital with a burst abdomen due to mesenteric ischemia. The patient underwent emergency laparotomy and bowel resection with jejunostomy. The patient developed wound dehiscence 2 d post surgery. Re-exploratory laparotomy with adhesiolysis was performed and broad-spectrum antibi-

otic treatment was commenced. He later developed fever and bleeding at the stoma site. At this time his peripheral blood findings were as follows: Hb: 7.5 g/dL; WBC: 4.5 x 109/L; neutrophils: 58.8%; lymphocytes: 26.5%; eosinophils: 0.7%; monocytes: 13%; basophils: 1%; platelet count: 87 x 109/L. Peripheral blood smear showed dimorphic anemia and thrombocytopenia, as well as neutrophilic predominance with mild left shift and thrombocytopenia. In addition, the smear showed that there were many extra-leukocytic budding yeasts, along with hyphal forms of Candida (Figure 1). The budding yeasts were phagocytized by the neutrophils (Figure 2). Aerobic blood culture showed growth of C. tropicalis and C. parapsilosis following overnight incubation. The organism was observed to be growing in other body fluids as well, including sputum and urine. The patient was aggressively treated with fluconazole and later with amphotericin B; however, he died due to multiple organ failure. Candidemia is associated with high crude mortality rates, ranging from 30% to 81% [4]. The most common pathogen is C. albicans, which accounts for 70% of fungemia cases. Nonetheless, the results of a retrospective study conducted at a tertiary care center in Northern India showed that most episodes were cause by species other than C. albicans, the most frequent of which was C. tropicalis, followed by C. albicans (21.5%) and C. parapsilosis (20%). In the presented case C. tropicalis and C. parapsilosis were noted. Various fungal infections have been diagnosed via direct detection of fungi in peripheral blood

Address for Correspondence: Anil HANDOO, M.D., Medanta The Medicity, Haematology, Gurgaon, India Phone: +91 9891280992 E-mail: ahhemat@gmail.com Received/Geliş tarihi : March 17, 2012 Accepted/Kabul tarihi : June 19, 2012

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Handoo A, et al : Intraleukocytic Candida is Diagnostic of Pathological Candidemia

Figure 1: Extra-leukocytic yeast and hyphal forms of candida. Giemsa 1000X

smears [5]. In addition, leukoagglutination in a smear has been described as the actual host response to fungal invasion. Observation of such should prompt the laboratory hematologist to look for the presence of hyphae and yeast forms of Candida [5]. Whereas intra-/extraleukocytic Candida—both budding yeast forms—and hyphal forms were seen in the presented case’s blood smear, leukoagglutination was not. Treatment and prevention of candidemia has many challenges, including correct species identification and appropriate treatment, because different species are susceptible to different antifungal drugs. In contrast to other IFIs, such as invasive aspergillosis,[6] mortality associated with invasive candidiasis has not decreased significantly during the previous decade, despite the introduction of new classes of antifungal agents [7]. In addition, such infections are associated with prolonged hospitalization and treatment cost, constituting an enormous financial burden. In conclusion, we think that careful observation of peripheral blood smears is important for the early detection of fungal infection. Conflict of Interest Statement The authors have no conflicts of interest, including specific financial interests, relationships, and/or affiliations, relevant to the subject matter or materials included.

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Figure 2: Phagocytosed candida in blood

References 1. Hajjeh RA, Sofair AN, Harrison LH, Lyon GM, ArthingtonSkaggs BA, Mirza SA, Phelan M, Morgan J, Lee-Yang W, Ciblak MA, Benjamin LE, Sanza LT, Huie S, Yeo SF, Brandt ME, Warnock DW. Incidence of bloodstream infections due to Candida species and invitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J Clin Microbiol 2004; 42:15191527. 2. Fridkin SK: Candidemia is costly: Plain and simple. Clin Infect Dis 2005; 41:1240-1241. 3. Ostrosky-Zeichner L: New approaches to the risk of Candida in the intensive care unit. Curr Opin Infect Dis 2003; 16:533537. 4. Anaissie EJ, Rex JH, Uzun O, Vartivarian S: Predictors of adverse outcome in cancer patients with candidemia. Am J Med 1998; 104:238-245. 5. Y Berrouane, H Bisiau, F Le Baron, C Cattoen, P Duthilleul, E Dei Cas. Candida albicans blastoconidia in peripheral blood smears from non-neutropenic surgical patients. J Clin Pathol 1998; 51:537-538. 6. Upton A, Kirby KA, Carpenter P, BoeckhM, Marr KA: Invasive aspergillosis following hematopoietic cell transplantation: outcomes and prognostic factors associated with mortality. Clin Infect Dis 2007; 44:531-540. 7. Fridkin SK: The changing face of fungal infections in health care settings. Clin Infect Dis 2005; 41:1455-1460.

Images in Hematology

DOI: 10.5505/tjh.2012.39259

Poland Syndrome Associated with Pernicious Anemia and Gastric Dysplasia Poland Sendromu ile İlişkili Pernisiyöz Anemi ve Gastrik Displazi Erman Aytaç1, Ali Vedat Durgun1, Deram Büyüktaş2, Sibel Erdamar3, Şeniz Öngören4 İstanbul University, Cerrahpasa School of Medicine, Department of General Surgery, İstanbul, Turkey İstanbul University, Cerrahpasa School of Medicine, Department of Internal Medicine, İstanbul, Turkey 3 İstanbul University, Cerrahpasa School of Medicine, Department of Pathology, İstanbul, Turkey 4 İstanbul University, Cerrahpasa School of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 1 2

A 55-year-old male with fatigue, dyspnea, and sweating presented to our clinic. Physical examination showed pectus carinatum deformity, flatness on the left side of the chest wall, breast asymmetry, and splenomegaly (Figure 1). Laboratory findings were as follows: macrocytic anemia (red blood cell count: 2x1012/L; hemoglobin: 8.4 g dL–1; hematocrit: 22.2%; mean corpuscular volume: 112 fL [normal range: 80-99]; mean corpuscular hemoglobin: 42 pg [normal range: 27-34]; very low B12 vitamin level: 0 pg mL–1 [normal range: 180-900]; LDH: 918 U L–1 [normal range: 125-243]; total bilirubin: 1.7 mg dL–1 [normal range: 0.2-1.3]). The serum folic acid concentration was normal. Peripheral blood smear showed marked anisocytosis, poikilocytosis, macro-ovalocytes, and hypersegmented neutrophils. Reticulocyte production index was 0.7. Histopathological analysis of a bone marrow biopsy specimen showed hypercellularity, low myeloid:erythroid ratio, and abnormal large RBC precursors with nuclearcytoplasmic asynchrony. Anti-parietal antibodies in the serum were positive at a serum dilution of 1:10. Antiintrinsic antibodies were also positive. Plain chest X-ray showed hyperlucency on the left side of the chest (Figure 2a). Pectus carinatum deformity, agenesis of the left pectoralis major muscle, and bilateral hypertrophy of the sternocleidomastoid muscles were observed via computed tomography (CT) (Figure 2b). The patient was diagnosed as PS. Abdominal ultrasonography showed splenomegaly. Vitamin B-12 treatment

was initiated (1000 μg d1 for 5 days, 1000 μg week1 for 4 weeks, and then 1000 μg month1 for life). The patient’s anemia responded well to the treatment and his symptoms began to improve after 2 weeks of the treatment. Lifelong parenteral vitamin B-12 treatment has

Figure 1: Asymmetric appearance of the nipples.

Address for Correspondence: Erman AYTAÇ, M.D., Cerrahpaşa Tıp Fakültesi, Genel Cerrahi Anabilim Dalı, 34098 İstanbul, Turkey Phone: +90 212 414 30 00 E-mail: eaytactr@yahoo.com Received/Geliş tarihi : July 23, 2011 Accepted/Kabul tarihi : September 19, 2011

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A

Turk J Hematol 2012; 29: 441-444

B

Figure 2: (A) Chest X-ray shows hyperlucency on the left side of the chest. (B) CT image shows agenesis of the left pectoralis major muscle.

type) in cardiac mucosa, low-grade chronic gastritis, and patching intestinal metaplasia in the antrum (Figure 3).

Figure 3: High-grade dysplasia in the cardiac mucosa (40x magnification with hematoxylin and eosin stain).

planned. Gastroscopic examination resulted in a polyp of cardia that was impossible to remove endoscopically for technical reasons. Histopathological examination of the biopsy specimen showed in situ adenocarcinoma and total gastrectomy was performed. Histopathological examination of the gastrectomy material showed foveolar highgrade dysplasia, elevated p53 expression with p53-positive staining, expansive intestinal metaplasia (complete

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PS is a rare congenital disease characterized by unilateral agenesis of the pectoral muscles, various ipsilateral deformities of the upper extremities, and malformations of the anterior chest wall. Genetic and teratogenic factors might play a role in its etiology. Clinical manifestations are variable. The incidence of PS varies from 1/7,000 to 1/100,000. The right side of the body and males are more commonly affected. PS is an autosomal dominant transition trait; nonetheless, some researchers reported that there is not a genetic association. Many cases are sporadic. Hypoplasia of the subclavian artery, and the vertebral arteries and their branches due to a momentary interruption or reduction in embryonic development is observed in the syndrome [1,2]. Absence of costal cartilage or the ribs, diaphragm hernias, heart anomalies such as dextrocardia, central nervous system and genitourinary system anomalies, and vertebral anomalies are also associated with PS [3]. An autoimmune disease with dysplastic mucosa of the stomach in association with this congenital anomaly has, to date, not been reported. PS can be present with various deformities of the thoracic wall and upper extremities, skin pathologies, and cardiac anomalies. Such malformations are associated with morbidity and limited social activity. Congenital anoma-

Turk J Hematol 2012; 29: 441-444

lies of the pectoral muscles cause little or no functional deficit in normal daily activities. The presented patient did not have any complaints about his extremities and had no functional deficits. Various surgical techniques have been described for the repair of chest wall defects in PS [4-6]. The presented patient admitted to our hospital with symptoms of anemia. Macrocytic anemia, very low vitamin B-12 level, low reticulocyte index, splenomegaly, and the presence of anti-parietal and anti-intrinsic factor antibodies were indicative of the diagnosis of pernicious anemia. Pernicious anemia is an autoimmune, heritable multifactorial disorder. The presence of a hemoglobin concentration <13 g dL–1 in men and <12 g dL–1 in women, mean corpuscular volume ≥100 fL, and low vitamin B-12 level, together with the concomitant presence of atrophic gastritis and intrinsic factor deficiency are the findings reminding pernicious anemia. The presence of intrinsic factor serum antibodies and hypergastrinemia can support the diagnosis of pernicious anemia; however, the absence of these findings does not exclude the diagnosis. It was reported that 10%-20% of patients with pernicious anemia also have atrophic gastritis of the antrum, with gastrin cell reduction and normal serum gastrin levels [7-9]. The sensitivity of intrinsic factor antibodies in the diagnosis of pernicious anemia is 50%-70% [10]. Pernicious anemia with elevated serum levels of parietal cell antibodies occurs in 60%-90% of cases and is considered a sequela of autoimmune atrophic gastritis (AMAG). Chronic atrophic gastritis with intestinal metaplasia, gastric polyps, and gastric dysplasia is frequently observed in patients with pernicious anemia. AMAG is associated with an increased risk of gastric cancer. Among patients with AMAG, the incidence of gastric carcinoma and carcinoids is 1%-3% and 1%-7%, respectively [9-12]. Armbrect et al. recommend performing endoscopy with multiple biopsies at least once at the time of diagnosis in all patients with pernicious anemia. They also favor removing polyps and re-examining patients with polyps [12]. As such, we performed gastroscopy in the presented patient following the diagnosis of pernicious anemia. Abnormal physical examination findings of the chest wall led us to perform thoracic CT and the patient was subsequently diagnosed with PS. Neoplastic pathologies are sometimes seen with PS. Wilms tumor, carcinoma of the breast, lung cancer, and neuroblastoma have been reported in some PS patients [13-15]. PS has been reported in association with hematological malignancies, such as acute leukemias and chronic granulocytic leukemia, and lymphomas [16]; however, to the best of our knowledge gastric neoplasia in a PS patient has not been previously reported.

Aytaç E, et al : Pernicious Anemia in Poland Syndrome

The etiology of PS remains unknown. Several etiopathogenic mechanisms underlying PS have been hypothesized, including vascular defect due to an insult during early embryologic stages, paradominant inheritance, and the presence of an autosomal lethal gene surviving via mosaicism. To the best of our knowledge this is the first report of an autoimmune disorder in a patient with PS. Additionally, there are no reports of gastric malignancy in PS. Due to a possible predisposition to autoimmune diseases, such as pernicious anemia, clinicians should consider autoimmune disorders and malignancies while examining patients with PS. Informed consent was obtained. Conflict of Interest Statement The authors declare that they have no conflicts of interest that could be perceived as having influenced the impartiality of the materials presented. Funding The present study received no grant from a funding agency in the public, commercial, or a profit sector. References 1. Fokin AA, Robicsek F. Poland’s syndrome revisited. Ann Thorac Surg 2002; 74:2218-2225. 2. Stevens DB, Fink BA, Prevel C. Poland’s syndrome in one identical twin. J Pediatr Orthop 2000; 20:392-395 3. Urschel HC Jr. Poland’s syndrome. Chest Surg Clin N Am 2000; 10:393-403. 4. Bainbridge LC, Wright AR, Kanthan R. Computed tomography in the preoperative assessment of Poland’s syndrome. Br J Plast Surg 1991;44: 604-607. 5. Dos Santos Costa S, Blotta RM, Mariano MB, Meurer L, Edelweiss MI. Aesthetic Improvements in Poland’s Syndrome Treatment with Omentum Flap. Aesthetic Plast Surg 2010; 34:634-639. 6. Lacorte D, Marsella M, Guerrini P. A case of Poland Syndrome associated with dextroposition. Ital J Pediatr 2010; 36:21. 7. Lahner E, Annibale B. Pernicious anemia: New insights from a gastroenterological point of view. World J Gastroenterol 2009;15:5121-5128. 8. Cattan D. Pernicious anemia: What are the actual diagnosis criteria? World J Gastroenterol 2011; 17:543-544. 9. Stockbrügger RW, Menon GG, Beilby JO, Mason RR, Cotton PB. Gastroscopic screening in 80 patients with pernicious anaemia. Gut 1983; 24:1141-1147. 10. Carmel R. How I treat cobalamin (vitamin B12) deficiency. Blood 2008; 112:2214-2221.

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11. Neesse A, Michl P, Barth P, Vieth M, Langer P, Ellenrieder V, Gress TM. Multifocal early gastric cancer in a patient with autoimmune atrophic gastritis and iron deficiency anaemia. Z Gastroenterol 2009; 47: 223-227. 12. Armbrecht U, Stockbrügger RW, Rode J, Menon GG, Cotton PB. Development of gastric dysplasia in pernicious anaemia: A clinical and endoscopic follow up study of 80 patients. Gut 1990; 31:1105-1109. 13. Athale UH, Warrier R. Poland’s syndrome and Wilms tumor: An unusual association. Med Pediatr Oncol 1998; 30:67-68.

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14. Katz SC, Hazen A, Colen SR, Roses DF. Poland’s syndrome and carcinoma of the breast: A case report. Breast J 2001; 7:56-59. 15. Ahn MI, Park SH, Park YH. Poland‘s syndrome with lung cancer. A case report. Acta Radiol 2000;41:432-434. 16. Parikh PM, Karandikar SM, Koppikar S, Pahuja R, Charak BS, Saikia T, Gopal R, Advani SH. Poland’s syndrome with acute lymphoblastic leukemia in an adult. Med Pediatr Oncol 1988; 16:290-292.