relationship between deoxycytidine kinase (dck) genotypic variants and fludarabine toxicity in...

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Leukemia Research 35 (2011) 431–437 Contents lists available at ScienceDirect Leukemia Research journal homepage: www.elsevier.com/locate/leukres Relationship between deoxycytidine kinase (DCK) genotypic variants and fludarabine toxicity in patients with follicular lymphoma Ana Rivero a , Inmaculada Rapado a , José F. Tomás b , Carlos Montalbán c , Raquel de O ˜ na b , José Paz-Carreira d , Miguel Canales e , Rafael Martínez f , Pedro Sánchez-Godoy g , Alberto Fernández de Sevilla h , Javier de la Serna a , Joaquín Martínez-López a,a Department of Haematology, Hospital 12 de Octubre, Madrid, Spain b Department of Haematology, MD Anderson, Madrid, Spain c Department of Internal Medicine, Hospital Ramón y Cajal, Madrid, Spain d Department of Haematology, Centro Oncológico de Galicia, A Coru˜ na, Madrid, Spain e Department of Haematology, Hospital La Paz, Madrid, Spain f Department of Haematology, Hospital Clínico San Carlos, Madrid, Spain g Department of Haematology, Hospital Severo Ochoa, Leganés, Spain h Department of Haematology, Hospital Duran i Reynals, L’Hospitalet de Llobregat, Barcelona, Spain article info Article history: Received 30 July 2010 Received in revised form 23 September 2010 Accepted 29 September 2010 Available online 27 October 2010 Keywords: DCK genotypic variants Toxicity Fludarabine Follicular lymphoma abstract DCK catalyzes the intracellular phosphorylation of fludarabine. The promoter and coding region of the DCK gene were analyzed in 74 follicular lymphoma (FL) patients receiving a therapeutic regimen that included fludarabine. DCK mRNA expression was quantified in a cohort of healthy donors. Four previously described genotypic variants, 360C>G, 201C>T (rs2306744), C28624T (rs11544786) and c.91+37G>C (rs9997790), as well as the new variant, 12C>G, were identified. Variant C28624T showed a lower risk of lymphopenia (P = 0.04), but a higher risk of neutropenia (P = 0.04). Statistical significance was found in bivariate logistic regression between lymphopenia and infectious episodes in the induction period (odds ratio 3.85, P = 0.04). © 2010 Elsevier Ltd. All rights reserved. 1. Introduction The challenge of treatment lies in achieving a complete response (CR) in the disease without causing host toxicity. There is inter- individual variability in optimum drug doses, which depends on many factors, including drug interactions, age, diet, concomitant illnesses and genetic traits [1]. Deoxycytidine kinase (DCK) catalyzes the rate-limiting step in the phosphorylation of 2 -deoxyadenosine, 2 -deoxyguanosine, 2 -deoxycytidine and their analogues, in the cytoplasm of most mammalian cells [2]. The DCK gene promoter region spans 1500 bp, and exerts its regulatory action through a 690 bp region, compris- ing part of the 5 -untranslated region (UTR), intron 1 and exon 1 [3]. This is a very GC rich region which contains GC and E boxes, Corresponding author at: Department of Haematology, Hospital Universitario 12 de Octubre, Avda de Cordoba, s/n. 28041, Madrid, Spain. Tel.: +34 91 390 84 95; fax: +34 91 390 87 92. E-mail address: [email protected] (J. Martínez-López). at which the specificity protein 1 and upstream stimulatory factor transcription factors bind [4,5]. Resistance to nucleoside analogues in acute myeloid leukemia (AML) and follicular lymphoma (FL) has been reported to be related to deficient DCK activity due to genetic alterations [6–9]. However, enhanced DCK activity may result in increased generation of cytotoxic compounds within cells [10]. Fludarabine monophosphate (FA) is a purine analogue with immunosuppressive and antineoplastic actions. Fludarabine is mainly metabolized by DCK, although it may also be phosphory- lated by deoxyguanosine kinase in the mitochondria [2]. The combination of fludarabine and cyclophosphamide, with or without rituximab, has been frequently used as second line chemotherapy in FL [11,12]. This therapeutic combination has been shown to provide high response rates in chronic lym- phoid leukemias [13]. However, some investigators have reported frequent hematological adverse events (AEs) when these combina- tions were used [14,15], and dose reduction or sequential regimens of these drugs have been proposed in other trials [16,17]. The LNHF-03 study is a multicenter phase II study conducted under the auspices of the Asociación Espa ˜ nola de Hematología y 0145-2126/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2010.09.026

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Page 1: Relationship between deoxycytidine kinase (DCK) genotypic variants and fludarabine toxicity in patients with follicular lymphoma

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Leukemia Research 35 (2011) 431–437

Contents lists available at ScienceDirect

Leukemia Research

journa l homepage: www.e lsev ier .com/ locate / leukres

elationship between deoxycytidine kinase (DCK) genotypic variants andudarabine toxicity in patients with follicular lymphoma

na Riveroa, Inmaculada Rapadoa, José F. Tomásb, Carlos Montalbánc, Raquel de Onab,osé Paz-Carreirad, Miguel Canalese, Rafael Martínez f, Pedro Sánchez-Godoyg,lberto Fernández de Sevillah, Javier de la Sernaa, Joaquín Martínez-Lópeza,∗

Department of Haematology, Hospital 12 de Octubre, Madrid, SpainDepartment of Haematology, MD Anderson, Madrid, SpainDepartment of Internal Medicine, Hospital Ramón y Cajal, Madrid, SpainDepartment of Haematology, Centro Oncológico de Galicia, A Coruna, Madrid, SpainDepartment of Haematology, Hospital La Paz, Madrid, SpainDepartment of Haematology, Hospital Clínico San Carlos, Madrid, SpainDepartment of Haematology, Hospital Severo Ochoa, Leganés, SpainDepartment of Haematology, Hospital Duran i Reynals, L’Hospitalet de Llobregat, Barcelona, Spain

r t i c l e i n f o

rticle history:eceived 30 July 2010eceived in revised form3 September 2010

a b s t r a c t

DCK catalyzes the intracellular phosphorylation of fludarabine. The promoter and coding region of theDCK gene were analyzed in 74 follicular lymphoma (FL) patients receiving a therapeutic regimen thatincluded fludarabine. DCK mRNA expression was quantified in a cohort of healthy donors. Four previouslydescribed genotypic variants, −360C>G, −201C>T (rs2306744), C28624T (rs11544786) and c.91+37G>C

ccepted 29 September 2010vailable online 27 October 2010

eywords:CK genotypic variantsoxicityludarabine

(rs9997790), as well as the new variant, −12C>G, were identified. Variant C28624T showed a lower riskof lymphopenia (P = 0.04), but a higher risk of neutropenia (P = 0.04). Statistical significance was found inbivariate logistic regression between lymphopenia and infectious episodes in the induction period (oddsratio 3.85, P = 0.04).

© 2010 Elsevier Ltd. All rights reserved.

ollicular lymphoma

. Introduction

The challenge of treatment lies in achieving a complete responseCR) in the disease without causing host toxicity. There is inter-ndividual variability in optimum drug doses, which depends on

any factors, including drug interactions, age, diet, concomitantllnesses and genetic traits [1].

Deoxycytidine kinase (DCK) catalyzes the rate-limiting stepn the phosphorylation of 2′-deoxyadenosine, 2′-deoxyguanosine,′-deoxycytidine and their analogues, in the cytoplasm of most

ammalian cells [2]. The DCK gene promoter region spans 1500 bp,

nd exerts its regulatory action through a 690 bp region, compris-ng part of the 5′-untranslated region (UTR), intron 1 and exon 13]. This is a very GC rich region which contains GC and E boxes,

∗ Corresponding author at: Department of Haematology, Hospital Universitario2 de Octubre, Avda de Cordoba, s/n. 28041, Madrid, Spain. Tel.: +34 91 390 84 95;ax: +34 91 390 87 92.

E-mail address: [email protected] (J. Martínez-López).

145-2126/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2010.09.026

at which the specificity protein 1 and upstream stimulatory factortranscription factors bind [4,5]. Resistance to nucleoside analoguesin acute myeloid leukemia (AML) and follicular lymphoma (FL) hasbeen reported to be related to deficient DCK activity due to geneticalterations [6–9]. However, enhanced DCK activity may result inincreased generation of cytotoxic compounds within cells [10].

Fludarabine monophosphate (FA) is a purine analogue withimmunosuppressive and antineoplastic actions. Fludarabine ismainly metabolized by DCK, although it may also be phosphory-lated by deoxyguanosine kinase in the mitochondria [2].

The combination of fludarabine and cyclophosphamide, withor without rituximab, has been frequently used as second linechemotherapy in FL [11,12]. This therapeutic combination hasbeen shown to provide high response rates in chronic lym-phoid leukemias [13]. However, some investigators have reported

frequent hematological adverse events (AEs) when these combina-tions were used [14,15], and dose reduction or sequential regimensof these drugs have been proposed in other trials [16,17].

The LNHF-03 study is a multicenter phase II study conductedunder the auspices of the Asociación Espanola de Hematología y

Page 2: Relationship between deoxycytidine kinase (DCK) genotypic variants and fludarabine toxicity in patients with follicular lymphoma

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emoterapia (AEHH). A total of 74 previously untreated patientsith a diagnosis of follicular non-Hodgkin’s lymphoma (NHL)

eceived rituximab plus fludarabine/cyclophosphamide inductionherapy and rituximab maintenance therapy. The results of thisrial, which will be published elsewhere, showed impressivelinical efficacy but was complicated by a high rate of severe neu-ropenia and lymphopenia.

The present study focuses on the correlation of DCK genotypicariants on the fludarabine toxicity experienced by a small cohortf Caucasian patients with FL. DCK promoter and coding regionolymorphisms were examined by high resolution melting (HRM)nalysis and sequencing. Expression of DCK mRNA was quantifiedn a control group to verify its association with DCK genotypic vari-nts.

. Patients and methods

.1. Patient population

From September 2004 to March 2006, 74 newly diagnosed Caucasian patientsith grades 1 and 2 FL were included in a prospective study designed to evaluate the

linical efficacy of 6 cycles of fludarabine (25 mg/m2 × 3 days), cyclophosphamide1 g/m2/day 1), and rituximab (375 mg/m2/day 1) (FCR) followed by maintenanceherapy with rituximab (375 mg/m2/week) for 4 weeks every 6 months during 2ears. The protocol was approved by the institutional ethics committee at each par-icipating center and the study was conducted in accordance with CEE regulationsnd the principles of the Declaration of Helsinki. Written informed consent wasbtained from all patients. Enrolled patients presented with FL CD20+ grade I–IIInd Ann Arbor stage II–IV, and had not received previous treatment. Diagnosis wasased on the established WHO criteria for NHL [18], and included either histological

nvestigation and/or positive CD20+ immunophenotyping. Patients were requiredo exhibit a performance status ≤2 on the ECOG scale. Patients were followed upor a median of 33.5 months (range 22.3–45.2). All registered events were gradedccording to the reference guide, Common Terminology for Criteria Adverse EventsCTCAE, v.3). Cheson criteria [19] were used to assess the response to treatment. Thetudy also included 28 healthy Caucasian blood donors as controls.

.2. DNA samples

Genomic DNA was extracted from peripheral blood (PB) or bone marrow of 74 FLatients at diagnosis, and from 28 healthy donors, using the automated MagNA PureC Nucleic Acid Extractor (Roche Applied Science, Mannheim, Germany). Mononu-lear cells were isolated from PB of healthy donors using Ficoll-PaqueTM Plus (GEealthcare Bio-Science AB, Uppsala, Sweden). Total RNA was extracted from theseells using TRIzol® reagent (Invitrogen, Prat de Llobregat, Spain) according to theanufacturer’s protocol, and was immediately converted to single-strand comple-entary DNA (cDNA) using a High Capacity cDNA Reverse Transcription Kit (Applied

iosystems, Foster City, CA, USA).

.3. DCK genotype and sequence analysis

The coding region of the DCK gene was analyzed by a single HRM reaction, exceptor exon 3 that showed multiple melting domains, and was split into two ampliconso be analysed. The promoter region was divided into three different fragments,wo of them were suitable for HRM analysis. However, the other one due to itsigh GC content had to be analyzed by sequencing. Therefore, this 672 bp fragmentas amplified by conventional PCR. The other two promoter amplicons of 258 and

17 pb, respectively, were analyzed in two different PCR reactions using the HRMechnique.

HRM analysis was performed using a LightCycler® 480 Instrument (Rochepplied Science) with the LightCycler High Resolution Master (Roche DiagnosticsmbH). Conventional PCR amplifications were performed using Takara PCR Ther-al Cycler Dice (Takara Bio Europe, Saint Germaine en Laye, France). Purified PCR

roducts were sequenced using the BigDye Terminator 3.1 Cycle Sequencing KitApplied Biosystems), with specific primers, on an ABI PRISM 3130 Avant (Appliediosystems). PCR products were purified using the ExoSap It PCR Clean-Up Kit (GEealthcare, Little Chalfont, UK) according to the manufacturer’s protocol.

All primers were designed using Primer Express V2.0 software (Applied Biosys-ems) and the sequences are presented in Supplemental data tables. The absencef multiple melting domains on selected amplicons was evaluated using Poland’s

lgorithm [20].

Variants predicted by HRM analysis were confirmed by sequencing. Four repre-entative samples of every large group with a particular melting curve profile werenalyzed. All samples belonging to small groups were sequenced. For exons 1, 2, 4,, and 7, purified HRM amplification products were used to confirm the polymor-hisms. For exon 6, new specific sequencing primers were designed. For exon 3, therimers Fex3.1 and Rex3.2 (Supplemental tables) were used.

arch 35 (2011) 431–437

2.4. DCK mRNA expression

The expression of the DCK gene was measured by (RQ-PCR) using the primer3.2F and a new specifically designed primer (Supplemental Table S2). Quantitativereal time PCR (RQ-PCR) to measure DCK mRNA expression was performed on anABI PRISM 7900 HT Fast Real Time PCR System (Applied Biosystems). PCR was per-formed in a total volume of 10 �L and included 5 �L of SYBR® GREEN PCR MasterMix (Applied Biosystems), 200 ng of each primer and 5–10 ng of cDNA. All sampleswere amplified in duplicate. Quantification of DCK gene expression relative to thatof the housekeeping ABL1 gene was performed according to the method previouslydescribed by Pfaffl [21].

3. Statistical analysis

Patients, who developed ≥grade 3 adverse events, were clas-sified into the following different categories: hemoglobin belowstandard value (≥grade 3), neutropenia (grade 4), thrombopenia,leukopenia, lymphopenia (≥grade 3), any hematological malig-nancy, infectious adverse events, and a category which involvedother toxicities. All toxicity variables were evaluated for the periodof FCR therapy, and also for the maintenance and follow-up periods.

Statistical analysis was performed with respect to each geno-typic variant. Patients who carried any genotypic variant of theDCK gene were compared with patients who did not, and statisticalanalysis was performed on their respective clinical data.

Associations between discrete and categorical numerical vari-ables were analyzed using �2 and Fisher’s exact tests. Forcomparison of mean values between different groups, unpairedStudent’s t-tests, analysis of variance and Kruskal–Wallis tests wereused. A P value less than 0.05 was considered significant.

Univariate overall survival (OS) and event free survival (EFS)were calculated using the Kaplan–Meier method, and differencesbetween curves were evaluated with the log-rank test. All toxicitycategories were evaluated separately as events. A bivariate logisticregression was performed in order to evaluate possible associa-tions among DCK genotypic variants and toxicity categories. Thelinkage disequilibrium test and estimation of haplotype frequencyamong single nucleotide polymorphisms (SNP) were performedusing Arlequin 2.0 software [22].

4. Results

4.1. DCK gene sequence analysis

Screening of the promoter and coding regions of the DCKgene, either by conventional PCR or HRM analysis, resulted in theidentification of five genotypic variants in 14 patients who wereheterozygous for these variants. The polymorphisms predicted byHRM analysis were confirmed by sequencing. Conventional PCRanalysis revealed the presence of two SNPs, while HRM anal-ysis revealed three additional SNPs (Table 1). All SNPs, exceptC28624T, were located in the regulatory region. The first nucleotideupstream of the ATG translation start site was defined as −1.A novel DCK variant was identified at position −12C>G in onepatient (Fig. 1). In addition, four known polymorphisms were iden-tified: −360C>G, −201C>T (rs2306744), C28624T (rs11544786) andc.91+37G>C (rs9997790). The c.91+37G>C and C28624T variantswere both present in the same patient.

The −360C>G and −201C>T promoter SNPs were in com-plete linkage disequilibrium (rho square, 1; P < 10−4) as previouslyreported by Shi et al. [23]. No other polymorphisms showed signif-icant linkage (P ≥ 0.2).

DCK mRNA expression was assessed in samples from 28 healthydonors. DCK mRNA expression levels showed no significant differ-entiation between donors who carried any genotypic variant andthose who did not. The allele frequencies and DCK mRNA expressionlevels for lymphoma patients and healthy donors are summarized

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A. Rivero et al. / Leukemia Research 35 (2011) 431–437 433

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n Table 1. This table also compares the allelic frequencies obtainedy Joerger et al. [32] in a Caucasian healthy population.

.2. Response

The therapeutic strategy resulted in a high clinical responseate after induction treatment in all patients, with completeesponse (CR) in 64/72 patients (89%) and partial response (PR)n 8/72 patients (11%). Some patients who initially showed partialesponses, showed CR after conclusion of the maintenance phase,nd 57 of the 62 evaluable patients (92%) showed CR at this time. 13f 14 patients with DCK variants showed CR in the induction phasend all continued to show CR in the maintenance phase. There wereot any differences in response based on the presence of geneticariants (Table 2).

.3. Toxic adverse events

Seventy-two of the 74 patients who started the induction phasef the study received at least 4 cycles of FCR therapy. Two patientsid not complete the three cycles required for evaluation. Sixty-twof the 72 patients who completed at least 4 cycles of FCR ther-py started maintenance treatment. Only 14 patients completedhe clinical trial treatment and received the fourth course of main-

enance therapy. Ten patients experienced cycle delays and doseeductions of >20%, eight experienced dose reduction and 29 expe-ienced cycle delays during FCR treatment.

Among the 1636 global AEs reported during the study, 1069ccurred during the sixth cycle of FCR therapy and during ini-

able 1able shows the allele frequencies and mRNA expression levels for genotypic variants of

Lymphoma (n = 74) Other frequeJoerger et al.

n Allelic frequency n All

−360CG/−201CTA 5 0.035 4 0.0−12CG 1 0.007 – –28624CTB 7 0.013 13 0.0+37CGC 2 0.048 – –Wild-typea,b,c,d 60 0.965a/0.993b/0.987c/0.952d 0.9

a −360CC/−201CC.b −12CC.c 28624CC.d +37CC.A rs2306744.B rs11544786.C rs9997790.

cted in the DCK gene promoter region. The melting profile of DNA from a wild-type

tial maintenance therapy. Remarkably, 328 severe adverse events(≥grade 3) occurred during induction and initial maintenance ther-apy. Adverse events are summarized in Table 2.

Over this period of time, 15 patients died: 5 during inductiontreatment and 10 during rituximab maintenance therapy and thefollow-up period. Of these, 8 patients died as a consequence ofinfectious complications: 5 due to respiratory AEs, 2 due to septicshock and 1 due to multi-organism infection. One patient died dueto non-infectious cranial neuritis. Three patients died due to car-diac failure, rectal carcinoma and hepatic cirrhosis, respectively. Inaddition, 2 patients died as a consequence of hematological AEs:two due to acute leukemia and one due to progression of disease.

Among the respiratory deaths, three patients presented withprofound and prolonged neutropenia and lymphopenia, andanother had aplastic anemia. One of the patients with septic shockand the patient with multi-organism infection also presented withsevere and prolonged neutropenia and lymphopenia. The patientwith heart failure showed a febrile syndrome. The two patientswho died due to AML developed secondary myelodysplastic syn-drome and AML, respectively, during the first cycle of maintenancetherapy with rituximab.

4.4. Relationship between genotypic variants, toxic adverse

events and outcomes

The toxicity variables and their association with DCK gene vari-ants during fludarabine, cyclophophamide and rituximab inductiontreatment and maintenance/follow-up with rituximab are pre-

the DCK gene in patients with follicular lymphoma and healthy donors.

ncies (n = 100)[32]

Healthy donors (n = 28)

elic frequency n Allelic frequency DCK expression

2 4 0.93/0.07 38.24 (11.39–58.08)– – –

65 3 0.946/0.054 37.01 (16.33–39.95)– – –

8a/0.935c 21 0.93a/0.946c 31.45 (4.055–61.82)

Page 4: Relationship between deoxycytidine kinase (DCK) genotypic variants and fludarabine toxicity in patients with follicular lymphoma

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Table 2Statistical analysis of toxic adverse events, response assessment and their association with DCK gene variants.

FCR treatment (n = 72) Maintenance and follow-up (n = 62)

−360CG/−201CTa

−12CG 28624CTb +37CGc +37CGc/28624CTb

WT Pe −360CG/−201CT

Pf 28624CT −360CG/−201CTa

28624CTb 37CGc +37CGc/28624CTb

WT Pe −360CG/−201CT

Pd 28624CT

Anemia (y/n) 1/4 1/0 1/5 0/1 0/1 5/53 0.40 0.51 0/5 0/6 0/1 0/1 1/48 0.91 0.51Neutropenia (y/n) 2/3 0/1 6/0 0/1 1/0 32/26 0.65 0.04 0/5 1/5 0/1 0/1 9/40 0.58 0.64Lymphopenia (y/n) 0/5 0/1 0/6 0/1 0/1 23/35 0.19 0.04 1/4 1/5 0/1 0/1 13/36 0.61 0.67Leucopenia (y/n) 0/5 0/1 1/5 0/1 0/1 8/50 0.49 0.67 0/5 1/5 1/0 0/1 9/40 0.58 0.64Thrombopenia (y/n) 0/5 1/0 0/6 0/1 0/1 4/54 0.71 0.63 0/5 0/6 0/1 0/1 3/46 0.74 0.67Any Citopenia (y/n) 3/2 1/0 6/0 0/1 1/0 44/14 0.59 0.33 1/4 2/4 1/0 0/1 23/26 0.37 0.44Infections (y/n) 1/4 0/1 0/6 0/1 0/1 14/44 0.58 0.61 1/4 2/4 0/1 0/1 12/37 0.65 0.57Haematological

toxicity non relatedto Citopenia (y/n)

0/5 0/1 0/6 0/1 0/1 2/56 0.85 0.8 1/4 0/6 0/1 0/1 2/47 0.26 0.58

Other toxicities (y/n) 0/5 0/1 0/6 0/1 0/1 6/52 0.49 0.58 0/5 1/5 0/1 0/1 5/44 0.60 0.57Response to treatment

(CR/PR)5/0 1/0 6/0 1/0 1/0 50/8 0.49 0.58 5/0 6/0 1/0 1/0 45/4 0.67 0.57

Exitus (y/n) 0/5 0/1 0/6 0/1 0/1 5/53 0.65 0.56 1/4 0/6 0/1 0/1 9/40 0.66 0.58Without any AE (y/n) 3/2 1/0 6/0 0/1 1/0 46/12 0.31 0.33 2/3 3/3 1/0 0/1 28/21 0.65 0.69Treatment (FCR)

delayed or decreased(Red/Del/Both/Nomod)d

1/0/2/2 0/0/0/1 0/4/0/2 0/1/0/0 1/0/0/0 6/24/8/20 0.21 0.10 – – – – –

a rs2306744.b rs11544786.c rs9997790.d Red: treatment dose reduced; Del: treatment delayed; Both: treatment delayed and reduced; No mod: treatment was not modified.e P calculated −360CG/−201CT versus WT.f P calculated 28624CT versus WT.

Page 5: Relationship between deoxycytidine kinase (DCK) genotypic variants and fludarabine toxicity in patients with follicular lymphoma

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ig. 2. Figure shows a Kaplan–Meier curve Event Free Survival for neutropeniaoxicity category during FCR therapy. This analysis shows an association between28624T variant and the development of neutropenia through this period (log rankest, P = 0.014).

ented in Table 2. Patients with DCK heterozygotic variants did notuffer adverse events such as lymphopenia, develop any unclassi-ed toxicities or hematological malignancies during FCR therapy.

n this period, 5 patients died and none of them carried any of theCK genotypic variants. Only one of the 10 patients who died during

he maintenance/follow-up period carried the −360C>G/−201C>Tolymorphisms.

Remarkably, none of the patients carrying DCK gene vari-nts developed episodes of lymphopenia during FCR therapy0/14), while 23/35 of patients without polymorphisms sufferedrom this toxicity. In contrast, severe neutropenia was similarn 360C>G/−201C>T patients (2/3) and WT patients (32/26), andigher in C28624T patients (7/0). �2 and Fisher’s exact tests didot show statistical differences for toxicity variables between60C>G/−201C>T and WT variants throughout any period. How-ver, statistical significances were found between C28624T and WTariants for lymphopenia (0.04) and neutropenia (0.04) toxicities.

Bivariate logistic regression was performed for60C>G/−201C>T versus WT variants and C28624T versus WTariants for each toxicity variable and also for relevant factors thatight have influenced this toxicity and the response of patientsith FL (ECOG status, lactate dehydrogenase, gammaglobulins, and

ollicular lymphoma international prognostic index (FLIPI)). Nonef these analyses revealed any association. An additional logisticegression, performed for the whole group of patients with noegarding to inherited variants, showed that lymphopenia duringCR therapy may play a role in the development of infections (oddsatio (OR) 3.85, 95% confidence interval (CI) 1.07–13.88, P = 0.04).

OS, did not reveal any influence of the DCK gene polymorphisms.FS analyses were performed for every toxicity category during FCRherapy and also over the FCR therapy and maintenance/follow-uperiods. These analyses showed an association between C28624Tariant and neutropenia, both for FCR period (log rank test,= 0.014) (Fig. 2) and, for FCR and maintenance/follow-up periods

log rank test, P = 0.016).

. Discussion

DCK activity is regulated at transcriptional and post-ranscriptional levels [4,24–27]. Genetic alterations may occur at

arch 35 (2011) 431–437 435

the DNA or RNA level and lead to decreased expression [6–9,28,29].As previously reported [30,31], HRM is a good method for revealingnew genetic variations or identifying predicted polymorphisms.HRM analysis was an adequate technique for complete screeningof the DCK gene coding region, since the HRM results matchedthose obtained by sequencing.

The frequencies of the DCK genotypic variants identified in thepresent study in a Caucasian population showed some differencescompared with those reported in previous studies. The −201C>Tand −360C>G SNPs occurred less frequently than in an Asian pop-ulation [23] but with a similar frequency to that reported for aCaucasian population [32]. However, other frequent genetic vari-ations in Caucasians were absent in the present population. Inaddition, a new genotypic variation at position −12C>G was iden-tified in this study (Fig. 1).

Our results did not show significant differences in DCK mRNAexpression in healthy patients who carried the detected polymor-phisms. In contrast, previous studies performed on cDNA eitherfrom cell lines [28] or cells of patients [23] showed differences inmRNA expression. Lamba et al. [28] showed that mRNA expres-sion and enzyme activity were decreased due to heterozygosity inB-lymphoblast cell lines. However, other studies showed increasedmRNA expression in limited numbers of heterozygous AML patients[23,33]. Increased DCK gene expression has been reported in dif-ferent malignant cells [28,34,35], but unfortunately cDNA fromlymphoma patients was not available for measurement of DCK geneexpression. Furthermore, the increase in DCK mRNA expressionwhen cells are exposed to high doses of purine analogues may dif-fer among genotypic variants [36,37]. Toxicity is a major problemin cancer therapy due to the narrow therapeutic index [1], and maydepend on individual polymorphisms carried by patients. Patientstreated under this therapeutic regimen showed a high rate of CR,although this was complicated by severe adverse events, whichmainly occurred at the end of the induction period and early inthe maintenance period. Despite the small sample size of this study,patients who inherited infrequent SNPs suffered, mainly during FCRtherapy, fewer hematological toxicities, except for neutropenia,and just one of 15 deaths carried the −360C>G/−201C>T genotypicvariant.

It is worthy note that none of the 14 patients carrying DCK genevariants developed episodes of lymphopenia during FCR therapy,while 40% of WT patients suffered from this toxicity. However,statistically significant decreased risk of lymphopenia was foundjust for C28624T variant due to low frequencies of variant groups.Besides logistic regression showed a correlation between lym-phopenia and infectious events during the induction period offludarabine treatment, that might be related to the fewer infec-tious episodes observed in these groups. The depletion of CD4+ andCD8+ T lymphocytes had been previously reported by Gamberaleet al. [10], in an in-vitro study analyzing the susceptibility of T cellsubsets after induction of fludarabine therapy in a cohort of patientswith CLL.

Univariate analysis and log rank test calculated in the EFS analy-sis showed a statistically significant risk of neutropenia for C28624Tgroup versus WT group. These results were apparently inconsis-tent with the above results obtained for lymphopenia, but it mightbe justify by a marked lower expression of DCK protein for theC28624T variant, though DCK mRNA quantification did not showexpression differences in healthy patients. In humans, granulocyterate turnover is >20% per day and lymphocyte turnover, 1–15% perweek [38], so the immediate availability of DCK protein may affect

granulocyte proliferation to a large extent.

As all patients received similar doses of the drugs, differencesin toxicities would not be merely related to a synergistic effect offludarabine, cyclophosphamide and rituximab, leading to potentimmunosuppression [2].

Page 6: Relationship between deoxycytidine kinase (DCK) genotypic variants and fludarabine toxicity in patients with follicular lymphoma

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36 A. Rivero et al. / Leukemi

This investigation allowed us to conclude that HRM analysisonstitutes a reliable method for screening the coding region ofhe DCK gene. Basal expression of DCK mRNA in healthy Caucasianonors was independent of inheritance of the reported DCK SNPs.enetic variation in the DCK gene may provide a slight protectiveffect against the development of lymphopenia in patients with FL,reated with fludarabine and other cytotoxic drugs. However at theame time, patients with C28624T DCK variant may present a slightncreased risk of suffering neutropenia. Because of the small sam-le size of the present study, this finding should be confirmed inlarger series of patients. Additional studies are required to iden-

ify new genes or polymorphisms that may explain the observedoxicities.

onflict of interest statement

The authors declare that there is no competing financial andersonal interest in relation to the work described.

cknowledgements

The authors would like to thank Tomas Serrano for technicalupport. A.R. was supported by a grant from Eugenio Rodriguezascual foundation. This work was partially supported by Rochearma.

Contributions. A.R. and I.R. contributed equally to this work. Theyontributed in the acquisition of data, analysis and interpretationf data. J.M.L. contributed to this work in the conception and designf the study, the analysis and interpretation of data. J.F.T., C.M.,.O., J.P.C., M.C., R.M., P.S.G., A.F.S., J.S. contributed in the acquisi-ion of data. All authors have revised critically the manuscript andpproved the final version submitted.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.leukres.2010.09.026.

eferences

[1] Evans WE, Relling MV. Pharmacogenomics: translating functional genomicsinto rational therapeutics. Science 1999;286:487–91.

[2] Van den Neste E, Cardoen S, Offner F, Bontemps F. Old and new insightsinto the mechanisms of action of two nucleoside analogs active in lymphoidmalignancies: fludarabine and cladribine (review). Int J Oncol 2005;27:1113–24.

[3] Song JJ, Walker S, Chen E, Johnson EE, Spychala J, Gribbin T, et al. Genomicstructure and chromosomal localization of the human deoxycytidine kinasegene. Proc Natl Acad Sci U S A 1993;90:431–4.

[4] Ge Y, Jensen TL, Matherly LH, Taub JW. Physical and functional interactionsbetween USF and Sp1 proteins regulate human deoxycytidine kinase promoteractivity. J Biol Chem 2003;278:49901–10.

[5] Chen EH, Johnson EE, Vetter SM, Mitchell BS. Characterization of the deoxy-cytidine kinase promoter in human lymphoblast cell lines. J Clin Invest1995;95:1660–8.

[6] Galmarini CM, Clarke ML, Jordheim L, Santos CL, Cros E, Mackey JR, et al. Resis-tance to gemcitabine in a human follicular lymphoma cell line is due to partialdeletion of the deoxycytidine kinase gene. BMC Pharmacol 2004;4:8.

[7] Owens JK, Shewach DS, Ullman B, Mitchell BS. Resistance to1-beta-d-arabinofuranosylcytosine in human T-lymphoblasts mediated bymutations within the deoxycytidine kinase gene. Cancer Res 1992;52:2389–93.

[8] Veuger MJ, Honders MW, Landegent JE, Willemze R, Barge RM. High incidenceof alternatively spliced forms of deoxycytidine kinase in patients with resistantacute myeloid leukemia. Blood 2000;96:1517–24.

[9] Kocabas NA, Aksoy P, Pelleymounter LL, Moon I, Ryu JS, Gilbert JA, et al.

Gemcitabine pharmacogenomics: deoxycytidine kinase and cytidylate kinasegene resequencing and functional genomics. Drug Metab Dispos 2008;36:1951–9.

10] Gamberale R, Galmarini CM, Fernandez-Calotti P, Jordheim L, Sanchez-AvalosJ, Dumontet C, et al. In vitro susceptibility of CD4+ and CD8+ T cell subsets tofludarabine. Biochem Pharmacol 2003;66:2185–91.

[

[

arch 35 (2011) 431–437

11] Sacchi S, Pozzi S, Marcheselli R, Federico M, Tucci A, Merli F, et al. Rituximabin combination with fludarabine and cyclophosphamide in the treatment ofpatients with recurrent follicular lymphoma. Cancer 2007;110:121–8.

12] Zinzani PL, Pulsoni A, Perrotti A, Soverini S, Zaja F, De Renzo A, et al. Fludarabineplus mitoxantrone with and without rituximab versus CHOP with and withoutrituximab as front-line treatment for patients with follicular lymphoma. J ClinOncol 2004;22:2654–61.

13] Tam CS, Wolf MM, Januszewicz EH, Prince HM, Westerman D, Seymour JF. Flu-darabine and cyclophosphamide using an attenuated dose schedule is a highlyeffective regimen for patients with indolent lymphoid malignancies. Cancer2004;100:2181–9.

14] Forstpointner R, Unterhalt M, Dreyling M, Bock HP, Repp R, Wandt H, et al. Main-tenance therapy with rituximab leads to a significant prolongation of responseduration after salvage therapy with a combination of rituximab, fludarabine,cyclophosphamide, and mitoxantrone (R-FCM) in patients with recurring andrefractory follicular and mantle cell lymphomas: results of a prospective ran-domized study of the German Low Grade Lymphoma Study Group (GLSG).Blood 2006;108:4003–8.

15] Flinn IW, Neuberg DS, Grever MR, Gordon WD, Bennett JM, Paietta EM, et al.Phase III Trial of Fludarabine Plus Cyclophosphamide compared with Fludara-bine for patients with previously untreated Chronic Lymphocytic Leukemia:US Intergroup Trial E2997. J Clin Oncol 2007:25.

16] Foon KA, Boyiadzis M, Land SR, Marks S, Raptis A, Pietragallo L, et al. Chemoim-munotherapy with low-dose fludarabine and cyclophosphamide and high doserituximab in previously untreated patients with chronic lymphocytic leukemia.J Clin Oncol 2009;27:498–503.

17] Lamanna N, Jurcic JG, Noy A, Maslak P, Gencarelli AN, Panageas KS, et al. Sequen-tial therapy with fludarabine, high-dose cyclophosphamide, and rituximab inpreviously untreated patients with chronic lymphocytic leukemia produceshigh-quality responses: molecular remissions predict for durable completeresponses. J Clin Oncol 2009;27:491–7.

18] Jaffe ES, Harris NL, Stein H, Vardiman JW. WHO Classification of Tumours. IARCWHO Classification of Tumours, vol. 3.

19] Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, et al.Report of an international workshop to standardize response criteria fornon-Hodgkin’s lymphomas. NCI Sponsored International Working Group. J ClinOncol 1999;17:1244.

20] Poland Melt Program. http://www.biophys.uni-duesseldorf.de/local/POLAND/poland.html.

21] Pfaffl MW. A new mathematical model for relative quantification in real-timeRT-PCR. Nucleic Acids Res 2001;29:e45.

22] Schneider S, Roessli D, Excoffier L. Arlequin: A software for population genet-ics data analysis Ver 2.000. Genetics and Biometry Lab, Dept of Anthropology,University of Geneva; 2002.

23] Shi JY, Shi ZZ, Zhang SJ, Zhu YM, Gu BW, Li G, et al. Association between singlenucleotide polymorphisms in deoxycytidine kinase and treatment responseamong acute myeloid leukaemia patients. Pharmacogenetics 2004;14:759–68.

24] Mitchell BS, Song JJ, Johnson EE, Chen E, Dayton JS. Regulation of human deoxy-cytidine kinase expression. Adv Enzyme Regul 1993;33:61–8.

25] Kroep JR, Loves WJ, van der Wilt CL, Alvarez E, Talianidis I, Boven E, et al. Pre-treatment deoxycytidine kinase levels predict in vivo gemcitabine sensitivity.Mol Cancer Ther 2002;1:371–6.

26] Lotfi K, Karlsson K, Fyrberg A, Juliusson G, Jonsson V, Peterson C, et al. Thepattern of deoxycytidine- and deoxyguanosine kinase activity in relation tomessenger RNA expression in blood cells from untreated patients with B-cellchronic lymphocytic leukemia. Biochem Pharmacol 2006;71:882–90.

27] Smal C, Vertommen D, Bertrand L, Ntamashimikiro S, Rider MH, Van Den NesteE, et al. Identification of in vivo phosphorylation sites on human deoxycy-tidine kinase. Role of Ser-74 in the control of enzyme activity. J Biol Chem2006;281:4887–93.

28] Lamba JK, Crews K, Pounds S, Schuetz EG, Gresham J, Gandhi V, et al. Pharmaco-genetics of deoxycytidine kinase: identification and characterization of novelgenetic variants. J Pharmacol Exp Ther 2007;323:935–45.

29] Al-Madhoun AS, van der Wilt CL, Loves WJ, Padron JM, Eriksson S, Talianidis I, etal. Detection of an alternatively spliced form of deoxycytidine kinase mRNA inthe 2′-2′-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancercell line AG6000. Biochem Pharmacol 2004;68:601–9.

30] Rapado I, Grande S, Albizua E, Ayala R, Hernandez JA, Gallardo M, et al. High res-olution melting analysis for JAK2 Exon 14 and Exon 12 mutations: a diagnostictool for myeloproliferative neoplasms. J Mol Diagn 2009;11:155–61.

31] Wu SB, Wirthensohn MG, Hunt P, Gibson JP, Sedgley M. High resolution meltinganalysis of almond SNPs derived from ESTs. Theor Appl Genet 2008.

32] Joerger M, Bosch TM, Doodeman VD, Beijnen JH, Smits PH, Schellens JH. Noveldeoxycytidine kinase gene polymorphisms: a population screening study inCaucasian healthy volunteers. Eur J Clin Pharmacol 2006;62:681–4.

33] Jordheim LP, Nguyen-Dumont T, Thomas X, Dumontet C, Tavtigian SV. Differen-tial allelic expression in leucoblast from patients with acute myeloid leukemiasuggests genetic regulation of CDA, DCK, NT5C2, NT5C3 and TP53. Drug MetabDispos 2008;36:2419–23.

34] Spasokoukotskaja T, Arner ES, Brosjo O, Gunven P, Juliusson G, LiliemarkJ, et al. Expression of deoxycytidine kinase and phosphorylation of2-chlorodeoxyadenosine in human normal and tumour cells and tissues. Eur JCancer 1995;31A:202–8.

35] Smal C, Van Den Neste E, Maerevoet M, Poire X, Theate I, Bontemps F.Positive regulation of deoxycytidine kinase activity by phosphorylation of

Page 7: Relationship between deoxycytidine kinase (DCK) genotypic variants and fludarabine toxicity in patients with follicular lymphoma

a Rese

[

A. Rivero et al. / Leukemi

Ser-74 in B-cell chronic lymphocytic leukaemia lymphocytes. Cancer Lett2007;253:68–73.

36] Antonsson BE, Avramis VI, Nyce J, Holcenberg JS. Effect of 5-azacytidine andcongeners on DNA methylation and expression of deoxycytidine kinase inthe human lymphoid cell lines CCRF/CEM/0 and CCRF/CEM/dCk-1. Cancer Res1987;47:3672–8.

[

[

arch 35 (2011) 431–437 437

37] Smal C, Cardoen S, Bertrand L, Delacauw A, Ferrant A, Van den Berghe G, etal. Activation of deoxycytidine kinase by protein kinase inhibitors and okadaicacid in leukemic cells. Biochem Pharmacol 2004;68:95–103.

38] Busch R, Neese RA, Awada M, Hayes GM, Hellerstein MK. Measurement of cellproliferation by heavy water labeling. Nat Protoc 2007;2:3045–57.