The interleukin-10 gene promoter polymorphisms influence ... file2 ABSTRACT The aim of the study was to investigate whether interleukin-10 (IL-10) genetic polymorphisms influence this

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Dominique Rigal, Krzystof Warzocha, Bertrand Coiffier and Gilles SallesEwa Lech-Maranda, Lucile Baseggio, Jacques Bienvenu, Carole Charlot, Francoise Berger, outcome of diffuse large B-cell lymphomaThe interleukin-10 gene promoter polymorphisms influence the clinical

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1

The interleukin-10 gene promoter polymorphisms

influence the clinical outcome of diffuse large B-cell lymphoma

Short title: IL-10 polymorphisms and lymphoma outcome

Ewa Lech-Maranda1,2, Lucile Baseggio 1,3, Jacques Bienvenu 1,4, Carole Charlot 1,3, Françoise

Berger 1, 5 Dominique Rigal 1, 6, Krzysztof Warzocha7, Bertrand Coiffier1,3 and Gilles Salles1,3

1Equipe d’Accueil 3738 «Pathologie des Cellules Lymphoïdes », Université Claude Bernard, Lyon,

France; 2Department of Hematology, Medical University of Lodz, Poland; 3Service d’Hématologie, 4Laboratoire d’Immunologie, 5Service d’Anatomie Pathologique, Centre Hospitalier Lyon-Sud, Pierre-

Bénite, France; 6 Etablissement Français du Sang, Lyon, France; 7Institute of Hematology and Blood

Transfusion, Warsaw, Poland

E. Lech-Maranda was supported by the UICC International Cancer Technology Transfer. This work

was supported by the Comité du Rhône de la Ligue contre le Cancer and les Hospices Civils de Lyon

(PHRC 2002).

Corresponding author:

Gilles Salles, MD. Ph. D.

Service d’Hématologie, Centre-Hospitalier Lyon-Sud, 69495 Pierre-Bénite, France

Tel: 33 (0)4 78 86 11 91; fax: 33 (0)4 78 86 65 66

e-mail: [email protected]

Word counts : Abstract 187 ; Text 2805

Scientific category : Neoplasma

Blood First Edition Paper, prepublished online December 30, 2003; DOI 10.1182/blood-2003-06-1850

Copyright (c) 2003 American Society of Hematology




2

ABSTRACT

The aim of the study was to investigate whether interleukin-10 (IL-10) genetic

polymorphisms influence this cytokine production as well as the incidence and outcome of

diffuse large B-cell lymphoma (DLBCL). The frequency of IL-10-1082G allele was found to be

higher in 199 DLBCL patients as compared to 112 controls (0.47 versus 0.39, P=.043).

Increased serum levels of IL-10 was associated with adverse prognostic factors and poor

DLBCL outcome. The frequencies of IL-10-819T and IL-10-592A alleles were lower in patients

with elevated IL-10 serum levels (0.155 versus 0.32, P=0.14). As compared to patients

carrying the IL-10-1082AA genotype, those with the IL-10-1082G allele (IL-10-1082GG/GA

genotypes) had higher complete remission rate (78% [CI 71%-85%] versus 65% [CI 52%-

78%], P=.07), 5-years freedom from progression (FFP) (60% [CI 52%-68%] versus 40% [CI

27%-53%], P=.013) and overall survival (OS) (63% [CI 55%-71%] versus 33% [CI 20%-

45%], P=.0009). Among factors of the International Prognostic Index, IL-10-1082G allele

remained an independent variable predicting longer FFP (RR [relative risk]=.76, P=.00035)

and OS (RR=.78, P=.0015). These results indicate that IL-10 production contributes to the

clinical course of DLBCL and that this phenomenon involves a substantial genetic

component.




3

INTRODUCTION

Diffuse large B-cell lymphoma (DLBCL) is the most frequent non-Hodgkin's

lymphoma (NHL) entity in the North America and Western Europe. The disease incidence is

increasing but etiological factors contributing to this phenomenon remain still largely

unknown.1 Although it is a curable disease, many patients do not achieve complete remission

(CR) or they relapse after conventional chemotherapy. The formulation of the International

Prognostic Index (IPI) has provided generally accepted criteria to identify specific risk groups

of DLBCL and to design appropriate therapies. The clinical prognostic features incorporated

in the IPI, including age, lactate dehydrogenase (LDH) level, performance status, clinical

stage and number of extranodal sites, mostly reflect the disease’s extension and the patient’s

characteristics.2,3 These tumour- and host-related parameters are likely to reflect some

underlying biological mechanisms. It has been suggested that immune system alterations may

be linked to the incidence and clinical course of lymphomas.4 Therefore, attempts to clarify

the mechanisms involved in immune system deregulation in lymphoma should contribute to a

better understanding of the clinical course of this malignancy.

Interleukin-10 (IL-10) is an important immuno-regulatory cytokine mainly produced

by monocytes and macrophages, T cells as well as normal and neoplastic B lymphocytes. IL-

10 plays a key role in controlling the balance between cellular and humoral immune

responses. IL-10 has strong immunosuppressive effects via the inhibition of pro-inflammatory

T helper 1 (Th1) lymphocytes, and conversely stimulates the proliferation and differentiation

of B and Th2 cells.5 Numerous studies have shown that IL-10 may be involved in the

pathogenesis of lymphoid disorders.5-8 It has been found to act as an autocrine growth factor

which up-regulates bcl-2 expression in some B-cell malignancies.7,8 Increased serum IL-10




4

levels were also found to be associated with poor prognosis and shorter survival of the

patients with NHL and Hodgkin’s lymphoma.9-11

The precise mechanisms engaged in the regulation of IL-10 production remain

undetermined, although inherited factors appear to play an important.12 The gene encoding

IL-10 is located on chromosome 1 (1q31-1q32).13,14 It has been reported that three single

nucleotide polymorphisms in the IL-10 gene promoter, including IL-10-1082, IL-10-819 and IL-

10-592, may influence IL-10 production in vitro.15 In addition, associations of these

polymorphisms with the incidence or clinical outcome of various infectious or inflammatory

disorders indicate that genetic variations within the IL-10 locus might be also functionally

relevant in vivo. 12, 16-20

In the present study we report that DLBCL patients have a higher frequency of

the IL-10-1082G allele than ethnically-matched healthy individuals in the French population.

Increased serum levels of IL-10 was associated with numerous adverse prognostic factors and

poor DLBCL outcome. Importantly, the frequencies of the IL-10-819T and IL-10-592A alleles

were lower in patients with elevated IL-10 serum levels and IL-10-1082G allele was associated

with longer freedom from progression (FFP) and overall survival (OS). These results indicate

that IL-10 production contributes to the clinical course of DLBCL and that this phenomenon

involves substantial genetic component.

MATERIALS AND METHODS

Subjects

The study comprised of 199 consecutive patients with DLBCL treated at the

Department of Hematology of the Centre Hospitalier Lyon-Sud and 112 unrelated ethnically-




5

matched healthy blood donors (Etablissement Français du Sang, Lyon, France). All samples

for genetic analysis were obtained after informed consent and coded. The patients’

confidentiality was preserved in accordance with the guidelines for studies of human subjects

in our institution.

The initial medical evaluation consisted of a complete history and physical

examination, computed tomographic scan of the chest, abdomen and pelvis, blood

morphology and chemistry. The extent of the disease was categorized according to the Ann

Arbor classification, and performance status was assessed using Eastern Cooperative

Oncology Group (ECOG) criteria. Clinical characteristics of the patients enrolled in the study

are shown in Table 1.

Treatment

All patients included in this study received anthracyclin-containing regimens,

consisting of CHOP (cyclophosphamide, adriamycine, vincristine, prednisone) or high-dose

CHOP 21 according to the age and number of IPI factors. No patients received rituximab as a

part of the first line regimen. Complete remission (CR) was defined as the disappearance of

all disease manifestations and normalization of all laboratory values. Freedom from

Progression (FFP) survival was determined from the onset of treatment until relapse, disease

progression, or the last follow-up evaluation. Overall survival (OS) was determined from the

onset of treatment until the last follow-up evaluation or death from any cause.

Among 199 patients, 148 (74%) achieved CR, whereas 51 (26%) did not. Eighty-two

patients (41%) have experienced disease progression, and 77 (39%) patients died. The median

follow-up for the patients remaining alive was 42 months (range: 9-196 months).

Genotyping analyses




6

Genomic DNA from peripheral blood mononuclear cells was extracted using High

Pure PCR Template Preparation Kit (Roche, Mannheim, Germany). IL-10-1082 polymorphism

was genotyped using an allele-specific polymerase chain reaction (ASPCR). The specific

primers pair S (5’-CCCCAGGTAGAGCAACACTCC) with B1 (5’-

CCTATCCCTACTTCCCCC) or B2 (5’-CCTATCCCTACTTCCCCT) were used to amplify

a 153-bp fragment of the IL-10 gene, which includes the polymorphic site at the nucleotide

position -1082.22 Each sample was tested with both primers pairs, i.e. S-B1 and S-B2. The

primers pair S with AS (5’-ACACCATCTCCAGCACATAG) was used to amplify the IL-10

gene’s fragment of 325 bp which served as an internal control and a genomic DNA template

for sequencing. Primer AS was also used as a competitor for the primer pairs S-B1 and S-B2

to improve the specificity of the ASPCR assay.23 After heating at 950 C for 10 minutes, PCR

amplification was performed with 30 cycles: 950 C for 30 seconds; 600 C for 60 seconds; 720

C for 60 seconds; followed by a final extension step at 720 C for 7 minutes. PCR products

were stained and visualized on a 2% agarose gel with ethidium bromide.

All IL-10-1082 genotyping results were independently confirmed using another allelic

discrimination assay with specific fluorescent dye-labeled (FAM and VIC) MGB probes

(Applied Biosystem, Foster City, CA, USA) and real time PCR analysis performed on an ABI

PRISM 7700 Sequence Detector (Applied Biosystem). The forward (5’-CAAATCCAAGAC

AACACTACTAAGGC) and reverse (5’-GGGTGGAAGAAGTTGAAATAACAAG)

primers’ pair, and MGB probes specific for the IL-10-1082G (CTTCCCCCTCCCAAA) and IL-

10-1082A (CTTCCCCTTCCCAAAG) alleles were used to amplify the 135 bp fragment of the

IL-10 promoter. PCR conditions were used as following: 500 for 2 minutes, 950 C for 10

minutes, and after 40 cycles with 920 C for 15 seconds and 620 C for 60 seconds.

IL-10-819 and IL-10-592 polymorphisms were genotyped using a PCR-based restriction

fragment length polymorphism (PCR-RFLP).24 PCR-amplified products of 588bp were




7

obtained with the use of upstream (5’-ATCCAAGACAACACTACTAA) and downstream

(5’-TAAATATCCTCAAAGTTCC) primers. PCR amplification was performed with 30

cycles: 950 C for 30 seconds; 540 C for 60 seconds; 720 C for 60 seconds; followed by a final

extension step at 720 C for 7 minutes. The PCR-amplified products were digested overnight

with 0.75 U of MaeIII (Roche Diagnostic GmbH, Mannheim, Germany) for the IL-10-819

polymorphism or with 8 U of RsaI (Promega Corporation, Madison, WI, USA) for the

IL-10-592 polymorphism. Digested PCR products were separated by electrophoresis on a 10%

polyacrylamide gel.

To confirm the accuracy of the ASPCR and PCR-RFLP assays, amplification products

from 2 individuals homozygous and 1 heterozygous for each polymorphic allele were purified

from the gel, ligated into pGEM-T vector (Promega) and subcloned. Recombinant plasmid

DNAs were sequenced using Alf ExpressTM DNA Sequencer (Pharmacia Biotech, Uppsala,

Sweden). In every studied case, the results of IL-10-1082, IL-10-819, IL-10-592 genotyping

obtained by sequencing were the same as using ASPCR or RFLP methods.

Evaluation of serum IL-10 levels

Blood samples from 149 newly diagnosed patients were collected before treatment

initiation using sterile tubes containing EDTA to prevent further release of cytokine from

circulating mononuclear cells. Serum samples were stored at -800 C and thawed immediately

before the determination of IL-10 level using an human enzyme-linked immunoabsorbent

assay (ELISA) (BioSource International, Inc., California, USA). The detection limit of the test

was 5 pg/mL.

Patients with active bacterial or fungal infection, and those who tested positive for the

human immunodeficiency virus as well as patients with a previous history of autoimmune




8

disease and those who had received recent corticosteroid therapy were excluded from the

analysis.

Statistical analysis

Associations between allele frequencies or genotype distributions and clinical or

biological variable were assessed with the χ2 test with Yates’s correction when a cell

frequency was less than 20 unless any expected frequency was less than 5, when Fisher’s

exact test was used. Survival (FFP and OS) were estimated by the Kaplan-Meier method and

compared using the log-rank test. A multivariate regression analysis with the Cox

proportional hazard model was used to adjust the effect of the IL-10 polymorphisms along

with variables of the IPI for potential independent prognostic factors. Only patients with

complete data were entered into the regression procedure (n=175). Statistical tests with P<.05

were considered significant. Statistical analysis was performed using the Statistica package

(StatSoft, Tulsa, OK). Confidence interval (95%) were calculated.

RESULTS

IL-10-1082, -819, - 592 polymorphisms in DLBCL patients and healthy controls

The frequency of IL-10-1082G allele was higher in lymphoma patients as compared to

healthy controls (0.47 versus 0.39, P=.043) which translated into a higher frequency of IL-10-

1082GG/GA genotypes in the former population (72%, [CI 66%-78%] versus 60%, [CI 51%-

69%], P=.023). Allelic frequencies and distributions at the IL-10-819 and IL-10-592

polymorphisms did not differ between both studied populations (Table 2).




9

IL-10-1082, -819, - 592 polymorphisms and IL-10 serum levels in patients with DLBCL

Only 149 DLBCL patients had available samples for IL-10 ELISA assays at diagnosis.

Of those, 117 patients (79%) had IL-10 serum levels below the detection limit (<5 pg/mL)

and 32 (21%) above this value (median=31.5 pg/mL, range: 5 to 2480 pg/mL). Detectable IL-

10 serum levels were associated with age>60 years, ECOG status ≥ 2, disease stage III/IV,

elevated serum LDH and β2-microglobulin levels, presence of B symptoms, anemia and low

serum albumin levels as well as with intermediate-high and high risk groups according to the

IPI (Table 1). As compared to the patients with IL-10 serum levels below the detection limit,

patients with elevated cytokine levels had significantly lower CR rate (50% [CI 33%-67%,]

versus 82% [CI 75%-89%], P=.0005), lower estimated 5-years FFP (24% [CI 10%-39%]

versus 66% [CI 57%-74%], P=.0015) and lower 5-years OS (32% [CI 16%-48%] versus 65%

[CI 56%-74%], P=.0009).

With respect to IL-10 gene polymorphisms, the frequencies of IL-10-819T and IL-10-

592A alleles were lower in the patients with elevated IL-10 serum levels versus below its

detection limit (0.155 versus 0.32, P=0.14). This translated into a higher frequency of IL-10-

819CC/IL-10-592CC genotypes in the former group (72% [CI 56%-87%] versus 45% [CI 36%-

54%], P=.014). No associations were found between IL-10 serum levels and allelic or

genotype variations within IL-10-1082 polymorphism (Table 3).

The fifty patients for which samples for IL-10 serum level evaluation were not

available did not differ from the remaining 149 patients in terms of genotyping, laboratory or

clinical profiles nor FFP and OS intervals (not shown).

IL-10-1082, -819, - 592 polymorphisms and DLBCL outcome




10

Among 199 patients with DLBCL, no associations were found between IL-10-1082G

allele and prognostic variables listed in Table 1 except of male gender (P=.026) and age <60

years (P=.032).

As compared to the patients carrying the IL-10-1082AA genotype, those with IL-10-1082G

allele (IL-10-1082GG/GA genotypes) had slightly higher complete remission rate (78% [CI 71%-

85%] versus 65% [CI 52%-78%], P=.07), and presented higher estimated 5-years FFP (60%

[CI 52%-68%] versus 40% [CI 27%-53%], P=.013) and higher 5-years OS (63% [CI 55%-

71%] versus 33% [CI 20%-45%], P=.0009) (Fig. 1). No associations were found between the

prognostic variables, FFP or OS intervals and IL-10-1082GG genotype or allele/genotype

distributions within the IL-10-819, -592 polymorphisms.

After incorporating IL-10-1082G allele and all prognostic variables of the IPI in a

multivariate Cox regression model, the IL-10-1082G allele was found to be an independent

variable predicting longer FFP survival (RR [relative risk]=.76, P=.00035), followed by LDH

serum levels within normal values (RR=.76, P=.0027), disease stage I/II (RR=.81, P=.043)

and number of extranodal sites <2 (RR=.83, P=.048) (total R2=.85, P<.0001). Using the same

variables, IL-10-1082G allele (RR=.78, P=.0015) followed by LDH serum levels within normal

values (RR=.78, P=.0076), disease stage I/II (RR=.78, P=.021), and number of extranodal

sites <2 (RR=.81, P=.033) were retained in the multivariate predictive model for longer OS

(total R2= .85, P<.0001).

DISCUSSION

IL-10 is a pleiotropic immunomodulatory cytokine which plays a crucial role in

normal ontogenesis and function of the immune system. It is also involved in the acute and




11

chronic inflammatory responses that accompany various infectious, autoimmune and

lymphoproliferative disorders.5 The results of the present study confirms previous

observations9-11,25,26 that elevated IL-10 serum levels are associated with adverse prognostic

factors and predict poor DLBCL outcome, and for the first time links this observation to IL-

10 genetic polymorphisms.

We observed that IL-10 serum levels mainly reflect tumor burden (advanced disease

stage, elevated LDH and β2-microglobulin serum levels) and host-tumor relationship

(presence of B symptoms, anemia, low serum albumin levels). These data suggest that IL-10

might be produced by both lymphoma as well as bystander reactive cells.27,28 It seems likely

therefore that increased serum levels of IL-10 reflects an enhanced activation of the immune

system upon more aggressive disease but its potential action as a growth factor for lymphoma

cells or as a suppressor of macrophages or T-cell functions should also be taken into

account.29

In vitro studies revealed that IL-10 production could be related to its gene promoter

polymorphisms.15 Several studies such as those assessing the susceptibility to Epstein-Barr

virus infection, the severity of autoimmune diseases or graft versus host disease (GVHD),

were performed in different ethnic groups and indicated that distinct IL-10 alleles or

haplotypes were a key factor of IL-10 production in vivo.16,17,22,30 These observations raise the

possibility that susceptibility and clinical course of disorders in which immune activation

plays an important pathogenic role could be related to the genetic control of IL-10 production.

In the present study, an moderate excess of the IL-10-1082G allele was found among the

patients with DLBCL as compared to ethnically-matched healthy controls. The allelic

frequencies and distributions of the IL-10-819 and IL-10-592 polymorphisms did not differ

between both cohorts. These data suggest that the presence of the IL-10-1082G allele (or

eventually another allelic variation genetically linked to the former) may contribute to the




12

genetic background of DLBCL occurrence. On the other hand, since the observed difference

in the prevalence of IL-10-1082G allele between patients and controls was only marginally

significant, it is not possible to exclude the presence of a bias towards an over-representation

of this particular subgroup of individuals in the present cohort of patients with DLBCL. Other

similar studies are therefore warranted.

The frequencies and distributions of the IL-10-1082, -819, -592 polymorphic alleles in the

present population of healthy controls were similar to those observed in bone marrow donors

from the French population independently obtained by Socié et al.31 These allelic frequencies

were different as compared to the healthy individuals from Manchester and South-East

England and particularly from Southern China.15,32-34 It seems likely therefore that highly

polymorphic IL-10 promoter variations reflects different prevalent haplotypes in various

ethnic groups.

In the present study, we observed the association between the presence of the IL-10-

819T or IL-10-592A alleles and low IL-10 serum levels at the time of initial DLBCL

presentation. This remains in line with observations of others indicating lower capability of

IL-10 production for the IL-10-819T or IL-10-592A alleles and higher for the IL-10-1082GG

genotype.15-20 Although we did not find an association between IL-10-1082GG genotype and

increased IL-10 serum levels, this could be limited by the low patients number in this

particular subgroup (n=8). Overall, these data suggest that genetic control of IL-10 production

influences the serum levels of this cytokine in DLBCL patients.

The most striking evidence for the clinical significance of IL-10 promoter gene

polymorphisms in DLBCL was the observed association of the IL-10-1082G allele and a

favorable disease outcome. Since no associations were found between the FFP or OS intervals

and allelic variations within IL-10-819,-592 polymorphisms, these results indicate that DLBCL




13

outcome was solely related to the IL-10-1082G and could not be explained by the remaining

alleles.

Because IL-10-1082G was previously found to be associated with high IL-10-producing

capability15-20, our data suggest that increased IL-10 production within tumor

microenvironment might be of protective value and conversely, that low IL-10-producing

capability makes individuals susceptible to more aggressive course of the disease. For

example, IL-10 was found to increase T-cell cytotoxicity, to inhibit tumor angiogenesis and to

antagonize the action of proinflammatory cytokines.35-37 Alternatively, the influence of

clinical course of DLBCL by the IL-10-1082G may imply others genes residing close to this

locus. Other studies regarding the role of tumor necrosis factor and HLA class II

polymorphisms in patients with lymphoma have also indicated that a single allele could not

account for the variability of the disease outcome.38,39 Similar observations were obtained in

GVHD and other clinical conditions.20, 22

In summary, our findings demonstrate that IL-10 production contributes to the clinical

course of DLBCL and that this phenomenon involves a substantial genetic component.

Further identification of inherited genetic markers associated with clinical aggressiveness of

lymphoma can guide the search for immunological mechanisms underlying that variability.40




14

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19

Table 1. Characteristics of 199 patients with DLBCL and their associations with

IL-10 serum levels in 149 patients who provided samples available for ELISA at

the time of initial presentation.

Characteristics All patients Patients with Patients with IL-10 (< 5 pg/mL) IL-10 (≥ 5 pg/mL) P values*

n=199 n=117 n=32

Sex nsFemale 104 (52 %) 63 (54%) 16 (50%)Male 95 (48 %) 54 (46%) 16 (50%)

Age .01

≤ 60 years 104 (52 %) 65 (56%) 9 (28%)> 60 years 95 (48 %) 52 (44%) 23 (72%)

Performance status (ECOG) .001< 2 137 (69 %) 89 (76%) 16 (50%)≥ 2 54 (27%) 21 (18%) 16 (50%) unknown 8 7

B symptoms .0004 absent 141 (71 %) 94 (80%) 15 (47%) present 58 (29 %) 23 (20%) 17 (53%)

Ann Arbor stage .007I,II 78 (39 %) 51 (43%) 6 (19%)III,IV 110 (55 %) 57 (49%) 26 (81%)unknown 11 9

Serum LDH .005 ≤ 1 x normal 77 (39 %) 56 (48%) 7 (22%)> 1 x normal 106 (53 %) 49 (42%) 24 (75%)




20

unknown 16 12 1

Serum β2-microglobulin <.0001 ≤ 3.0 mg/L 125 (63 %) 81 (69%) 12 (37.5%)> 3.0 mg/L 50 (25 %) 20 (17%) 20 (62.5%)unknown 24 16

No of extranodal sites ns < 2 116 (58 %) 71 (61%) 16 (50%)≥ 2 67 (34 %) 35 (30%) 13 (41%)unknown 16 11 3

Serum albumin level <.0001 ≤ 35 g/L 53 (27 %) 30 (26%) 22 (69%)> 35 g/L 123 (62 %) 74 (63%) 8 (25%)unknown 23 13 2

Hemoglobin .03 ≤ 12 g/dL 87 (44 %) 47 (40%) 21 (66%)

> 12 g/dL 99 (50 %) 59 (50%) 11 (34%)unknown 13 11

Bulky tumor (≥ 10 cm) ns absent 111 (56 %) 65 (55%) 13 (41%)present 77 (39 %) 43 (37%) 18 (56%)unknown 11 9 1

Complete remission rate .0005 yes 148 (74%) 96 (82%) 16 (50%)

no 51 (26%) 21 (18%) 16 (50%)

International Prognostic Index risk groups .003 low / intermediate low 96 (48 %) 64 (55%) 8 (25%)intermediate high / high 79 (40 %) 39 (33%) 20 (62%)unknown 24 14 4

* The associations were compared with χ2 test; ns denotes not significant




21

Table 2. Allele frequency and genotype distribution of the IL-10-1082, IL-10-819, IL-10-592

polymorphisms in 199 DLBCL patients and 112 healthy controls

Control DLBCL patients

n=112 n=199

Allele frequency

IL-10-1082G 0.39 0.47*

IL-10-1082A 0.61 0.53

IL-10-819C 0.68 0.745

IL-10-819T 0.32 0.255

IL-10-592C 0.68 0.745

IL-10-592A 0.32 0.255

Genotype distribution

IL-10-1082GG 20 (18%) 44 (22%) †

IL-10-1082GA 47 (42%) 100 (50%)

IL-10-1082AA 45 (40%) 55 (28%)

IL-10-819CC 53 (47%) 107 (54%)

IL-10-819CT 46 (41%) 81 (41%)

IL-10-819TT 13 (12%) 11 (5%)

IL-10-592CC 53 (47%) 107 (54%)

IL-10-592CA 46 (41%) 81 (41%)

IL-10-592AA 13 (12%) 11 (5%)




22

* P=.043, frequency of the IL-10-1082G allele in DLBCL patients as compared to controls; †

P=.023, frequency of the IL-10-1082GG/GA genotypes in DLBCL patients as compared to

controls. P value refers to the χ2 test




23

Table 3. The associations of IL-10 levels with allele frequency and genotype distribution of

the IL-10-1082, IL-10-819, IL-10-592 polymorphisms in 149 DLBCL patients who provided

samples available for ELISA at the time of initial presentation.

IL-10 (< 5 pg/mL) IL-10 (≥ 5 pg/mL)

n=117 n=32

Allele frequency

IL-10-1082G 0.45 0.53

IL-10-1082A 0.55 0.47

IL-10-819C 0.68 0.845

IL-10-819T 0.32 * 0.155

IL-10-592C 0.68 0.845

IL-10-592A 0.32 * 0.155

Genotype distribution

IL-10-1082GG 26(22%) 8(25%)

IL-10-1082GA 54 (46%) 18 (56%)

IL-10-1082AA 37 (32%) 6 (19%)

IL-10-819CC 53 (45%) 23 (72%) †

IL-10-819CT 54 (46%) 8 (25%)

IL-10-819TT 10 ( 9%) 1 (3%)

IL-10-592CC 53 (45%) 23 (72%) †

IL-10-592CA 54 (46%) 8 (25%)

IL-10-592AA 10 ( 9%) 1 (3%)

* P=.014, frequency of the IL-10-819T or IL-10-592A alleles in DLBCL patients with elevated IL-10

serum levels (≥ 5 pg/mL) versus below its detection limit (<5 pg/mL); † P=.014, frequency of the

IL-10-819CC or IL-10-592CC genotypes in DLBCL patients with elevated IL-10 serum levels (≥ 5

pg/mL) versus below its detection limit (<5 pg/mL). P value refers to the χ2 test




24

Legend to Figure 1. Freedom from progression (A) and overall survival (B) of 199 DLBCL

patients according to the IL-10-1082 polymorphism. The continuous lines denote the patients

carrying the IL-10-1082G allele (n=144), including IL-10-1082GG (n=44) and IL-10-1082GA (n=100)

genotypes, while the dotted lines denote the patients carrying IL-10-1082AA genotype (n=55); P

value refers to the LogRank test.




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A)

(B)




26




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