Abstract

Background: Recently published data indicate that host germline variations in immune genes can influence the outcome of lymphoma patients. Interleukin (IL)-4 and IL13 are crucial immune factors and may influence the course of the disease. Both cytokines signal through the interleukin-4 receptor (IL4R). Therefore, we investigated whether polymorphisms of IL4, IL13 and IL4R genes could predict the outcome of diffuse large B-cell lymphoma (DLBCL) patients.

Methods: In 228 DLBCL samples of the German High-Grade Non-Hodgkin's Lymphoma Study Group, the polymorphisms of IL4 (-524CT, rs2243250), IL13 (-1069CT, rs1800925) and IL4R (I75V, rs1805010; S503P, rs1805015; Q576R, rs1801275) were analyzed and the soluble interleukin-4 receptor (sIL4R) serum level was measured before the start of chemotherapy.

Results: Patients harboring IL4R V75 (IL4RI75V-AG and IL4RI75V-GG) had shorter overall survival (OS) (P = 0.044) and event-free survival (EFS) (P = 0.056) periods compared with I75 carriers (IL4RI75V-AA). Multivariate analysis adjusted to the International Prognostic Index revealed a relative risk of 1.9 for carriers of the IL4R V75 (P = 0.011) in relation to OS. DLBCL patients homozygous for the IL4R I75 and low sIL4R serum levels have the most favorable OS and EFS.

Conclusions: These data support the role for host germline gene variations of immunologically important factors like the IL4R I75V gene variation to predict the survival in DLBCL patients.

introduction

Diffuse large B-cell lymphoma (DLBCL) is the major subtype of non-Hodgkin's lymphoma (NHL), accounting for approximately one-third of all lymphoma cases in the western world. Although cure rates are high in DLBCL, many patients do not achieve complete remission or relapse after induction treatment. The introduction of the International Prognostic Index (IPI) has improved risk stratification of NHL patients but is still insufficient for outcome prediction, mainly for the group of the nonresponders [1]. Therefore, factors not included in the IPI have to be further validated in order to better predict patients’ outcome, to stratify patients’ treatment strategies as well as to understand the underlying pathological mechanisms of lymphoma. Cytokine gene variations have been shown to influence the susceptibility to NHL as well as the clinical course of lymphoma [2–6]. In a cohort of 500 NHL patients from the NHL-B study of the German High-Grade Non-Hodgkin's Lymphoma Study Group (DSHNHL), we recently found an association of distal IL10 gene variations to the clinical course of high-grade NHL [2]. However, in this study, the subgroup of DLBCL patients showed neither association of the analyzed IL10 gene variations to the clinical outcome nor the susceptibility of DLBCL, leading us to question the influence of other genetic variations in immune genes in DLBCL patients [2].

Cytokines and their receptors play an essential role in regulation of the host immunity and could act as potential autocrine or paracrine factors for lymphoma initiation or progression. The expression of cytokines and their respective receptors on different cell types regulate the communication between the components of immune system and could be responsible for the host–lymphoma interactions as well. Interleukin-4 (IL4) and IL13 are crucial cytokines to polarize naïve CD4 T lymphocytes to Th2 cells and also to further trigger the humoral Th2 immune responses. Both cytokines use the interleukin-4 receptor (IL4R) chain to signal through different receptor heterodimers. IL4 signals by the use of the common gamma chain together with the IL4R chain (type I receptor) and both IL4 and IL13 can signal through the dimer of the IL13RA1 chain in combination with the IL4R (type II receptor). Moreover, a soluble form of the IL4R chain (sIL4R) has been described [7]. The expression of soluble cytokine receptors is believed to alter the properties of the respective cytokine signaling cascade. Serum levels of soluble cytokine receptors have been reported to be of prognostic significance in patients with NHL [8, 9].

In this exploratory study, we analyzed the association of IL4, IL13 and IL4R gene polymorphisms and the respective sIL4R serum levels to the clinical outcome in a cohort of 228 DLBCL patients of the NHL-B1/B2 study of the DSHNHL. Our results, based on clinical data of a large prospective clinical trial, indicate for the first time an association of the IL4R I75V gene polymorphism with the treatment outcome of DLBCL patients.

methods

study design

The study was conducted in accordance with the Declaration of Helsinki. This polymorphism analysis was approved by the Ethics Committee of the University of Göttingen (4 November 2001). All patients gave written informed consent to the inclusion into the NHL-B trial. Patients were eligible for inclusion if they had previously untreated, biopsy-confirmed DLBCL according to the Revised European–American Lymphoma Classification (translated into the World Health Organization classification). Within the NHL-B1/B2 trials of the DSHNHL 1399 high-grade NHL patients were included, thereof 913 patients with DLBCL [10, 11]. Data and samples from 228 DLBCL patients were selected from the DLBCL NHL-B1/B2 study population based on the availability of samples and representativeness for all DLBCL patients of the trials, as described recently [2, 3, 10, 11]. The selected patients were equally distributed across the different study arms of the NHL-B1/B2 trials. Clinical characteristics and histology of the patients eligible for this study are shown in Table 1 in comparison with all DLBCL patients of the NHL-B1/B2 studies. The respective histology is based on a review by a panel of expert hematopathologists as described before [10, 11].

Table 1.

Clinical characteristics and histolopathology of study patients with DLBCL

 All DLBCL patients NHL-B1/B2 trials (n = 913) DLBCL patients analyzed for gene variations (n = 228) 
Patient's characteristics   
    Sex   
        Male 507 (55.5) 126 (55.3) 
        Female 406 (44.5) 102 (44.7) 
    Age, median (minimum; maximum), years 61 (18; 75) 60.5 (22; 75) 
    Serum LDH >N 225 (24.6) 49 (21.5) 
    Age >60 years 489 (53.6) 114 (50.0) 
    Performance status ECOG >1 110 (12.0) 28 (12.3) 
    Ann Arbor stages III, IV 371 (40.6) 91 (39.9) 
    No. of extranodal sites >1 166 (18.2) 38 (16.7) 
    IPIa   
        Low (IPI = 0, 1) 531 (58.2) 140 (61.4) 
        Low intermediate (IPI = 2) 164 (18.0) 44 (19.3) 
        High intermediate (IPI = 3) 131 (14.3) 24 (10.5) 
        High (IPI = 4, 5) 87 (9.5) 20 (8.8) 
    Bulky tumor (≥7.5 cm) 296 (32.4) 75 (32.9) 
    B symptoms 247 (27.1) 54 (23.7) 
    Extranodal involvement 448 (49.1) 109 (47.8) 
Histology   
    DLBCL 913 (100.0) 228 (100.0) 
        Centroblastic diffuse 687 (75.2) 165 (72.4) 
            Monomorphic 83 (9.1) 25 (11.0) 
            Multilobulated 57 (6.2) 11 (4.8) 
            Polymorphic 349 (38.2) 86 (37.7) 
            Not otherwise specified 198 (21.7) 43 (18.9) 
        Immunoblastic 114 (12.5) 32 (14.0) 
        T-cell rich B-cell 24 (2.6) 7 (3.1) 
    Anaplastic 28 (3.1) 11 (4.8) 
    Not otherwise specified 60 (6.6) 13 (5.7) 
 All DLBCL patients NHL-B1/B2 trials (n = 913) DLBCL patients analyzed for gene variations (n = 228) 
Patient's characteristics   
    Sex   
        Male 507 (55.5) 126 (55.3) 
        Female 406 (44.5) 102 (44.7) 
    Age, median (minimum; maximum), years 61 (18; 75) 60.5 (22; 75) 
    Serum LDH >N 225 (24.6) 49 (21.5) 
    Age >60 years 489 (53.6) 114 (50.0) 
    Performance status ECOG >1 110 (12.0) 28 (12.3) 
    Ann Arbor stages III, IV 371 (40.6) 91 (39.9) 
    No. of extranodal sites >1 166 (18.2) 38 (16.7) 
    IPIa   
        Low (IPI = 0, 1) 531 (58.2) 140 (61.4) 
        Low intermediate (IPI = 2) 164 (18.0) 44 (19.3) 
        High intermediate (IPI = 3) 131 (14.3) 24 (10.5) 
        High (IPI = 4, 5) 87 (9.5) 20 (8.8) 
    Bulky tumor (≥7.5 cm) 296 (32.4) 75 (32.9) 
    B symptoms 247 (27.1) 54 (23.7) 
    Extranodal involvement 448 (49.1) 109 (47.8) 
Histology   
    DLBCL 913 (100.0) 228 (100.0) 
        Centroblastic diffuse 687 (75.2) 165 (72.4) 
            Monomorphic 83 (9.1) 25 (11.0) 
            Multilobulated 57 (6.2) 11 (4.8) 
            Polymorphic 349 (38.2) 86 (37.7) 
            Not otherwise specified 198 (21.7) 43 (18.9) 
        Immunoblastic 114 (12.5) 32 (14.0) 
        T-cell rich B-cell 24 (2.6) 7 (3.1) 
    Anaplastic 28 (3.1) 11 (4.8) 
    Not otherwise specified 60 (6.6) 13 (5.7) 

The total numbers of patients and (in parentheses) respective percentages are presented.

a

LDH >N, age >60 years, ECOG >1, stage III, IV and no. of extranodal sites ≥2.

DLBCL, diffuse large B-cell lymphoma; NHL, non-Hodgkin's lymphoma; LDH, lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group; IPI, International Prognostic Index.

genotyping analyses

Genomic DNA from lymphoma patients was isolated as described previously [2, 3, 12]. The polymorphisms of IL4 (-524CT, rs2243250), IL13 (-1069CT, rs1800925) and IL4R (I75V, rs1805010; S503P, rs1805015; and Q576R, rs1801275) genes were analyzed. For the analysis of the genetic polymorphisms (listed in Table 3 and supplemental Table 1, available at Annals of Oncology online), an Applied Biosystems ABI 7900HT instrument was used based on the sequence data and assay conditions of the National Cancer Institute SNP500 database (http://snp500cancer.nci.nih.gov/).

serum analysis

Sera were collected and stored frozen at the trial office biomaterials storage facility center at −70°C until measurement of serum factors. Serum concentrations of sIL4R were determined with Quantikine ELISA Kits (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) according to the manufacturer's instruction. All samples and standards were run in duplicates; 115 pg/ml of sIL4R were set to discriminate high and low sIL4R levels based on upper quartile value of the whole cohort.

statistical analysis

Statistical analysis was carried out as described before [2]. Event-free survival (EFS) was defined as the time from first day of treatment to progressive disease under therapy, or failure to achieve a complete remission or complete remission unconfirmed (i.e. no change or partial remission associated with additional salvage therapy), additional therapy in excess of that prescribed in the protocol, relapse or death from any cause, whatever came first. Overall survival (OS) was defined as the time from first day of treatment to death from any cause. Patients without an event in EFS or OS were censored at the last day with valid information for the respective end point. EFS and OS were estimated according to Kaplan—Meier test and compared by log-rank test. For univariate analyses, P values < 0.05 were considered significant. Estimators at 3 years are given with 95% confidence intervals. Multivariate analyses were carried out with the use of Cox proportional hazard models to estimate hazard ratios for evolving an event. All factors with P < 0.1 were included in the multivariate analysis. We are aware of the problem of multiple comparisons between different combinations of heterozygous and homozygous groups within each genotype and have chosen to extract the most prominent aspect; therefore, this study is labeled as an exploratory study. Statistical analyses were carried out with SPSS 15.0.

results

IL4R I75V polymorphism is associated with survival of DLBCL patients

Within this cohort of DLBCL patients, we analyzed polymorphisms of IL4 (-524CT), IL13 (-1069CT) and IL4R (I75V, S503P and Q576R) genes. Genotype distribution and Hardy–Weinberg equilibrium of the analyzed polymorphisms are shown in Table 2 and supplemental Table 1 (available at Annals of Oncology online). Subsequently, the association of the polymorphisms with the OS and EFS of DLBCL patients was analyzed. Univariate analysis revealed for the IL4R I75V variation differences in the survival of DLBCL patients. Individuals with at least one V75 variant (represented by IL4RI75V-GG and IL4RI75V-AG genotypes) had decreased OS (P = 0.044) and EFS (P = 0.056) periods compared with homozygous A-allele carrier representing the I75 variant (IL4RI75V-AA) (Figure 1A and B). The 3-year survival rate of patients with I75 variant (IL4RI75V-AA) was 80.0% (OS) and 71.4% (EFS) compared with 66.8% (OS) and 58.2% (EFS) in patients with V75 variants (IL4RI75V-AG and IL4RI75V-GG) (Table 3). The two other IL4R polymorphisms (S503P and Q576R) showed also shorter survival periods for homozygous carriers of the respective minor alleles. However, this was not significant probably due to their low frequency (Table 3). No relation of the promoter polymorphisms of IL4 (-524CT) and IL13 (-1069CT) to the survival of DLBCL patients was found (supplemental Table 2, available at Annals of Oncology online).

Table 2.

IL4R SNPs, genotype distribution and HWE

SNP name Ref. SNP ID Genotype AA variant No. of patients % of patients HWE P value (χ2) 
IL4R I75V rs1805010 AA I75/I75 70 30.7 0.265 
  AG I75/V75 105 46.1  
  GG V75/V75 53 23.2  
IL4R S503P rs1805015 TT S503/S503 156 68.4 0.603 
  TC S503/P503 66 28.9  
  CC P503/P503 2.6  
IL4R Q576R rs1801275 AA Q576/Q576 140 61.4 0.754 
  AG Q576/R576 79 34.6  
  GG R576/R576 3.9  
SNP name Ref. SNP ID Genotype AA variant No. of patients % of patients HWE P value (χ2) 
IL4R I75V rs1805010 AA I75/I75 70 30.7 0.265 
  AG I75/V75 105 46.1  
  GG V75/V75 53 23.2  
IL4R S503P rs1805015 TT S503/S503 156 68.4 0.603 
  TC S503/P503 66 28.9  
  CC P503/P503 2.6  
IL4R Q576R rs1801275 AA Q576/Q576 140 61.4 0.754 
  AG Q576/R576 79 34.6  
  GG R576/R576 3.9  

IL4R, interleukin-4 receptor; SNP, single-nucleotide polymorphisms; HWE, Hardy–Weinberg equilibrium.

Table 3.

Association between IL4R nonsynonymous coding SNPs and OS and EFS of DLBCL patients

Genotype  3-year rate (95% CI) P 3-year rate (95% CI) P Genotype 3-year rate P 3-year rate P 
 OS  EFS    OS EFS  
IL4R I75V           
    AA n = 70 I75/I75 80.0 (70.6–89.4) 0.121 71,4 (60.0–82.0) 0.157 AA n = 70 80.0 0.044 71.4 0.056 
    AG n = 105 I75/V75 67.4 (58.4–76.4)  59.0 (49.6–68.4)  AG and GG n = 158 66.8  58.2  
    GG n = 53 V75/V75 65.6 (52.7–78.5;)  56.5 (43.2–69.8)  AA and AG n = 175 72.4 0.662 64.0 0.604 
      GG n = 53 65.6  56.5  
IL4R S503P           
    TT n = 156 S503/S503 71.1 (64.0–78.2) 0.250 62.8 (55.2–70.4) 0.534 TT n = 156 71.1 0.618 62.8 0.832 
    TC n = 66 S503/P503 72.4 (61.6–83.2)  62.1 (50.3–73.9)  TC and CC n = 72 70.5  61.1  
    CC n = 6 P503/P503 50.0 (10.0–90.0)  50.0 (10.0–90.0)  TT and TC n = 222 71.4 0.150 62.6 0.264 
      CC n = 6 50.0  50.0  
IL4R Q576R           
    AA n = 140 Q576/Q576 70.6 (63.0–78.2) 0.330 62.1 (54.1–70.1) 0.508 AA n = 140 70.6 0.414 62.1 0.899 
    AG n = 79 Q576/R576 73.1 (63.3–82.9)  63.3 (52.7–73.9)  AG and GG n = 188 71.3  62.5  
    GG n = 9 R576/R576 55.6 (23.1–88.1)  55.6 (23.1–88.1)  AA and AG n = 219 71.5 0.312 62.5 0.279 
      GG n = 9 55.6  55.6  
Genotype  3-year rate (95% CI) P 3-year rate (95% CI) P Genotype 3-year rate P 3-year rate P 
 OS  EFS    OS EFS  
IL4R I75V           
    AA n = 70 I75/I75 80.0 (70.6–89.4) 0.121 71,4 (60.0–82.0) 0.157 AA n = 70 80.0 0.044 71.4 0.056 
    AG n = 105 I75/V75 67.4 (58.4–76.4)  59.0 (49.6–68.4)  AG and GG n = 158 66.8  58.2  
    GG n = 53 V75/V75 65.6 (52.7–78.5;)  56.5 (43.2–69.8)  AA and AG n = 175 72.4 0.662 64.0 0.604 
      GG n = 53 65.6  56.5  
IL4R S503P           
    TT n = 156 S503/S503 71.1 (64.0–78.2) 0.250 62.8 (55.2–70.4) 0.534 TT n = 156 71.1 0.618 62.8 0.832 
    TC n = 66 S503/P503 72.4 (61.6–83.2)  62.1 (50.3–73.9)  TC and CC n = 72 70.5  61.1  
    CC n = 6 P503/P503 50.0 (10.0–90.0)  50.0 (10.0–90.0)  TT and TC n = 222 71.4 0.150 62.6 0.264 
      CC n = 6 50.0  50.0  
IL4R Q576R           
    AA n = 140 Q576/Q576 70.6 (63.0–78.2) 0.330 62.1 (54.1–70.1) 0.508 AA n = 140 70.6 0.414 62.1 0.899 
    AG n = 79 Q576/R576 73.1 (63.3–82.9)  63.3 (52.7–73.9)  AG and GG n = 188 71.3  62.5  
    GG n = 9 R576/R576 55.6 (23.1–88.1)  55.6 (23.1–88.1)  AA and AG n = 219 71.5 0.312 62.5 0.279 
      GG n = 9 55.6  55.6  

Patients characterized by the IL4R genotypes I75/V75 and V75/V75 had a poorer prognosis comparing them with homozygous I75/I75 genotypes. Significant P values of the IL4R I75V gene variation are shown in italics. For continuative description of significant results, see also Figure 1.

IL4R, interleukin-4 receptor; SNP, single nucleotide polymorphisms; OS, overall survival; EFS, event-free survival; DLBCL, diffuse large B-cell lymphoma; CI, confidence interval.

Figure 1.

Overall (A) and event-free survival (B) of DLBCL patients in relation to IL4R gene variation I75V. Comparison of I75V genotypes reveals differences of homozygous I75/I75 carriers (black lines) toward the other genotypes with regards to OS (A) (P = 0.044) and EFS (B) (P = 0.056) of DLBCL patients. Patients with I75/V75 (gray lines) or V75/V75 (light gray lines) variations have a poor prognosis compared with patients with I75/I75 (black lines) genotype. For 3-year rates and multivariate analysis, see Table 2 and Table 3, respectively.

Figure 1.

Overall (A) and event-free survival (B) of DLBCL patients in relation to IL4R gene variation I75V. Comparison of I75V genotypes reveals differences of homozygous I75/I75 carriers (black lines) toward the other genotypes with regards to OS (A) (P = 0.044) and EFS (B) (P = 0.056) of DLBCL patients. Patients with I75/V75 (gray lines) or V75/V75 (light gray lines) variations have a poor prognosis compared with patients with I75/I75 (black lines) genotype. For 3-year rates and multivariate analysis, see Table 2 and Table 3, respectively.

multivariate analysis of IL4R I75V according to IPI factors

In a multivariate analysis adjusting for the IPI, we investigated the effect of the IL4R I75V gene variation on the survival of DLBCL patients. The presence of IL4R V75 variants (IL4RI75V-AG and IL4RI75V-GG) is a statistically independent predictor of OS [relative risk (RR) 1.9, P = 0.011] and a relevant, though not significant, predictor for EFS (RR 1.5, P = 0.076) (Table 4). These effects are also consistent when adding the treatment results of the NHL-B1/B2 trials to the multivariate analysis. These results indicate a strong association of this gene variation with the outcome of DLBCL patients.

Table 4.

Multivariate analysis of IL4R I75V gene variation IL4R V75 versus I75 (IL4RI75V-AG and IL4RI75V-GG versus IL4RI75V-AA) adjusted to the IPI (Cox model) in relation to OS and EFS

Factor OS
 
EFS
 
RR (95% CI) P RR (95% CI) P 
Serum LDH >N 2.1 (1.3–3.6) 0.003 1.7 (1.1–2.8) 0.024 
Age >60 years 2.6 (1.5–4.5) 0.001 2.1 (1.3–3.3) 0.002 
Performance status ECOG >1 2.5 (1.5–4.1) 0.001 2.6 (1.6–4.2) <0.001 
Ann Arbor stage III/IV 1.4 (0.9–2.3) 0.123 1.5 (1.0–2.2) 0.054 
Extranodal involvement >1 1.6 (1.0–2.7) 0.064 1.3 (0.8–2.0) 0.321 
IL4R V75 versus I75 1.9 (1.2–3.2) 0.011 1.5 (1.0–2.3) 0.076 
Factor OS
 
EFS
 
RR (95% CI) P RR (95% CI) P 
Serum LDH >N 2.1 (1.3–3.6) 0.003 1.7 (1.1–2.8) 0.024 
Age >60 years 2.6 (1.5–4.5) 0.001 2.1 (1.3–3.3) 0.002 
Performance status ECOG >1 2.5 (1.5–4.1) 0.001 2.6 (1.6–4.2) <0.001 
Ann Arbor stage III/IV 1.4 (0.9–2.3) 0.123 1.5 (1.0–2.2) 0.054 
Extranodal involvement >1 1.6 (1.0–2.7) 0.064 1.3 (0.8–2.0) 0.321 
IL4R V75 versus I75 1.9 (1.2–3.2) 0.011 1.5 (1.0–2.3) 0.076 

Significant P values of the IL4R I75V gene variation are shown in italics. IL4R, interleukin-4 receptor; IPI, International Prognostic Index; OS, overall survival; EFS, event-free survival; RR, relative risk; CI, confidence interval; LDH, lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group.

impact of IL4R I75V in combination with sIL4R serum level on survival of DLBCL patients

Cytokine and respective soluble cytokine receptor serum levels are parameters that could to some extent reflect the actual host–lymphoma interaction and thereby influence the prognosis for DLBCL patients. We therefore measured sIL4R serum levels at the time of admission to the hospital before the first treatment and combined sIL4R serum levels with IL4R I75V genotypes in the survival analysis because we found that an elevation of sIL4R serum levels [high (>115 pg/ml) versus low (≤115 pg/ml)] showed adverse effects on the OS (P < 0.001) and EFS (P < 0.001) of all 228 DLBCL patients. Univariate analysis of DLBCL patients with I75 (IL4RI75V-AA) genotype and sIL4R levels ≤115 pg/ml showed the most favorable OS (Figure 2A) and EFS (Figure 2B) outcome. However, when they had serum levels >115 pg/ml, their OS and EFS were less favorable. The 3-year rates of patients with I75 variant (IL4RI75V-AA) with low sIL4R levels compared with I75 variant patients with high sIL4R levels are 84.3% versus 68.4% (OS, P = 0.003) and 74.5% versus 63.2% (EFS, P = 0.023). DLBCL patients with IL4R V75 variants (IL4RI75V-AG and IL4RI75V-GG) and high serum levels of sIL4R have a worse clinical course compared with those with low sIL4R levels (Figure 2). The 3-year rates of patients with IL4R V75 variants (IL4RI75V-AG and IL4RI75V-GG) with low sIL4R levels compared with V75 variant patients with high sIL4R levels are 71.9% versus 49.7% (OS, P = 0.001) and 63.9% versus 38.9% (EFS, P = 0.002). All genotypes of the IL4R I75V variation showed significant differences in the survival of DLBCL patients taking sIL4R serum level into account (Table 5). Our data indicate a strong association of the IL4R I75V polymorphism to the survival of DLBCL patients and show that joint effects of this gene variation with serum levels of sIL4R are observed. These may further subdivide patients into groups with slower or faster disease progression. Including sIL4R serum levels (≤115 pg/ml versus >115 pg/ml) into the multivariate analysis adjusted to the IPI factors, the effect of the IL4R I75V gene variation remains unchanged (OS: RR = 1.9, P = 0.012 and EFS: RR = 1.5, P = 0.077) whereas the sIL4R serum level (>115 pg/ml) is not an independent factor (OS: RR 1.1, P = 0.722 and EFS: RR 1.0, P = 0.998). This analysis strongly supports the hypothesis that, besides clinical and molecular lymphoma characteristics of the lymphoma itself, the genetic background may be an important additional factor predicting the outcome of DLBCL patients.

Table 5.

OS and EFS periods of DLBCL patients in relation to IL4R I75V gene polymorphism combined with sIL4R serum level

Genotype
 
3-year rate (95% CI) 3-year rate (95% CI) 
IL4R I75V AA variant sIL4R serum level (pg/ml) No. of patients OS EFS 
AA I75/I75 ≤115 51 84.3 (74.3–94.3) 74.5 (62.5–86.5) 
AG I75/V75 ≤115 79 73.1 (63.3–82.9) 64.6 (54.0–75.2 
GG V75/V75 ≤115 43 69.6 (55.9–83.3) 62.7 (48.2–77.2) 
AA I75/I75 >115 19 68.4 (47.4–89.4) 63.2 (41.4–85.0 
AG I75/V75 >115 26 50.0 (30.8–69.2) 42.3 (23.3–61.3) 
GG V75/V75 >115 10 48.0 (15.9–80.1) 30.0 (1.6–58.4) 
Genotype
 
3-year rate (95% CI) 3-year rate (95% CI) 
IL4R I75V AA variant sIL4R serum level (pg/ml) No. of patients OS EFS 
AA I75/I75 ≤115 51 84.3 (74.3–94.3) 74.5 (62.5–86.5) 
AG I75/V75 ≤115 79 73.1 (63.3–82.9) 64.6 (54.0–75.2 
GG V75/V75 ≤115 43 69.6 (55.9–83.3) 62.7 (48.2–77.2) 
AA I75/I75 >115 19 68.4 (47.4–89.4) 63.2 (41.4–85.0 
AG I75/V75 >115 26 50.0 (30.8–69.2) 42.3 (23.3–61.3) 
GG V75/V75 >115 10 48.0 (15.9–80.1) 30.0 (1.6–58.4) 

Combining IL4R genotypes with serum level of sIL4R reveals detailed differences in survival of DLBCL patients (global P < 0.001). Carriers of IL4R V75 (I75V-AG and -GG) variants with high serum levels of sIL4R have lowest survival periods. IL4R I75/I75 carriers with low sIL4R levels have the highest survival periods.

OS, overall survival; EFS, event-free survival; DLBCL, diffuse large B-cell lymphoma; sIL4R, soluble interleukin-4 receptor; CI, confidence interval.

Figure 2.

Overall (A) and event-free survival (B) of DLBCL patients in relation to IL4R gene variation I75V genotypes separated in high and low serum levels of sIL4R. Patients with high levels of sIL4R and the IL4R I75/V75 or V75/V75 (dotted gray lines) variants have lowest survival rates, whereas homozygous carriers of the I75/I75 variant and low sIL4R serum levels (black lines) have the best survival rates. Patients with high sIL4R serum levels and I75/I75 genotype (gray lines) range together with patients with low sIL4R levels and the unfavorable genotypes I75/V75 and V75/V75 (black dotted lines).

Figure 2.

Overall (A) and event-free survival (B) of DLBCL patients in relation to IL4R gene variation I75V genotypes separated in high and low serum levels of sIL4R. Patients with high levels of sIL4R and the IL4R I75/V75 or V75/V75 (dotted gray lines) variants have lowest survival rates, whereas homozygous carriers of the I75/I75 variant and low sIL4R serum levels (black lines) have the best survival rates. Patients with high sIL4R serum levels and I75/I75 genotype (gray lines) range together with patients with low sIL4R levels and the unfavorable genotypes I75/V75 and V75/V75 (black dotted lines).

discussion

Recent evidence indicates that gene variations of cytokines and their receptors are associated with the risk of developing hematological malignancies [3–6, 13]. However, only a limited number of studies have analyzed the clinical course and treatment outcome of lymphoma in association with inherited gene polymorphisms. This is mainly due to the fact that a sufficient number of samples from patients included in controlled prospective clinical trials are difficult to obtain. Therefore, the DSHNHL collection of patient data and pretreatment samples provides a unique opportunity to assess these correlations.

For the IL10 gene, different promoter gene variations have been associated with the clinical course of lymphoma patients [2, 5]. We could recently show an association of far-distal IL10 promoter variations with the OS of lymphoma patients, however not in the subgroup of DLBCL patients [2]. Due to the absence of an association with IL10 polymorphisms in DLBCL patients, we focused here on other host immune factors with relation to DLBCL. During our study, our candidate gene approach was supported by observations made by other groups that polymorphisms within the chosen genes are associated with the risk of NHL development but may also reflect treatment response [4].

To our knowledge, this study is the first analysis of nonsynonymous coding polymorphisms of the IL4R gene in DLBCL patients within a prospective multicenter clinical trial. We show that the I75V genetic variant of IL4R is associated with the survival in this cohort of DLBCL patients. Homozygous A-allele carriers reassembling the I75 variation have a better prognosis compared with the other genotypes. The multivariate analysis of the IL4R I75V variation revealed a 1.9-fold increased risk for carriers of V75 variant to have a poor prognosis (OS). This observed RR is comparable to those estimated for clinical parameters included in the IPI, the current standard risk stratification model for patients with NHL. As this model is based on clinical factors only, its power to describe the biological heterogeneity of DLBCL is limited. A refinement of this prognostic model will be achieved by the inclusion of biological risk factors. Other variations, such as IL4R S503P, IL4R Q576R, IL4 -524CT and IL13 -1069CT gene variations, had no impact on the survival of DLBCL patients in this exploratory study.

The sIL4R serum level study shows a strong negative impact of levels >115 pg/ml. Therefore, we were interested in combining the sIL4R serum level in our analysis with IL4R I75V gene variations in order to further distinguish DLBCL patients. Patients with high levels of sIL4R and the unfavorable IL4R V75 variants had worse survival rates, whereas homozygous carriers of the I75 variant and low sIL4R serum levels had the best survival rates. With this combined analysis of serum sIL4R and IL4R I75V, for ∼22% of DLBCL patients (IL4R I75 and sIL4R ≤115 pg/ml), a good clinical outcome could be revealed, whereas for ∼16% of the patients (IL4R V75 and sIL4R >115 pg/ml), a significantly worse outcome is observed. However, in a multivariate IPI risk factor analysis, the effect of the IL4R I75V gene variation remained unchanged in contrast to the sIL4R serum level which is not an independent factor.

Recently, Habermann et al. [14] provided evidence that the IL4R regulatory single nucleotide polymorphisms (SNP) (rs2057768) could be a predictor of long-time survival in DLBCL patients as part of a risk score combination of four cytokine SNPs. The T allele of this SNP is in linkage disequilibrium to the IL4R I75 variant (IL4RI75V-AA), both associated with better survival of the DLBCL patients [15]. This further supports our hypothesis that variations in the IL4R gene could be an important prognostic factor predicting the outcome of DLBCL patients. Nevertheless, the functional consequences of these genetic variations are not understood yet. Previous studies indicate that the nonsynonymous coding polymorphisms are likely to change IL4R functions in some in vitro assays, but the in vivo influence on the immune system has been controversially discussed [16–19]. The IL4R I75 variant seems to have a higher signal transduction capacity toward the V75 variant and was therefore referred to as ‘gain-of-function’ variant [16, 19]. Recently, a higher expression of the IL4R in the germinal center B-cell-like DLBCL subgroup was shown and distinct functions of IL4 in the DLBCL subgroups were proposed [20, 21]. Two publications based on gene expression profiling of DLBCL patients raised the possibility that an active immune response is favorable for DLBCL patients [22, 23]. Thus, the favorable impact of the IL4R I75 variant on DLBCL outcome may be related to a more efficient immune response capacity against the lymphoma cells by enhanced IL4R expression or signal transduction capacities of the IL4R.

Only few studies have so far analyzed IL4R polymorphisms in patients with malignant diseases other than NHL. In a Japanese study on sporadic renal cell carcinoma, the IL4R I75V polymorphism was described as a useful genetic marker for assessing the risk of the tumor development and progression [24]. No increased risk for colorectal cancer but an increased risk for cervical cancer was found for the IL4R V75 allele [25, 26]. Cervical cancer is related to human papillomavirus infection and Th1 immune responses are essential for viral clearance. The IL4R I75V polymorphism was also related to other viral diseases like acquired immunodeficiency syndrome or Rous sarcoma virus bronchiolitis and thus may alter the Th1/Th2 balance [27, 28]. An altered host–tumor interaction by an imbalance of Th1/Th2 cytokine expression or an altered response to these cytokines may lead to harmful processes in DLBCL patients and thereby affect the clinical course in the way shown here for the IL4R I75V gene variation.

In summary, we report here for the first time that the nonsynonymous IL4R gene variation I75V is associated with the treatment outcome of DLBCL patients. Additional studies will have to prove if the IL4R I75V gene variation will indeed become a prognostic factor for DLBCL outcome. The I75 variation showed a significant favorable survival prognosis compared with V75 variants. The incorporation of the serum level of sIL4R together with the IL4R I75V genotypes will give further insights into the clinical pathophysiology of DLBCL. A comprehensive review incorporating genetic markers and inherited gene variations is underway in a collaborative analysis of the Ricover60 trial together with the molecular mechanisms of malignant lymphomas consortium [29, 30]. This analysis will also evaluate the influence of rituximab-containing regimens on the gene polymorphism-based risk factor analysis.

funding

Deutsche Forschungsgemeinschaft e. V. (Graduiertenkolleg 1034, www.gcpg.de), BMBF (NGFN-1) and Deutsche Krebshilfe e. V. (NHL-B1/B2).

The authors are grateful to the colleagues from the GRK1034 for helpful discussions and the José Carreras Leukämie-Stiftung e.V. for further support.

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