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Metabolic pathway enzymes, such as Cytochrome P450 (CYP), glutathione S-transferase (GST), and N-Acetyltransferases (NAT) are involved in activation and detoxification of environmental carcinogens as well as drug metabolism. We hypothesized that the genetic variations in such metabolic pathways may affect NHL prognosis and survival.
Follow-up information of 469 female NHL incident cases diagnosed during 1995-2000 in Connecticut were abstracted from Connecticut Tumor Registry in 2008; survival analyses were conducted using the Kaplan-Meier method, and hazard ratios (HR) were estimated by Cox Proportional Hazard models adjusting for demographic and tumor characteristics which were suggested by previous studies to be determinants of NHL survival. Our results identified nine SNPs from five metabolism genes (CYP2E1, GSTP1, GSTT1, NAT1 and NAT2) that were associated with NHL survival. Specifically, polymorphisms in NAT1 and NAT2 genes were associated with diffuse large B-cell lymphoma survival; polymorphisms in GSTT1 was associated with follicular lymphoma survival; and polymorphisms in CYP2E1, GSTP1 and NAT1 were associated with survival of chronic lymphocytic leukemia/small lymphocytic lymphoma.
Our study suggests that genetic polymorphisms in metabolic pathways may help improving the prediction of NHL survival and prognosis.
The incidence of non-Hodgkin lymphoma (NHL) has been increasing worldwide. In 2005, there were approximately 401, 697 NHL survivors—207,821 men and 193,876 women—in the U.S., representing the 7th biggest cancer burden in the U.S. population . NHL is a group of heterogeneous malignancies of the lymphoid cells. Different NHL histological subtypes have different incidence patterns and different underlining etiology . Studies also have shown that the NHL survival patterns varied by histological subtype [3,4], suggesting different prognostic risk factors for NHL histological subtypes.
Established adverse prognostic factors for NHL, as delineated in the International Prognostic Factor Index (IPI), include older age at diagnosis, higher tumor stage, poor performance score, extranodal involvement and above-normal lactate dehydrogenase . The presence of B-symptom was also found to be associated with worse prognosis of NHL cases in population studies [3,4]. However, these clinical features could not predict NHL survival completely, and recent gene-expression studies suggested that the knowledge about molecular characteristics of the tumor and its microenvironment may improve prediction of NHL survival [6,7]. Metabolic pathway enzymes, such as Cytochrome P450 (CYP), glutathione S-transferase (GST), and N-Acetyltransferases (NAT) are involved in activation and detoxification of environmental carcinogens as well as drug metabolism. Therefore, they may play an important role in determination of the susceptibility to the toxic effects of chemicals, and may also influence tumor-response to anticancer drugs. We hypothesized that the genetic variations in such metabolic pathways may affect NHL prognosis and survival, and we examined the associations by NHL subtype.
The study population has been described elsewhere [8,9]. In brief, a total of 1,122 potential female NHL cases aged between 21 and 84 years were identified between 1995 and 2000 through the Yale Comprehensive Cancer Center's Rapid Case Ascertainment Shared Resource (RCA), a component of the Connecticut Tumor Registry (CTR). Among those cases, 167 died before they could be interviewed and 123 were excluded because of doctor refusal, previous diagnosis of cancer, or inability to speak English. Out of 832 remaining eligible cases, 601 completed an in-person interview. Pathology slides or tissue blocks were obtained from the hospitals where the cases had been diagnosed. The specimens were reviewed by two independent study pathologists. All NHL cases were classified according to the World Health Organization (WHO) classification system [10,11].
Vital status for these NHL cases was abstracted at the CTR in May-October 2008. Other follow-up information was also abstracted, including date of death, most recent follow-up date, type and date of treatments, dates of relapse and/or secondary cancer, B-symptoms, and tumor stage. Of the 601 cases, 13 were not able to be identified in the CTR system, 42 were found to have a cancer history prior to diagnosis of NHL, and 77 had genotyping data missing, yielding 469 NHL patients in the final analyses. Of these, 150 had diffuse large B-cell lymphoma (DLBCL); 112 had follicular lymphoma (FL); 51 had chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL); 32 had marginal zone B-cell lymphoma (MZBL); and 35 had T/NK-cell lymphoma (T-cell).
The study was approved by the Human Investigation Committee at Yale University and the Connecticut Department of Public Health.
The methods for evaluating genotypes in our study population have been described previously [12,13]. Briefly, DNA was extracted from blood or buccal cell samples using phenol-chloroform extraction. Twenty one Tag single-nucleotide polymorphisms (SNPs) from 11 candidate genes involved in metabolic pathway, including CYP1A1 (rs1048943), CYP1A2 (rs762551), CYP1B1 (rs1056836), CYP2C9 (rs1799853), CYP2E1 (rs2070673, rs2031920), CYP21A2 (rs6474), GSTM3 (rs1799735), GSTP1 (rs1695, rs1138272), GSTT1 (EX5-49+>-), NAT1 (rs1057126, rs15561, rs4987076, rs4986782), NAT2(rs1208, rs1799931, rs1041983, rs1801280, rs1799929, rs1799930), were chosen from the designable set of common SNPs (minor allele frequency >5%) genotyped in the Caucasian (CEU) population sample of the HapMap Project (Data Release 20/Phase II, NCBI Build 35 assembly, dpSNPb125) using the software Tagzilla (http://tagzilla.nci.nih.gov/), which implements a tagging algorithm based on the pairwise binning method of Carlson et al. . For each gene, SNPs within the region 20kb 5’ of the ATG-translation initiation codon and 10kb 3’ of the end of the last exon were binned using a binning threshold of r2 > 0.80. When there were multiple transcripts available for genes, the primary transcript was assessed. Genotyping was conducted at the National Cancer Institute Core Genotyping Facility (Advanced Technology Center, Gaithersburg, MD; http://snp500cancer.nci.nih.gov)  using a real-time PCR assay (20 SNPs) and a custom-designed GoldenGate assay (Illumina, www.illumina.com) (1 SNP). Duplicate samples and replicate samples were genotyped for quality control, and blinded to laboratory personnel. The concordance rates were 99-100% for all assays.
Survival analyses were done for both overall survival (OS) and disease-free survival (DFS). In OS analysis, deaths were events and being alive was censoring. In DFS analysis, deaths, relapses and occurrences of secondary cancer were events and otherwise were censorings.
We used Cox proportional hazards (PH) regression to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) for the association of each individual genotype with OS and DFS. The homozygote of the most common allele was used as the reference group and coded as 0, and the heterozygote and homozygote variant genotypes were grouped together and coded as 1. Age (continuous), education (high school or less, some college, and college graduate or more), stage (I, II, III, IV, and unknown), B-symptom (yes, no, unknown) and initial treatment (none, surgery, radiation, chemotherapy, and other) were adjusted as a priori confounder variables. Adjustments for race did not result in material changes for the observed associations and thus were not included in the final model.
A multi-SNP model was fitted for NHL overall and subtypes respectively. First, the significant SNPs (p-value <0.05) from above single-SNP analysis were selected and fit in a Cox model adjusting for age, education, stage, B-symptom and initial treatment. Then we ran stepwise selection for the Cox model to retain a parsimonious number of SNPs while keeping a priori confounders in the model. Thus a group of SNPs that independently affects NHL survival was identified and the deleterious genotypes were known from the single-SNP analysis. Kaplan-Meier survival curves were plotted by the number of deleterious genotypes for NHL overall and subtypes. Log-rank tests were conducted to examine the difference.
Multivariate analysis was further conducted by NAT1 genotype and NAT2 phenotype for NHL overall and subtypes using Cox PH regression adjusting a priori confounders. NAT1*10 was designated as the at-risk allele based on previous etiology studies . We thus calculated HRs, 95% CIs, and a P-value for the linear trend comparing individuals with one or two copies of NAT1*10 with individuals with no copies of NAT1*10. The NAT2 genotype-phenotype relationship is well-established. For analysis of NAT2 phenotypes, individuals homozygous for NAT2 rapid- and slow-acetylator alleles were designated as rapid and slow acetylators, respectively; individuals possessing one rapid- and one slow-acetylator allele (heterozygotes) were designated as intermediate-acetylators . We calculated ORs, 95% CIs and a P-value for the linear trend comparing individuals with intermediate or rapid NAT2 phenotype with slow NAT2 phenotype.
Statistical analyses were performed using SAS, version 9.1 (SAS Institute, Cary, NC).
Demographic and tumor characteristics for 469 NHL cases are presented in Table 1. During the follow-up, 195 deaths, 11 recurrence of NHL, and 41 secondary cancers were occurred. The mean was 6.92 years (SD = 3.14, range: 0.38 - 11.68) for OS and 6.60 years (SD = 3.22, range: 0.04 - 11.68) for DFS.
Frequencies of SNPs, NAT1 genotypes and NAT2 phenotypes in NHL overall and subtypes are presented in Table 2. The non-genotyped rates were ranged from 4.5% (CYP2C9 rs1799853) to 29.0% (GSTT1 Ex5-49+>-).
As shown in Table 3, there were one SNP in CYP2E1, two SNPs in NAT1 and three SNPs in NAT2 significantly associated with risk of death in NHL cases after adjusted for demographic and clinical risk factors (Table 3). Carriers of A-allele for CYP2E1 (rs2070673), NAT1 (rs1057126) or NAT1 (rs15561) had a 34-43% reduced risk of death compared to those without A-allele. Carriers of variant alleles for NAT2 (rs1208) (AG or GG), NAT2 (rs1801280) (CT or CC) or NAT2 (rs1799929) (CT or TT) had a 46-61% increased risk of death compared to those with wild-type alleles. When NHL subtypes were examined separately, four SNPs in NAT1 and NAT2 (rs1057126, rs15561, rs1208 and rs1041983) were found to significantly associated with risk of death in DLBCL patients; one insertion/deletion polymorphism in GSTT1 was significantly associated with risk of death in FL patients; one SNP in CYP2E1 (rs2070673), one SNP in GSTP1 (rs1695) and two SNPs in NAT1 (rs1057126 and rs15561) were significantly associated with risk of death in CLL/SLL patients (Table 3). No significant result was found for MZBL and T-cell lymphoma (data not shown).
One SNP in CYP2E1 (rs2070673) and two SNPs in NAT1 (rs1057126 and rs15561) were found to be significantly associated with risk of death, relapse or secondary cancer in overall NHL cases (Table 4). The carriers of variant alleles had a 27-33% reduced risk of death, relapse or secondary cancer compared to the homozygote wild-type carriers. When NHL subtypes were examined separately, carrying of G-allele for NAT2 (rs1208) was associated with a 2-fold risk of death, relapse or secondary cancer in DLBCL survivors (HR=1.94, 95% CI: 1.08-3.48); carrying of null allele for GSTT1 (+- or --) was associated with a 62% reduced risk of death, relapse or secondary cancer in FL survivors (HR=0.38, 95% CI: 0.19-0.77); carrying of A-allele for CYP2E1 (rs2070673), NAT1 (rs1057126) and NAT1 (rs15561) was associated with 63-80% reduced risks, while carrying of G-allele for GSTP1 (rs1695) was associated with 3-fold risk of death, relapse or secondary cancer in CLL/SLL survivors (Table 4). No significant result was found for MZBL and T-cell lymphoma (data not shown).
With stepwise selection for OS models, four out of six SNPs were identified as risk SNPs for NHL OS: CYP2E1 (rs2070673), NAT1 (rs15561), NAT2 (rs1208) and NAT2 (rs1801280); two out of four SNPs were identified as risk SNPs for DLBCL OS: NAT1 (rs15561) and NAT2 (rs1208); GSTT1 was identified as risk SNP for FL OS; and three out of four SNPs were identified as risk SNPs for CLL/SLL OS: CYP2E1 (rs2070673), GSTP1 (rs1695) and NAT1 (rs1057126). The OS curves by number of deleterious genotypes and P-values for log-rank tests for NHL overall and subtype cases were shown in Figure 1.
With stepwise selection for DFS models, two out of three SNPs were identified as risk SNPs for NHL DFS: CYP2E1 (rs2070673) and NAT1 (rs15561); NAT2 (rs1208) was identified as a risk SNP for DLBCL DFS; GSTT1 was identified as risk SNP for FL DFS; and three out of four SNPs were identified as risk SNPs for CLL/SLL DFS: CYP2E1 (rs2070673), GSTP1 (rs1695) and NAT1 (rs1057126). The DFS curves by number of deleterious genotypes and P-values for log-rank tests for NHL overall and subtype cases were shown in Figure 2.
For NAT1*10 genotype, we observed a decreased risk of death among overall NHL cases and CLL/SLL cases with at lease one copy of NAT1*10 compared to those with no copy of NAT1*10 (HR= 0.63 and 95% CI: 0.45-0.89 for NHL overall; HR=0.25 and 95% CI: 0.07-0.88 for CLL/SLL) (Table 5); we also observed decreased risk of death, relapse or secondary cancer among overall NHL cases and CLL/SLL cases with at least one copy of NAT1*10 compared to those with no copy of NAT1*10 (HR= 0.73 and 95% CI: 0.54-1.00 for NHL overall; HR=0.21 and 95% CI: 0.06-0.70 for CLL/SLL) (Table 5).
For NAT2 phenotype, no significant results were found between slow-acetylators and intermediate/rapid-acetylators (Table 5).
Using a population-based sample of female NHL cases diagnosed from 1995 to 2000 in Connecticut and followed through mid 2008, we identified nine SNPs from 5 metabolism genes (CYP2E1, GSTP1, GSTT1, NAT1 and NAT2) that were associated with overall survival of NHL overall or histological subtypes. Five of these nine SNPs were further found to be associated with disease-free survival of NHL overall or histological subtypes. Our study offers the first analysis of the impact of genetic variations in the CYP and NAT genes on the survival of NHL by subtype, and our results should be confirmed by other studies.
CYPs, GSTs and NAT are all important enzymes involved in metabolism of exogenous and endogenous compounds. The family of CYPs activates environmental carcinogens to electrophilic metabolites capable of binding to DNA. The GST family of enzymes detoxifies the reactive compounds by converting them to inactive, water soluble metabolites. NAT catalyzes aromatic and heterocyclic amines via N- or O-acetylation thus involved in both activation and detoxification of numerous drugs and carcinogens. The roles of their genetic polymorphisms in cancer risk have been the subject of numerous studies. Evidence, although not so extensive, did show that genetic variations in CYP, GST and NAT might be associated with risk of NHL overall and/or subtypes. For example, polymorphisms in CYP1A1 were found to be associated with risk of DLBCL  and CLL ; distributions of CYP2E1 genotypes were found to be different among NHL patients and healthy controls [20-22]; single reports also suggest some relevance of genetic variants in CYP1B1, CYP2C9, and CYP17A1 and risk of NHL or its subtypes [19,20,23]. Multiple studies reported higher risk of NHL , DLBCL , and MZBL [25,26] associated with GSTT1 null; two GSTP1 polymorphisms were found to be associated with DLBCL risk in two studies [18,27]; studies also showed that GSTM1 null is associated with increased risk of NHL [19,28]. A large study found increased risk of NHL associated with NAT1*10/*10 genotype and intermediate and rapid NAT2 acetylators . These findings supported that the genetic variations in such metabolic pathways may play a role in lymphomagenesis.
We observed a favorable effect of carrying A allele in CYP2E1 (rs2070673) on NHL survival, especially pronounced in CLL/SLL patients. As far as we know, this is the first time that the relationship of CYP genetic variations and NHL survival is studied. As a member of CYP family expressed in liver and extrahepatic tissues, CYP2E1 metabolizes a variety of nitrosamines and low-molecular-weight chemicals including drugs, solvents, toxins and environmental pollutants . It is known that CYP2E1 mediates metabolism of several drugs such as acetaminophen, chlorzoxazone, and enflurane, but its full potential has not been well characterized, especially with drug candidates in development . Our results suggest that CYP2E1 might play a role in the metabolism of drugs used in CLL/SLL treatment, such as Fludarbine, Rituximab, Cyclophosphamide, Alemtuzumab, Bendamustine, Chlorambucil.
We observed that GSTT1 null was associated with a 59% reduced risk of death and a 52% reduced risk of relapse, secondary cancer or death in FL patients. This is in line with previous studies on lymphoma prognosis: Stanulla et al. found that GSTT1 null was associated with a reduced risk of relapse in childhood acute lymphoblastic leukemia patients , and Hohous et al. found that GSTT1 null was more prevalent in low-stage HL patients than in high-stage HL patients. However, a recent study of 89 FL by Hohaus et al. found that GSTT1 null was associated with worse event-free survival . Several possible reasons could explain the discrepancy between our finding and theirs: first, since different drugs are used in treating low-stage and high-stage cancer patients, GST may play different drug-metabolism roles in these two types of patients. There are more low-stage FL patients in our study than in theirs (52% vs. 7% Stage I); when we limited our analysis to FL patients with tumor stage higher than I, no survival difference for GSTT1 deletion was observed (P-value for log-rank test: 0.8447 for OS and 0.9856 for DFS). Another possible reason is that given the evidence of sex-related differences in the expression of various GST isozymes [34,35] and the facts that GST could be induced by various hormones [36,37], GSTT1 may play different roles in different genders. While our study subjects were all female, the gender composition of their study was about half and half.
We also observed impacts of polymorphisms on NAT1 and NAT2 genes on the prognosis of NHL overall, DLBCL and CLL/SLL. There has been few studies linked NAT genetic variations to NHL survival or prognosis. Active NAT1 overexpression was shown to enhance cell growth and etoposide resistance , and NAT1 genotype was found to be associated with the risk of relapse or death among children with neuroblastoma . Although expression of NAT2 was recently found to alter the toxicity of CB1954, the component of an attractive therapy for tumor treatment , NAT2 polymorphism studies among patients with breast cancer , gastric cancer  and colorectal cancer  did not find any prognostic significance. Our study in NHL patients was the first one that observed NAT2 polymorphisms’ prognostic effects in cancer patients.
In order to investigate the generalizability of the study results, we compared its overall survival curve with that of 13,899 Female NHL patients aged 21-84 diagnosed during 1996-2000 at 17 Surveillance, Epidemiology and End Results (SEER) registries . The two survival curves were parallel throughout the follow-up period except during the first half year, during which 15.8% of SEER patients died, while none of the patients died in our study. Considering 167 out of 1,122 (14.9%) identified cases were not able to be enrolled in the CT study because they died before interview , the overall survival of our case series is comparable to the survival observed by SEER. Our results might not apply to the most aggressive NHL cases with short-term survivals since there were limited cases in our study who died within a half year.
We utilized CTR to abstract follow-up information. Being the oldest tumor registry in the U.S. and functioning as one of the SEER registries since 1973, CTR is a reliable source for vital status, recurrence and occurrence of secondary cancer. According to the recently submitted SEER database (Nov 2007) , among those microscopically confirmed female NHL patients diagnosed in 1996-2000 in CT and aged 21-84, 99.2% were actively followed by CTR through 12/31/2004. Several published studies using CTR as follow-up method have shown its validity [47-49]. Through CTR, we were also able to obtain the information on tumor stage, B-symptoms, and initial treatment and adjusted them as a priori confounders in our analysis. However, the treatment information collected by CTR is not so detailed and comprehensive, which limited our analysis because further exploration of the metabolism interactions of the investigated genes and specific drugs was not possible.
Compared to most of the published clinical reports on NHL survival, our study has a relatively larger sample size, which provided power to detect differences among NHL subtypes, especially for the two most common subtypes: DLBCL and FL. Although we did not find any significant effects of metabolism genetic polymorphisms on prognosis and survival for MZBL and T-cell lymphoma patients, it could be due to the small number of patients with these subtypes in our study. We also cannot rule out the possibility that some significant findings were due to chance.
In conclusion, this study shows that genetic polymorphisms in metabolism pathway genes of CYP, GST, and NAT may affect NHL prognosis and survival. They might be useful factors in prediction of NHL prognosis and treatment selection. A better understanding of the underlying biological mechanisms should be pursued.
This study is supported by grant CA62006 from the National Cancer Institute (NCI) and by a pilot grant from the Yale Cancer Center. This publication was made possible by CTSA Grant number UL1 RR024139 from the National Center for Research Resources (NCRR), a component of the NIH, and NHL roadmap for medical Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the NCRR. This research was approved by the DPH HIC. Certain data used in this study were obtained from the Connecticut Department of Public Health. The authors assume full responsibility for analyses and interpretation of these data.