It has not been well established whether genetic variations can be biomarkers for clinical outcome of gemcitabine therapy. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) of gemcitabine metabolic and transporter genes that are associated with toxicity and efficacy of gemcitabine-based therapy in patients with locally advanced pancreatic cancer.
The authors evaluated 17 SNPs of the CDA,dCK, DCTD, RRM1, hCNT1-3, and hENT1 genes in 149 patients with locally advanced pancreatic cancer who underwent gemcitabine-based chemoradiotherapy. The association of genotypes with neutropenia, tumor response to therapy, overall survival, and progression-free survival (PFS) was analyzed by logistic regression, log-rank test, Kaplan-Meier plot, and Cox proportional hazards regression.
The CDA A-76C, dCK C-1205T, RRM1 A33G, and hENT1 C913T genotypes were significantly associated with grade 3 to 4 neutropenia (P = .020, .015, .003, and .017, respectively).The CDA A-76C and hENT1 A-201G genotypes were significantly associated with tumor response to therapy (P = .017 and P = .019). A combined genotype effect of CDA A-76C, RRM1 A33G, RRM1 C-27A, and hENT1 A-201G on PFS was observed. Patients carrying 0 to 1 (n = 64), 2 (n = 50), or 3 to 4 (n = 17) at-risk genotypes had median PFS times of 8.3, 6.0, and 4.2 months, respectively (P = .002).
Pancreatic cancer is the third most common gastrointestinal malignancy and the fourth leading cause of cancer deaths in the United States.1 At diagnosis, only 20% of patients have a surgically resectable tumor, 30% have a locally advanced tumor, and 50% present with distant metastasis.2 Over the past decade, gemcitabine (2′,2′-difluorodeoxycytidine) has been the standard agent for first-line chemotherapy of advanced pancreatic cancer, producing limited clinical benefit and improved overall survival (OS) as compared with 5-fluorouracil.3 Recent studies have reported the efficacy of a combination therapy of gemcitabine plus radiation for unresectable locally advanced pancreatic cancer.4, 5 However, factors that can predict tumor response and survival have not been well elucidated.6 In addition, although 1 major side effect caused by gemcitabine is hematological toxicity such as neutropenia, available biomarkers for severe toxicity have not yet been established.
Gemcitabine is a specific analogue of the native pyrimidine nucleotide deoxycytidine and a prodrug that requires cellular uptake and intracellular phosphorylation (Fig. 1).7-9 Gemcitabine intracellular uptake is mediated mainly by human equilibrative nucleoside transporter (hENT1, also known as solute carrier family 29 A1) and, to a lesser extent, by human concentrative nucleoside transporters (hCNT) 1 and hCNT3 (also known as solute carrier family 28 A1 or A3).9 Inside cells, gemcitabine is phosphorylated to its monophosphate form by deoxycytidine kinase (dCK), and this step is essential for further phosphorylation to its active triphosphate form.10 The active diphosphate metabolite of gemcitabine is also active and inhibits DNA synthesis indirectly by inhibiting ribonucleotide reductase (RRM1).8, 11, 12 Gemcitabine is inactivated primarily by deoxycytidine deaminase (CDA) into 2′,2′-difluorodeoxyuridine, and gemcitabine monophosphate is inactivated by deoxycytidylate deaminase (DCTD) into 2′,2′-difluorodeoxyuridine, monophosphate form.8, 9
Previous studies have demonstrated the relationship between gemcitabine metabolic or transport enzymes and clinical outcome. One study showed that low expression of CDA was associated with severe hematologic toxicity of gemcitabine.13 Other studies in cell lines or tumor tissues have established the association between resistance to gemcitabine and decreased nucleoside transport into cells,14-16 decreased expression of activation enzymes such as dCK,17-20 increased expression of degradation enzymes such as CDA and DCTD,21, 22 as well as increased expression of RRM1.23-26 In clinical studies of pancreatic cancer, high expression of hENT1 in tumors has been associated with improved survival in patients treated with gemcitabine.15, 16, 23, 27
Single nucleotide polymorphisms (SNPs) of enzymes in gemcitabine's pharmacologic pathway have been previously identified.8 The activity of these enzymes has been correlated with polymorphic gene variations by in vivo and in vitro studies.9, 28-30 However, only a few clinical studies have shown a positive association between the enzyme SNPs and gemcitabine toxicity.31-33 We have previously shown that genetic variations in gemcitabine metabolism and transport are associated with drug toxicity and OS in patients with resectable pancreatic cancer.34 In the current study, we tried to validate the previous findings in 149 patients with locally advanced pancreatic cancer who had undergone gemcitabine-based therapy.
MATERIALS AND METHODS
Patient Recruitment and Data Collection
A single-institution retrospective analysis was completed. We identified 149 patients with biopsy-confirmed locally advanced pancreatic cancer at the time of diagnosis. Locally advanced pancreatic cancer was defined as unresectable tumors that extended to the celiac axis or the superior mesenteric artery or tumors that occluded the superior mesenteric venous-portal venous confluence based on a review of the computed tomography (CT).35 All patients were required to be treatment naive and underwent gemcitabine-based chemotherapy as first-line therapy as a single agent or in combination at The University of Texas M. D. Anderson Cancer Center (Houston, Tex) from February 1999 to June 2007. The median dose of gemcitabine therapy was 750 mg/m2 (range, 450-1000 mg/m2). In 75 (50.3%) patients, either cisplatin or oxaliplatin was administered with gemcitabine. In 125 (83.9%) patients, gemcitabine therapy was followed by consolidative radiotherapy at a dose of 30 grays. Patient observation continued through June 2009. Information on treatment provided, toxicity, tumor response to therapy, tumor progression, and survival time was collected by reviewing patients' medical records in an electronic database. This study was approved by the institutional review board of The University of Texas M. D. Anderson Cancer Center.
Neutropenia, the most common hematologic toxicity caused by gemcitabine, was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.0. Tumor response to therapy was evaluated by comparing CT at the time of diagnosis with CT at 6 to 8 weeks after chemoradiotherapy or chemotherapy, and was defined according to the Response Evaluation Criteria in Solid Tumors as partial response, stable disease, or progressive disease. OS and progression-free survival (PFS) were calculated from the date of diagnosis to the date of death and progression or last follow-up date, respectively. Twelve patients were excluded from PFS analysis because they were lost to follow-up on disease progression. Performance status was evaluated by Eastern Cooperative Oncology Group criteria.
Extracting and Genotyping DNA
We selected 17 SNPs of the CDA, dCK, RRM1, DCTD, hCNT1-3, and hENT1 genes according to the following criteria: 1) minor allele frequency of the SNP was >10% among Caucasians, 2) they were coding SNPs including nonsynonymous or synonymous SNPs, and 3) they were SNPs that have been associated with cancer risk or clinical outcome in prior studies. Table 1 summarizes the genes, nucleotide substitutions, function (such as encoding amino acid changes), reference SNP identification numbers, and minor allele frequencies of the 17 SNPs evaluated in this study.
SNP indicates single-nucleotide polymorphism; RS No., reference SNP identification number.
The data observed in current study.
The reported minor allele frequency (Caucasian) was from the SNP500 cancer database.
Peripheral blood lymphocytes before chemotherapy were obtained from 149 locally advanced pancreatic cancer patients with informed consent, and DNA was extracted using Qiagen DNA isolation kits (Valencia, Calif). Taqman 5′ nuclease assay was performed to determine all genetic variants. Primers and TaqMan MGB probes were provided by TaqMan SNP Genotyping Assay Services (Applied Biosystems, Foster City, Calif). The probes were labeled with the fluorescent dye VIC or FAM for each allele at the 5′ end. Polymerase chain reaction (PCR) was performed in a 5-μL total volume consisting of TaqMan Universal PCR Master Mix, 20 ng of genomic DNA (diluted with dH2O), and TaqMan SNP Genotyping Assay Mix. Allele discrimination was accomplished by running endpoint detection using the ABI Prism 7900HT Sequence Detection System and SDS 2.3 software (Applied Biosystems).
The genotype distribution was tested for Hardy-Weinberg equilibrium using the goodness-of-fit chi-square test. The genotype association with grade 3 to 4 neutropenia toxicity, and tumor response to therapy was analyzed by logistic regression. Gemcitabine dose intensity by genotype was compared using t test. OS and PFS were analyzed by log-rank test, Kaplan-Meier plot, and Cox proportional hazards regression model. The heterozygous and homozygous genotypes were combined in these analyses if the frequency of the homozygous mutant was low or if the homozygous and heterozygous genotypes had the same direction of effect on toxicity, tumor response, or survival. Multivariate analyses were performed with adjustment for clinical predictors that were statistically significant. All statistical testing was conducted with SPSS software, version 17.0 (SPSS Inc, Chicago, Ill), and statistical significance and borderline significance were defined as P < .05 and P < .20, respectively.
We estimated the false-positive report probability for the observed statistically significant associations using the methods described by Wacholder et al.36 False-positive report probability is the probability of no true association between a genetic variant and a phenotype given a statistically significant finding. False-positive report probability is determined not only by the observed P value but also by both the prior probability that the association between the genetic variant and the phenotype is real and the statistical power of the test. In the current study, odds ratio (OR) and hazard ratio (HR) values of 2.0 to 4.0 were considered as a likely threshold value. The prior probability used was 0.25 for all SNPs. The false-positive report probability value for noteworthiness was set at 0.2.
Patients' Characteristics and Clinical Predictors
Table 2 shows the patients' characteristics, clinical features of their tumors, and treatment. The median age of the 149 patients was 62 years (range, 38-86 years). Non-Hispanic whites comprised 92% of the patients. After a median follow-up of 16.8 months (range, 2-60 months), the median survival time of all patients was 15.2 ± 0.8 months (95% confidence interval [CI], 13.6-16.9). Tumor response to therapy was significantly associated with OS (P < .001). Eastern Cooperative Oncology Group performance status and presence of diabetes as a comorbidity had a borderline significant association with OS (P = .143 and P = .081) by log-rank test. Although 24 (16.1%) patients had not undergone radiotherapy, that factor was not associated with OS (P = .503). Concurrent therapy with a platinum drug also did not impact OS (P = .745).
Table 2. Patient Characteristics and Overall Survival (N = 149)
We successfully amplified the 17 genotypes in 97.3% to 100% of the samples. Approximately 10% of total samples were analyzed in duplicate, and no discrepancies were seen. Genotype frequencies of the 17 SNPs were found to be in Hardy-Weinberg equilibrium (chi-square = 0.001–2.097, P = .148 to.973). No significant racial differences in genotype frequency were observed (data not shown). The 2 SNPs (IVS12 -201A>G and IVS2 -549T>C) of the hENT1 gene were in linkage disequilibrium (D′ = 0.774, P < .01).
Association of Genotypes With Toxicity
None of the clinical factors including concurrent treatment with platinum drug (P = .457) or radiotherapy (P = .126) was associated with neutropenia, the most common hematologic toxicity caused by gemcitabine. The CDA A-76C, dCK C-1205T, RRM1 A33G, and hENT1 C913T genotypes, individually and jointly, were significantly associated with severe (grade 3-4) neutropenia (Table 3). For example, 39 (43.8%) of the CDA-76 AC/CC carriers compared with only 15 (25.0%) of the AA carriers had severe neutropenia (P = .020). Patients carrying 2 or 3 to 4 at-risk alleles had a significantly higher frequency of severe neutropenia than did patients carrying only 0 to 1 at-risk alleles (OR, 3.24; 95% CI, 1.19–8.82; P = .021 and OR, 11.0; 95% CI, 4.02–30.1, P < .001, respectively; Table 3). The false-positive report probability was .02 for patients carrying 3 to 4 at-risk genotypes, indicating noteworthiness. No significant association of toxicity was observed in the remaining SNPs (data not shown).
Table 3. Neutropenia Toxicity and Genotype (N = 149)
Association of Genotypes With Tumor Response to Therapy
One hundred forty-nine locally advanced pancreatic cancer patients were analyzed on treatment effect. Radiation therapy and platinum drug use did not correlate with tumor response (P = .858 and P = .562). Two SNPs, CDA A-76C and hENT1 A-201G, were significantly associated with tumor response in radiological evaluation after adjusting for age (P = .017 and P = .019; Table 4). For example, 41 (48.2%) of the CDA-76 AC/CC carriers compared with 16 (27.6%) of the AA carriers had a poor response to gemcitabine-based chemotherapy. Patients carrying 1to 2 at-risk alleles had a significantly worse response to therapy than did patients carrying no at-risk alleles (OR, 3.40; 95% CI, 1.49–7.78; P = .004). The false-positive report probability was 0.097 for patients carrying 1 to 2 at-risk genotypes, indicating noteworthiness. Gemcitabine dose intensity was slightly lower in CDA CC/AC variant carriers (683 ± 31 mg/m2) than in the AA carriers (752 ± 46 mg/m2), but the difference was not statistically significant (P = .217).
Table 4. Tumor Response to Therapy and Genotype (N = 149)
Genotype Frequency and Its Association With OS and PFS
None of the examined 17 SNPs was associated with OS (data not shown). The data of 137 locally advanced pancreatic cancer patients were available for PFS analysis. Individually, 2 SNPs (RRM1 A33G, RRM1 C-27A) showed significant association with PFS (P = .048 and P = .042, respectively; Table 5). In addition, when the CDA A-76C and hENT1 A-201G variants were analyzed in combination with RRM1 A33G and RRM1 C-27A, a gene-dosage effect on PFS was observed. As the number of at-risk alleles increased, the PFS decreased (Fig. 2). Patients carrying 0 to 1 (n = 64), 2 (n = 50), or 3 to 4 (n = 17) at-risk alleles had median PFS times of 8.3, 6.0, and 4.2 months (Table 5), as well as 6-month PFS rate of 76.5%, 52.0%, and 29.4%, respectively. The HR (95% CI) of progression was 1.79 (1.20-2.66) and 3.25 (1.79-5.90) for patients carrying 2 and 3–4 at-risk genotypes (P = .004 and P < .001; Table 5), respectively, after adjusting for performance status and tumor size. The false-positive report probabilities for patients carrying 2 and 3 to 4 at-risk genotypes were .017 and .006, respectively, indicating noteworthiness.
Table 5. Progression-Free Survival and Genotype (n = 137)
Our results in this study support the hypothesis that SNPs of gemcitabine metabolic and transporter genes are associated with clinical outcome in patients with locally advanced pancreatic cancer. The gene variants of CDA A-76C,dCK C-1205T, RRM1 A33G, and hENT1 C913T correlated with severe neutropenia. In addition, the CDA A-76C and hENT1 A-201G genotypes were significantly associated with tumor response to gemcitabine-based therapy and were marginally associated with PFS. These genotype effects remained significant after adjusting for clinical predictors in statistics.
CDA is involved in the salvage pathway of pyrimidine and plays a key role in detoxifying gemcitabine.9 Three main SNPs have been identified in the CDA gene: C111T (T145T), A-76C (K27Q), and G208A (A70T).8, 37, 38 Although the CDA 208AA homozygote allele and its related haplotype have been associated with severe drug toxicity in Japanese cancer patients treated with gemcitabine plus cisplatin, we excluded this SNP from our study because CDA G208A had not been detected in Caucasians.29, 31, 32 The CDA A-76C variant C allele (Gln27) has been reported to have moderately or significantly lower deaminase activity for gemcitabine or cytosine arabinoside than the wild-type genotype.28, 39 Our data showed significantly higher toxicity in the CDA-76 CC/AC variant than in the AA wild-type, suggesting lower deaminase activity of the C allele (Gln27) variant, which is consistent with previously reported data from in vitro studies.28, 39 Although our results indicated that the CDA -76 CC/AC variant was also associated with poorer tumor response, we do not believe this is because of dose reductions, as there was no significant difference in the gemcitabine dose intensity in the CDA -76 CC/AC variant carriers as compared with the AA carriers. Nevertheless, there were controversial findings on this SNP in previous studies. The CDA A-76C variant A allele (Lys27) had significantly lower deaminase activity than the C allele (Gln27) in a study conducted in 90 patients with lung cancer.40 The Lys27 haplotype did not show any significant effect on gemcitabine pharmacokinetics in a study of 256 Japanese patients.32 Future studies are warranted to clarify the functional and clinical importance of this SNP in gemcitabine therapy.
dCK is the rate-limiting enzyme for intracellular activation of gemcitabine and was therefore thought to play an important role in sensitivity to gemcitabine.9 Some studies have shown that the enzyme activity or expression level of dCK was associated with sensitivity to gemcitabine and survival of pancreatic cancer patients.17, 41 Shi et al reported that the haplotype containing dCK C-360G and C-201T had a significant association with higher levels of dCK mRNA and longer survival time in patients with acute myeloid leukemia treated with cytosine arabinoside.20 Our study showed a significantly higher toxicity in patients with the dCK−1205 TT variant than the CC/CT variant. Because this SNP is located in the intronic region, it is not clear whether it directly affects dCK enzyme activity or whether it is in linkage disequilibrium with other functional SNPs or other genes.
RRM1 is essential for DNA synthesis and repair.9 Davidson et al reported that the increased mRNA level of RRM1 resulted in drug resistance.26 In a different study, Rha et al demonstrated a strong association between gemcitabine-induced neutropenia and the RRM1 haplotype containing 2 SNPs (A2455G and G2464A).33 Our data showed that the RRM1 33AA variant was significantly associated with severe toxicity, suggesting a high susceptibility of this variant to gemcitabine. RRM1 A33G is a synonymous SNP (T741T) that does not produce amino acid change. However, Kimchi-Sarfaty et al reported that a synonymous SNP in the MDR1 gene yielded a protein product with altered drug and inhibitor interactions.42 Thus, the functional consequence of RRM1 A33G SNP should be further investigated.
Nucleoside transporters have been thought to have an important role in gemcitabine cytotoxicity and efficacy.16 Gemcitabine intracellular uptake is mediated mainly by hENT1 and, to a lesser extent, by hCNT1 and hCNT3,9 supporting our current observations that the hENT1 C913T genotype was significantly associated with neutropenia toxicity and the hENT1 A-201G genotype with tumor response to gemcitabine and PFS. Although 2 previous studies on the nonsynonymous SNPs of hENT1 failed to demonstrate functional diversity,43, 44 it was reported that the CGG/CGC haplotypes of the hENT1 promoter region containing the C-1345G, G-1050A, and G-706C SNPs showed moderately higher expression of hENT1.45 The functional significance of the polymorphic variants investigated in our current study has not yet been demonstrated. Considering that hENT1 expression has been associated with survival of patients with pancreatic cancer,27 further genotype-phenotype analysis would be needed to clarify whether the hENT1 genotype can be used as a surrogate marker for hENT1 activity.
In this study, we focused on locally advanced pancreatic cancer, because metastatic pancreatic cancer is associated with greater clinical and biological heterogeneity, and in most instances patients were seen in consultation at our institute but their primary treatments for metastatic disease were administered at other referring facilities. Compared with findings of our previous study in patients with potentially resectable pancreatic cancer who underwent neoadjuvant gemcitabine-based chemoradiation,34 although the clinical characteristics of the 2 study populations are quite different, the association of dCK-1205 T allele with severe gemcitabine toxicity and hENT1-201 A allele with better survival were observed in both studies, suggesting the robustness of these findings. In most locally advanced pancreatic cancer cases, tissue samples are unavailable for measurement of protein expression. Therefore, if genotyping data from peripheral blood DNA is validated and found to be a reliable predictor for gemcitabine toxicity and efficacy, application of such data would be widely beneficial for patients with unresectable advanced pancreatic cancer.
In conclusion, genotypes of gemcitabine metabolic and transporter genes have potential as predictive biomarkers for toxicity and treatment effects of gemcitabine-based therapy in locally advanced pancreatic cancer patients. Our observations still need to be confirmed in separate and larger patient populations. If confirmed, these findings may be helpful in stratifying patients to individualized therapy.
CONFLICT OF INTEREST DISCLOSURES
Supported by the National Institutes of Health (NIH) R01 grant CA098380 (D.L.), SPORE P20 grant CA101936 (J.L.A.), NIH Cancer Center Core grant CA16672, Lockton Research Funds research grant (D.L.), and the Urbieta Family Cancer Fund (C.E.).