Despite improvements in the early detection of gastric cancer, a significant proportion of patients present with inoperable stages where chemotherapy is required. 5-fluorouracil (5-FU) remains the main chemotherapeutic agent for the treatment of gastric cancer, and combination chemotherapy with 5-FU has shown an improved clinical outcomes 1. 5-FU with cisplatin showed an effective clinical outcome 2, however, toxicities were considerable 1. Oxaliplatin, another platinum based agent, has a more favorable tolerability profile than cisplatin. Hence, a combination chemotherapy of 5-FU with oxaliplatin has been investigated in numerous phase II studies, using different doses and schedules 34567. However it remains to be clarified which is the best combination, with the highest efficacy and lowest toxicity. Thus, we conducted a phase II trial of 5-FU, folinic acid and oxaliplatin (a modified FOLFOX-6 regimen) in advanced gastric cancer (AGC) patients as a first line palliative chemotherapy.

Another problem in chemotherapy of AGC is the selection of patients who might benefit from specific chemotherapy. One promising therapeutic challenge is to identify genetic markers based on pharmacogenomics. Genomic polymorphism can influence drug transport, metabolism and cellular response, and lead to individual variations in terms of the response and toxicity and even to overall survival 89. A number of studies have investigated the relationships between treatment outcomes and individual genetic polymorphisms which will determine the efficacies and toxicities of chemotherapeutic agents, especially of 5-FU and platinum agents.

The antitumor effect of 5-FU has ascribed to the competitive inhibition of thymidylate synthase (TS) 10. A high intratumoral TS expression has been correlated with resistance to 5-FU and a poor clinical outcome in colorectal cancer 11121314. Several polymorphisms in TS may influence TS mRNA transcription, stability, or protein expression. Polymorphisms with double or triple repeats of a 28-base pair (bp) sequence in the enhancer region (ER) are known to be associated with the efficacy and toxicity of 5-FU 151617. The -6 bp/-6 bp deletion polymorphism in the 3'UTR of TS is associated with decreased mRNA stability in vitro and lower intratumoral TS expression in vivo. Further, the 6 bp polymorphism varies greatly within different ethnic populations and is in linkage disequilibrium with the TS 5' tandem repeat enhancer polymorphism 18. A functional G/C single nucleotide polymorphism (SNP) within a second repeat of triple repeat (3R) allele was found to determine two additional alleles (3G or 3C) at this locus 19. In vitro, the 3G containing genotype showed a higher TS mRNA expression 1920.

Oxaliplatin has antitumor activity by virtue of its ability to form platinum-DNA adducts. Bulky platinum-DNA adducts are mainly repaired by the nucleotide excision repair pathway, in which proteins of the excision repair cross-complementation 1 (ERCC1), xeroderma pigmentosum group D (XPD, also known as ERCC2) and X-ray repair cross-complementing group (XRCC), have important roles 1321. ERCC, XPD and XRCC contain SNPs that may confer different activities to platinum agents, thus modifying the clinical outcome 222324. Glutathione S- transferase π 1 (GSTP1), which is involved in platinum detoxification, also has a polymorphism that is associated with prolonged survival in cisplatin-treated gastric cancer 2526.

The primary endpoint of this study was to evaluate the efficacy in terms of response rate and the secondary endpoints of this study were to evaluate the efficacy in terms of time to progression, overall survival and toxicity of modified FOLFOX-6 chemotherapy in AGC patients. Exploratory pharmacogenomic collateral study was performed to identify the predictive or prognostic value of germline polymorphisms of candidate genes associated with 5-FU and oxaliplatin.

In this work we evaluated the efficacy of modified FOLFOX-6 chemotherapy in AGC patients, and examined the relevance of the relationship between germline genetic polymorphisms and the clinical outcome. The results indicate that a modified FOLFOX-6 chemotherapy appears to be active and well tolerated.

With an overall RR of 43.8%, our results compare favorably with other phase II studies of FOLFOX chemotherapy, which range from 38% to 56% 34567. By contrast to the FOLFOX-6 regimen for AGC 3, the regimen here omitted the 5-FU bolus injection in order to reduce myelosuppression. In terms of toxicities, the modified FOLFOX-6 regimen showed an 11.0% occurrence of grade 3 or 4 neutropenia, which is lower than the 38% level shown with the FOLFOX-6 regimen 3. Grade 3 or 4 peripheral sensory neuropathy occurred in only 1.4% of the patients. This was lower than original FOLFOX-6 using oxaliplatin of 100 mg/m² with a median cumulative dose of 901 mg/m² for oxaliplatin. In our study the median cumulative dose of 570 mg/m² for oxaliplatin which is lower than original FOLFOX-6. With considering median cumulative dose of oxaliplatin, this was comparable to other lower dose oxaliplatin-based regimen or omitting the 5-FU bolus 47. In our study, dose modification of oxaliplatin to 85 mg/m² was performed if the patient experienced grade 2 peripheral neuropathy and we strictly followed the protocol which permitted the initiation of chemotherapy after recovery from all toxicities less than grade 2.

In the present study, TS and XPD polymorphisms were found to be associated with RR, and these polymorphisms could be used as predictive markers. TS, the target of 5-FU, has been investigated repeatedly in various aspects to predict the response to 5-FU, in terms of polymorphism, mRNA and protein expressions 1016. In our study, the 6-bp deletion polymorphism in TS-3'UTR was found to be correlated with favorable clinical outcome. That means that the patients with -6 bp/-6 bp were found to have a higher RR and prolonged time to progression. TS-3'UTR plays a role as a post transcriptional regulator, mainly by controlling mRNA stability and/or translational efficiency. The 6-bp deletion in TS-3'UTR reduces mRNA stability and lowers intratumoral TS mRNA levels 18. A reduced level of TS mRNA might also lead to a lower TS protein expression, and make tumors more sensitive to 5-FU based chemotherapy 133637. Previous studies in colorectal cancer provide strong evidence that a lower TS expression appears to be associated with a higher RR and prolonged survival with 5-FU based chemotherapy 111314. These findings are supported by in vitro data that -6 bp/-6 bp in TS-3'UTR affect to lower TS expression and higher sensitivity to 5-FU in gastric cancer cell lines. In gastric cancer, we confirmed that a 6-bp deletion is associated with better clinical outcome in homogenous patients treated with a first line modified FOLFOX-6 regimen.

However, some studies conducted in Western countries 2526 failed to find a correlation between the 6-bp deletion polymorphism and 5-FU sensitivity in gastric cancer, while our results are concordant with those of another study conducted in an Asian 38. One possible explanation for this contradictory result is ethnic difference.

Caucasian patients had higher proportion of favorable 2R/2R genotype (16.0% to 26.4%) 263940, lower proportion of unfavorable 3R/3R or 3G containing genotypes in 5'UTR (28.8% to 39.2%) 263440 and lower proportion of favorable -6 bp/-6 bp in 3'UTR (7.8% to 22.0%) 18263440. In contrast, Asian patients showed lower proportion of favorable 2R/2R (0.0% to 6.0%) 3441 higher proportion of unfavorable 3R/3R (66.4% to 72.3%) 3442 and higher proportion of favorable -6 bp/-6 bp (36.4% to 56.0%) 183438. These differences between favorable and unfavorable genotype frequencies might make different results according to ethnic diversity.

The clinical influence of TS polymorphism in 5-FU based chemotherapy are often controversial because of different clinical settings (adjuvant vs. palliative), tumor type (colon vs. gastric cancer), and different 5-FU infusion times 1625263942434445. These experimental variables should be considered when interpreting predictive values of the TS polymorphism.

Another factor to be taken into consideration is the possible impact on outcome of other drugs combined with 5-FU. Even antitumor activity of 5-FU may be decreased by TS polymorphism, another combined drug might compensate the decreased antitumor activity and make sufficient tumor response. This would make the meaningful polymorphism in single agent protocol less significant in combination regimens 46. Thus this could account for the differing results with prior reports. A meta-analysis could perhaps provide some clarity in this area.

XPD (also known as ERCC2) encodes DNA helicase, which is a member of the nuclear excision repair pathway and plays a role in repairing platinum-DNA adducts. The C/C genotype of XPD-Arg156Arg SNP showed lower RR than C/A or A/A genotype (26.1% vs. 52.0%, p = 0.038) and shorter TTP (4.1 months vs. 6.2 months, p = 0.022). The lower RR with a modified FOLFOX-6 chemotherapy might result from the tendency that the C/C genotype of XPD- Arg156Arg had a higher DNA repair capacity than C/A or A/A genotype 32. However, it did not affect to prolongation of survival. The other polymorphisms examined failed to show any relation with clinical outcome.

In the presented study, we analyzed germline genotype. Hence the correlation between germline genotype from peripheral blood and somatic genotype from tumor tissues should be considered. Few studies focused on comparing both genotypes. In terms of 28-bp repeat polymorphism, tumor specific loss of heterozygosity at the TS locus has been reported 47. In contrast, some studies have reported that there is no difference in TS ER polymorphism between genotypes of tumor and normal tissues in gastric 41 and colorectal cancer 40.

Herein, we presume that germline genotypes are nearly identical to somatic genotypes, even though it might not always reflect the tumor genotype. However, germline genotypes determined from peripheral blood mononuclear cells have many strong points. The germline genotypes offer the better clinical accessibility and applicability, compared to tumor tissue DNA or mRNA, which present difficulties in obtaining and handling samples. In our results, germline genotypes, which were obtained by simple blood test, have shown a good association with the clinical outcome and are easily interpreted. In this phase II study, the small sample size (n = 73) might remains a limitation to clarify the role of polymorphism. However, our patient population was considerably homogeneous in terms of ethnicity and chemotherapeutic regimen, and this protocol was prospective.

We found that modified FOLFOX-6 chemotherapy appears to be effective. A 6-bp deletion homozygote of TS-3'UTR was found to be a predictive marker of the response and was also found to be associated with a prolonged TTP in AGC patients on modified FOLFOX-6 chemotherapy. Moreover, our results suggest that germline genetic polymorphisms of TS and XPD may be useful candidates for a pharmacogenomic prediction of the response to modified FOLFOX-6 chemotherapy in AGC. Alternative treatment other than 5-FU or platinum based therapy should be considered for the patients who have unfavorable genotype. We conclude that testing for the TS-3'UTR 6-bp deletion and XPD polymorphisms might be a candidate pharmacogenomic factors to be explored in the future larger scale study to identify the gastric cancer patients who might benefit from 5-FU based first line chemotherapy.

The authors declare that they have no competing interests.

BK collected the data, performed the statistical analysis and drafted the manuscript. S–AI designed the concept of this study, performed the statistical analysis with interpretation and approved the final manuscript. S–WH performed the statistical analysis and critically revised the manuscript. HSH carried out the genotyping and management of the samples. D–YO, JHK, S–HL, D–WK, T–YK, DSH and Y–JB performed the chemotherapy for patients and revised the manuscript. All authors read and approved the final manuscript.