Low Thymidylate Synthase Research Articles

Thymidylate Synthase as a Prognostic Biomarker for Locally Advanced Rectal Cancer after multimodal Treatment

PurposeFor years, 5-fluorouracil (5-FU) has been the backbone of radiochemotherapy (RCT) of locally advanced rectal cancer. Its main target, thymidylate synthase (TS), is speculated to be an important biomarker for response prediction and long-term prognosis. In this study, we analyzed TS expression in the rectal cancer tissue of 208 patients to evaluate its predictive/prognostic potential.MethodsAll patients included were diagnosed with locally advanced adenocarcinoma of the rectum (UICC II and III) and were treated within randomized clinical trials of the German Rectal Cancer Study Group. Preoperative RCT (50.4 Gy and concomitant either 5-FU or 5-FU and oxaliplatin) was administered in 167 patients followed by surgical resection with total mesorectal excision (TME). Another 41 patients received postoperative RCT. TS levels and further clinicopathological parameters were assessed in univariate and multivariate analyses. Additionally, a TS gene polymorphism was analyzed with respect to the intratumoral protein levels.ResultsLow TS expression in pretreatment biopsies correlated with impaired patient survival (p = 0.015). Analysis of a 28-bp repeat revealed a correlation between the *3/*3 genotype and high TS expression in pretherapeutic biopsies. In this study, a correlation of TS expression and grade of RCT-induced tumor regression was not found. Histopathological examination confirmed a complete tumor remission in 16 patients (9.6%).Analyses of the resection specimen indicated an unfavorable prognosis for patients with low intratumoral TS expression in case of detected lymph node metastases (p = 0.04).ConclusionsTS can serve as a prognostic biomarker indicating an unfavorable prognosis for patients with low TS expression.Electronic supplementary materialThe online version of this article (doi:10.1245/s10434-011-1608-4) contains supplementary material, which is available to authorized users.

Biomarker study in rectal cancer patients after 5FU-based radiochemotherapy: Evaluation of the prognostic capacity of thymidylate synthase in pretreatment biopsies and resected adenocarcinoma.

435 Background: Fluorouracil (5FU) remains the backbone of neoadjuvant radiochemotherapy (RCT) as well as adjuvant therapeutic strategies in multimodal treatment of rectal cancer patients. Due to its central role as the major target of 5FU thymidylate synthase (TS) is a promising biomarker in rectal cancer. We assessed TS in 208 patients with regard to its predictive/prognostic capacity for disease free DFS and overall cancer specific survival (CSS). Methods: 167 patients cUICC stages II (28%) and III (72%) received preoperative 5FU based RCT followed by total mesorectal excision (TME) A comparison group n = 41 received postoperative RCT after primary TME. All patients were treated after standardized protocols within phase-II/-III trials of the German Rectal Cancer Study Group. TS levels from pretreatment biopsies and corresponding resection specimens were assessed by immunohistochemical staining for their impact on DFS and CSS. Additionally, a TS gene polymorphism (28 bp repeat) was analysed in respect to intracellular protein expression levels and prognostic significance. Results: Patients with low TS expression in pre-treatment biopsies showed a correlation with impaired CSS (p = 0.015). After neoadjuvant RCT there was evidence of lymph node metastases ypUICC stage III in 32.6%. Complete histopathologically confirmed tumor regression TRG 4 was achieved in 16 patients (9.5%). During follow-up (median 57 months) patients with low intratumoral TS expression and positive nodal status were at high risk for local and/or distant metastatic recurrence (p = 0.040). Analysis of the 28bp repeat revealed a correlation of *3/*3 genotype with high TS expression in pretherapeutical biopsies (p = 0.05). Conclusions: TS represents a prognostic biomarker in locally advanced rectal cancer indicating an unfavourable outcome for patients with low TS expression and might help to adapt adjuvant therapy regimens by stratifying patients according to their risk for cancer recurrence. No significant financial relationships to disclose.

Low Thymidylate Synthase, Thymidine Phosphorylase, and Dihydropyrimidine Dehydrogenase mRNA Expression Correlate With Prolonged Survival in Resected Non–Small-Cell Lung Cancer

BackgroundThymidylate synthase (TS), thymidine phosphorylase (TP), and dihydropyrimidine dehydrogenase (DPD) are key enzymes in the 5-fluorouracil (5-FU) pathway. The aim of this study was to investigate the mRNA expression of TS, TP, and DPD in tumor and nontumor lung tissue of patients with NSCLC and to determine the potential of these genes as molecular biomarkers. Materials and MethodsThe TS, TP, and DPD mRNA expression was analyzed in tumor and nontumor tissue of 91 patients with NSCLC by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) with β-actin as the internal control. All tumors were R0 resected. The median follow-up was 85.9 months. ResultsThe mRNA expression of TS, TP, and DPD was detectable in both tumor and nontumor tissue. Tumor TP (tTP) seems to correlate with tumor TS (tTS) and tumor DPD (tDPD) mRNA expression, but no correlation in the mRNA expression of tTS and tDPD was found. The TS and TP mRNA expression levels were significantly associated with patient prognosis. The 5-year survival probability was 58.7% (TS), and 59.6% (TP) for patients with a low TS and TP mRNA expression and 33.4% (TS), and 31.8% (TP) for patients with a high mRNA expression (P = .04 [TS]; P = .03 [TP]; log-rank). The probability of survival was significantly different among patients with no and any 1 highly expressed gene compared with patients with any 2 or more of the 3 investigated genes highly expressed (P = .012). ConclusionHigh TS, TP, and DPD mRNA expression are biomarkers for a more severe malign NSCLC biology. Quantitation of the mRNA expression of these genes seems to be helpful in differing patients with unequal malign tumor entities and therefore possibly helpful in selecting tailored additional therapies to control the disease.

Predictive Factors of Complete Response to Neoadjuvant Chemoradiotherapy in Patients with Rectal Cancer

Purpose: Pathological complete response (pCR) after neoadjuvant chemoradiotherapy is a favorable prognosticator in rectal cancer patients. We investigated whether the biological features of the primary tumor affect pCR. Materials and Methods:Forty-six patients treated with capecitabine-oxaliplatin and pelvic conformal radiotherapy were considered. Forty-three patients underwent surgery, and the pathologic response was scored according to the tumor regression grade (TRG) scale. Epidermal growth factor receptor (EGFR), vascular endothelial growth factor, poly(adenosine diphosphate-ribose) polymerase-1, X-ray cross-complimenting, thymidylate synthase (TS) and Ki67 expression were evaluated by immunohistochemistry on rectal biopsies obtained before chemoradiotherapy, and scored as the percentage of positive cells. Cutoffs were selected based on ROC analysis. The correlation between the biological factors and the TRG coded as TRG1 (pCR) versus TRG ≧2 (no pCR) was assessed by the χ² test and logistic regression analysis. Results: Low EGFR (p = 0.007), high TS (p = 0.002), and high Ki67 (p = 0.05) were strongly associated with pCR. Upon univariate analysis, TRG significantly affected disease-free survival (p = 0.03). Conclusions: pCR was significantly associated with high TS, high Ki67 and low EGFR expression. Patients with pCR have a significantly lower incidence of relapse.

Correlation between chemosensitivity of pancreatic carcinoma cells and thymidylate synthase protein expression

Objective To investigate the relationship between the thymidylate synthase (TS) protein expression levels and chemosensitivity of different pancreatic cancer cells to 5-Fu. Methods The chemosensitivity of seven pancreatic carcinoma cell lines to 5-Fu in vitro was evaluated with CCK-8 kit. TS protein expression of pancreatic carcinoma cells was detected by Western blotting. Results Pancreatic carcinoma cells AsPC-1, BxPC-3, Capan-1, SU86.86 and T3M4 expressed low amounts of TS protein (low TS protein group). Cells of PANC-1 and COLO357 expressed high amounts of TS protein (high TS protein group) in seven cell lines. The inhibition rate (IR) of low TS protein group cells for low, middle and high doses to 5-Fu was 0.683 ± 0.131, 0.769 ± 0.114 and 0.836 ± 0.094, respectively. The IR of high TS protein group for low, middle and high doses to 5-Fu was 0.370 ± 0.157, 0.548 ± 0.018 and 0.638 ± 0.020, respectively. IR of 5-Fu tended to be lower in the cells with high TS protein expression than those with low TS protein expression ( P < 0.05). Conclusion TS protein expression levels were significantly correlated to 5-Fu sensitivity in seven pancreatic carcinoma cell lines. Key words: Pancreatic carcinoma; Thymidylate synthase; Chemosensitivity; Fluorouracil

Characterization of a novel lymph node metastasis model from human colonic cancer and its preclinical use for comparison of anti‐metastatic efficacy between oral S‐1 and UFT/ LV

Although lymph node metastasis (LNM) is the most critical prognostic factor in colorectal cancer patients, the anti-LNM efficacy of chemotherapeutic agents is largely unknown because of the limitations of reproducible human colorectal cancer LNM models. Here, we developed a new LNM model from a recently established colorectal cancer cell line (COLM-5) and compared the anti-LNM efficacy of two oral formulations of 5-fluorouracil (5-FU) derivatives, S-1 and UFT/leucovorin (LV). COLM-5 cells is a poorly differentiated adenocarcinoma cell line with unique features such as left-sided, beta-catenin cytoplasmic localization, and microsatellite stable phenotype. COLM-5 cells expressed vascular endothelial growth factor (VEGF-C) and exhibited peritumoral lymphangiogenesis. Consequently, they showed high LNM potential at an incidence of approximately 90% when subcutaneously injected into nude mice, allowing use for preclinical study. When chemotherapy with S-1 or UFT/LV started from the micrometastasis stage, not the advanced macroscopic metastasis stage, anti-LNM efficacy of S-1 was significantly higher than that of UFT/LV at the dosage in which antitumor activity of the two drugs against primary subcutaneous tumor was comparable. COLM-5 cells showed expression pattern of 5-FU metabolizing enzymes such as high dihydropyrimidine dehydrogenase (DPD) and low thymidylate synthase (TS)/orotate phosphoribosyltransferase (OPRT) both in vitro and in vivo. These results suggest that the preferential anti-LNM activity of S-1 compared with UFT/LV against high-DPD COLM-5 tumors is due to the higher DPD inhibitory activity of 5-chloro-2, 4-dihydroxypyrimidine (CDHP) present in S-1 than uracil in UFT. The COLM-5 model would be an excellent tool for understanding the basic mechanism of LNM and for preclinical study on the anti-LNM efficacy of the drugs.

Emerging role of thymidylate synthase for the pharmacogenomic selection of patients with thoracic cancer

The use of antifolate drugs, and particularly of pemetrexed, is emerging as a relevant therapeutic strategy for the treatment of some specific thoracic cancers. Pemetrexed is a novel multitargeted antifolate which has been shown in vitro to inhibit at least three different enzymes in the folate pathway: thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT) of which TS is its main target [1]. These enzymes are involved in the synthesis of nucleotides and interference with their function will ultimately hinder the synthesis of RNA and DNA. Pre-clinical studies have demonstrated the cytotoxic activity of this agent in a broad range of tumor types including non-small cell lung cancer (NSCLC). In vitro studies indicated that tumor cell lines expressing high levels of TS or DHFR have reduced sensitivity to pemetrexed, suggesting that increased expression levels might correlate with reduced clinical efficacy [2]. In NSCLC, which accounts for more than 80% of all lung cancer, two recent independent Phase III trials showed that pemetrexed alone or in combination with cisplatin demonstrated a statistically superior activity in patients with non-squamous histotypes [3, 4]. Although histology has not consistently been associated with clinical outcomes in almost every trial testing the role of platinum-based chemotherapy in advanced NSCLC, it has now emerged as a potential predictive factor due to the evidence of the differential expression of TS between the different tumor histotypes, which seems to play an important role [3]. Preliminary molecular evidence, in fact, has previously shown that TS expression is significantly higher in squamous cell carcinoma compared with adenocarcinoma by means of both Real-Time PCR gene quantification and of immunohistochemistry for the detection of the protein levels [5]. In undifferentiated large cell carcinoma, a subsequent analysis consistently reported significantly higher TS levels in desmocollin 3 (DSC-3)-positive as compared to DSC-3negative tumors (indicating a squamous and adenocarcinoma origin of the tumors, respectively), reporting that lack of DSC-3 immunoreactivity in this histologic subtype is associated with lower TS expression [8]. In the same experimental setting, TS expression levels were tested in 22 cases having a cytological diagnosis of NSCLC with no clear histological definition, and in the corresponding histological samples obtained at the time of subsequent surgical resection. As a result, a significantly high degree of concordance in terms of TS protein expression in matched specimens was detected. This result raises the possibility of using the protein levels, evaluated by immunohistostaining on cytological specimens, to select the patients with unresectable advanced disease to be treated with TS-inhibiting agents. On the other hand, the small cell carcinoma (SCLC) group of patients, comprising approximately 13% of all lung cancers, has shown minimal response to pemetrexed in several studies including a large Phase III study [6]. Again, the explanation for these results may be found at the molecular level, in that SCLC has been shown to have very high TS expression when compared with either well-differentiated pulmonary neuroendocrine tumors or NSCLC [7]. These data once again support the hypothesis that elevated TS expression decreases the response to pemetrexed, and that the determination of TS levels can be used in to exclude those patients who are most likely to be unaffected from the treatment. A clear example of this is found in a very recent retrospective study from our group carried out on malignant pleural mesothelioma (MPM), a disease in which pemetrexed has been previously shown to be clinically active. We showed that TS protein and gene expression levels are significantly correlated with the clinical outcome in a consecutive series of 60 patients with advanced MPM treated with pemetrexed, with or without plat

A multicenter, randomized phase II study of second-line maximum androgen blockade with an alternative antiandrogen combined with tegafur-uracil (UFT) for prostate cancer with relapse after initial hormonal therapy.

e15030 Background: Management of advanced prostate cancer with relapse after initial hormonal therapy is still challenging. Some reports have shown that the second line maximum androgen blockade with an alternative antiandrogen (2nd MAB) is still effective. However 2nd MAB has relatively low PSA response rate of about 35%. We sought to determine whether the combination therapy of 2nd MAB and UFT would be even more effective than 2nd MAB alone and also to correlate the treatment effect with the thymidylate synthase (TS) mRNA expression levels. Methods: Patients who were histologically confirmed with prostate cancer and relapsed after LH-RH analogue alone or MAB were included in this study. ECOG performance status was 0-2. All patients were randomly allocated into two groups, 2nd MAB (group A) or 2nd MAB combined with UFT orally at a dose of 260 mg/m2/day (group B) until recurrence. The primary endpoint was PSA response rate (effectiveness defined as > 50% decrease of PSA) and one of the secondary endpoint was PFS. TS mRNA expression levels in formalin fixed paraffin embedded biopsy specimens were measured by RT-PCR method. Results: Fifty three patients were enrolled in this study and clinical data for 38 patients were available so far. Median age was 76 (47-89) years. There were no severe AEs in both groups. PSA response rate in group B (70.6%) tended to be higher than that in group A (36.8%) (p=0.0543). Group B (16.1 months) had significantly longer median PFS than group A (6.4 months) (p=0.0028). Biopsy samples were available in 33 patients for TS mRNA measurement. Interestingly, among patients who were treated with 2nd MAB combined with UFT, those with low TS (88%) had higher PSA response rate than those with high TS (57%). Conclusions: After relapse with initial hormonal therapy, 2nd MAB along with UFT is an effective and well-tolerated treatment option before introducing docetaxel, especially for elderly patients with prostate cancer with low TS expression level. No significant financial relationships to disclose.

Association of thymidylate synthase (TS) expression with response to S-1 plus carboplatin (CBDCA) in non-small cell lung cancer (NSCLC) patients (pts).

e21025 Background: S-1, a novel oral fluoropyrimidine derivative, is active against NSCLC. Phase II studies of S-1 plus platinum compounds have shown activity for first-line treatment of advanced NSCLC. It is important to design an optimal therapeutic strategy according to tumor biology to improve the treatment of NSCLC. In the present study, we evaluated the correlations between immunohistochemical expression of TS, orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD), and response to S-1 plus CBDCA in advanced NSCLC pts. Methods: Chemotherapy-naïve pts with advanced stage (IIIB/ IV) NSCLC, ECOG PS of 0-1, adequate organ functions and archival tumor tissue were assigned to receive either CBDCA AUC 5 mg/mL/min on day 1 plus oral S-1 80 mg/m2/day (40 mg/m2 b.i.d.) on days 1 to 14 (22 pts) or CBDCA AUC 6 mg/mL/min plus paclitaxel (PTX) (200 mg/m2) on day 1 every 21 days (26 pts). Association between TS, OPRT and DPD protein expression levels in tumors and response to chemotherapy was evaluated. Results: There was no statistical difference in TS, OPRT and DPD expression level between S-1 plus CBDCA and PTX plus CBDCA. In the S-1 plus CBDCA groups, TS expression of responding tumors was significantly lower than that of nonresponding ones (p = 0.006), whereas no significant difference was observed in the PTX plus CBDCA groups. Furthermore, patients with low TS expression survived longer than those with high TS expression in patients receiving combination therapy with S-1 and CBDCA, but not in those treated with PTX plus CBDCA. Conclusions: Our results suggest that tumor TS expression levels is predictive markers for response to S-1 plus CBDCA chemotherapy in advanced NSCLC pts. No significant financial relationships to disclose.

Thymidylate synthase expression and response to irinotecan/5-fluorouracil therapy in advanced colorectal cancer (CRC).

e14123 Background: Molecular diagnostics are at the forefront of cancer therapy. Defining cellular profiles of malignancies facilitates selecting the most efficacious chemotherapy regimen. Thymidylate synthase (TS) is a critical enzyme in DNA synthesis and the target of 5-fluorouracil (5FU). Previous studies have identified an inverse relationship between tumoral TS expression and 5FU efficacy in colorectal cancer. Saltz et al described irinotecan (CPT-11) as an effective agent even in patients with high TS levels when combined with 5FU. Noda et al confirmed the relationship in 27 Japanese patients treated with IFL (5FU, CPT-11 and leucovorin). This association highlights the ability to treat patients with high TS expression using 5FU and CPT-11 combinations. Our study was designed to confirm these findings. Methods: TS gene expression was measured from metastatic tumors (primary tumor tissue was used if metastatic was not available) via RT-PCR extraction of formalin-fixed, paraffin-embedded samples. No clear cutoff exists for high and low TS expression; therefore, 3 separate cutoffs were assessed as previously published in the literature. A total of 36 patients treated with an CPT-11/5FU based regimen for advanced colorectal cancer were evaluated. Fifteen tissue blocks had limited tissue and analysis was not done. Seventeen patients had full follow-up data and are included in this interim analysis. The TS gene expression was then correlated to the response of the CPT-11- 5FU based regimen. Primary endpoints included clinical benefit (CR+PR+SD) and time to treatment failure (TTF). Results: Clinical benefit and TTF were found to be comparable in patients treated with an CPT-11-5FU combination regardless of TS expression (Table). Conclusions: This study provides additional data confirming that CPT-11-5FU combination therapies for advanced CRC can be effective even in patients with elevated TS mRNA values across all three reported TS cutoff values. TS ≥ 1.6 TS < 1.6 P value TS ≥ 2.76 TS < 2.76 P value TS ≥ 4 TS < 4 P value CB (%) 50 100 0.10 50 77.8 0.34 50 63.6 0.60 PD (%) 50 0 50 22.2 50 36.4 TTF (weeks) 44.3 16.8 0.93 40.9 34.8 0.72 32.0 39.9 0.66 Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics

Thymidylate Synthase But Not Excision Repair Cross-Complementation Group 1 Tumor Expression Predicts Outcome in Patients With Malignant Pleural Mesothelioma Treated With Pemetrexed-Based Chemotherapy

The relationship between thymidylate synthase (TS) expression and outcome in patients with malignant pleural mesothelioma (MPM) treated with pemetrexed (P) was retrospectively evaluated. Sixty histologically confirmed patients with MPM previously treated with P and platinum (45 of 60) or as single agent (15 of 60) were retrospectively considered. Eighty-one control patients with MPM not P-treated were also evaluated. TS and excision repair cross-complementation group 1 (ERCC1) gene expression levels were evaluated by real-time polymerase chain reaction and by immunohistochemistry using the H-score. Median TS H-score value was 90 (range, 5 to 240). A significant correlation between low TS protein expression and longer time to progression (TTP; 17.9 v 7.9 months; hazard ratio [HR], 2.05, 95% CI, 1.19 to 3.77; P = .02) or overall survival (OS; 30 v 16.7 months; HR, 2.38; 95% CI, 1.15 to 4.91; P = .019) was found when patients were divided according to median H-score. Conversely, TS mRNA levels were not significantly correlated with outcome. In platinum-treated patients (n = 45), no correlation was found with survival according to ERCC1 median H-score, but patients in the lower tertile had a significantly shorter survival (HR, 3.06; 95% CI, 1.08 to 8.69; P = .035). In control MPMs, TS had no prognostic role. At multivariate analysis, TS protein levels were the only independent prognostic factor for both TTP (HR, 2.71; 95% CI, 1.13 to 6.49; P = .02) and OS (HR, 6.91; 95% CI, 1.90 to 25.07; P = .003). In patients with MPM treated with P-based chemotherapy, low TS protein levels are predictive of improved TTP and OS. The role of TS assessment is worth of prospective validation in future studies on MPM.

Expression of thymidylate synthase determines the response of gastric cancer patients undergoing gastrectomy to 5-fluorouracil-based adjuvant chemotherapy

We investigated whether the intensity of thymidylate synthase (TS) staining in tissue samples obtained from gastric cancer (GC) patients undergoing gastrectomy could predict response to 5-FU-based adjuvant chemotherapy after gastrectomy. Clinicopathological features of 124 patients with histologically proven GC who underwent radical gastrectomy were retrospectively reviewed. Tissue samples obtained from these patients were immunohistochemically stained for assessing TS expression. We arbitrarily classified the TS staining results as low (<20% cytoplasmic immunostaining) and high (> or =20% cytoplasmic immunostaining) TS expression. The clinicopathological features of the low TS expression group patients were typically similar to those of the high TS expression group patients. However, multivariate forward stepwise logistic regression analysis revealed that low TS expression was independently associated with females and responders to 5-FU-based adjuvant chemotherapy. The median follow-up duration for the 124 GC patients who had undergone curative resection was 41.3 months. The GC patients who showed poor tumor differentiation and high TS expression had short disease-free survival (DFS) and overall survival (OS). Low TS expression is significantly associated with female GC patients and responders to 5-FU-based adjuvant chemotherapy. It predicts longer DFS and OS in selected GC patients treated with 5-FU-based adjuvant chemotherapy after curative resection. The results suggest that prospective assessment of TS staining intensity in tissue samples obtained from GC patients undergoing gastrectomy would be useful to predict the patients who would be benefited from 5-FU-based adjuvant chemotherapy after gastrectomy.

Evaluation of prognostic factors for the response to S-1 in patients with stage II or III advanced gastric cancer who underwent gastrectomy

Many studies have reported that low intratumoral mRNA expression of thymidylate synthase (TS) is an important biomarker of response to chemotherapy in patients with unresectable advanced gastric cancer. However, the role of gene expression profile of patients who received postoperative adjuvant chemotherapy remains unclear. In this study, we evaluated how TS and other associated genes related to outcome. Seventy-nine patients with stage II or III advanced gastric cancer who underwent gastrectomy were analyzed. Thirty-nine patients received adjuvant chemotherapy with S-1 after surgery (S-1 group) and 40 patients underwent surgery only (surgery group). Formalin-fixed, paraffin-embedded tumor tissues were dissected by the laser-captured microdissection technique and analyzed for target gene expressions using a quantitative real-time polymerase chain reaction. There were no significant differences between the S-1 group and the surgery group in gene expressions except TS (P=0.034). In the S-1 group, recurrence-free survival (RFS) and overall survival (OS) were significantly longer in patients with low TS expression compared with patients with high TS expression (P=0.021 and 0.016), whereas there were no correlations in the surgery group. Furthermore, RFS and OS were both correlated with extent of lymph node metastasis (N) (P=0.038 and 0.020) and TS expression (P=0.021 and 0.032). On multivariate analysis it was found that TS expression and N were significant independent prognostic factors of RFS and OS (TS: P=0.027 and 0.050, N: P=0.048 and 0.032). Our results suggested that intratumoral TS expression is an independent prognostic factor in patients with gastric cancer who received postoperative adjuvant chemotherapy with S-1.

Impacts of fluorouracil‐metabolizing enzymes on the outcomes of patients treated with S‐1 alone or S‐1 plus cisplatin for first‐line treatment of advanced gastric cancer

A phase III trial of S-1 plus cisplatin (SP) versus S-1 alone, for first-line treatment of advanced gastric cancer (SPIRITS trial), has shown that overall survival was better in patients treated with SP than with S-1 alone. In the present retrospective biomarker study, we aimed to develop a methodology to identify the patients with advanced gastric cancer who would respond better to S-1 alone than SP. We studied 120 patients who received S-1 alone or SP for first-line chemotherapy for advanced gastric cancer, and quantitatively evaluated mRNA levels of thymidylate synthase (TS), thymidine phosphorylase (TP), orotate phosphoribosyltransferase (OPRT), dihydropyrimidine dehydrogenase, vascular endothelial growth factor-A, and epidermal growth factor receptor in paraffin-embedded specimens of primary tumors. Multivariate survival analysis in patients who received S-1 monotherapy (66 patients) demonstrated that low TP expression (hazard ratio: 2.55 (95% CI: (1.33 to 4.89)), low TS (2.71 (1.36 to 5.37)), and high OPRT (0.33 (0.13 to 0.86)) were significant predictors of long overall survival. In patients with lower expression of both TP and TS (n = 23) than their cutoff values, the S-1 alone group (n = 15) had longer overall survival than the SP group (n = 8; median overall survival, 18.2 months vs. 9.4 months), whereas the frequency of overall adverse events in the S-1 alone group tended to be lower than that in SP group. Our results suggest that these biomarkers are useful for selection of patients with advanced gastric cancer in whom treatment with S-1 alone will yield survival benefit.

Clinical relevance of thymidylate synthase (TS) activity for S-1-based chemotherapy in squamous cell carcinoma of the oral cavity

S-1 is a newly developed oral fluoropyrimidine derivative that is now widely used as a chemotherapeutic agent in the treatment of oral squamous cell carcinoma (SCC). Thymidylate synthase (TS) is the rate-limiting enzyme in the de novo DNA biosynthetic pathway, and improves clinical response to chemotherapy with fluoropyrimidines. We have retrospectively evaluated the predictive value of thymidylate synthase activity in 75 patients with oral SCC with an enzyme-linked immunosorbent assay (ELISA). Mean (SD) activity (pmol/mg) in the specimens was 0.078 (0.080) (median 0.059). The median value was taken as the cut-off value based on which the patients were divided into high and low activity groups. Both the clinical and histopathological responses to chemotherapy and radiochemotherapy were higher in the group with low TS activity. The group with low TS activity also differed significantly in their clinical response to S-1-based chemotherapy ( p < 0.05). However, there was no significant difference in cause-specific survival. Measurement of TS activity may aid in predicting the clinical response to chemotherapy including S-1 for oral SCC.

Thymidylate Synthase and Dihydropyrimidine Dehydrogenase Are Upregulated in Pancreatic and Biliary Tract Cancers

Purpose: To investigate a rationale of pancreatobiliary cancers, thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) concentrations in cancer tissues were evaluated. Patients and Methods: Twenty-nine patients with pancreatic cancer (PC) and 43 with biliary tract cancer (BTC; 27 cases of bile duct cancer, 8 of gallbladder cancer and 8 of cancer of the papillary area) were included. ELISA was used to assess the concentrations of TS and DPD in cancerous and noncancerous tissues. All 82 patients were followed up at least 12 months after surgery. We compared patient survivals between the 2 groups, taking TS and DPD cutoff as median values, high versus low DPD and high versus low TS in the cancerous tissue of PC and BTC patients. Results: As for PC, DPD and TS concentrations were significantly higher in cancerous than in noncancerous tissues (p = 0.016 vs. 0.036). In BTC, TS concentration was higher in cancerous than in noncancerous tissue (p = 0.0001). Patients with low TS and DPD concentrations had a tendency to have a better disease-free survival than those with high TS and DPD concentrations in PC groups; especially, BTC patients’ disease-free survival with high TS was significantly longer than that with low TS (p = 0.0083). Discussion: TS and DPD levels had a tendency to be high in PC and BTC cancerous tissues. High TS in the cancerous tissue was associated with better disease-free survival in BTC.

Impact of dihydropyrimidine dehydrogenase status of biopsy specimens on efficacy of irinotecan plus cisplatin, S-1, or 5-FU as first-line treatment of advanced gastric cancer patients in JCOG9912

4535 Background: JCOG9912 (randomized phase III trial) showed a significant non-inferiority of S-1 to 5-FU (P&lt;0.001); however, either S-1 or irinotecan plus cisplatin (IP) failed to show superiority to 5-FU (P=0.034 and 0.055, respectively) in overall survival. Excision repair cross-complementing group 1 (ERCC1), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are reportedly related to outcome of advanced gastric cancer (AGC) patients (pts) treated with IP or 5-FU. Efficacy analyses are performed to evaluate the status of ERCC1, TS, DPD and 5 biomarkers related to anticancer drug sensitivity in first-line pts treated with IP, S-1, or 5-FU monotherapy under controlled conditions. Methods: Blocks from endoscopic biopsy specimens of primary lesions before chemotherapy were available from 365 of 704 pts in JCOG9912. Using laser-captured microdissection and real-time RT-PCR, we analyzed mRNA expression of ERCC1, TS, DPD in paraffin-embedded specimens. Expression levels of each gene were categorized into low and high values at each median. Results: The subjects with available tissue for analysis were representative of all randomized pts; 232 samples were assessable for TS, 168 for DPD, and 235 for ERCC1. There were no statistical differences in patient numbers between 3 arms. Pts with high TS showed worse progression-free survival (PFS) compared with those with low TS (hazard ratio (HR):1.26 [95%CI: 0.97–1.63]) in all pts; there was no difference in PFS between DPD and ERCC1 expression level. IP showed better PFS than S-1 in low DPD (HR: 0.57 [95%CI: 0.32–1.01]) but not in high DPD (HR: 1.24 [95%CI: 0.76–2.04]); there was no clear difference in PFS between pts treated with IP and S-1 regardless of TS and ERCC1 expression status. S-1 showed almost consistently better PFS than 5-FU regardless of mRNA expression status. Conclusions: This large analysis showed the prognostic value of TS status in AGC pts and the predictive value of DPD status for IP and S-1 as first-line treatment. Studies of DPD as a predictive marker for IP activity in AGC are warranted in future personalized phase III. [Table: see text]

Pharmacogenomic analysis from a phase III study of pemetrexed plus carboplatin (PC) versus etoposide plus carboplatin (EC) in chemonaive patients (pts) with extensive-stage disease small cell lung cancer (ED-SCLC)

8030 Background: We have reported results of a randomized phase III trial in chemonaive patients with ED-SCLC comparing PC to EC. Study enrollment was closed for futility but pharmacogenomic endpoints were not reported. Here we report the results of immunohistochemistry (IHC) analysis from tumor tissues and single nucleotide polymorphism (SNP) analysis of selected genes implicated in pemetrexed, carboplatin, or etoposide activity. Methods: Proteins studied by validated IHC on Benchmark XT were thymidylate synthase (TS), excision repair cross complementing-1 (ERCC1), glycinamide ribonucleotide formyl transferase (GARFT), and folylpolyglutamate synthetase (FPGS). SNP data from whole blood were genotyped by massArray mass spectrometry from 611 samples. One hundred fifty-eight SNPs were genotyped for TS, FPGS, γ-glutamyl hydrolase (GGH), methylene tetrahydrofolate reductase (MTHFR), folate receptor- α (FR-α), solute carrier 19A1 (SLC19A1) and adjacent regions. Allele frequency and Hardy-Weinberg equilibrium were calculated for each SNP. Results: Of 408 tissue samples (from 908 enrolled pts) available for IHC, 336 samples were assayed for &gt; 1 assay target. The IHC analyses showed improved OS in the EC arm relative to the PC arm for patients with low TSnuclear (12.9 vs 7.5 month, p &lt; 0.001, interaction p value = 0.017). There was no differential treatment effect within the PC arm for low vs. high TS. High vs low ERCC1 levels did not predict outcome following C exposure. GARFT was not associated with outcome by treatment. SNP rs2838952 (adjacent to SLC19A1) was significant for improved OS (HR = 0.590, p = 0.01) in both study arms. SNP rs12379987 (FPGS) was significantly associated with treatment for OS (interaction p value = 0.036). Exploratory analyses found associations between additional SNPs and PFS within and across treatments. Conclusions: Pharmacogenomic analyses of tumor samples show that low TSnuclear expression correlates with OS in the EC arm. Two germline SNPs in regions including FPGS and SLC19A1 were associated with better OS. Further analysis of these pharmacogenomic results is ongoing. [Table: see text]

Significance of dihydropyrimidine dehydrogenase and thymidylate synthase mRNA expressions in hepatocellular carcinoma

The significance of dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) gene expressions for the post-surgical prognosis of hepatocellular carcinoma (HCC) has not yet been determined. In the present study, we clarified the significance of DPD and TS gene expressions for the prognosis of HCC. Seventy-four patients, who underwent curative hepatic resection for primary HCC, were evaluated. The DPD and TS mRNA levels of the resected HCC specimens were evaluated using a microdissection technique and quantative real-time RT-PCR. The patients were categorized into high and low groups for each mRNA based on the median value. Various clinicopathological factors, including prognosis, and proliferation index using Ki-67 staining were evaluated in association with the DPD and TS mRNA expression levels. The low DPD mRNA expression was related to younger age, advanced clinical stage, undifferentiated histology, and microscopic intrahepatic metastasis. The overall and recurrence-free survival were significantly lower in the low DPD group than in the high DPD group (P < 0.05). Furthermore, the proliferation index in the low DPD group was significantly higher than that in the high DPD group (P < 0.01). On the other hand, the high TS group showed a tendency of better prognosis than the low TS group, although it was not statistically significant. The low DPD mRNA expression is a significant poor prognostic factor. after curative resection of HCC.

Liver-only metastatic colorectal cancer patients and thymidylate synthase polymorphisms for predicting response to 5-fluorouracil-based chemotherapy

We investigated the association between thymidylate synthase (TS) germline polymorphisms and response to 5-fluorouracil-based chemotherapy in 80 patients with liver-only metastatic colorectal cancer (MCRC). The tandem repeat polymorphism (VNTR) in TS 5′-untranslated region (5′-UTR), which consists of two (2R) or three (3R) 28-bp repeated sequences, with or without a G/C nucleotide change in 3R carriers (3G or 3C) and a 6-bp insertion/deletion (6+/6−) in the TS 3′-UTR, was studied. The distinction between high (2R/3G, 3C/3G and 3G/3G) and low (2R/2R, 2R/3C and 3C/3C) TS expression genotypes according to the 5′-UTR VNTR+G/C nucleotide change showed significant association with tumour response (P=0.01). In particular, high TS expression genotypes were found in 8 out of 34 patients (23.5%) with complete or partial response and in 24 out of 46 patients (52%) with stable disease and disease progression. Liver-only MCRC patients are a homogeneous and clinical relevant subgroup that may represent an ideal setting for studying the actual influence of TS polymorphisms.