Low Thymidylate Synthase Research Articles

Expression of folate pathway regulators and pemetrexed (pem) activity in patients (pts) with advanced non-small cell lung cancer (NSCLC).

7585 Background: Thymidylate synthase (TS) expression has been reported to predict pem activity in pts with advanced NSCLC. Besides TS, pem inhibits multiple enzymes of the folate pathway including dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GART) and aminoimidazole carboxamide ribonucleotide formyltransferase (AICAR). Aim of present study was to investigate whether these or other biomarkers influence pem activity in NSCLC pts. Methods: Advanced pretreated NSCLC pts who received at least two cycles of single agent pem were considered eligible if they had available tumor tissue to assess at least one of the proposed biomarkers. TS, DHFR, GART and AICAR protein expression was assessed by immunohistochemistry (IHC) in FFPE tumor samples. Pts were grouped as IHC+ and IHC– according to staining intensity. Additionally, presence of EGFR and KRAS mutations was investigated. Results: Ninety-six pts were included in the study. The majority of subjects was male (72%), smokers (93%), with adenocarcinoma (72%). Response rate (RR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS) were 12%, 47%, 2.3 and 9.5 months, respectively. Most pts resulted TS-(72%), AICAR-(82%), DHFR+ (68%), GART- (61%). EGFR and KRAS mutations were identified in 15% and 25% of assessable cases. None of the biomarkers was significantly associated with pts characteristics (gender, histology, smoking status), nor with RR or DCR. GART- pts experienced longer PFS when compared with the GART+ group (HR 0.56, p=.052), while no difference was observed according to TS, DHFR and AICAR status. A significantly longer OS was observed for TS- (HR 0.52, p=.012), GART- (HR 0.50, p=.037) and AICAR- (HR 0.40, p=.028) pts. No significant difference in the distribution of EGFR mutant pts receiving EGFR TKIs after pem failure was observed between IHC+ and IHC- groups. Conclusions: Low TS, GART and AICAR protein expression predicts significantly prolonged survival in single agent pem-treated pts with advanced NSCLC. The lack of PFS difference according to TS and AICAR status suggests prognostic rather than predictive relevance for these biomarkers.

Thymidylate synthase (TS) gene expression in patients with ALK positive (+) non-small cell lung cancer (NSCLC): Implications for therapy.

7582 Background: ALK+ NSCLC represents a molecular target-defined patient population highly responsive to the ALK inhibitor crizotinib. Previous reports have also suggested increased sensitivity of ALK+ NSCLC to the chemotherapeutic agent pemetrexed. Thymidylate synthase (TS) is a candidate predictive biomarker for pemetrexed activity. Here we report analysis of the Response Genetics Inc. (RGI) database for this association and implications for therapy. Methods: ALK fusion was identified by a novel RT-PCR assay (Danenberg et al: ASCO 2010). For TS, RNA from microdissected formalin-fixed paraffin-embedded tumors was analyzed as previously described, reported as the ratio of gene expression to β-actin. For reference, a TS level <2.33 is the cutpoint for sensitivity. Results: TS levels were available from 63 ALK+ patients and 1,698 ALK- control lung adenocarcinoma patients. All ALK+ patients had adenocarcinomas without EGFR or KRAS mutations. Median age: 59.0 (range 33-88), gender (male/female) 32/31 (51%/49%). Median TS RNA level in ALK+ patients was 2.02, range (0.55-19.44), and in ALK- patients was 3.32 (0.36-53.51), p<0.0001 (Mann-Whitney test). The majority of ALK+ patients (N=43, 68%) had a TS level <2.33 cutpoint, compared to only 32% of ALK- patients (N=551, p<0.0001). Conclusions: This analysis demonstrates relatively low TS gene expression in ALK+ patient tumors as determined by RT-PCR. These data provide a mechanism of action supportive of pemetrexed sensitivity for ALK+ NSCLC. [Table: see text]

Abstract 1713: Folylpoly-α-glutamate synthetase and thymidylate synthase are associated with clinical outcome from pemetrexed-based therapy in advanced non-small cell lung cancer (NSCLC)

Abstract Background: The antifolate pemetrexed targets multiple enzymes involved in pyrimidine and purine synthesis including thymidylate synthase (TS). After entry into cells, pemetrexed is converted to polyglutamated forms by folylpoly-α-glutamate synthetase (FPGS), a critical step to achieve full target inhibition. We hypothesized that FPGS and TS protein expression may predict outcome following pemetrexed-treatment of patients with advanced NSCLC, like in malignant pleural mesotheliomas (Christoph et al., J Clin Oncol. 2011: 29 suppl.). This is the largest report on pemetrexed-treatment outcome based on TS and FPGS in Caucasian patients with advanced NSCLC. Methods: Pretreatment tumor samples from 161 patients (pts) with metastatic NSCLC, treated with PMX (82 pts (51%)), a combination of PMX with platinum (74 pts (46%)) or within other combinations (5 pts (3%)), were retrospectively analyzed. FPGS and TS protein expression levels were evaluated by IHC using the H-Scoring system (0-300), which relies on the product of intensity (range 0 to 3) and the percentage of positive tumor cells (0-100%). Radiographic evaluation of response was performed according to RECIST criteria (version 1.1). Results: Median pretreatment H-scores were 180 for FPGS (range: 0-280) and 205 (range: 120-290) for TS. Using the log-rank test and the median H-score as cut-off, we found a significant association between low TS protein expression and improved progression-free survival (PFS) (median PFS of 5.5 months vs 3.4 months; hazard ratio [HR] 0.66, 95% CI, 0.45 to 0.96; P=0.03) or prolonged overall survival (median OS of 33.9 months vs 15.0 months; hazard ratio [HR] 0.52, 95% CI, 0.31 to 0.86; P=0.01). Moreover, high FPGS protein expression was only associated with better PFS (median PFS of 5.5 months vs 3.4 months; hazard ratio [HR] 0.58, 95% CI, 0.37 to 0.89; P=0.03). Considering exclusively patients suffering from adenocarcinomas (110 pts (68%)), TS was associated with objective response to pemetrexed-based treatment (mean H-score 192 for responders vs 210 for non-responders, P=0.03). Conclusions: We have investigated FPGS and TS protein expressions in tumor specimens from the largest series of PMX-treated Caucasian NSCLC patients. Baseline determination of TS and FPGS expression by IHC using the H-score system is associated with clinical outcome from PMX-based therapy in advanced NSCLC. Further prospective validation studies are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1713. doi:1538-7445.AM2012-1713

Abstract 5420: The effect of folate deficiency on chemosensitivity of colon cancer cells to 5-Fluorouracil

Abstract Background: Folate is an essential cofactor in one-carbon transfer reactions including nucleotide biosynthesis, thereby playing an important role in DNA replication and repair. In cancer cells, folate depletion interrupts DNA synthesis, thereby causing cancer cell death. This has been the basis for chemotherapy using antifolates and 5-Fluorouracil (5FU). We determined the effect of folate deficiency on the sensitivity of colon cancer cells to 5FU in a well established in vitro model of folate deficiency. Methods: Three human colon cancer cell lines (HCT116, Caco-2, HT29) were grown in RPMI1640 medium containing 0 (deficient) and 2.3 μM (replete) of folic acid. Proliferation was measured with growth curves every 3 days for 12 days and intracellular folate concentrations were determined at day 0, 6 and 12. Functional folate depletion was determined by the deoxyuridine suppression test. In vitro chemosensitivity of these colon cancer cell lines to 5FU with leucovorin (LV; 5 μM) was determined using a modified SRB protein assay. In HCT116 and Caco-2, thymidylate synthase (TS) activity was measured and TS, p53 and p21 protein expression was determined by immunoblotting. Results: All three colon cancer cell lines grown in the folate-deficient medium exhibited significantly slower growth rates than the corresponding cell lines grown in the folate-replete medium (p<0.05). Intracellular folate concentrations were significantly lower in all three cell lines grown in the folate-deficient medium than those grown in the folate-replete medium (p<0.05). Intracellular folate depletion induced in these cells was functionally significant as indicated by the deoxyuridine suppression test (p<0.05). All cells grown in the folate-deficient medium showed progressively lower intracellular folate concentrations over time (p<0.05). All three cell lines grown in the folate-deficient medium exhibited significantly higher chemosensitivity to 5FU+LV, albeit to a different degree, compared with those grown in the folate-replete medium (p<0.05). Caco-2, but not HCT116, cells grown in the folate-deficient medium showed significantly lower TS activity (p<0.05). Caco-2 cells grown in the folate-deficient medium had significantly lower protein expression of p53 and p21 but not TS. No differences in protein expression of TS, p53, and p21 were observed in HCT116 cells. Conclusion: Our data suggest that folate deficiency significantly enhances the sensitivity of colon cancer cells to 5FU based chemotherapy. Dietary or other strategies to deplete intracellular folate concentrations in colon cancer cells to enhance chemosensitivity to 5FU are worthy of further investigation. Given the dramatically increased dietary intake and blood levels of folate in North America resulting from folic acid fortification and supplementation, whether or not high folate status would affect chemotherapy for colon cancer also warrants future studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5420. doi:1538-7445.AM2012-5420

Assessment of thymidylate synthase expression in biopsy specimens and corresponding resection specimens of non‐small‐cell lung cancer

Pemetrexed (Pem) is a potent inhibitor of thymidylate synthase (TS), and has shown efficacy in the treatment of non-small-cell lung cancer (NSCLC). TS expression in NSCLC biopsy specimens may correlate with the tumour responses to TS-inhibiting agents. As TS is not homogeneously expressed, our aim was to test whether TS expression in biopsy specimens may be representative for the whole tumour and can be used for therapeutic assessment. We immunohistochemically analysed TS expression in biopsy and corresponding resection specimens of 100 consecutive NSCLCs. The samples were subgrouped according to TS expression levels, and the degree of agreement between the biopsy and the corresponding resection specimen was calculated. TS expression-based grouping according to one cut-off criterion (high or low expression) led to concordant results between biopsy and resection specimens. When more than one cut-off criterion was used, the results were significantly different. Thymidylate synthase expression levels in NSCLC biopsy specimens may correspond to TS expression of the whole tumour when robust scoring systems are applied. Further studies are needed to determine whether high or low TS expression in NSCLC biopsy specimens is of predictive value, and whether it may allow the selection of patients who will benefit from Pem treatment.

Abstract B6: Folylpoly-glutamate synthetase and thymidylate synthase are associated with clinical outcome from pemetrexed-based therapy in non-small cell lung cancer (NSCLC)

Abstract Background: The antifolate pemetrexed targets multiple enzymes involved in pyrimidine and purine synthesis including thymidylate synthase (TS). After entry into cells, pemetrexed is converted to polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS), a critical step to achieve full target inhibition. We hypothesized that FPGS and TS protein expression may predict outcome following pemetrexed-treatment of patients with advanced NSCLC, like in malignant pleural mesotheliomas (Christoph et al., J Clin Oncol. 2011: 29 suppl.). This is the largest report on pemetrexed-treatment outcome based on TS and FPGS in Caucasian patients with advanced NSCLC. Methods: Pretreatment tumor samples from 161 patients (pts) with metastatic NSCLC, treated with pemetrexed combined with platinum (74 pts (46%)) or as single agent (82 pts (51%)) or within other combinations (5 pts (3%)), were retrospectively analyzed. FPGS and TS protein expression levels were evaluated by IHC using the H-Scoring system (0–300), which relies on the product of intensity (range 0 to 3) and the percentage of positive tumor cells (0–100%). Radiographic evaluation of response was performed according to RECIST criteria (version 1.1). Results: Median pretreatment H-scores were 180 for FPGS (range: 0–280) and 205 (range: 120–290) for TS. Using the log-rank test and the median H-score as cut-off, we found a significant association between low TS protein expression and improved progression-free survival (PFS) (median PFS of 5.5 months vs. 3.4 months; hazard ratio [HR] 0.66, 95% CI, 0.45 to 0.96; P=0.03) or prolonged overall survival (median OS of 33.9 months vs. 15.0 months; hazard ratio [HR] 0.52, 95% CI, 0.31 to 0.86; P=0.01). Moreover, high FPGS protein expression was only associated with better PFS (median PFS of 5.5 months vs. 3.4 months; hazard ratio [HR] 0.58, 95% CI, 0.37 to 0.89; P=0.03). Considering exclusively patients suffering from adenocarcinomas (110 pts (68%)), TS was associated with objective response to pemetrexed-based treatment (mean H-score 192 for responders vs. 210 for non-responders, P=0.03). Conclusions: We have investigated FPGS and TS protein expressions in tumor specimens from the largest series of pemetrexed-treated Caucasian NSCLC patients. Baseline determination of TS and FPGS expression by IHC using the H-score system is associated with clinical outcome from pemetrexed-based therapy in advanced NSCLC. Further prospective validation studies are warranted.

Role of thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expressions in gallbladder carcinoma

Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are important enzymes in the metabolism of 5-fluorouracil, which have been examined as possible predictive markers. We conducted this study to clarify the role of TS and DPD expressions in gallbladder carcinoma (GBC). The subjects were 28 patients who underwent surgical resection of GBC. We examined intratumoral TS and DPD mRNA expressions, using the Danenberg tumor profile method. The expression levels were classified into two groups, based on median values. Clinicopathological variables, including prognosis, were then compared between the high and low expression groups. There was a significant difference in the incidence of lymph node metastasis between the high and low TS expression groups. The incidence of advanced clinical stage was higher in the low TS expression group than in the high TS expression group. However, no clear correlation was observed between the DPD mRNA expression and any clinicopathological variable. There was no significant difference in the postoperative survival rates between the groups, in accordance with the expression of TS or DPD genes. Low TS mRNA was correlated with a high incidence of lymph node metastasis and advanced clinical stage. Therefore, TS gene expression may help identify patients at increased risk of the progression of GBC.

Expressions of thymidylate synthase, thymidine phosphorylase, class III β-tubulin, and excision repair cross-complementing group 1 predict response in advanced gastric cancer patients receiving capecitabine plus paclitaxel or cisplatin

Toevaluate the role of class III β-tubulin (TUBB3), thymidylate synthase (TS), thymidine phosphorylase (TP), and excision repair cross-complementing group 1 (ERCC1) in clinical outcome of advanced gastric cancer patients receiving capecitabine plus paclitaxel or cisplatin. The clinical data and tumor specimens from 57 advanced gastric cancer patients receiving first-line capecitabine plus paclitaxel (cohort 1, n=36) and capecitabine plus cisplatin (cohort 2, n=21) were retrospectively collected, and TUBB3, TS, TP, and ERCC1 expressions were detected by real-time quantitative PCR. The associations between expressions of biomarkers and response or survival were analyzed statistically. The median age of 57 patients was 57 years (range: 27-75 years) with 38 males and 19 females. Of all patients, the response rates of patients with high TP, low TP and high TS, low TS expressions were 57.1%, 27.6% (P=0.024), and 55.2%, 28.6% (P=0.042), respectively. Among cohort 1, the response rates and median overall survivals of patients with low and high TUBB3 expressions were 61.1% vs. 33.3% (P=0.095) and 13.8 months vs. 6.6 months (P=0.019), respectively; the response rate (87.5%) of patients with low TUBB3 and high TP expressions was higher than that (14.3%) of patients with high TUBB3 and low TP expressions (P=0.01). Among cohort 2, the response rates of patients with low ERCC1 and high ERCC1 expressions were 45.5% and 20.0% respectively (P=0.361). TUBB3, TS and TP expressions could predict the response of advanced gastric cancer patients receiving capecitabine-based and paclitaxel-based chemotherapy. These results will be further confirmed in future large samples.

Response prediction in metastasised colorectal cancer using intratumoural thymidylate synthase: Results of a randomised multicentre trial

BackgroundMolecular markers to predict response to 5-fluorouracil (FU)-based treatment of recurrent or metastasised colorectal cancer (mCRC) are not established. The aim of this trial was to determine the value of thymidylate synthase (TS), a key enzyme of DNA synthesis and target of 5-FU, to predict response to chemotherapy of mCRC. MethodsTumour tissue was obtained from 168 patients with mCRC for relative thymidylate synthase (TS) mRNA quantitation. Patients were randomised to receive either 5-FU/folinic acid (FA, FUFA) alone or in combination with irinotecan 5-fluorouracil/folinic acid and irinotecan (FOLFIRI) stratified by TS (low versus high). Primary end-point was overall response to first-line treatment among TS high patients. All parties, except for the randomisation centre, were blinded for TS status. ResultsBiopsies (n=168) were taken without complications. TS levels were available for 147 patients (87.5%). Analysing response to FUFA and FOLFIRI in the per protocol set (n=119) after un-blinding TS in the data base revealed a trend to better overall response to FOLFIRI (9/19, 47%) in TS high compared to FUFA (5/23, 22%, p=0.077). In patients with biopsies taken from liver lesions (n=91) overall response to FOLFIRI and FUFA in TS high was 53% (9/17) and 18% (3/17), respectively (p=0.035). In patients with low TS, no remarkable difference in overall response to FOLFIRI and FUFA was observed. ConclusionsTaking a pre-treatment biopsy is a safe and feasible procedure in mCRC. After validation of our data in a larger group TS determination may have the potential to better help direct systemic treatment in patients with primarily non-resectable mCRC.

Successful Long-Term Treatment With Pemetrexed of NSCLC Associated With EML4-ALK and Low Thymidylate Synthase Expression

Fusion between echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes has recently been identified in non–small cell lung cancer (NSCLC). The dual MET-ALK inhibitor crizotinib has shown promising activity in patients whose tumors harbor this oncogene,1 but it has remained unclear whether such patients manifest similar sensitivity to cytotoxic chemotherapy. Two recent retrospective studies have suggested that EML4-ALK–positive patients may have a superior progression-free survival (PFS) with treatment using pemetrexed-based therapies.

Predictive values of 5-fluorouracil pathway genes for S-1 treatment in patients with advanced gastric cancer

Determination of significant associations between gene expression and predefined endpoints might improve treatment tailoring for advanced gastric cancer. We investigated the mRNA expression of 5-fluorouracil (5-FU) pathway genes in prechemotherapeutic tumor samples of primary gastric cancer to try to predict the treatment outcome of S-1 monotherapy. 5-FU pathway genes, dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), thymidylate synthase (TS), and thymidine phosphorylase (TP), were analyzed using quantitative real-time PCR of RNA extracted from archived formalin-fixed paraffin-embedded tissues. We selected the median value for each gene as a cutoff to separate patients into high and low gene expression groups. High OPRT gene expression was significantly associated with tumor response (P = 0.014). In a combined analysis including OPRT, patients with high OPRT and TP showed a higher overall response rate than did the remaining patients (40 vs. 10%, respectively; P = 0.002). For survival, patients with high OPRT and low TS levels showed prolonged survival in both progression-free survival (3.4 vs. 2.4 months, P = 0.024) and overall survival (11.0 vs. 8.2 months, P = 0.007). In a multivariate analysis, the combinations of OPRT and TP for response and OPRT and TS for both progression-free survival and overall survival were independent variables. To conclude, mRNA expression levels of molecular markers in formalin-fixed paraffin-embedded specimens of primary gastric tumors can be useful for identifying patients with advanced gastric cancer who would most likely benefit from S-1 treatment.

Significance of Thymidylate Synthase and Thyroid Transcription Factor 1 Expression in Patients with Nonsquamous Non-small Cell Lung Cancer Treated with Pemetrexed-Based Chemotherapy

This study is to evaluate whether thymidylate synthase (TS) or thyroid transcription factor 1 (TTF1) protein expression can predict clinical outcomes for pemetrexed-based chemotherapy in patients with nonsquamous non-small cell lung cancer (NSCLC). Two hundred eighty-five consecutive patients with nonsquamous NSCLC treated with pemetrexed-based chemotherapy were immunohistochemically analyzed for the expressions of TS and TTF1. TS and TTF1 expression were successfully analyzed in 193 and 284 cases, respectively. Tumors with TS-negativity or TTF1-positivity were more frequent in patients who were female, younger, had adenocarcinoma, or had never smoked. Higher response rates for pemetrexed-based chemotherapy were associated with TS-negativity (33.7% versus 14.1%, p = 0.002) and TTF1-positivity (28.1% versus 9.8%, p < 0.001). In univariate analysis, progression-free survival for pemetrexed-based chemotherapy was significantly longer in groups with adenocarcinoma (2.9 versus 1.4 months, p = 0.001), TS-negativity (4.1 versus 2.0 months, p = 0.001), and TTF1-positivity (3.8 versus 1.3 months, p < 0.001). In multivariate analysis, TS-negativity (hazard ratio [HR] = 0.70; 95% confidence interval [CI], 0.51-0.97) and TTF1-positivity (HR = 0.51; 95% CI, 0.35-0.73) were associated with longer progression-free survival. Patients with TTF1-positive tumors also had significantly longer overall survival times than patients with TTF1-negative tumors (25.4 versus 14.2 months, HR = 0.55; 95% CI, 0.39-0.77). Low TS or high TTF1 protein expression was significantly associated with better clinical outcomes in nonsquamous NSCLC patients who were treated with pemetrexed-based chemotherapy. The predictive role of TS or TTF1 expression should be further validated in a prospective randomized study.

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

4624 Background: Some reports have shown that the second-line maximum androgen blockade with an alternative antiandrogen (2nd MAB) is still effective. However 2ndMAB has relatively low PSA response rate. We sought to determine whether the combination therapy of 2nd MAB and UFT would be even more effective and to correlate the treatment efficacy with mRNA expressions of the enzymes that are related to 5-FU metabolism. Methods: Patients who were histologically confirmed with prostate cancer and who relapsed after initial hormonal therapy were included in this study. All patients were randomly allocated into two groups, 2nd MAB (group A) or 2nd MAB combined with UFT (group B). mRNA expressions of four enzymes including thymidylate synthase (TS), dihydropyrimidine dehydrogenase, orotate phosphoribosyl transferase, thymidine phosphorylase in prostate cancer cells obtained by laser capture microdissection were measured by real time RT-PCR. The primary endpoint was PSA response rate as defined as ≥ 50% decrease from the baseline. The secondary endpoints included progression-free survival (PFS), adverse events (AEs) and the correlation of treatment efficacy with mRNA expressions of the four enzymes. Results: Fifty two patients were enrolled in this study. Median age was 77 (47-92) years old. PSA response rate in group B (61.5%) tended to be higher than that in group A (34.6%) (p=0.095). The median percent PSA changes from baseline at 12 weeks were -8.4% in group A and -70.2% in group B. Group B (15.9 months) had significantly longer median PFS than group A (6.4 months) (p=0.0137). Prostate biopsy specimens were available in 49 patients for mRNA measurement. In group B, those with low TS (81.8%) tended to have higher PSA response rate than those with high TS (46.2%). In addition, among the patients with low TS, group B had significantly longer PFS than group A (p=0.0175). There were no severe AEs in both groups. Conclusions: After relapse with initial hormonal therapy, an alternative antiandrogen (2nd MAB) along with UFT is an effective and well-tolerated treatment option.

Significance of thymidylate synthase and thyroid transcription factor 1 expression in patients with nonsquamous non-small cell lung cancer treated with pemetrexed-based chemotherapy.

7579 Background: To evaluate whether thymidylate synthase (TS) or thyroid transcription factor 1 (TTF1) protein expression can predict clinical outcome for pemetrexed-based chemotherapy in patients with nonsquamous non-small cell lung cancer (NSCLC). Methods: Two hundred eighty-five consecutive patients with nonsquamous NSCLC treated with pemetrexed-based chemotherapy were immunohistochemically analyzed for the expression of TS and TTF1. Results: TS and TTF1 expression were successfully analyzed in 193 and 284 cases, respectively. Tumors with TS-negativity or TTF1-positivity was more frequent in groups of female, younger age, adenocarcinoma, or never-smoker. Higher response rates for pemetrexed-based chemotherapy were associated with TS-negativity (33.7% vs. 14.1%, P = 0.002) and TTF1-positivity (28.1% vs. 9.8%, P < 0.001), respectively. In univariate analysis, progression-free survival (PFS) for pemetrexed-based chemotherapy was significantly longer in groups of adenocarcinoma (2.9 vs. 1.4 months, P = 0.001), TS-negativity (4.1 vs 2.0 months, P = 0.001), and TTF1-positivity (3.9 vs. 1.3 months, P < 0.001). In multivariate analysis, TS-negativity (HR = 0.69; 95% CI, 0.50–0.96) and TTF1-positivity (HR = 0.51; 95% CI, 0.35–0.73) were associated with longer PFS, respectively. Patients with TTF1-positive tumors also had significantly longer overall survival time than patients with TTF1-negative tumors (25.4 vs. 14.2 months, HR = 0.55; 95% CI, 0.39–0.77). Conclusions: Low TS or high TTF1 protein expression was significantly associated with better clinical outcomes in nonsquamous NSCLC patients who were treated with pemetrexed-based chemotherapy. The predictive role of TS or TTF1 expression is required to be further validated in a prospective randomized study.

Predictive value of thymidylate synthase and folylpoly-glutamate synthetase for clinical benefit from pemetrexed in malignant pleural mesothelioma.

7024 Background: The antifolate pemetrexed (PMX) targets multiple enzymes involved in pyrimidine and purine synthesis including thymidylate synthase (TS). After entry into cells, PMX is converted to more potent polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS), a critical step to achieve full target inhibition. We hypothesized that FPGS and TS protein expression may predict response and outcome following PMX treatment of patients (pts) with malignant pleural mesothelioma (MPM). Methods: Pre-treatment tumor samples from 64 pts with MPM, treated with PMX combined with platinum (58/64) or as single agent (6/64), were retrospectively analyzed. TS and FPGS protein expression levels were evaluated by immunohistochemistry using the H-Scoring system (ranging from 0 to 300), which relies on the product of intensity of specific tumor cell immunoreactivity (range 0 to 3) and the percentage of positive tumor cells. Radiologic evaluation of response was performed according to RECIST. Results: Median pre-treatment H-scores were 220 (range: 50-300) for TS and 240 for FPGS (range: 110-300). Using the log-rank test and the median H-score as cut-off, we found a significant association of improved progression-free survival (PFS) with low TS protein expression (median PFS of 223 vs 181 days; hazard ratio [HR] 0.574, 95% CI, 0.330 to 0.997; P=0.049). High FPGS protein expression was associated with objective response (mean H-score 254 for responders vs 233 for non-responders, P=0.026) as well with absolute clinical benefit meaning objective response and disease control (mean H-score 248 for responders and stable disease pts vs 220 for progressive disease pts, P=0.005). We found an association of high FPGS protein expression with prolonged PFS (median PFS of 199 vs 97 days; HR 0.487, 95% CI, 0.093 to 1.068; P=0.013), additionally. Conclusions: This is the first report of FPGS examination in MPM. Based on our retrospective study, base line determination of TS and FPGS expression may be predictive for clinical benefit of PMX-based therapy in MPM. Confirmation of the prognostic and predictive value of TS and FPGS expression in MPM by prospective studies is warranted.

Abstract 5041: Thymidylate synthase (TS) in malignant pleural mesothelioma (MPM): Correlation with clinical outcome and pharmacological role in the synergistic interaction of vandetanib with pemetrexed/carboplatin combination

Abstract Pemetrexed improves the effect of platinum-compounds against MPM, but biomarkers of response and novel effective combinations are warranted. The present study evaluated the 1) correlation between expression and polymorphisms of pemetrexed's key target TS and outcome of MPM patients treated upfront with carboplatin/pemetrexed, and 2) pharmacological interaction of new targeted compounds (gefitinib, erlotinib, sorafenib, vandetanib, and enzastaurin) with carboplatin-pemetrexed in MPM cell lines. Analysis of TS TSER-2R/3R, mRNA and protein expression was performed by PCR and immunohistochemistry (using H-score) in tumors from 99 patients. The role of TS and other molecular determinants in the interaction of carboplatin, pemetrexed with new targeted compounds was investigated in the H2052, H2452, H28 and MSTO-211H cells. Drug interaction was studied using MTT and SRB assays and evaluated with combination index method. EGFR, Akt and Erk phosphorylation, as well as VEGF secretion, were analyzed with ELISA, whereas RT-PCR and western blot were performed to assess modulation of the expression of TS, E2F-1 and genes involved in DNA repair (ERCC1 and XPD). A significant correlation between low TS protein expression and response (odd ratio, 4.2; p=0.023), longer PFS (8vs6 months; hazard ratio [HR]:0.60: p=0.023), or OS (18vs9 months; HR:0.59; p=0.029) was found when patients were categorized according to median H-score. Similarly, patients with TS mRNA below the median had longer PFS and OS (p&amp;lt;0.001). The higher tertile of TS mRNA expression also correlated with higher risk of progressive disease (OR:2.5; p=0.044). At multivariate analysis, TS mRNA level and H-score confirmed their independent prognostic role for PFS and OS. No correlations were observed for TSER polymorphisms. Vandetanib emerged as the targeted compound with the most potent cell growth inhibitory effects, and interacted synergistically with carboplatin and pemetrexed in all cell lines, increasing apoptotic indices. Pemetrexed enhanced EGFR phosphorylation, but reduced Akt phosphorylation and ERCC1 and XPD expression. Conversely, vandetanib significantly downregulated EGFR, Erk and Akt phosphorylation, as well as E2F-1 and TS expression (eg, H28 cells had 4.8-fold TS mRNA decrease). In conclusion, low TS protein and mRNA levels were associated to response, longer PFS and OS. Furthermore, vandetanib improved pemetrexed-carboplatin activity against MPM cells, through modulation of critical molecular mechanisms, such as EGFR-Akt phosphorylation and TS expression. These data support further prospective trials for the validation of the prognostic/predictive role of TS in patients treated with pemetrexed-based regimens, as well as future studies on the integration of vandetanib in the treatment of MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5041. doi:10.1158/1538-7445.AM2011-5041

Thymidylate Synthase and Excision Repair Cross-Complementing Group-1 as Predictors of Responsiveness in Mesothelioma Patients Treated with Pemetrexed/Carboplatin

The pemetrexed/platinum agent combination represents the standard of care in first-line treatment for malignant pleural mesothelioma (MPM). However, there are no established indicators of responsiveness that can be used to optimize the treatment. This retrospective study aimed to assess the role of excision repair cross-complementing group-1 (ERCC1) and thymidylate synthase (TS) in tumors, and correlate expression levels and polymorphisms of these key determinants of drug activity with the outcome of MPM patients treated with carboplatin/pemetrexed in first-line setting. Analysis of TS and ERCC1 polymorphisms, mRNA and protein expression was done by PCR and immunohistochemistry [with the H-score (histologic score)] in tumor specimens from 126 MPM patients, including 99 carboplatin-/pemetrexed-treated patients. A significant correlation between low TS protein expression and disease control (DC) to carboplatin/pemetrexed therapy (P = 0.027), longer progression-free survival (PFS; P = 0.017), and longer overall survival (OS; P = 0.022) was found when patients were categorized according to median H-score. However, patients with the higher tertile of TS mRNA expression correlated with higher risk of developing progressive disease (P = 0.022), shorter PFS (P < 0.001), and shorter OS (P < 0.001). At multivariate analysis, the higher tertile of TS mRNA level and TS H-score confirmed their independent prognostic role for DC, PFS, and OS. No significant associations were found among ERCC1 protein expression, TS and ERCC1 polymorphisms, and clinical outcome. In our series of carboplatin-/pemetrexed-treated MPM patients, low TS protein and mRNA levels were significantly associated to DC, improved PFS, and OS. Prospective trials for the validation of the prognostic/predictive role of TS in MPM patients treated with pemetrexed-based regimens are warranted.

1284 LOW DOSE DOCETAXEL ENHANCES THE SENSITIVITY OF S-1 IN A XENOGRAFT MODEL OF HUMAN CASTRATION RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111284 LOW DOSE DOCETAXEL ENHANCES THE SENSITIVITY OF S-1 IN A XENOGRAFT MODEL OF HUMAN CASTRATION RESISTANT PROSTATE CANCER Masanori Hasegawa, Akira Miyajima, Takeo Kosaka, Yota Yasumizu, Nobuyuki Tanaka, Takahiro Maeda, Suguru Shirotake, Hiroki Ide, Eiji Kikuchi, and Mototsugu Oya Masanori HasegawaMasanori Hasegawa Tokyo, Japan More articles by this author , Akira MiyajimaAkira Miyajima Tokyo, Japan More articles by this author , Takeo KosakaTakeo Kosaka Tokyo, Japan More articles by this author , Yota YasumizuYota Yasumizu Tokyo, Japan More articles by this author , Nobuyuki TanakaNobuyuki Tanaka Tokyo, Japan More articles by this author , Takahiro MaedaTakahiro Maeda Tokyo, Japan More articles by this author , Suguru ShirotakeSuguru Shirotake Tokyo, Japan More articles by this author , Hiroki IdeHiroki Ide Tokyo, Japan More articles by this author , Eiji KikuchiEiji Kikuchi Tokyo, Japan More articles by this author , and Mototsugu OyaMototsugu Oya Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.970AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES No standard therapy is currently available for castration resistant prostate cancer (CRPC) patients who have disease progression during docetaxel (DOC) treatment. This study focused on S-1, oral 5-fluorouracil (5-FU)-based antitumor drug which demonstrated high maximum plasma 5-FU levels with mild gastrointestinal toxicity. In addition, an oral formulation has resulted in an improvement in patient quality of life. S-1 is now used as a front-line chemotherapeutic agent for gastric cancer in Japan. The aims of the present study were to determine the efficacy of S-1 or S-1 combined with low dose DOC against CRPC cell line and to explore their clinical potential for treating CRPC patients. METHODS The androgen dependent prostate cancer (ADPC) cell line LNCaP and its hormone refractory sub-line C4-2, and a subcutaneous tumor xenograft model prepared with C4-2 in SCID mice were used. Specimens obtained from ADPC and CRPC patients were also evaluated. To investigate mechanisms potentially underlying the synergistic inhibition of cancer growth, we examined thymidylate synthase (TS), a target of 5-FU. A high level of TS is thought to be related to resistance against 5-FU chemotherapy. RESULTS The CRPC specimens and C4-2 showed significantly lower TS expression than the ADPC specimens and LNCaP. In vivo, daily oral administration of S-1 (3 mg/kg, adjusted for human dose) significantly suppressed tumor growth. Although a single intraperitoneal injection of low dose DOC (2 mg/kg) did not show tumor suppression, low dose DOC enhanced anti-tumor effect of S-1. In vitro, low dose DOC, which did not induce G2/M arrest, increased p53 and p21, suppressed CDK4 activity, and resulted in down regulation of TS in C4-2. 5-FU enhanced TS expression, and combined with low dose DOC suppressed the TS which was enhanced by 5-FU. In addition, we also confirmed in vivo using immunostaining that S-1 enhanced TS expression, low dose DOC suppressed the expression of TS, and combined S-1/DOC suppressed the TS, which was enhanced by S-1. CONCLUSIONS The present findings indicate that CRPC patients with androgen ablation may be good candidates for 5-FU based chemotherapy, and these regimens have attractive therapeutic potential for clinical practice, and they may have a significant impact on therapeutic options. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e514 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masanori Hasegawa Tokyo, Japan More articles by this author Akira Miyajima Tokyo, Japan More articles by this author Takeo Kosaka Tokyo, Japan More articles by this author Yota Yasumizu Tokyo, Japan More articles by this author Nobuyuki Tanaka Tokyo, Japan More articles by this author Takahiro Maeda Tokyo, Japan More articles by this author Suguru Shirotake Tokyo, Japan More articles by this author Hiroki Ide Tokyo, Japan More articles by this author Eiji Kikuchi Tokyo, Japan More articles by this author Mototsugu Oya Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Thymidylate synthase and dihydrofolate reductase expression in non-small cell lung carcinoma: The association with treatment efficacy of pemetrexed

Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are target enzymes of inhibition by pemetrexed, an antifolate for treatment of advanced non-small-cell lung cancer (NSCLC). This study is to evaluate the association of TS and DHFR expressions and the treatment efficacy of pemetrexed in NSCLC patients. From January 2006 to October 2008, patients with advanced NSCLC treated with pemetrexed after prior chemotherapy were included. The TS and DHFR expressions in tumor tissues were examined by immunohistochemistry and evaluated by a semiquantitative histologic score ( H-score). The H-score was derived from the degrees of intensity of tumor cells multiplied by the percentage of positive neoplastic cells. The medical records were reviewed and analyzed with respect to patients’ characteristics, histology types, treatment responses and survivals. Among 268 NSCLC patients treated with pemetrexed, 49 had tumor specimens available for TS and DHFR evaluation. The TS expression was positively correlated with DHFR expression ( r 2 = 0.11, p = 0.02). Patients with low TS (≤150) expression had a longer median progression-free survival (PFS) than those with high TS (>150) expression (4.8 vs. 3.4 months; p = 0.01). Patients with low DHFR expression (≤120) also had a longer median PFS than patients with high DHFR expression (>120), which was not statistically significant (5.8 vs. 3.6 months; p = 0.33). In patients with adenocarcinoma, the low TS patient group also had a longer median PFS and a longer median overall survival (OS) as compared with patients with high TS expression (PFS, 4.8 vs. 3.8 months, p = 0.03; OS, 21.4 vs. 10.0 months, p = 0.03). Nevertheless, the association of DHFR expression level and median PFS as well as OS were not statistically significant. TS expression, rather than DHFR, may be an important predictive factor for treatment efficacy of pemetrexed in NSCLC patients.

Impact of intratumoral thymidylate synthase expression on prognosis after surgical resection for ampullary carcinoma

This study evaluated possible prognostic value of expression level of intratumoral thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT) in patients with resected ampullary carcinoma. Immunohistochemical staining evaluated intratumoral TS, DPD, and OPRT expression in 52 surgical specimens of patients with ampullary carcinoma. Relationships between clinicopathological factors including intratumoral TS, DPD, or OPRT expressions and survival was evaluated by univariate and multivariate analysis. High intratumoral DPD, OPRT, and TS expressions were found in 19 (37%), 30 (58%), and 23 (44%) of cases, respectively. Patients with high TS expression had more advanced-stage disease than those with low TS expression. Univariate analysis revealed that gender, perineural invasion, lymph node status, UICC pT factor, UICC stage, OPRT expression, and TS expression correlated significantly (P < 0.05) with disease-specific survival. Only perineural invasion (P = 0.043) and TS expression (P = 0.019) were independent prognostic factors of disease-specific survival by multivariate survival analysis. Five-year disease-specific survival rates of patients with high and low intratumoral TS expression were 47% and 91%, respectively. Intratumoral TS expression level may be a potent predictor of prognosis after surgical resection for ampullary carcinoma.