High Thymidylate Synthase Expression Research Articles

Bax Predicts Outcome in Gastric Cancer Patients Treated with 5-fluorouracil, Leucovorin, and Oxaliplatin Palliative Chemotherapy

Platinum and 5-fluorouracil (5-FU)-based regimens have been used the most frequently in palliative chemotherapy for gastric cancer. The present study evaluated the prognostic significance of Bax, excision repair cross-complementation group 1 (ERCC1), and thymidylate synthase (TS) in advanced gastric cancer patients treated with 5-FU, leucovorin, and oxaliplatin (FOLFOX) palliative chemotherapy. Seventy-two patients with metastatic or recurrent gastric cancer were treated with FOLFOX regimen. Pretreatment tumor biopsy specimens were analyzed for Bax, ERCC1, and TS expression by immunohistochemistry. High expression of Bax, ERCC1, and TS was observed in 31 (43%), 33 (46%), and 35 (49%) patients, respectively. The median overall survival (OS) of patients was 12 months. Low expression of Bax was associated with poor OS (median, 9 months vs. 18 months; 2-year, 10% vs. 48%; p=0.0005) in univariate analysis, while expression of ERCC1 and TS was not correlated with patient outcome. In multivariate analysis, low expression of Bax was a significant independent predictor of poor OS (p=0.028). Low expression of Bax was significantly associated with poor survival of patients with metastatic or recurrent gastric cancer treated with FOLFOX chemotherapy. Immunohistochemical staining for Bax with pretreatment biopsy specimen may be useful in selecting FOLFOX regimen as a treatment option for advanced gastric cancer patients.

Expression of drug-resistance-related proteins in nasopharyngeal cancer (NPC) patients treated with cisplatin-based concurrent chemoradiotherapy.

5551 Background: We evaluated the prognostic significance of excision repair cross-complementation group 1 protein (ERCC1), thymidylate synthase (TS), and Bax in patients with nasopharyngeal cancer (NPC) treated with concurrent chemoradiotherapy (CCRT). Methods: Pretreatment tumor biopsy specimens from 41 patients with locally advanced NPC (stage I: 1, II: 10, III: 9, IV: 21 patients) were analyzed for ERCC1, TS and Bax expression by immunohistochemistry. All patients were treated with one cycle of induction chemotherapy (5-fluorouracil 1,000 mg/m2/day and cisplatin 20 mg/m2/day, days 1-4) followed by CCRT starting on day 22. CCRT consisted of radiotherapy (70Gy/35 fractions for 7 weeks) with cisplatin 20mg/m2/day for 4 days on weeks 1, 4, 7 of radiotherapy. Results: High expression of ERCC1, TS and low expression of Bax was observed in 25 (60%), 21 (51%) and 21 (51%) patients, respectively. High expression of ERCC1 was associated with WHO type 1 or 2 histology (p=0.045). Complete response was achieved in 34 patients (83%) after CCRT. With median follow up duration of 106 months (32-152 months) in survivors, 5-year overall survival (OS) of all patients was 53%. In univariate analysis, 5-year OS (75% versus 28%, p=0.005) was significantly inferior in patients with high expression of ERCC1, while high expression of TS and Bax were not correlated with patients outcome. In multivariate analysis, high expression of ERCC1 was a significant independent prognostic factor for poor OS (p=0.029) along with WHO type 1 or 2 histology. Conclusions: High expression of ERCC1 protein may be a useful prognostic factor for poor outcome in patients with locally advanced NPC treated with CCRT. 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

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 (TS) gene copy number in NSCLC.

e21063 Background: Studies suggest that high TS expression associates with poor prognosis and confers resistance to antifolates, particularly pemetrexed. TS is essential for de novo production of dTMP and for DNA synthesis. The aim of this study was to determine TS gene copy number in NSCLC, especially with regard to histology, and evaluate the prognostic impact of TS gene copy number. Methods: Using an experimental silver in situ hybridization assay (Ventana Medical Systems, Inc., Tucson, AZ), TS was evaluated on a tissue microarray containing triplicate samples from 189 patients (pts) who underwent surgery for NSCLC. Results were obtained for 124 pts (66%). This group included 83% males, 60% squamous cell carcinomas (SCC) and 25% adenocarcinomas (AC). The pathological stages were I: 39%, II: 25% and III: 30%, IV: 5% and unknown: 1%. A certified pathologist counted signals in 50 nuclei/core and determined the mean TS copies/nucleus/core. The average of the triplicate cores was compared to the highest core of same pt. The highest core was used for further analyses due to better relationship to parameters tested. Results: Median gene copy number in the population was 2.5 (1.4-9.6) while mean was 2.7 (± 1.0). Nine cases (7%) had ≥ 4 genes/nucleus and 1 had clusters. Gene copy number significantly differed (p = 0.04) by histology: AC median 2.2 (1.6-4.5) mean 2.5 (± 0.7); SCC 2.5 (1.4-9.6), 2.8 (± 1.2); NOS/other 2.9 (1.5-3.4), 2.7 (± 0.6). Using 2.5 as a cut-off, 32% of ADs, 50% of SCCs and 68%of NOS/others were positive. For TS ≥ 4, 7/9 pts were SCC and 2/9 AD. Kaplan-Meier analysis using median cut-off revealed no relationship between TS copy number and PFS (p = 0.45) or OS (p = 0.40). Similar results were obtained using adjusted Cox regression analysis and examining TS as a continuous variable; PFS (HR 0.96, p = 0.77) or OS (HR 0.94, p = 0.70). No relationship between TS and PFS (p = 0.56) or OS (p = 0.37) was found after stratification by histology (SCC vs. non-SCC). Conclusions: TS gene copy number is increased in primary tumors from NSCLC pts. Many SCC tumors had high TS gene copy number, but others did not. TS gene copy number is not associated with prognosis. Future studies should test whether TS gene copy number predicts clinical benefit from folate-targeted therapeutics, particularly pemetrexed. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ventana Medical Systems Roche Ventana Medical Systems Ventana Medical Systems, Roche

Thymidilate synthase expression predicts longer survival in patients with stage II colon cancer treated with 5-flurouracil independently of microsatellite instability

5-Fluorouracil (5-FU) is the most commonly used therapeutic agent for colon cancer treatment. Several studies have evaluated in patients with colon cancer, either the role of genes involved in the 5-FU pathway, such as thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) or the role of microsatellite instability (MSI) as prognostic or predictive markers for adjuvant chemotherapy efficacy, with discordant results. In this study we investigated the combined effect of TS, TP, DPD mRNA expression and MSI status in primary tumors of patients with colon cancer, all treated with 5-FU adjuvant therapy. TS, TP and DPD expression levels were investigated by real-time quantitative RT-PCR on RNA extracts from formalin-fixed and paraffin-embedded tissues of 55 patients with colon adenocarcinoma. In the same case study MSI status was assessed on DNA extracts. A higher TS expression was significantly associated with a longer survival for patients with cancers of stage II (P<0.01), but not for those with stage III (P=0.68). In addition, in multivariate analysis, a higher TS expression was significantly associated with a decreased risk of death (HR 0.13, 95% CI 0.03-0.59, P<0.01), while the MSI status did not have effects on patients' survival. This retrospective investigation suggests that TS gene expression at mRNA level can be a useful marker of better survival in patients (especially of those with cancers of stage II) receiving 5-FU adjuvant chemotherapy, independently of the MSI status.

Immunohistochemical characterization of pyrimidine synthetic enzymes, thymidine kinase-1 and thymidylate synthase, in various types of cancer

Thymidine kinase-1 (TK-1) and thymidylate synthase (TS) are key enzymes for salvage and de novo pyrimidine synthesis, respectively. Numerous studies have suggested that increased TS levels are associated closely with resistance to fluoropyrimidine-based chemotherapy. TAS-102 is a novel drug containing trifluorothymidine, which is phosphorylated by TK-1 to its active monophosphated form, that in turn can inhibit TS. TAS-102 has been shown to exhibit antitumor activity in fluoropyrimidine-resistant human cancer cells. TAS-102 is currently undergoing clinical trials for use in gastrointestinal cancers. In the present study, we used immunohistochemistry to investigate the expression of TK-1 and TS in various types of cancer. TK-1 and TS expression was markedly different between cancer types. High TK-1 expression was detected prominently in gastrointestinal adenocarcinomas and esophageal and uterine squamous cell carcinomas. Gastrointestinal adenocarcinomas and squamous cell uterine carcinomas were often accompanied by high TS expression, indicating activation of pyrimidine synthesis through both the salvage and de novo pathways. These results led us to consider that TAS-102 may also be effective for esophageal and uterine squamous cell carcinomas, as well as for gastrointestinal adenocarcinomas, even in fluoropyrimidine-resistant cases with high TS expression. In contrast, thyroid papillary carcinomas, lung adenocarcinomas, hepatocellular carcinomas, pancreatic ductal carcinomas, and renal cell carcinomas, which exhibit low TK-1 expression, may be resistant to TAS-102. In non-small cell lung cancers, high TK-1 expression was demonstrated in squamous cell carcinomas, but not in adenocarcinomas. This result suggests that TAS-102 efficacy and the pyrimidine synthetic pathway may differ depending on histological type. Our results indicate that administration of TAS-102 could be selected on the basis of the immunohistochemical evaluation of TK-1 and TS.

High expression of excision repair cross-complementation group1 protein predicts poor outcome in patients with nasopharyngeal cancer

We evaluated the prognostic significance of excision repair cross-complementation group 1 protein (ERCC1) and thymidylate synthase (TS) in patients with nasopharyngeal cancer (NPC) treated with concurrent chemoradiotherapy (CCRT). Pre-treatment tumor biopsy specimens from 41 patients with locally advanced NPC (stage I: 1, II: 10, III: 9, IV: 21 patients) were analyzed for ERCC1 and TS expression by immunohistochemistry. All patients were treated with one cycle of induction chemotherapy (5-fluorouracil 1000 mg/m(2)/day and cisplatin 20mg/m(2)/day, days 1-4) followed by CCRT starting on day 22. CCRT consisted of radiotherapy (70 Gy/35 fractions for 7 weeks) with cisplatin 20mg/m(2)/day for 4 days on weeks 1, 4, and 7 of radiotherapy. High expression of ERCC1 and TS was observed in 25 (60%) and 21 (51%) patients, respectively. High expression of ERCC1 was associated with WHO type 1 or 2 histology (p=0.045). With a median follow-up duration of 106 months (32-152 months) in survivors, the 5-year overall survival (OS) of all patients was 53%. In univariate analysis, 5-year OS (73% versus 39%, p=0.005) was significantly inferior in patients with high expression of ERCC1, while high expression of TS was not correlated with patient outcome. In multivariate analysis, high expression of ERCC1 was a significant independent prognostic factor for poor OS (p=0.029) along with WHO type 1 or 2 histology. High expression of ERCC1 protein may be a useful prognostic factor for poor outcome in patients with locally advanced NPC treated with CCRT.

Histological and genetic markers for non-small-cell lung cancer: Customizing treatment based on individual tumor biology

To describe how molecular and genetic markers influence the response to therapy in patients with advanced non-small-cell lung cancer (NSCLC). Cisplatin resistance has been associated with high rates of expression of endonucleases, a family of DNA repair proteins that includes ERCC1 and BRCA1. Gemcitabine is thought to produce its antitumor effects by several mechanisms, including inhibition of ribonucleotide conversion to deoxyribonucleotide through the inactivation of ribonucleotide reductase. One type of gemcitabine resistance occurs when the inactivation of ribonucleotide reductase decreases, which correlates with increased expression of the M1 subunit of ribonucleotide reductase. Taxanes produce their anti-tumor effects by binding to and stabilizing intracellular microtubules, which are necessary for DNA replication and cell division. High expression of beta-tubulin III, a microtubule subunit with low taxane binding affinity, appears to confer resistance to taxane therapy. High expression of thymidylate synthase, an enzyme that is important in purine synthesis and DNA replication, has been associated with decreased response to uracil antimetabolites, and may also be an important prognostic factor in patients receiving pemetrexed. The epidermal growth factor receptor (EGFR) is a cell- surface receptor with an intracellular tyrosine kinase domain that is thought to modulate numerous cellular functions that contribute to tumorigenicity, tumor invasiveness, and resistance to therapy. Recent clinical trials have demonstrated that treatment response to small-molecule and monoclonal antibody inhibitors of EGFR is greatest for patients who have a higher EGFR gene copy number. Several genetic, molecular, and histological markers may affect treatment response in patients with NSCLC. Evaluating patients for such markers is important not only for treatment efficacy, but also to improve safety and tolerability by avoiding exposure to treatments that are unlikely to produce significant benefits. Nearly all of the available information regarding the predictive value of these markers has been derived from retrospective studies. Prospective clinical trials are important to validate marker evaluation methodology and the prospective utility of biomarkers in clinical decision making.

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.

Expression of excision repair cross-complementation group 1 protein and outcome in patients with nasopharyngeal cancer

6085 Background: We evaluated the prognostic significance of thymidylate synthase (TS), and Excision Repair Cross-Complementation Group 1 protein (ERCC1) in patients (pts) with nasopharyngeal cancer (NPC) treated with concurrent chemoradiotherapy (CCRT). Methods: Pretreatment tumor biopsy specimens from 41 pts with locally advanced NPC were analyzed for TS and ERCC1 expression by immunohistochemistry. All patients were treated with 1 cycle of induction chemotherapy (5-fluorouracil 1,000 mg/m2/day and cisplatin 20 mg/m2/day, days 1–4) followed by CCRT starting on day 22. CCRT consisted of radiotherapy (70Gy/35 fractions for 7 weeks) with cisplatin 20mg/m2/day for 4 days on weeks 1, 4, 7 of radiotherapy. Results: Complete response and partial response were achieved in 34 pts (83%) and 6 pts (15%), respectively. Within median follow up duration of 101 months (26–147months) in survivors, 5-years overall survival (OS) of all pts was 50%. High expression of TS and ERCC1 was observed in 21 (51%) and 25 (60%) pts, respectively. High expression of ERCC1 was associated with WHO type 1 or 2 histology (p = 0.045). In univariate analysis, high expression of ERCC1 was associated with poor OS (5-year: 73% versus 35%; p = 0.005), while high expression of TS was not correlated with pts outcome (p = 0.867). In multivariate analysis, high expression of ERCC1 was a significant independent predictor of poor OS (p = 0.041) along with WHO type 1 or 2 histology (p = 0.004). Conclusions: High expression of ERCC1 protein may be useful for prediction of poor outcome in pts with NPC treated with CCRT. No significant financial relationships to disclose.

Molecular profiling in patients (pts) with upper gastrointestinal (UGI) cancers correlated with clinical outcome

e22042 Background: Recent efforts have expanded our understanding of the molecular pathogenesis of UGI cancers and how oncogenes and tumor suppressor genes play a role in both carcinogenic and metastatic processes. These markers may serve as prognostic and/or predictive factors for recurrence following resection and resistance to radiotherapy and chemotherapy. Gene expression levels of thymidylate synthase (TS), thymidylate phosphorylase (TP), epidermal growth factor receptor (EGFR) and excision repair cross complementing (ERCC-1) have been shown to be associated with outcomes in lung, gastric and colon cancer. We evaluated TS, ERCC-1, EGFR and TP gene expression levels in patients with UGI cancers. Methods: This was a retrospective study of 80 pts with UGI cancers evaluated at USC who underwent molecular profiling. The primary objective was to determine a correlation between TS, TP, ERCC1 and EGFR and correlate with clinical outcome. Characteristics of these 80 pts: (16 females, 64 males ); median age 61 years (range 34–85); tumor types evaluated - 32 (40%) esophageal, 24(30%) gastric, 24(30%) GE junction; stages- 53 % IV, 21% III, 12% II, 2% I were evaluated for intratumoral gene expression of TS, TP, ERCC-1, &amp; EGFR by real time quantitative PCR using Taqman technology from microdisected paraffin-embedded tumor sections. Results: High TS expression was associated with shorter OS (12.8 months vs. 23.7 months p=0.036). A significant correlation was found between TP &amp; ERCC-1 (p=0.0078, r=0.37); TP &amp; TS (p=0.0128, r=0.35); TP &amp; EGFR (p=0.0065, r=0.39); TS &amp; ERCC-1 (p=0.0004, r=0.39); ERCC-1 &amp; EGFR (p=0.025, r=0.30). No statistically significant relationship was found between TS &amp; EGFR (p=0.06,r=0.25). There was no correlation of ERCC-1, TP, &amp; EGFR with OS that reached statistical significance. Conclusions: TS mRNA levels were shown to be associated with OS in UGI tumors, consistent with data reported in colon cancer. TS gene expression was significantly associated with expression levels of ERCC-1. In addition, ERCC1 was associated with EGFR. These data show for the first time that molecular pathways of cytotoxic agents are linked to the EGFR pathway suggesting that sensitivity to fluoropyrimidines, oxaliplatin, and EGFR inhibitors may be associated. No significant financial relationships to disclose.

Nuclear thymidylate synthase expression in sporadic colorectal cancer depends on the site of the tumor

Colorectal carcinoma (CRC) is a heterogeneous disease with specific epidemiological, pathological, molecular, and clinical characteristics that depend on the location of the tumor relative to the splenic flexure. Thymidylate synthase (TS) is a major target of 5-fluorouracil-based chemotherapy for CRC and high expression of this enzyme in tumor cells can influence the effect of therapy. We examined differences in TS protein expression in nuclei of tumor cells between CRCs located proximal and distal to the splenic flexure. Nuclear TS was detected by immunohistochemistry with a TS 106 monoclonal antibody on tissue microarrays constructed from 269 CRCs. The median histological score of nuclear TS expression of all proximal tumors was two times higher (p = 0.0003) and in men three times higher (p = 0.00023) than that found in distal tumors. In multivariate analysis which included age, sex, Astler-Coller stage, histological grade, and site, only proximal location of the tumor was identified as an independent factor associated with higher TS expression (odds ratio 2.46, 95% confidence interval = 1.29-4.70, p = 0.0062). These results demonstrate significant differences in nuclear TS expression between proximal and distal cancers and suggest the potential importance of the site of the tumor for proper stratification of patients for chemotherapy.

Thymidylate synthase was associated with patient prognosis and the response to adjuvant therapy in bladder cancer

To investigate the expression of thymidylate synthase (TS), a key enzyme in DNA synthesis that is over-expressed in several cancer cells, in bladder cancer and its association with patient prognosis and the response to adjuvant therapy. In all, 67 bladder tissue specimens were obtained from patients who had undergone transurethral resection (TUR). TS expression in bladder cancer and normal bladder tissue was analysed by immunohistochemistry. Of the 67 bladder tissue specimens, 47 (70%) and 10 (15%) had positive expression for TS in cancer and normal tissues, respectively. TS expression was greater in patients with Grade 3 (16/17, 94%) than in Grade 1 and 2 (31/50, 64%; P = 0.002). It was also greater in Stage T1 (14/14) than in Stage Ta (33/53, 62%; P = 0.001). Furthermore, patients with negative TS expression had a longer postoperative recurrence-free survival (RFS) than those with positive expression during the 5 year follow-up (P = 0.028). In the patients with positive TS-expressing tumours, adjuvant therapy significantly improved RFS (P < 0.001). High TS expression might be a marker of poor prognosis for patients with bladder cancer. In addition, patients with high TS expression might also be benefit from adjuvant therapy.

Phase II Study of UFT with Leucovorin Plus Hepatic Arterial Infusion with Irinotecan, 5-Fluorouracil and Leucovorin for Non-Resectable Liver Metastases of Colorectal Cancer

Background: Compared with systemic therapy, hepatic arterial infusion (HAI) increases the response to fluoropyrimidines. Methods: Thirty-one patients with non-resectable, colorectal cancer (CRC) liver metastases received irinotecan 120 mg/m², followed by leucovorin (LV) 20 mg/m² and 5-fluorouracil (5-FU) 500 mg/m² administered by HAI every 2 weeks, plus UFT (tegafur-uracil) 200 mg/m²/day with LV 30 mg/day on days 1–22, followed by a 6-day rest. Results: The objective response rate was 65% (all 20 patients achieving a partial response). Ten patients (32%) had stable disease. The median time to progression (TTP) and overall survival (OS) were 12 and 36 months. OS was similar in patients with low versus high expression of thymidylate synthase (TS) and/or dihydropyrimidine dehydrogenase (DPD). The regimen was well tolerated. Conclusions: UFT with LV plus HAI irinotecan and 5-FU/LV was a feasible and effective treatment for non-resectable CRC liver metastases, increasing response, TTP and OS. TS and DPD levels in liver metastases did not predict outcome.

Personalized Medicine in Advanced Non–Small-Cell Lung Cancer: Reality or Wishful Thinking?

TO THE EDITOR: We read with interest the recent manuscript of Scagliotti et al reporting results of a noninferiority study comparing cisplatin plus gemcitabine to cisplatin and pemetrexed in advanced stage non–small-cell lung cancer (NSCLC). The authors are to be congratulated for completing the largest ever reported randomized trial in advanced NSCLC with more than twice as many patients assigned to treatment arms compared with other pivotal phase III trials. We have some reservations in relation to the conclusion recommending cisplatin plus pemetrexed as a reference regimen for future studies in nonsquamous NSCLC testing molecular-targeted therapies. While the prespecified analysis demonstrated the superiority of cisplatin plus pemetrexed over cisplatin and gemcitabine in terms of response rate, progression-free survival (PFS) and overall survival in nonsquamous histology, this trial was neither designed nor powered to answer this question. Rather, it should be considered hypothesis generating. The preclinical rationale derives from a study showing high expression of thymidylate synthase (therapeutic target of pemetrexed) in squamous carcinoma, indicating resistance to pemetrexed. However, the clinical evidence currently available is not overwhelming. The majority of published studies of chemotherapy in NSCLC have not shown any significant difference with respect to histology. Recently Pang et al reported a combined analysis of two phase II trials. Patients with adenocarcinoma had better response rates to pemetrexed-based first-line chemotherapy but this did not transform to any significant differences in PFS and overall survival. It appears that response rates were not independently reviewed. A retrospective analysis of a phase III study of second-line therapy comparing docetaxel with pemetrexed suggested worse survival in pemetrexed arm with squamous histology and, conversely, a benefit in nonsquamous histology. Another study comparing standardand low-dose of pemetrexed in second and third line showed on retrospective analysis that patients with nonsquamous histology did remarkably better. Finally Ciuleanu et al presented better PFS with pemetrexed maintenance after first-line therapy in comparison to placebo in nonsquamous histology. This data could be interpreted in various ways. It may mean that pemetrexed is less effective in squamous histology with similar efficacy in nonsquamous in comparison with other platinum doublets. Therefore, we believe the definite answer can only be provided by a specifically designed prospective randomized trial.

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.

Intense cytoplasmic ezrin immunoreactivity predicts poor survival in colorectal cancer

Ezrin is a membrane-cytoskeleton anchor, which, in experimental models, regulates tumor cell invasion and metastatic ability. We carried out immunohistochemical analysis of ezrin in 74 advanced colorectal cancer patients and correlated it to clinicopathologic variables and disease outcome. In contrast to the predominantly membraneous immunoreactivity of normal colorectal epithelium, ezrin expression in the colorectal cells was typically cytoplasmic. Altogether, 16.2% (12/74) of the tumors showed negative/weak ezrin staining, 35.1% (26/74) had moderate staining, and 48.6% (36/74) had intense staining. The expression was more intense in colon than in rectal carcinomas (P = .003). Increased ezrin expression was associated with adverse outcome, that is, shorter disease-specific survival; 48.3 months and 36.6 months for negative-weak versus intense expression (P = .041) as well as shorter survival with metastases at 36 months (P = .030); the metastases(36) rates in ezrin(neg/weak), ezrin(moderate), ezrin(intense) are 58.3%, 25.0%, and 18.4%, respectively. In univariate survival analysis, dichotomized (negative/weak versus moderate/strong) ezrin expression significantly predicted both the 5-year disease specific survival (P = .035) and 5-year metastases (P = .018) but lost this predictive power in multivariate (Cox) analysis. High ezrin expression was also related to high E-cadherin (cytoplasmic) expression, DNA aneuploidy, and high thymidylate synthase expression (P = .046, P = .042, P = .046, respectively). These results suggest that ezrin may play a role in colorectal cancer progression and that ezrin expression might provide clinically valuable information in predicting the biological behavior of colorectal cancer.

Evaluating the drug-target relationship between thymidylate synthase expression and tumor response to 5-fluorouracil: Is it time to move forward?

Thymidylate synthase is a target of 5-fluoruracil, a pyrimidine analog used to treat gastrointestinal and other cancers. The 5-fluorouracil metabolite, fluoro-deoxyuridine monophosphate, forms a ternary complex with thymidylate synthase and 5,10-methylene tetrahydrofolate. The purpose of this study was to evaluate the time-honored connection between thymidylate synthase and 5-fluorouracil. From our literature search spanning reports from 1995 to 2007 published in journals having an impact factor greater than 2, we stratified the tumors within each article, according to low versus high thymidylate synthase expression. These groups were subdivided into responders, stable disease, or disease progression. The relationship between thymidylate synthase expression and 5-fluorouracil response was analyzed for the overall group, as well as for subsets. Overall, the literature supported an approximately 2-fold inverse relationship between thymidylate synthase expression and response to 5-fluoruracil. We found no change in the trend for a relationship between thymidylate synthase and 5-fluorouracil when the literature was stratified by date of publication, impact factor of the journal in which the report was published, or substrate (mRNA versus protein) for measuring thymidylate synthase expression. Of note, there is no significant change in the trend when comparing 5-fluorouracil treatment alone or in combination with leucovorin. We found a decline of this trend when certain chemotherapeutics were used in combination with 5-fluorouracil. In sum, the connection between thymidylate synthase expression and patient response to 5-fluorouracil does not satisfy expectations for an effective drug-target relationship; and thus, studies of the thymidylate synthase tandem repeat status might only be clinically valuable in regards to patient toxicity. Thus, we question the reliability of thymidylate synthase expression as a clinical predictor of 5-fluorouracil response. Future research could perhaps be directed towards alternate targets and metabolites of 5-fluorouracil, in an effort to find a clinically relevant biomarker panel for response and to optimize fluoropyrimidine-based therapy.