Low Thymidylate Synthase Levels Research Articles

Measurement of Nucleic Acid Metabolizing Enzymes in Stage III Colorectal Cancer Adds Precision to Adjuvant Fluorouracil and Leucovorin Therapy

1. Abstract 1.1. <span style=font-size:10.0pt;mso-fareast-font-family: ms= mincho=>Background <span style=font-size:10.0pt;mso-fareast-font-family: ms= mincho=>: Recurrence of colorectal cancer has been shown to be inhibited by adjuvant chemotherapy. To achieve effective adjuvant chemotherapy, a target molecule is necessary . Therefore, we performed a prospective clinical trial to determine the relationship between adjuvant chemotherapy outcomes and prognostic factors in colorectal cancer based on nucleic acid metabolizing enzyme levels. 1.2. Methods: A total of 147 patients with stage IIIA, IIIB, and IIIC cancer were selected for adjuvant chemotherapy. We analyzed Disease-Free Survival (DFS) as the primary endpoint and Overall Survival (OS) as the secondary endpoint in relation to the Thymidylate Synthase (TS) and Dihydropyrimidine Dehydrogenase (DPD) levels as measured by enzyme-linked immunosorbent assay. According <span style=letter-spacing: -.15pt>to the levels of the mean preliminary TS of 21.0 ng/mg protein and DPD of 115 ng/mg protein, the patients were divided into 4 groups. They were then administered postoperative adjuvant chemotherapy that primarily included fluorouracil and Leucovorin (FL) or Uracil/Tegafur (UFT) and Leucovorin (UZEL) for 6 months. 1.3. Results: The 5-year DFS in advanced stage III cancer was 63.9%. A significant difference was observed between the LL (low TS and low DPD levels) and HL (high TS and low DPD levels) groups and the LH (low TS and high DPD levels) and HH (high TS and high DPD levels) groups. Multivariate<span style=letter-spacing: -.5pt> analysis revealed a significant difference<span style=letter-spacing: -.1pt> in stage, TS level, and TS and DPD levels. The OS was 79.6% for all the groups. A significant difference was observed between the LL, LH, and HL groups and the HH groups. Multivariate analysis revealed a significant difference<span style=letter-spacing: -.5pt> in the stage, age, TS level, and<span style=letter-spacing: -.55pt> TS and DPD levels. <span style=font-size:10.0pt;letter-spacing: -.15pt>1.4. Conclusions: Measurement of the TS and DPD levels of a primary colorectal cancer lesion as target molecules may be helpful for achieving an effective postoperative adjuvant chemotherapy. TS and DPD can potentially serve as more accurate predictors of OS than age and cancer stage. 2. Keywords: Adjuvant Chemotherapy; Colorectal Cancer; Metabolizing <span style=font-size: 10.0pt;mso-fareast-font-family: ms= mincho=> <span style=font-size: 10.0pt>Enzyme; Precision Medicine

Higher Tissue Levels of Thymidylate Synthase Determined by ELISA Are Associated with Poor Prognosis of Patients with Lung Cancer.

Thymidylate synthase (TS) is essential in thymidylate biosynthesis and DNA replication. Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in pyrimidine catabolism and is important in catabolism of 5-fluorouracil (5-FU). The significance of TS and DPD expressed in lung cancer remains controversial. Here we analyzed the relationship between TS and DPD expression and clinicopathological features of lung cancer. Enzyme-linked immunosorbent assays (ELISAs) were used to measure TS and DPD levels in paired tumor and non-tumor lung tissues obtained from 168 patients (107 adenocarcinomas, 39 squamous cell carcinomas, and 22 others), who had operations at the Shinshu University Hospital from 2004 to 2007 and were followed up for a median of 57.0 months. TS and DPD expression levels were higher in tumor tissues, and TS expression levels were significantly lower in adenocarcinomas than those in other subtypes. In addition, patients with low TS levels survived longer compared with patents with high TS levels. By contrast, DPD expression levels were not correlated with overall patient survival. Importantly, patients with low TS and DPD levels exhibited significantly prolonged survival than those with high TS and DPD. Among the 168 patients, 59 patients were treated with tegafur-uracil (UFT), a DPD-inhibitory fluoropyrimidine, and the UFT-treated patients with high TS and high DPD levels showed worst prognosis. Our study demonstrates a significant correlation between low TS expression levels and long-term prognosis of patients with lung cancer. Thus, ELISA is a clinically useful method to measure TS and DPD expression in lung cancer tissues.

Intrahepatic cholangiocarcinoma and gallbladder cancer: distinguishing molecular profiles to guide potential therapy

BackgroundChemotherapy regimens for intrahepatic cholangiocarcinoma (ICC) and gallbladder adenocarcinoma (GC) remain interchangeable; however, response rates are frequently suboptimal. Biomarkers from ICC and GC patients were interrogated to identify actionable differences with potential therapeutic implications. MethodsFrom 2009 to 2012, pathological specimens from 217 ICC and 28 GC patients referred to Caris Life Sciences were evaluated. Specific testing by immunohistochemical analysis for 17 different biomarkers was performed. ResultsIn the collective cohort (n= 245), actionable targets included: 95% low thymidylate synthase (TS), 82% low ribonucleotide reductase subunit M (RMM) 1 and 74% low excision repair cross complementation group (ERCC) 1, indicating potential susceptibility to fluoropyrimidines/capecitabine, gemcitabine and platinum agents, respectively. Additional targets included TOPO1 (53.3% high, Irinotecan), MGMT (50.3% low, temozolomide), TOP2A (33% high, anthracyclines) and PGP (30.1% low, taxanes). Subgroup analysis by tumour origin demonstrated a differential biomarker expression pattern with a higher frequency of ICC tumours showing low levels of TS (99% versus 72%, P < 0.01), and RRM1 (85% versus 64%, P = 0.02) when compared with GC. Conversely a greater frequency of GC demonstrated high levels of TOPO1 (76% versus 50%, P = 0.02) versus ICC, indicating a potential increased benefit from irinotecan. DiscussionDifferences in the molecular profiles between ICC and GC provide evidence that the two are distinct diseases, requiring different treatment strategies to optimize a response.

Molecular Profiling of Angiosarcomas of the Breast

ABSTRACT Aim: Angiosarcoma of the breast is a rare, aggressive soft tissue neoplasm occurring as either a primary or secondary malignancy due to previous radiotherapy for breast carcinoma. There is an unmet medical need for targeted therapies for angiosarcoma. Methods: Seventeen patients with angiosarcoma of the breast were identified (Caris Life Sciences) and profiled for biomarkers of drug response using multiplatform methodologies (Tumor DNA sequencing, gene copy number alterations, RNA/microarray and protein/IHC expression). Results: Eight primary, 5 post-radiation and 4 mammary angiosarcomas of unknown etiology were identified in women (age 31-85 years), all exhibiting multiple biomarker alterations. Eight out of 17 cases (47%) harbored overexpression (mRNA and/or protein) of the genes [VEGFR2 (5/17), PDGFRA/PDGFRB (3/17), c-KIT (2/17)] that can be targeted by multikinase inhibitor Sunitinib. Based on the VEGFR2 overexpression, one patient with recurrent, refractory angiosarcoma was treated with Sunitinib resulting in a complete remission and is disease free at 14 months. Also, multiplatform molecular profiling revealed useful predictors of various other treatment modalities including anthracyclines (overexpression of topoisomerase 2a in 69%), topo 1 inhibitors (topoisomerase 1 overexpression in 44%), and fluoropyrimidines (low levels of thymidylate synthase in 29%). One case devoid of the Sunitinib-associated genetic alterations harbored a KRAS mutation indicating a potential benefit of MEK inhibitors. Conclusions: A comprehensive molecular profiling of breast angiosarcomas enables identification of various molecular alterations that can be treated by both targeted and conventional treatment modalities. Disclosure: All authors have declared no conflicts of interest.

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.

Effect of leucovorin on the antitumor efficacy of the 5-FU prodrug, tegafur-uracil, in human colorectal cancer xenografts with various expression levels of thymidylate synthase

The combination of oral tegafur-uracil (UFT) with leucovorin (LV) is used to treat patients with stage II to III colon cancer based on the results of postoperative randomized studies in which UFT/LV treatment showed an equivalent efficacy to intravenous 5-FU plus LV therapy. However, whether the addition of LV to UFT can elevate the antitumor activity of UFT in colorectal tumors with high expression levels of thymidylate synthase (TS), which affects 5-FU efficacy, remains to be clarified. This study investigated the effect of LV on the antitumor activity of UFT and/or 5-FU prodrugs in low folate diet-fed nude mice using human colorectal cancer xenografts with various expression levels of TS. The addition of LV to UFT resulted in a 55-79% inhibition of tumor growth among 11 types of colorectal tumor xenograft, whereas UFT alone showed 23-67% antitumor activity. Although there was an inverse relationship between the antitumor effect of UFT alone and UFT plus LV and tumoral TS activity, UFT plus LV appeared to have a more potent antitumor effect than UFT alone on colorectal tumors such as Co-3 and KM12C/5-FU with high expression levels of TS. This finding was confirmed by the significant positive correlation between the relative inhibition ratio of UFT/LV to UFT alone and TS levels in tumors. To investigate the reason for the higher efficacy of UFT/LV on colorectal cancer xenografts with high TS activity, intratumoral levels of reduced folates and a ternary complex of TS after oral UFT with or without LV were measured using Co-3 xenografts. Elevated levels of reduced folates and an increased ternary complex of TS in LV-treated tumors were noted. Our results indicate that a combined therapy of UFT with LV may contribute to the treatment of colorectal cancer patients with low and high expression levels of tumoral TS by increased formation of the ternary complex of TS leading to potentiated antitumor efficacy of UFT.

ERCC1 mRNA levels and survival of advanced gastric cancer patients treated with a modified FOLFOX regimen

Molecular markers involved in DNA repair can help to predict survival in gastric cancer patients treated with 5-FU plus platinum chemotherapy. Excision repair cross-complementing 1 (ERCC1) and thymidylate synthase (TS) mRNA expression levels were assessed in advanced gastric cancer tumour samples using real-time quantitative PCR in 76 patients treated with a modified FOLFOX (biweekly oxaliplatin plus 5-FU and folinic acid) regimen. Median survival time in patients with low ERCC1 levels was significantly longer than in those with high levels (15.8 vs 6.2 months; P<0.0001). Patients with high TS levels had longer survival than those with low levels (12.2 vs 10.1 months; P=0.01). Forty-eight patients with low ERCC1 and high TS levels had a median survival of 16.1 months (P<0.0001). The hazard ratio for patients with high ERCC1 expression was 9.4 (P<0.0001). In patients with high mRNA levels of ERCC1, alternative chemotherapy regimens should be considered.

New Data Integrating Multitargeted Antifolates into Treatment of First-line and Relapsed Non–Small-Cell Lung Cancer

Non-small-cell lung cancer (NSCLC) represents approximately 80% of all lung cancers. With modern platinum- based combination regimens, overall median survival has reached 9-12 months. Antifolates are active against several solid tumors and hematologic malignancies. The cytotoxic action of antifolates is mainly related to their ability to inhibit several different folate-dependent enzymes involved in DNA synthesis. Pemetrexed is a novel multitargeted antifolate that inhibits at least 3 of the enzymes involved in purine and pyrimidine synthesis: thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). Pemetrexed was approved for the treatment of relapsed NSCLC as it produced equivalent response and survival rates and less toxicity compared with docetaxel. Pemetrexed in combination with platinum analogues or with gemcitabine showed equivalent clinical impact compared with standard combinations of platinum plus third-generation agents. We analyze the potential implications of pemetrexed's role in first-line chemotherapy of NSCLC as well as hints of differential cytotoxic action according to histology, new schedules of vitamin supplementation, and target enzymes expression levels. Issues of pharmacogenomics are becoming relevant in defining pemetrexed efficacy. Chemosensitivity was significantly linked to low levels of TS, GARFT, and DHFR in preclinical models. Consequently, the differential expression of TS according to histology might explain the different activity of pemetrexed according to histology, as recently postulated.

Bcl-2 expression significantly correlates with thymidylate synthase expression in colorectal cancer patients

To examine the expression of thymidylate synthase (TS) and oncoprotein Bcl-2 in advanced colorectal cancer (CRC) patients, and to determine their mutual relationship, association to therapeutic response and impact on disease outcome. Tumor samples from 67 patients with CRC, who were treated at advanced stage with either irinotecan alone or in combination with 5-fluorouracil/leucovorin, were analyzed for expression of TS and Bcl-2 using immunohistochemistry. A significant linear correlation between lower expression levels of Bcl-2 and lower levels of TS expression was found (P = 0.033). Patients with high levels of both TS and Bcl-2 expression had a significantly longer disease-free survival (DFS) (42.6 mo vs 5.4 mo, n = 25) than those with low TS/Bcl-2 index (P = 0.001). Tumors with low levels of both TS and Bcl-2 were associated with a longer survival with metastasis (WMS) interval in the whole patients group (n = 67, P = 0.035). TS/Bcl-2 index was not significantly related to disease-specific survival. The present data suggest that CRC patients with low TS/Bcl-2 demonstrate a significantly shorter DFS and longer WMS.

Thymidylate synthase and microsatellite instability in colorectal cancer: Implications for disease free survival, treatment response and survival with metastases

Background. Colorectal cancer (CRC) cell lines displaying microsatellite instability (MSI) are resistant to 5-fluorouracil (5-FU) in vitro, which can be overcome by restoring DNA mismatch repair (MMR) competence. Thymidylate synthase (TS) is inhibited by 5-FU, being another potential mediator of therapeutic resistance to 5-FU. The clinical relevance of these observations remains unclear. Objective. We examined the expression of TS and two MMR proteins (hMLH1 and hMSH2) in advanced CRC patients, to determine a) their mutual relationship, b) association to therapeutic response and c) impact on disease outcome. Material and methods. Tumour samples from 73 patients CRC who were treated in advanced stage with either irinotecan alone or in combination with 5-FU/leucovorin, were analysed for expression of TS, hMLH1 and hMSH2 using immunohistochemistry (IHC). Results. TS expression was closely correlated with hMLH1 expression (negative-weak/moderate-strong) (p=0.0001). TS-MMR expression was significantly (p=0.029 for whole series; p=0.004 for the 5-FU treated cases) related to response to treatment; tumours with low levels of both TS and MMR responded better (n=14/27, 51.8%) than those with high TS and MMR (n=3/18, 16.6%). Patients with high TS-MMR expression had a significantly longer DFS (47 months vs. 9months, n=26) than those with low TS-MMR index (p=0.015), while the reverse was true concerning survival with metastases (WMS) (p=0.018) in all the patients (n=73). Conclusions. The present data suggest that MSI patients with low TS and deficient MMR demonstrate a significantly shorter DFS and longer WMS than patients with high expression of both markers, and they are also more likely to obtain the greatest benefit from 5-FU based chemotherapy.

In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression

mRNA expression of genes involved in the mechanism of action of pemetrexed was correlated with in vitro chemosensitivity of freshly explanted human tumor specimens. Chemosensitivity to pemetrexed was studied in soft-agar. Multiplex rtPCR experiments for reduced folate carrier (RFC), folate receptor-alpha (FR-alpha), folylpolyglutamate synthetase (FPGS), thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyl transferase (GARFT), mrp4, and mrp5 were performed in parallel. Correlations, threshold optimization, sensitivity, specificity, and efficiency were analyzed using the appropriate statistical methodologies. In 61 samples, low levels of TS, GARFT, DHFR, and mrp4 gene expression significantly correlated with chemosensitivity to pemetrexed. Optimization analyses demonstrated threshold values of 144 copies for TS and six copies for mrp4 relative to 10(4) copies of beta-actin. These results form a rational basis for the design of clinical trials to evaluate the expression of these enzymes as predictors for treatment outcome.

Thymidylate synthase and thymidine phosphorylase gene expression as predictive parameters for the efficacy of 5-fluorouraci-based adjuvant chemotherapy for gastric cancer

To investigate the prognostic role of thymidylate synthase (TS) and thymidine phosphorylase (TP) mRNA levels in T3 or T4 gastric cancer treated with 5-fluorouracil-based adjuvant chemotherapy. Fifty-one patients with T3 or T4 gastric cancer received systemic 5-fluorouracil-based adjuvant chemotherapy, and intratumoral expression of TS and TP in 51 gastric cancer tissue samples was tested by real-time quantitative PCR. The median disease-free survival (DFS) time was 10.2 mo in the patients. There were no significant differences in DFS between the groups with high and low levels of TP. However, the group with low level of TS had a longer DFS (14.4 mo vs 8.3 mo, P = 0.017). The median overall survival (OS) time was 18.5 mo, and there were significant differences in OS between the groups with high and low levels of TS or TP (for TS, 17.0 mo vs 21.3 mo, P = 0.010; for TP, 16.6 mo vs 22.5 mo, P = 0.009). Moreover, the coupled low expression of these two genes was strongly associated with a longer survival time of patients as compared with that of a single gene. Expression of TS and TP mRNA is a useful predictive parameter for the survival of postoperative gastric cancer patients after 5-fluorouracil-based adjuvant chemotherapy.

Prognostic and predictive significance of 5-fluorouracil metabolic enzymes in colorectal cancer

2021 Background: Low levels of the 5-Fluorouracil (5-FU) metabolic enzymes, thymidylate synthase (TS), dihydropyrimdine dehydrogenase (DPD) and thymidine phosphorylase (TP) are linked to improved survival in colorectal cancer (CRC) patients receiving 5-FU chemotherapy. However, whether they are prognostic (related to tumor biology) or predictive (drug sensitivity) indicators is not clarified. This study aimed to (1) discriminate the prognostic and predictive significance of TS, DPD and TP and (2) ascertain the clinical and molecular subtype associations of the proteins in a large sample series. Methods: Tissue arrays containing sections from 956 stage 2/3 CRC cases were stained immunohistochemically for TS, DPD and TP. Associations with clinical and molecular characteristics and survival according to treatment status were assessed by Kruskall-Wallis and Kaplan-Meier analysis. Results: Low TS levels were associated with late stage, proximal tumor location and absence of microsatellite instability, low DPD with younger age and late stage and low TP with proximal tumor location. Low levels of all three enzymes associated with absence of tumor infiltrating lymphocytes. There were no associations with gender, grade or ras and p53 mutation. In stage 2 patients treated by surgery alone, those with low DPD levels had a worse survival than those with high levels (p&lt;0.01). Stage 3 patients treated with chemotherapy had a better survival than those without in subgroups of patients with low TS (p=0.05), DPD (p&lt;0.01) and TP (p=0.03) but not high levels of the proteins. Conclusions: Our results suggest the improved outcome of 5FU-treated patients with low tumor TS, DPD and TP levels may be due primarily to a benefit from adjuvant treatment rather than a favorable prognosis. TS, DPD and TP levels may be useful indicators for identifying CRC patients likely to benefit from 5-FU treatment. No significant financial relationships to disclose.

Thymidylate synthase and MDR1 mRNA levels predict response to chemotherapy with S-1 in metastatic colorectal cancer

13107 Background: To test the hypotheses of whether the relative mRNA expression of the thymidylate synthase (TS) gene and multidrug resistance 1 (MDR1) gene are associated with response to chemotherapy with S-1 in metastatic colorectal cancer. Methods: Eighteen patients with progressive stage IV CRC were treated with S-1 twice daily (BSA = 1.5 m2, 60 mg/day) for 28 days, followed by a 2-week period rest. cDNA was derived from frozen tumor specimens to determine TS and MDR1 mRNA expression relative to the internal reference gene GAPDH using fluorescence-based, real-time reverse transcriptase polymerase chain reaction (Taqman) system. Results: The median TS gene expression level from 18 CRC tumors was 0.94 × 10(−3) (minimum expression, 0.15 × 10(−3); maximum expression, 8.0 × 10(−3)), and the median MDR1 gene expression level was 7.75 × 10(−3) (minimum, 0.83; maximum, 41.5 × 10(−3)). The gene expression cutoff values for chemotherapy nonresponse were 1.0 x 10(-3) for TS and 10.0 × 10(−3) for MDR1. The response rate for patients with TS &lt; or = 1.0 × 10(−3) (10 of 18 patients) was 60% (6/10), compared with 0% (0/8) for patients with TS greater than 1.0 × 10(−3) (P = 0.013). Patients with MDR1 expression &lt; or = 10.0 × 10(−3) (13 of 18 patients) had a response rate of 46% (6/13), compared with 0% (0/5) for patients with MDR1 expression greater than 10.0 × 10(−3) (P = 0.11). Regarding the combination of TS and MDR1 Expression, low TS and MDR1 expression levels were detected in 8 (44%) of the patients, and 10 patients (56%) had a high TS and/or MDR1 expression level. The response rate was 75% (6/8) for the low TS and MDR1 expressors and 0% (0/10) for the high TS and/or MDR1 expressors (P = 0.001). Conclusions: These data suggest that intratumoral TS and MDR1 mRNA expression levels are independent predictive markers of response to S-1 chemotherapy in metastatic colorectal cancer. No significant financial relationships to disclose.

Polymorphisms in the Thymidylate Synthase Gene Predict Response to 5-Fluorouracil Therapy in Colorectal Cancer

Introduction Advances in the field of pharmacogenomics have enhanced our knowledge of the molecular basis of cancer and provided a link between detection of genetic polymorphisms and response to treatment. Different variations of genetic polymorphisms, such as tandem repeats, single nucleotide polymorphisms, or nucleotide deletions, can lead to alterations in the transcription, translation, or stability of certain gene products that could be critical for the clinical activity and/or toxicity of chemotherapeutic drugs. One such target is thymidylate synthase (TS), an enzyme that catalyzes the reductive methylation of deoxyuridine-5'monophosphate (dUMP) to deoxythymidine-5'-monophosphate (dTMP) in the cell and generates the sole intracellular de novo source of thymidylate, a key precursor needed for DNA biosynthesis. This folate-dependent enzyme is an important target for chemotherapy drugs such as 5fluorouracil (5-FU), fluorodeoxyuridine (FUDR), oral 5-FU prodrugs (eg, uracil/tegafur, S-1, and capecitabine), and other novel folate-based drugs (eg, raltitrexed and pemetrexed). These agents directly inhibit TS enzymatic function, thereby resulting in thymidylate depletion with subsequent DNA damage and cell death. In the treatment of colorectal cancer (CRC), 5-FU–based chemotherapeutic regimens have yielded overall response rates ranging from 10% to 30% for advanced disease1 and provide approximately 15% improvement in 5year survival as adjuvant therapy for stage III disease.2 One key question that remains, however, is how to best predict which patients will benefit from such a therapeutic treatment strategy. Several studies have observed that high levels of TS expression at the protein or messenger RNA (mRNA) level, in the primary tumor or at the metastatic sites, have correlated with a lack of response to 5-FU–based chemotherapy. In contrast, low expression levels of TS have been associated with a positive clinical response. In order for these drugs to be effective in killing a tumor cell, active enzyme levels have to be reduced for a sufficient period for cell cycle arrest to be initiated. If TS expression is increased, then standard drug doses would be ineffective in attenuating enzyme activity. This reasoning has been supported in a retrospective analysis of TS levels, as measured by immunohistochemistry, in tumor specimens of patients with advanced CRC. Among 48 patients who received 5-FU–based therapy, the overall response rate was lower (24% vs. 67%; P = 0.003), and median time to progression (6.2 months vs. 9.6 months; P = 0.005) and median overall survival (OS) times (15.4 months vs. 18.4 months; P = 0.02) were shorter in patients with high tumor TS expression compared with patients with low TS expression.3 Discrepancies have been reported in TS expression levels detected in the primary colorectal tumor versus hepatic metastases. In 14 patients with CRC and hepatic metastases matched samples, TS mRNA levels, as measured by semiquantitative reverse-transcriptase polymerase chain reaction, were higher in the primary tumor (mean, 1.14 ± 0.34) compared with the liver metastases (mean, 0.76 ± 0.37; Wilcoxon test; P < 0.05).4

Phase II study of tailored chemotherapy for advanced colorectal cancer with either 5-fluouracil and leucovorin or oxaliplatin and irinotecan based on the expression of thymidylate synthase and dihydropyrimidine dehydrogenase

Background: Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are essential enzymes for 5-fluorouracil (5-FU) metabolism. In patients with advanced colorectal cancer (ACRC), retrospective studies have shown that low expression levels of TS and DPD correlated with response to 5-FU. We performed a prospective study in which the choice of first-line chemotherapy with either 5-FU or a non-5-FU containing regimen was based on TS and DPD expression.Patients and methods: Fresh-frozen samples of metastases were obtained from 58 previously untreated patients with ACRC. mRNA expression of TS and DPD was quantified using an RT–PCR assay. Patients with low tumor expression of both TS and DPD received weekly bolus 5-FU/leucovorin (LV) 500mg/m2 (group A); patients with high TS and/or DPD received 3-weekly oxaliplatin 85mg/m2 and irinotecan 200mg/m2 (group B). After progression, cross-over to the alternative regimen was attempted.Results: Of 53 eligible patients, 31 had tumors with both low TS and low DPD, and were treated in group A. A response was observed in 11 patients [35%; 95% confidence interval (CI) 19% to 54%]. Cross-over to second-line oxaliplatin/irinotecan resulted in a partial response in two out of 16 patients (13%; 95% CI 1% to 38%). In group B, four out of 22 patients responded (18%; 95% CI 5% to 40%), while no responses were observed in 12 patients after cross-over to 5-FU/LV (0%; 95% CI 0% to 28%).Conclusions: Prospective selection of 5-FU/LV chemotherapy based on low TS and DPD expression in patients with ACRC did not confirm the high response rates reported in retrospective studies. The procedure of obtaining metastatic tissue and quantitation of enzymes appeared feasible but cumbersome. Before assessing the clinical utility of a predictive marker in a randomized trial, future studies should focus on prospective validation of the assay in a large and well defined population.

Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), ERCC1, &amp; thymidine phosphorylase gene expression in primary &amp; metastatic (met) colorectal cancer (CRC) tissue in patients (pts) treated on a phase I trial of oxaliplatin (OX) &amp; capecitabine (Xel)

3549 Background: Overexpression of TS/DPD & ERCC1 in tumor tissue have been associated with insensitivity to 5-FU and OX therapy, respectively, while high TP levels predict for increased sensitivity to Xel. Methods: When feasible, biopsies of met tumor were taken in pts before OX (130 mg/m2 d1)/Xel (1200–3000 mg/m2 d1–5,8–12) q 3wk. Microdissected met & archival primary tumors were analyzed for gene expression (vs b-actin) of these 4 targets by real-time QRT-PCR. Survival (Surv) & time to treatment failure (TTF) were analyzed by log-rank. Gene expression v outcome was assessed using median (med), 25th & 75th percentiles for each target. Results: 90 pts participated. The med # prior chemotherapies was 2; 96% had prior 5-FU; 78% had prior irinotecan. Med mRNA levels were higher in met v primary tumor (table); rank sum analysis of paired samples showed (med): TS, 4.2 vs 2.2, p<0.001; DPD, 1.7 vs 0.4, p=0.005; ERCC1, 2.3 vs 1.1, p=0.002; TP, 6.3 vs 3.9 p=0.018. Pts whose tumor had ERCC1 >75th % had shorter TTF (med 162 d vs 85 d, p=0.026); pts with TP >75th % tended to have longer TTF (med 194 d vs 146 d, p=0.072). Individual mRNA levels in met tumor did not correlate with Surv, but pts whose tumors had both TS & ERCC1 < med had longer Surv (med 502 vs 296 days, p=0.038). Conclusions: In this study, target gene expression in primary tumor did not reflect levels in met tissue. In pre-treated CRC pts, mRNA levels of TP & ERCC1 in met tumor had some predictive value for TTF with OX/Xel therapy. Low levels of TS & ERCC1 predicted for improved Surv. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics, Sanofi-Synthelabo Response Genetics

Indifferently Pursued or Unowned Drugs: Who Should Lead Where Companies Do Not Tread?

The essence of a successful drug development program for cancer is the demonstration of safety and efficacy (defined as clinical benefit) in the intended population. As others have observed, molecularly targeted agents having potential noncytotoxic end points have, for the most part, proceeded through fairly standard decision-making algorithms, in which responses early in an agent’s development serve as an important signal of potential clinical benefit and form the basis for subsequent development. With even a hint of such responses, it is possible to map out a strategy that may lead to expeditious regulatory approval. Recent good examples of agents of this type vigorously pursued by industry include bortezomib in multiple myeloma and gefitinib in lung cancer. In the case of agents that modulate tumor-cell resistance to chemotherapy or biologics that act in concert with cytotoxic agents, such as cetuximab and bevacizumab, more complicated strategies, including randomized approaches in precisely defined study populations, are necessary and appropriate. Such studies clearly benefit from industry-based organizational drive and expertise for their expeditious design and completion. Pharmaceutical companies tend to want such post– phase I programs to proceed expeditiously and generally demonstrate a lessening of ardor if the plan for definitive registrational trials lasts beyond a 3to 5-year time frame; that latter number would be considered generous by many. The problem is that this time table ill serves the cause of agents that have a development strategy that arises from academic investigators working with hypothesis-directed, rather than market-oriented, goals. The compounds studied may not actually belong to a particular company, or the relevant company may not see the basis for facile regulatory approval, even with interesting clinical results from such small investigator-driven trials. Therefore, are we risking the loss of useful opportunities? How can such agents, which might lie at the fringe or indeed outside the envelope of corporate sponsorship, be usefully pursued? Is all oncology drug discovery and development to be driven by a corporate timetable, potentially not benefiting from informed maturation of a drug’s intellectual and practical basis for use that might come from study in academically oriented laboratory programs and their derivative clinical trials? In this issue, Kortmansky et al report a phase I trial of escalating doses of fluorouracil (FU) in conjunction with the protein kinase antagonist 7-hydroxystaurosporine (UCN-01; NSC 638850). The rationale for this study came from prior laboratory investigations from this group, which demonstrated that UCN-01 caused downregulation of thymidylate synthase (TS), potentially as the result of UCN-01–induced decline in the activity of the E2F transcription factor. Because the level of TS expression has been linked by a number of (but not all) prior studies to the ultimate efficacy of FU-based therapies, with low levels of TS correlating with increased evidence of drug efficacy, a molecular basis for favorable modulation of FU efficacy by UCN-01 could be imagined. The results of the phase I study are encouraging. The study by Kortmansky et al clearly shows that a full dose of 24-hour infusional FU can be administered with the previously identified maximumtolerated dose of UCN-01 without exacerbating either FUrelated toxicities or the expected toxicities of UCN-01. Although no formal responses were observed in this generally heavily pretreated population, interesting oncophenomena occurred, including the perception of stable disease beyond 2 months in approximately one quarter of the assessable patients, which mirrors a prior suggestion of disease stabilization in a fraction of refractory lung cancer patients treated with UCN-01 and carboplatin. An effort in Kortmansky et al’s study to discern modulation of TS mRNA in peripheral-blood mononuclear cells was not informative, although the authors themselves note that these JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 23 NUMBER 9 MARCH 2

Thymidylate synthase expression pattern is a prognostic factor in patients of colorectal cancer treated with 5-fluorouracil.

High intratumoral expression of thymidylate synthase (TS) has been reported as a factor of poor prognosis in patients with advanced colorectal cancer (CRC), but such association is unclear in some studies. Also, TS has been stated as a typical cytosolic enzyme, but nuclear location has been occasionally reported, and data on the clinical meaning of TS intracellular location are scarce. A retrospective study was performed in paraffin-embedded sections of primary tumor from 77 CRC patients treated with surgical resection and adjuvant 5-FU-based chemotherapy. TS levels and expression patterns were determined by immunohistochemistry (IHQ) using TS-106 antibody. Qualitative and quantitative variables were compared respectively by chi2 and Kruskal-Wallis tests; overall survival (OS) and disease-free survival (DFS) were analyzed with the Kaplan-Meier method and compared using the log-rank and Wilcoxon tests. TS was cytoplasmic in 27.1% of positive tumors and both, nuclear and cytoplasmic in 72.9%; specimens from seven patients (9.1%) lacked TS expression. TS levels were high in 21.6% of tumors with nuclear expression and low in 5.6%, whereas 68.4% of cytoplasmic ones showed low immunostaining intensity (p=0.02); cytoplasmic pattern was also associated to longer OS (p<0.009) and DFS (p=0.003). In patients with nuclear expression, low TS expression was associated to shorter OS (p<0.003) and DFS (p<0.04). These results indicate that, in our study, TS immunostaining patterns were related with OS and DFS, the best prognostic corresponding to cytoplasmic one, and, within the subset of patients with nuclear expression, low TS levels were associated to worse clinical outcome.

Thymidylate synthase expression and prognosis of patients with gastrointestinal cancers receiving adjuvant chemotherapy: a review

Many studies have been published that report an association between thymidylate synthase (TS) and response to fluoropyrimidine-based chemotherapy and the overall outcome of patients with gastrointestinal cancer. The results have given rise to the possibility that, by determination of TS levels, the physician may decide if the patient has a potential benefit from fluoropyrimidine-based treatment, similar to measurements of oestrogen receptors in breast cancer. The purpose of this review is to summarize critically the reports on TS measurement in gastrointestinal cancer, focusing on the adjuvant fluoropyrimidine treatment situation. We reviewed more than 20 studies that reported the association of TS with the clinical outcome in patients with gastrointestinal cancer who had undergone complete resection of the primary tumour only or were receiving additional adjuvant chemotherapy. Patients with metastasized disease who expressed high TS levels display a low probability of responding to fluoropyrimidine-based treatment and have a poorer survival rate. Patients with high TS levels who undergo complete surgical resection of the primary tumour also have a poorer prognosis than those with tumours with low TS expression. In contrast to advanced disease and to surgery alone, patients with high TS levels appear to benefit, especially, from adjuvant fluoropyrimidine-based chemotherapy after complete primary tumour resection, while patients with low TS levels do not. Patients with gastrointestinal cancers that express high TS levels have a poor prognosis with regard to fluoropyrimidine-based palliative chemotherapy or complete primary tumour resection. In contrast, patients with high TS levels might benefit from adjuvant fluoropyrimidine-based treatment after primary tumour resection. However, additional prospective studies are mandatory to define the precise role of TS in adjuvant therapy.