Thymidine Phosphorylase Gene Expression Research Articles

2062 Increase in thymidine phosphorylase gene expression in tumor tissues is associated with response to preoperative chemoradiotherapy including S-1 or UFT in patients with rectal cancer

2062 Increase in thymidine phosphorylase gene expression in tumor tissues is associated with response to preoperative chemoradiotherapy including S-1 or UFT in patients with rectal cancer

Thymidine phosphorylase expression is associated with time to progression in patients with metastatic colorectal cancer.

Background5-Fluorouracil (5-FU) is the cornerstone of chemotherapeutic treatment for patients with colorectal cancer. The enzyme thymidine phosphorylase (TP) catalyzes the conversion of 5-FU to its active metabolite, 5-fluoro-2’-deoxyuridine. TP is expressed in tumour epithelial cells and stromal cells, particularly in tumour-associated macrophages. These macrophages may affect sensitivity to chemotherapy. Previously, we identified TP as a predictive factor in microdissected tumour samples of patients with advanced colorectal cancer. In the present study, we analysed TP expression in tissues and associated stromal cells from patients with advanced colorectal cancer and associated TP levels to tumour response and time-to-event variables during first-line chemotherapy treatment. We also investigated the association between serum TP levels at the time of surgery and gene expression in primary tumour tissues.MethodsThis study included 125 patients with metastatic colorectal cancer treated with first-line 5-FU-based chemotherapy. To quantify TP gene expression levels in tumour tissues, real-time polymerase chain reaction was performed using the 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). TP protein concentration in matched serum samples was determined using an enzyme-linked immunosorbent assay system (USCN Life Science Inc.).ResultsThe tumour response rate was 31%, and 30% of patients exhibited stable disease. No associations between TP expression level and age or gender were observed. Levels of TP mRNA in mucosa and tumours were positively correlated (r = 0.41, p < 0.01). No correlation between TP expression and tumour response rate was observed. Time to progression was significantly longer in patients with high TP expression (p < 0.01). Serum TP protein levels were not associated with tumour response or time-to-event variables and did not correlate with gene expression in tumour tissues.ConclusionsHigh TP gene expression in non-microdissected tumour tissues of patients with advanced colorectal cancer correlates with longer time to progression, which could be related to treatment. These results are in contrast to previous studies where microdissected tumour cells were analysed and may be due to the presence of adjacent stromal cells. Serum TP protein expression does not correlate to TP gene expression in tissues of patients with advanced colorectal cancer.

Comparison of site-specific gene expression levels in primary tumors and synchronous lymph node metastases in advanced gastric cancer

Many malignant tumors consist of heterogeneous subpopulations of cells. This heterogeneity is associated with genetic characteristics. However, it remains unclear whether gene expression levels differ among specific sites of tumors in gastric cancer. We studied differences in gene expression levels among specific sites of primary tumors and synchronous lymph node metastases, using formalin-fixed, paraffin-embedded specimens resected surgically from 48 patients with previously untreated advanced gastric cancer. Specimens were obtained by laser-captured microdissection from five regions: (1) nonneoplastic mucosa, (2) surface layer (mucosa) of the primary tumor (surface sections), (3) middle layer (submucosa) of the primary tumor (middle sections), (4) the deepest layer of the primary tumor (muscularis propria or deeper) at the site of deepest invasion (deep sections), and (5) level 1 synchronous lymph node metastasis (lymph node metastases). Expression levels of the following target genes were determined by quantitative real-time polymerase chain reaction: thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1α (HIF1α). TP, DPD, EGFR, and HIF1α gene expression levels were significantly higher in deep sections than in surface sections. TP, EGFR, VEGF, and HIF1α gene expression levels were significantly higher in lymph node metastases than in surface sections. TP, DPD, EGFR, VEGF, and HIF1α gene expression levels were positively correlated with the specific samples harvested from the tumors. Our results show that the expression levels of some genes in tumor cells can change in specific sites of tumors and can become higher in association with tumor progression.

Association of right-sided tumors with high thymidine phosphorylase gene expression levels and the response to oral uracil and tegafur/leucovorin chemotherapy among patients with colorectal cancer

To identify useful predictive factors for the response to 5-fluorouracil (5-FU)/leucovorin (LV) and oral uracil and tegafur (UFT)/LV chemotherapy among patients with colorectal cancer, we investigated the association between the gene expression levels of pyrimidine and folate metabolism-related enzymes in colorectal cancer (CRC) tissues and the response to UFT/LV neoadjuvant chemotherapy. The subjects were 76 CRC patients who were scheduled to undergo surgery. UFT (300mg/m(2)/day) and LV (75mg/body/day) were administered for 2 weeks before surgery. Biopsy samples were endoscopically obtained before drug administration. The gene expression levels of 14 genes in the biopsy samples were quantitatively evaluated using a real-time polymerase chain reaction (RT-PCR) assay. Fifteen patients (19.7%) with marked pathological regression were judged to be responders. Thymidine phosphorylase (TP) gene expression levels among the responders were significantly higher than those among the non-responders. Right-sided tumors with high TP gene expression levels were associated with a significantly higher response rate to UFT/LV chemotherapy than left-sided tumors. TP gene expression levels in primary CRC tissues and the primary tumor site may be useful predictors of the efficacy of oral UFT/LV chemotherapy.

Phase II and gene expression analysis trial of neoadjuvant capecitabine plus irinotecan followed by capecitabine-based chemoradiotherapy for locally advanced rectal cancer: Hoosier Oncology Group GI03-53

We designed this study in locally advanced rectal cancer to determine the pathological response, toxicity, and disease-free survival (DFS) with induction capecitabine plus irinotecan followed by capecitabine-based chemoradiotherapy (CRT) and analyze the gene expression of enzymes involved in the metabolism of capecitabine and irinotecan for associations with response and toxicity. Patients with T3/T4 or node positive rectal cancer were treated with capecitabine 1,000 mg/m(2) twice daily (BID) days 1-14, and irinotecan 200 mg/m(2) on day 1 every 21 days for 2 cycles, followed by capecitabine 825 mg/m(2) BID days 1-5 per week with concurrent radiotherapy 50.4 Gy in 28 fractions. Surgical resection occurred a median of 7.4 weeks after CRT. Gene expression levels or sequencing were used to analyze carboxylesterase-converting enzymes (CES1, CES2), thymidylate synthase (TS), thymidine phosphorylase (TP), dehydropyrimidine dehydrogenase (DPD), topoisomerase I (TOPO I), and uridine-diphosphate (UDP) glucuronosyl transferase 1A1 in pre- and post-treatment tumor and normal tissue samples. Twenty-two patients were enrolled, and 18 completed neoadjuvant therapy and underwent R0 resection. Two patients with UGT1A1 7/7 had grade 3 and 4 neutropenic fever and sepsis. Pathological complete response (pCR) occurred in 6 of 18 patients (33 %) and 10 (56 %) had tumor and/or nodal downstaging. The 3-year DFS was 75.5 % (95 % CI, 39.7-91.8 %). Locoregional control rate was 100 %. We observed higher TP gene expression in pCR patients, but no correlations with toxicity. This neoadjuvant regimen was safe and demonstrated significant antitumor activity. High TP tumor gene expression was associated with obtaining pCR.

Thymidine Phosphorylase Gene Expression in Stage III Colorectal Cancer

BackgroundThe thymidine phosphorylase (TP) enzyme has several tumor-promoting functions. The aim of this study was to explore TP gene expression in relation to clinical and histopathological data obtained from patients with stage III colorectal cancer.Methods and resultsTP gene expression was analyzed by real-time quantitative PCR in tumor and mucosa samples from 254 patients. TP gene expression in tumors correlated with lymph node staging, with higher expression relating to a higher number of positive nodes and a worse N-stage. Higher TP expression was also associated with a worse histological tumor grade. Patients with rectal cancer had significantly higher TP expression in mucosa and tumors compared with patients having colon cancer.ConclusionHigher intratumoral TP expression appears to be related to a worse N stage, and thus, with a worse prognosis. TP gene expression measured in a preoperative biopsy could be of interest in preoperative staging.

6119 POSTER High Level of Thymidine Phosphorylase Gene Expression in Tumour Tissues is Associated With Response to Oral Uracil and Tegafur/leucovorin Chemotherapy in Patients With Colorectal Cancer

6119 POSTER High Level of Thymidine Phosphorylase Gene Expression in Tumour Tissues is Associated With Response to Oral Uracil and Tegafur/leucovorin Chemotherapy in Patients With Colorectal Cancer

Histone deacetylase inhibitors induce thymidine phosphorylase expression in cultured breast cancer cell lines

Thymidine phosphorylase (TP) is an enzyme involved in thymidine synthesis and degradation. The expression of this enzyme has been proposed as a predictive factor for the therapeutic benefit of capecitabine, which is a precursor of the drug 5'-fluorouracil. In fact, TP is the rate-limiting enzyme in the activation of capecitabine. Therefore, higher levels of TP are expected to sensitize cancer cells to capecitabine treatment. In the present study, using breast cancer cell lines, we found a correlation between TP mRNA and protein levels, suggesting that compounds able to increase TP gene expression also increase protein levels. Accordingly, we demonstrated that treatment of breast cancer MCF7 and MDA231 cell lines with histone deacetylase inhibitors, tricostatin A and suberoylanilide hydroxamic acid, increased TP both at the mRNA and protein level. The effects of histone deacetylase inhibitors were not found to occur via the cytokine TNFα, a known inducer of TP expression. Our findings suggest a strategy to sensitize breast cancer cells to capecitabine treatment.

Abstract 5249: Analysis of gene expression levels associating with metabolism of 5-fluorouracil and folate in primary colorectal cancer and corresponding synchronous or metachronous liver metastasis

Abstract Background Gene expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) have been shown to be associated with response to 5-fluorouracil-based therapies. Folylpolyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH) and dihydrofolate reductase (DHFR) are associated with folate metabolism which regulates intracellular folate level. Analyzing these gene expression levels in liver metastases is important to obtain the prediction of therapy, however, only surgical specimens of the primary tumor are readily available for analysis in many cases. Our aim is to determine how TS, DPD, TP, OPRT, FPGS, GGH and DHFR gene expression levels in primary colorectal cancer (CRC) are related to those in liver metastases. Methods Formalin-fixed, paraffin-embedded tumor specimens from 50 pairs of primary CRC and corresponding liver metastases, including 30 synchronous and 20 metachronous metastases, were dissected by using laser-captured microdissection. Total RNA was extracted and cDNA was prepared by reverse-transcription. Quantitation of target genes and internal reference gene, beta-2-microglobulin, was performed using real-time RT-PCR. Results Significant difference was seen between mRNA expression levels of TS, DPD, FPGS and DHFR in primary tumor and those in corresponding synchronous liver metastases (median value: TS 1.18 vs. 0.05; p&amp;lt;0.0001, DPD 0.18 vs.0.05; p&amp;lt;0.0001, FPGS 0.07 vs. 0.02; p=0.0005, DHFR 0.31 vs. 0.03; p&amp;lt;0.0001), TS, DPD, TP, OPRT, FPGS and DHFR in primary tumor and those in corresponding metachronous liver metastases (TS 0.23 vs. 0.06; p&amp;lt;0.0001, DPD .0.31 vs. 0.03; p&amp;lt;0.0001, TP 0.21 vs. 0.14, p=0.021, OPRT 0.35 vs. 0.17; p=0.016, FPGS 0.11 vs. 0.04; p=0.0003, DHFR 0.41 vs. 0.03, p&amp;lt;0.0001). When matched tissue sets were compared on an individual basis, there was a significant correlation for TP, OPRT and FPGS mRNA expression between primary tumor and corresponding synchronous liver metastases (TP rs=0.64; p=0.018, OPRT rs=0.62; p=0.006, FPGS rs=0.51; p=0.004), DPD and GGH in primary tumor and corresponding metachronous liver metastases (DPD rs=0.54; p=0.014, GGH rs=0.59; p=0.007). Conclusion A good prediction of TP, OPRT and FPGS mRNA levels in synchronous liver metastases as well as DPD and GGH mRNA levels in metachronous liver metastasis can be obtained by measuring those of primary CRC, although no correlation was seen for other genes. We are currently investigating more number of cases and genes related with sensitivity to antitumor drugs. 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 5249. doi:10.1158/1538-7445.AM2011-5249

Efficacy of laser capture microdissection plus RT-PCR technique in analyzing gene expression levels of human gastric cancer

e15669 Background: We reported the efficacy of laser capture microdissection plus RT-PCR (LCM+RT-PCR) technique in analyzing gene expression levels of human colon cancer (Makino, BMC cancer, 2008). In the present study, gene expression level of angiogenic factors, such as vascular endothelial growth factor (VEGF), thymidine phosphorylase (TP) and cyclooxygenase-2 (COX-2) in human gastric cancer was studies using LCM+RT-PCR methods. Methods: Gene expression level of VEGF, TP and COX-2 were separately quantified in cancer cells and in cancerous stroma of 51 gastric cancers specimen by LCM+RT-PCR methods (Danenberg Tumor Profile). Results: All the gene expressions were successfully estimated separately in cancer cells and cancerous stroma. VEGF and TP gene higher expressed in cancer cell than in cancerous stroma (P&lt;0.0001, Wilcoxon), whereas COX-2 gene expression was higher in cancerous stroma (P=0.0004, Wilcoxon). Only in TP, positive correlation of gene expression was observed between in cancer cells and in cancerous stroma (rs=0.494, P=0.0014, Spearman). And a linear relationship was observed between VEGF and TP gene expression in cancerous stroma (rs=0.768, P&lt;0.0001, Spearman). Conclusions: Gene expression level of angiogenic factor was different between in cancer cells and cancerous stroma. By using LCM+RT-PCR method, gene expression level of angiogenic factors can be separately estimated in cancer cells and cancerous stroma. No significant financial relationships to disclose.

Molecular determinants of efficacy for 5‐FU‐based treatments in advanced colorectal cancer: mRNA expression for 18 chemotherapy‐related genes

5-Fluorouracil (5-FU)-based regimens remain a cornerstone in the treatment of colorectal cancer (CRC). However, the attendant toxicity prevents these regimens from reaching maximum therapeutic potential. In this retrospective analysis, we examined the pretreatment expression of 18 genes in archival tumor bank samples from patients with advanced CRC to determine if one or more of the selected genes showed promise as either a prognostic or predictive marker of 5-FU-based treatment outcomes. One hundred and forty-four CRC patient samples (collected from 1983 to 2004) were analyzed via real-time PCR for gene expression. Univariate analyses were used to correlate gene expression with efficacy and time-to-event variables. Low thymidine phosphorylase (TP), dihydrofolate reductase, dihydropyrimidine dehydrogenase (DPD), excision repair cross-complementing 1 (ERCC1) and thymidylate synthase gene expression were associated with better time-to-progression in the entire population. Low TP, DPD and ERCC1 expression were independently associated with improved overall survival. Low TP gene expression was also predictive of response. This study suggests that TP gene expression in particular is a predictive as well as a prognostic biomarker for advanced CRC patients. Gene panels assessing pretreatment TP, DPD, ERCC1, dihydrofolate reductase and thymidylate synthase gene expression may help improve the therapeutic potential of 5-FU- or other novel antifolate-based regimens. Further analysis of the prognostic or predictive value of these genes in prospective trials in CRC patients seems warranted.

Thymidylate synthase, dihydropyrimidine dehydrogenase, ERCC1, and thymidine phosphorylase gene expression in primary and metastatic gastrointestinal adenocarcinoma tissue in patients treated on a phase I trial of oxaliplatin and capecitabine

BackgroundOver-expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in tumor tissue is associated with insensitivity to 5-fluorouracil (5-FU). Over-expression of ERCC1 correlates with insensitivity to oxaliplatin (OX) therapy, while high thymidine phosphorylase (TP) levels predict for increased sensitivity to capecitabine (Xel).MethodsBiopsies of metastatic tumor were taken before OX (130 mg/m2 day 1) given with Xel (1200–3000 mg/m2 in two divided doses days 1–5 and 8–12) every 3-weeks. Micro-dissected metastatic and primary tumors were analyzed for relative gene expression by real-time quantitative polymerase chain reaction. The clinical protocol prospectively identified the molecular targets of interest that would be tested. Endpoints for the molecular analyses were correlation of median, first and third quartiles for relative gene expression of each target with response, time to treatment failure (TTF), and survival.ResultsAmong 91 patients participating in this trial; 97% had colorectal cancer. The median number of prior chemotherapy regimens was 2, and most had prior 5-FU and irinotecan. In paired samples, median mRNA levels were significantly higher in metastatic versus primary tumor (-fold): TS (1.9), DPD (3.8), ERCC1 (2.1) and TP (1.6). A strong positive correlation was noted between DPD and TP mRNA levels in both primary (r = 0.693, p < 0.0005) and metastatic tissue (r = 0.697, p < 0.00001). There was an association between TS gene expression and responsive and stable disease: patients whose intratumoral TS mRNA levels were above the median value had significantly greater risk of early disease progression (43% vs 17%), but this did not translate into a significant difference in TTF. ERCC1 gene expression above the third quartile was associated with a shorter TTF (median 85 vs 162 days, p = 0.046). Patients whose TS mRNA levels in metastatic tumor tissue were below the median had a longer overall survival (median 417 vs 294 days, p = 0.042).ConclusionTarget gene expression in primary tumor was significantly lower than that in paired metastatic tissue. High ERCC1 mRNA levels in metastatic tumor was associated with a shorter TTF. Lower expression of TS mRNA correlated with a lower chance of early PD with XelOX therapy and improved overall survival.

Role of tumor angiogenesis in gallbladder carcinoma: with special reference to thymidine phosphorylase

Angiogenesis plays an important role in tumor growth and metastasis. Thymidine phosphorylase (TP), as well as vascular endothelial growth factor (VEGF), is known to be an angiogenic factor. The aim of this study was to clarify whether TP regulates angiogenesis and to determine the clinicopathological role of TP expression in gallbladder cancer. Thirty-seven patients with gallbladder cancer who underwent curative surgical resection were included in this study, and we evaluated results in 21 of these patients in whom TP gene expression could be measured. Intratumoral TP gene expression was evaluated using the Danenberg tumor profile method. TP gene expression was classified into two groups according to median values: high and low. Tissue TP expression was classified into two groups (positive and negative) by a pathologist. Clinicopathological variables were compared between groups with high and low TP gene expression (TP high and TP low groups). No correlation was observed between TP gene expression (high and low groups) and any clinicopathological variables except survival rate. However, the survival rate was significantly lower in the TP high group (P<0.05). TP gene expression was associated with tissue TP expression evaluated by immunohistochemical staining (P<0.05). In addition, tumor microvessel density was significantly higher in the TP high group (P<0.05). Expression of the TP gene in gallbladder carcinoma is associated with angiogenesis and may be a new independent prognostic parameter.

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.

Thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase expression in colorectal cancer and normal mucosa in patients

To compare thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) gene polymorphism and expression in colorectal cancer (CRC), and normal mucosa in chemotherapy-naïve patients. TS, DPD and TP mRNA expression was analysed by real-time reverse-transcription polymerase chain reaction in primary CRC and adjacent normal tissues from 53 patients with glyceraldehyde-3-phosphate dehydrogenase as housekeeping gene. TS promoter (TSER and C/G SNP) and DPD IVS14+1G>A genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism assays. Moreover, the correlation between TS, DPD and TP expression and cytotoxicity of 5-fluorouracil was evaluated in Colo 320, HT-29, CaCo-2 and SW620 human CRC cell lines. TP and DPD mRNA expression was significantly different in tumour and normal tissue (7.51+/-13.50 vs. 1.10+/-0.57, P<0.05 and 0.60+/-0.63 vs. 1.17+/-0.55, P<0.0001, respectively), whereas no differences were observed in TS mRNA levels. High-grade, undifferentiated tumours (WHO grade 3) had significantly higher mRNA levels of TS with respect to moderately differentiated (WHO grade 2) carcinomas (0.38+/-0.37 vs. 0.00+/-0.44, respectively; P<0.05). Noteworthy, TS mRNA expression was significantly decreased (P<0.05) in homozygous TSER*3G/3G (-0.35+/-0.35) with respect to pooled homozygous TSER*2/2 and heterozygous TSER*2/3 genotypes (0.14+/-0.41). In-vitro results showed a higher sensitivity to 5-FU of cell lines with the lowest TS expression. The present results demonstrated significant differences in DPD and TP gene expression between normal mucosa and tumour samples, while TSER*3G/3G and high-grade histology were associated with significant variation in TS gene expression in tumour samples.

Thymidine Phosphorylase Expression Is Associated With Response to Capecitabine Plus Irinotecan in Patients With Metastatic Colorectal Cancer

To evaluate the clinical activity and toxicity of capecitabine plus irinotecan as first-line therapy for patients with metastatic colorectal cancer (mCRC), and to describe the association of expression of thymidine phosphorylase (TP), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) with antitumor activity. Patients with previously untreated mCRC received irinotecan days 1 and 8 intravenously, and capecitabine days 2 to 15 orally in 21-day cycles. Doses were irinotecan 125 mg/m2 and capecitabine 1,000 mg/m2 bid (n = 15; cohort 1), or irinotecan 100 mg/m2 and capecitabine 900 mg/m2 bid (n = 52; cohort 2). Tissues from primary and metastatic sites were assessed for TP, TS, and DPD gene and protein expression. An unacceptable level of GI toxicity in the first 15 patients led to a protocol modification in starting doses. The response rate was 45% (30 of 67 patients). Overall survival was associated with TP expression assessed by immunohistochemistry in both primary tumors (P = .045) and metastases (P = .001). Objective tumor response was associated with TP expression in primary tumors (odds ratio, 4.77; 95% CI, 1.25 to 18.18), with a similar trend in metastases (odds ratio, 8.67; 95% CI, 0.95 to 79.1). TP gene expression in primary tumors was also associated with response. These data indicate that capecitabine plus irinotecan is an active regimen against mCRC. The biomarker analysis (including metastatic tissue) was feasible in a multicenter setting, and provides preliminary evidence that TP expression may be a predictive marker for response.

Lymph Node Status and TS Gene Expression Are Prognostic Markers in Stage II/III Rectal Cancer After Neoadjuvant Fluorouracil-Based Chemoradiotherapy

According to the CAO/ARO/AIO-94 trial of the German Rectal Cancer Study Group, preoperative combined fluorouracil (FU) -based long-term chemoradiotherapy (CT/RT) is recommended for patients with International Union Against Cancer (UICC) stage II/III rectal cancer. However, despite the local benefit of neoadjuvant treatment, the overall prognostic value remains uncertain in comparison with adjuvant CT/RT. Furthermore, the prognostic value of molecular biomarkers, such as thymidylate synthase (TS), thymidine phosphorylase (TP), and dihydropyrimidine dehydrogenase (DPD), all of which are involved in the FU metabolism, is unknown in neoadjuvant settings. We assessed the impact of standardized preoperative CT/RT and intratumoral TS, TP, and DPD levels on patient outcome. Forty patients with rectal cancer pretherapeutic UICC stage II/III, receiving preoperative FU-based CT/RT (CAO/ARO/AIO-94 trial) followed by standardized surgery, including total mesorectal excision, were investigated. Downsizing, downstaging, tumor regression, as well as TS, TP, and DPD gene expression of post-treatment surgical specimens were correlated with disease-free survival (DFS) and overall survival (OS). Significant downsizing (P < .001) and downstaging (P = .001) were achieved with preoperative CT/RT. During a median follow-up of 49 months (95% CI, 43 to 58 months), the cancer recurrence rate was 28.2%. DFS and OS were significantly increased in patients with downstaging (P < .001 and P = .003, respectively), compared with patients without downstaging. All patients who developed cancer recurrence had a persistent positive lymph node status after preoperative CT/RT (P < .001) and a significantly higher TS gene expression (P = .035) compared with those patients without recurrence. Persistent positive lymph node status and high intratumoral TS expression after preoperative CT/RT are predictive of an unfavorable prognosis in rectal cancer UICC stage II/III.

5‐fluorouracil‐related gene expression levels in primary colorectal cancer and corresponding liver metastasis

Gene expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT) have been shown to be associated with response to 5-fluorouracil-based therapies. Analyzing these gene expression levels in liver metastases is important to obtain the best prediction of therapy. Our aim was to determine how TS, DPD, TP and OPRT gene expression levels in primary colorectal cancer (CRC) were related to those in liver metastases. Formalin-fixed, paraffin-embedded tumor specimens from 31 pairs of primary CRC and corresponding liver metastases were dissected by using laser-captured microdissection. RNA was extracted and cDNA was prepared by reverse-transcription. Quantitation of target gene and internal reference gene was performed using real-time PCR. No significant difference was seen between median mRNA expression levels of TS, DPD, TP and OPRT in primary cancer and those in corresponding liver metastases (median value: TS 1.48 vs. 1.43; p=0.92, DPD 0.19 vs.0.12; p=0.10, TP 1.20 vs. 0.98; p=0.39, OPRT 1.17 vs. 0.95; p=0.10). When matched tissue sets were compared on an individual basis, there was a significant correlation for TS mRNA expression between primary cancer and corresponding liver metastases (rs=0.52, p=0.0026). However, no correlation was seen between matched sets for DPD, TP or OPRT. Significant correlation was seen between DPD and TP expression levels in both primary CRC (rs=0.38, p=0.03) and liver metastases (rs=0.72, p<0.0001). A good prediction of TS mRNA levels in liver metastases can be obtained by measuring those of primary CRC, although no correlation was seen for DPD, TP and OPRT.

Molecular factors of 5-fluorouracil metabolism in colorectal cancer: Analysis of primary tumor and lymph node metastasis

Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) are predictive markers for tumor response to 5-fluorouracil-based therapies. To determine whether gene expression values measured in primary cancer tissue would be useful for prediction of response of lymph node metastases, the expressions of these genes were quantitatively analyzed in 35 pairs of primary colorectal cancer (CRC) and corresponding lymph node metastases using real-time PCR. DPD and TP mRNA levels were significantly lower in the primary colorectal tumor and lymph node metastases compared with the normal adjacent stroma tissue (p<0.01), whereas TS mRNA levels were significantly higher in the primary tumor and lymph node metastases than in the normal adjacent tissue (p<0.001). Median gene expression levels of TP and TS did not differ significantly between primary colorectal tumor and corresponding lymph node metastasis but median DPD gene expression levels in the lymph node metastases were significantly higher compared to matched primary colorectal tumors (p=0.015). There was a significant correlation for DPD, TP and TS gene expression levels between primary colorectal tumor specimens and the matched lymph node metastasis. These results suggest that biopsies of the tumor of origin may be valid for determining predictive markers for chemotherapy response in patients with metastatic CRC.

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