Dihydropyrimidine Dehydrogenase Expression Levels Research Articles

Dihydropyrimidine Dehydrogenase-Mediated Resistance to 5-Fluorouracil: Mechanistic Investigation and Solution.

5-Fluorouracil (5-FU) is one of the most widely used chemotherapeutics for the treatment of cancers associated with the aerodigestive tract, breast, and colorectal system. The efficacy of 5-FU is majorly affected by dihydropyrimidine dehydrogenase (DPD) as it degrades more than 80% of administered 5-FU into an inactive metabolite, dihydrofluorouracil. Herein we discuss the molecular mechanism of this inactivation by analyzing the interaction pattern and electrostatic complementarity of the DPD-5-FU complex. The basis of DPD overexpression in cancer cell lines due to significantly distinct levels of the miRNAs (miR-134, miR-27b, and miR-27a) compared to normal cells has also been outlined. Additionally, some kinases including sphingosine kinase 2 (SphK2) have been reported to correlate with DPD expression. Currently, to address this problem various strategies are reported in the literature, including 5-FU analogues (bypass the DPD-mediated inactivation), DPD downregulators (regulate the DPD expression levels in tumors), inhibitors (as promising adjuvants), and formulation development loaded with 5-FU (liposomes, nanoparticles, nanogels, etc.), which are briefly discussed in this Review.

Importance of Rare DPYD Genetic Polymorphisms for 5-Fluorouracil Therapy in the Japanese Population.

Dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is the rate-limiting enzyme in 5-fluorouracil (5-FU) degradation. In Caucasians, four DPYD risk variants are recognized to be responsible for interindividual variations in the development of 5-FU toxicity. However, these risk variants have not been identified in Asian populations. Recently, 41 DPYD allelic variants, including 15 novel single nucleotide variants, were identified in 3,554 Japanese individuals by analyzing their whole-genome sequences; however, the effects of these variants on DPD enzymatic activity remain unknown. In the present study, an in vitro analysis was performed on 41 DPD allelic variants and three DPD risk variants to elucidate the changes in enzymatic activity. Wild-type and 44 DPD-variant proteins were heterologously expressed in 293FT cells. DPD expression levels and dimerization of DPD were determined by immunoblotting after SDS-PAGE and blue native PAGE, respectively. The enzymatic activity of DPD was evaluated by quantification of dihydro-5-FU, a metabolite of 5-FU, using high-performance liquid chromatography-tandem mass spectrometry. Moreover, we used 3D simulation modeling to analyze the effect of amino acid substitutions on the conformation of DPD. Among the 41 DPD variants, seven exhibited drastically decreased intrinsic clearance (CL int ) compared to the wild-type protein. Moreover, R353C and G926V exhibited no enzymatic activity, and the band patterns observed in the immunoblots after blue native PAGE indicated that DPD dimerization is required for its enzymatic activity. Our data suggest that these variants may contribute to the significant inter-individual variability observed in the pharmacokinetics and pharmacodynamics of 5-FU. In our study, nine DPD variants exhibited drastically decreased or no enzymatic activity due to dimerization inhibition or conformational changes in each domain. Especially, the rare DPYD variants, although at very low frequencies, may serve as important pharmacogenomic markers associated with the severe 5-FU toxicity in Japanese population.

Human equilibrative nucleoside transporter‐1 expression is a predictor in patients with resected pancreatic cancer treated with adjuvant S‐1 chemotherapy

The high expression of human equilibrative nucleoside transporter‐1 (hENT1) and the low expression of dihydropyrimidine dehydrogenase (DPD) are reported to predict a favorable prognosis in patients treated with gemcitabine (GEM) and 5‐fluorouracil (5FU) as the adjuvant setting, respectively. The expression of hENT1 and DPD were analyzed in patients registered in the JASPAC 01 trial, which showed a better survival of S‐1 over GEM as adjuvant chemotherapy after resection for pancreatic cancer, and their possible roles for predicting treatment outcomes and selecting a chemotherapeutic agent were investigated. Intensity of hENT1 and DPD expression was categorized into no, weak, moderate or strong by immunohistochemistry staining, and the patients were classified into high (strong/moderate) and low (no/weak) groups. Specimens were available for 326 of 377 (86.5%) patients. High expression of hENT1 and DPD was detected in 100 (30.7%) and 63 (19.3%) of 326 patients, respectively. In the S‐1 arm, the median overall survival (OS) with low hENT1, 58.0 months, was significantly better than that with high hENT1, 30.9 months (hazard ratio 1.75, P = 0.007). In contrast, there were no significant differences in OS between DPD low and high groups in the S‐1 arm and neither the expression levels of hENT1 nor DPD revealed a relationship with treatment outcomes in the GEM arm. The present study did not show that the DPD and hENT1 are useful biomarkers for choosing S‐1 or GEM as adjuvant chemotherapy. However, hENT1 expression is a significant prognostic factor for survival in the S‐1 arm.

Dihydropyrimidine Dehydrogenase Levels in Colorectal Cancer Cells Treated with a Combination of Heat Shock Protein 90 Inhibitor and Oxaliplatin or Capecitabine.

Purpose: Dihydropyrimidine dehydrogenase (DPD) is the principal enzyme in the catabolism of fluoropyrimidine drugs including capecitabine. A recent report has suggested that oxaliplatin chemotherapy is associated with elevated DPD levels and chemoresistance pattern. As a newly developed chemotherapeutic agent, 17-allyloamino-17-demethoxy-geldanamycin (17-AAG) can be effective in combination therapy with oxaliplatin and capecitabine in colorectal cancer (CRC). DPD expression level can be a predictive factor in oxaliplatin and capecitabine-based chemotherapy. We evaluated DPD in mRNA and protein levels with new treatments: 17-AAG in combination with oxaliplatin and capecitabine in HT-29 and HCT-116 cell lines. Methods: Drug sensitivity was determined by the water-soluble tetrazolium-1 assay in a previous survey. Then, we evaluated the expression levels of DPD and its relationship with the chemotherapy response in capecitabine, oxaliplatin, and 17-AAG treated cases in single and combination cases in two panels of CRC cell lines. DPD gene and protein expression levels were determined by real-time polymerase chain reaction and western blotting assay, respectively. Results: DPD gene expression levels insignificantly increased in single-treated cases versus untreated controls in both cell lines versus controls. Then, the capecitabine and oxaliplatin were added in double combinations, where DPD gene and protein expression increased in combination cases compared to pre-chemotherapy and single drug treatments. Conclusion: The elevated levels of cytotoxicity in more effective combinations could be related to a different mechanism apart from DPD mediating effects or high DPD level in the remaining resistance cells (drug-insensitive cells), which should be investigated in subsequent studies.

Dihydropyrimidine dehydrogenase overexpression correlates with potential resistance to 5-fluorouracil-based treatment in head and neck squamous cell carcinoma

Background: Although dihydropyrimidine dehydrogenase (DPD) expression has been reported to correlate with 5-fluorouracil (5-FU) resistance in several cancers, the relationship between DPD expression and chemotherapeutic resistance to 5-FU remains unclear in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to determine the impact of DPD expression on 5-FU sensitivity and survival in HNSCC patients receiving 5-FU-based treatment. Methods: To study the correlation between DPD expression and clinical outcome of HNSCC patients who had undergone concurrent chemoradiotherapy with 5-FU-based regimens, we first examined DPD mRNA expression of these patient samples. We next developed a 5-FU-resistant HNSCC cell line (HSC-3R) to clarify the association between DPD expression and 5-FU resistance. Clonogenic survival assay was performed to determine the sensitivity of HSC-3 cells and HSC-3R cells to 5-FU. DPD expression levels in parental HSC-3 and HSC-3R cell lines were then examined by Western blotting and real-time quantitative polymerase chain reaction analysis. Lastly, we performed WST-8 assay to examine the effects of 5-Chloro-2,4-dihydroxypyridine (CDHP), a 5-FU modulator to competitively inhibit DPD, on 5-FU cytotoxicity in the HSC-3 and HSC-3R cells, to evaluate if inhibition of DPD can restore the sensitivity of HNSCC cells to 5-FU. Results: Nineteen HNSCC patients who had undergone concurrent chemoradiotherapy with 5-FU-based regimens were enrolled in this study. The cut-off value for DPD mRNA expression calculated from the ROC curve was 8.53 against cancer-specific survival of these patients. The high-level DPD expression group showed significantly shorter overall and cancer-specific survival compared to the low-level DPD expression group (P=0.0018 and 0.0004, respectively). Both of protein and mRNA expression levels were greater in the HSC-3R cells than that in the HSC-3 cells. While HSC-3R cells showed 12.64-fold greater resistance to 5-FU compared with HSC-3 cells, the combination of 5-FU with CDHP had a significant inhibitory effect on 5-FU cytotoxicity in HSC-3R cells in CDHP exposure. Conclusions: In this study, we clarified that the high-level DPD expression group of HNSCC patients undergoing concurrent chemoradiotherapy with 5-FU-based regimens showed significantly shorter overall survival. The 5-FU-resistant cells established from HNSCC cells showed increased DPD expression and attenuated 5-FU resistance induced by CDHP. Our results suggested that a high level of DPD expression was correlated with 5-FU resistance and that DPD expression level might be a predictive biomarker in 5-FU-based chemoradiotherapy for HNSCC patients.

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.

The Impact of the Expression Level of Intratumoral Dihydropyrimidine Dehydrogenase on Chemotherapy Sensitivity and Survival of Patients in Gastric Cancer: A Meta-Analysis.

The potential impact that the intratumoral expression level of dihydropyrimidine dehydrogenase (DPD) has on chemotherapy sensitivity and long-term survival for gastric cancer (GC) patients remains controversial; therefore, this study seeks to clarify this issue. Our meta-analysis was performed using Review Manager (RevMan) 5.3 software. In vitro drug sensitivity tests, correlation coefficients between sensitivity to 5-fluorouracil (5-FU), and expression levels of intratumoral DPD were used as effective indexes to analyse. Overall survival (OS) and progression-free survival (PFS) were used as endpoints for patient outcome, and hazard ratios (HRs) and 95% confidence intervals (CIs) were noted as measures of effect. There were 15 eligible studies including 1805 patients for the final analysis. The analysis revealed a statistically significant difference between the expression level of intratumoral DPD activity, DPD mRNA levels, and sensitivity to 5-FU in GC patients, with high expression levels of intratumoral DPD resulting in low sensitivity to 5-FU. However, no matter what therapeutic regimens were used, there was no significant difference for patient outcomes between high and low DPD expression groups, either in OS or in PFS. In conclusion, high levels of intratumoral DPD expression have a negative impact on sensitivity to 5-FU in GC patients, but no prognostic value for long-term survival was uncovered.

Changes in expression levels of excision repair cross-complementing group 1 (ERCC1) and dihydropyrimidine dehydrogenase (DPD) after first-line oxaliplatin-based chemotherapy for metastatic colorectal cancer: Results of a multicenter study.

463 Background: Our previous single-center study showed that expression levels of ERCC1 (a nucleotide excision repair pathway gene) and DPD (a pyrimidine catabolic pathway gene) in liver metastases from colorectal cancers (CRCs) were significantly higher in patients given oxaliplatin as first-line chemotherapy than those not given oxaliplatin. We conducted this multicenter study to validate our findings and better define a basic rationale for second-line therapy for metastatic CRC resistant to first-line therapy. Methods: Irinotecan-naive patients with mCRC (n = 190) were enrolled at 17 sites in Japan. Resected liver metastases were available for 91 patients who had received FOLFOX (or FOLFOX plus bevacizumab; FOLFOX group) and 99 who had not received oxaliplatin-based chemotherapy (non-oxaliplatin group). We measured mRNA expression levels of ERCC1 and DPD by real-time RT-PCR. Protein expression levels of ERCC1 and DPD were evaluated immunohistochemically. The methylation level of the long interspersed nucleotide element-1 (LINE-1; a surrogate m arker of global DNA methylation) was determined by pyrosequencing. Results: The median age at liver resection was 64 years (range, 32–89). Baseline features (e.g., sex, histology) were similar in the groups. Expression levels of DPD at the mRNA and protein levels were significantly higher in the FOLFOX group than in the non-oxaliplatin group (p < 0.05). The mRNA expression level of ERCC1 was slightly, but not significantly higher in the FOLFOX group than in the non-oxaliplatin group (p = 0.053). Interestingly, ERCC1 expression levels correlated with DPD expression levels (r = 0.38, p< 0.0001). LINE-1 methylation levels were similar in the groups. Conclusions: This multicenter study confirmed increased expression levels of DPD after first-line oxaliplatin-based chemotherapy, which may affect chemosensitivity to subsequent therapy. ERCC1 expression and epigenetic control by mechanisms such as aberrant promoter methylation of specific genes warrant further investigation.

Correlated analysis of 5 fluorouracil metabolic enzymes with tumor response after SOX regimen neoadjuvant chemotherapy in advanced gastric cancer

To analyze the impact of mRNA expression of oral fluoropyrimidine (S-1) metabolism on treatment outcomes in locally advanced gastric cancer patients on preoperative S-1 oxaliplatin-based chemotherapy. Between June 2012 and March 2013, 32 patients with preoperative AJCC stage II-III gastric cancer patients were enrolled. They received S-1 (80 mg·m⁻² × d⁻¹, days 1-14) and oxaliplatin (130 mg/m², day 1) every 3 weeks and subsequently underwent gastrectomy with D2 lymphadenectomy. Paired tumor and normal fresh frozen tissues were collected to evaluate the mRNA levels of thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and OPRT with quantitative reverse transcription(RT) -PCR. Among them, 21 (65.6%) patients had clinical tumor response and histological response occurred in 10 (31.3%) patients. Quantitative RT-PCR results showed that OPRT mRNA expression was significantly higher in clinical tumor responders than non-responders (3.95 ± 0.81 vs 1.79 ± 0.64, P = 0.005). Diffuse-type gastric cancer patients (n = 22) demonstrated higher OPRT expression levels than intestinal-type(n = 10) ones (2.54 ± 0.75 vs 1.49 ± 0.56, P = 0.014). The mRNA expressions of TS and TP in gastric cancer tissues with lymph node (LN) metastasis (n = 13) were significantly higher than those in gastric cancer tissues without LN metastasis (n = 19, both P < 0.05) .Similar results were not found for comparing dihydropyrimidine dehydrogenase expression levels (all P > 0.05). OPRT, TS and TP may become potential predictive biomarkers in advanced gastric cancer patients on oral fluoropyrimidine (S-1)-based chemotherapy.

Clinical significance of the thymidylate synthase, dihydropyrimidine dehydrogenase, and thymidine phosphorylase mRNA expressions in hepatocellular carcinoma patients receiving 5-fluorouracil-based transarterial chemoembolization treatment

PurposeTo determine whether 5-fluorouracil (5-FU) sensitivity is associated with the mRNA expressions of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and thymidine phosphorylase (TP) in patients with hepatocellular carcinoma (HCC) treated with 5-FU-based transarterial chemoembolization (TACE).MethodsFormalin-fixed, paraffin-embedded tumor specimens from 40 patients treated with 5-FU-based TACE were selected for the examination of TS, DPD, and TP expression level by a quantitative real-time reverse transcription- polymerase chain reaction (PCR) technique. Patients were categorized into high and low expression groups according to the median expression level of each enzyme. Associations between the mRNA expression levels of TS, DPD, and TP and clinical parameters including treatment efficacies, clinicopathological factors, and prognosis were assessed.ResultsHigh DPD expression was associated with worse treatment outcome, including intrahepatic disease progression rate (hazard ratio [HR] for high DPD versus low DPD, 2.212; 95% confidence interval [CI], 1.030–4.753; P = 0.042), extrahepatic disease progression rate (HR for high versus low DPD, 3.171; 95% CI, 1.003–10.023; P = 0.049), and progression-free survival (HR for high versus low DPD, 2.308; 95% CI, 1.102–4.836; P = 0.027). No correlation was found between the mRNA expression of TS/TP and treatment outcome.ConclusionDPD mRNA expression level was negatively correlated with the clinical outcomes of HCC patients treated with 5-FU-based TACE. These results provide indirect evidence that high DPD mRNA expression is a predictive marker of treatment resistance for 5-FU.

The upregulation of dihydropyrimidine dehydrogenase in liver is involved in acquired resistance to 5-fluorouracil

BackgroundAcquired resistance to 5-fluorouracil (5-FU) is one of the important reasons for failure in 5-FU-based chemotherapy. The upregulation of dihydropyrimidine dehydrogenase (DPD) in tumours was reported as an important factor for acquired 5-FU resistance. The aim of this study is to examine whether intra-hepatic DPD was involved in acquired 5-FU resistance. MethodsHT-29 human colorectal xenograft tumours were established in nude mice. After long-term exposure to 5-FU, some of the tumour became “resistant” and the others remained “sensitive” to 5-FU. DPD expression levels in the livers and tumours of “resistant”, “sensitive” or untreated mice were examined, and pharmacokinetics of 5-FU in rats’ plasma were investigated. Gimeracil, a DPD inhibitor, was checked whether it could reverse the reduced bioavailability of 5-FU. ResultsDPD expression was upregulated obviously in tumours of “resistant” mice as reported previously. Importantly, DPD expression was also upregulated significantly in livers of “resistant” mice, compared with those of “sensitive” or untreated mice. Furthermore, the upregulation of DPD expression in livers led to accelerated metabolism of 5-FU. Gimeracil was found to reverse the reduced serum 5-FU concentration. The cultured tumour cells from 5-FU treated mice showed relative sensitivity to higher concentration of 5-FU, even the “resistant” tumour cells. ConclusionOur study suggested that the upregulation of DPD in liver may be involved in acquired resistance to 5-FU, and DPD inhibitors or increasing 5-FU dosage may have potential application in overcoming 5-FU acquired resistance.

OPRT Is a Potential Predictive Factor for the Response to S-1 in Gastric Cancer

Objective: To analyze the impact of mRNA expression of oral fluoropyrimidine (S-1) metabolism (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase [OPRT]), on treatment outcomes in locally advanced gastric cancer patients receiving preoperative S-1 combined with oxaliplatin chemotherapy. Methods: Preoperative stage III gastric cancer patients received S-1 (80 mg/m2/day; days 1-14) and oxaliplatin (130 mg/m2; day 1) every 3 weeks and subsequently received gastrectomy with D1/D2 lymphadenectomy. Paired tumor and normal fresh frozen tissues were collected to evaluate mRNA levels of thymidylate synthase, thymidine phosphorylase, dihydropyrimidine dehydrogenase, and orotate phosphoribosyltransferase using quantitative reverse-transcriptase polymerase chain reaction. Results: Between December 2009 and October 2010, thirty-five patients were enrolled in this study. 24 (68.5%) patients had clinical tumor response and 10 (28.6%) patients achieved histological response. Quantitative reverse-transcriptase polymerase chain reaction results showed that orotate phosphoribo-syltransferase (OPRT) mRNA expression was significantly higher in histological responders than non-responders (3.75 vs. 1.81, P = 0.005). Diffuse-type gastric cancer patients demonstrated higher orotate phosphoribosyltransferase (OPRT) expression levels than intestinal-type ones (2.79 vs. 1.60, P = 0.014). Similar results were not found when comparing thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase expression levels. Conclusion: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy. Mini Abstract: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy.

Upregulation of ERCC1 and DPD expressions after oxaliplatin-based first-line chemotherapy for metastatic colorectal cancer

Background:The updated randomised phase 2/3 FIRIS study demonstrated the noninferiority of IRIS (irinotecan and S-1) to FOLFIRI (irinotecan, folinic acid, and 5-FU) for metastatic colorectal cancer. Meanwhile, in the subset analysis including patients who previously have undergone oxaliplatin-containing chemotherapy, the IRIS group showed longer survival than the FOLFIRI group. However, the molecular mechanism underlying this result is still unknown.Methods:The National Cancer Institute 60 (NCI60) cell line panel data were utilised to build the hypothesis. A total of 45 irinotecan-naive metastatic colorectal cancer patients who had undergone hepatic resection were included for the validation study. The mRNA expressions of excision repair cross-complementing group 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), and topoisomerase-1 (TOP1) were evaluated by quantitative RT–PCR. The expressions of ERCC1 and DPD were also evaluated by immunohistochemistry.Results:Sensitivity to oxaliplatin in 60 cell lines was significantly correlated with that of 5-FU. Resistant cells to oxaliplatin showed significantly higher ERCC1 and DPD expression than sensitive cells. In validation study, ERCC1 and DPD but not TOP1 expressions in cancer cells were significantly higher in FOLFOX (oxaliplatin, folinic acid, and 5-FU)-treated patients (N=24) than nontreated patients (N=21). The ERCC1 and DPD protein expressions were also significantly higher in FOLFOX-treated patients.Conclusion:The ERCC1 and DPD expression levels at both mRNA and protein levels were significantly higher in patients with oxaliplatin as a first-line chemotherapy than those without oxaliplatin. The IRIS regimens with the DPD inhibitory fluoropyrimidine may show superior activity against DPD-high tumours (e.g., tumours treated with oxaliplatin) compared with FOLFIRI.

Suppression of heat shock protein 27 expression promotes 5-fluorouracil sensitivity in colon cancer cells in a xenograft model

The present study investigates whether the expression levels of heat shock protein27 (HSP27) in colon cancer cells are associated with 5-fluorouracil (5-FU) sensitivity in a xenograft model, as well as the mechanism responsible for regulating 5-FU sensitivity. HCT116 cells which have a high expression of HSP27 were stably transfected with specific short hairpin RNA (shRNA) in order to suppress HSP27 expression. The association between HSP27 protein expression levels and 5-FU sensitivity was evaluated in a mouse xenograft model. The mRNA expression of 5-FU metabolic enzymes and cell apoptosis were also analyzed in the transfected cells. The suppression of HSP27 protein expression led to enhanced 5-FU sensitivity. The mRNA expression levels of dihydropyrimidine dehydrogenase and orotate phosphoribosyltransferase, but not those of thymidylate synthase, and the number of apoptotic cells increased in the transfected cells after 5-FU exposure. In conclusion, the suppression of HSP27 expression in colon cancer cells may promote 5-FU sensitivity by inducing apoptosis, despite the acceleration in 5-FU metabolism.

The role of TP, TS, and DPD as potential predictors of outcome following capecitabine plus oxaliplatin (XELOX) versus bolus 5-fluorouracil/leucovorin (5-FU/LV) as adjuvant therapy for stage III colon cancer: Biomarker findings from study NO16968 (XELOXA).

3578 Background: In NO16968, XELOX was superior in terms of disease-free survival (DFS) and overall survival (OS) to bolus 5-FU/LV as adjuvant therapy for stage III colon cancer (Schmoll et al. ASCO GI 2012). Three key enzymes appear to have the potential to predict efficacy and/or safety of fluoropyrimidine-based treatment: thymidine phosphorylase (TP), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD). We evaluated the association between baseline TP, TS and DPD and outcome (DFS and OS). Methods: Pts with stage III colon cancer received either XELOX (8 cycles, 24w) or bolus 5-FU/LV (Mayo Clinic, 6 cycles, 24w; Roswell Park, 4 cycles, 32w). The primary study endpoint was DFS; secondary endpoints included OS. TP, TS and DPD expression levels were determined in formalin-fixed, paraffin-embedded tissues by RT-PCR, and the median used as a cut-off point: high (above median) vs. low (below median). Results: The biomarker population included 498 (26%) of 1886 pts entered (XELOX, n=242; 5-FU/LV, n=256). Baseline demographics, tumor characteristics, cancer history and efficacy (DFS and OS) were similar to those in the main study population. Cox regression analysis for DFS (Table). In the XELOX group pts with low DPD and TP levels and a high TP/DPD ratio appeared to have significantly better DFS; this effect was not observed with 5-FU/LV. Subgroup analysis shows that the difference between XELOX and 5-FU/LV was also higher in pts with low DPD levels. Conclusions: These exploratory findings suggest that tumor DPD and TP RNA levels could be used to predict outcomes of adjuvant treatment with fluoropyrimidine/oxaliplatin combinations, and should be validated prospectively. Analysis of the current dataset is ongoing and further details on potential biomarkers will be available. [Table: see text]

Changes in expression levels of excision repair cross-complementing group 1 (ERCC1) and dihydropyrimidine dehydrogenase (DPD) during first-line oxaliplatin-based treatment of advanced metastatic colorectal cancer (mCRC).

520 Background: We previously reported the updated results of the FIRIS study, reconfirming the non-inferiority of IRIS (irinotecan/S-1) to FOLFIRI with progression-free survival (PFS) as the primary endpoint (ASCO2011 #3562). Moreover, in patients who had received prior oxaliplatin-based chemotherapy, median OS was significantly longer in the IRIS group than in the FOLFIRI group (adjusted HR=0.755, 95% CI: 0.580–0.987). The aim of this retrospective study was to elucidate molecular mechanisms underlying the better survival in the IRIS group among patients who had previously received oxaliplatin. Methods: Irinotecan-naive mCRC patients (ECOG PS 0–1, adequate organ function, resectable liver metastases) were enrolled. Resected liver metastases were available for 17 patients who had received FOLFOX and 21 patients who had received no prior oxaliplatin-based chemotherapy. Using laser captured microdissection and real-time RT-PCR, we analyzed mRNA expression of excision repair cross complementing group 1 (ERCC1) and dihydropyrimidine dehydrogenase (DPD), factors related to drug resistance and metabolism. Results: Baseline factors (sex, age, histology) of the subjects were similar in the two groups. Expression levels of nucleotide excision repair pathway gene ERCC1 and pyrimidine catabolic pathway gene DPD differed significantly between the FOLFOX group and non-oxaliplatin group. Mean expression levels of ERCC1 and DPD were respectively 1.9 and 7.4 times higher in the FOLFOX group than in the non-oxaliplatin group (ERCC1: 1.58 vs. 0.85, P=0.0005; DPD: 0.580 vs. 0.078, P=0.01). Conclusions: Increased expression of ERCC1 and DPD after first-line oxaliplatin-based chemotherapy is probably related to chemosensitivity to subsequent therapy. Tumor heterogeneity and clonal selection of tumor cells resistant to oxaliplatin may also be involved.

Roles of thymidylate synthase and dihydropyrimidine dehydrogenase expression in blood as predictors of response to multimodal therapy in esophageal cancer

Thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) RNA expression in peripheral blood was examined as a noninvasive molecular predictor of response to neoadjuvant radiochemotherapy in patients with locally advanced cancer of the esophagus. Blood samples were drawn from 29 patients with esophageal cancer (10 squamous cell carcinomas and 19 adenocarciomas) before neoadjuvant radiochemotherapy. After extraction of cellular tumor RNA from blood samples, quantitative expression analysis of TS and DPD was performed with quantitative real-time reverse-transcription polymerase chain reaction. Twenty of 29 (68%) of patients had a minor histopathologic response, and 9 of 29 (32%) had a major response to neadjuvant radiochemotherapy. RNA expression in the blood of patients was detectable for TS in 86%, for DPD in 97%, and in 100% for β-actin. No significant associations were detected between TS and DPD expression levels and clinical variables of the patients. A high expression level for TS was associated with a minor response to neoadjuvant treatment (P = .046), while there was no significant association between DPD and response to therapy. Combined analysis of TS and DPD expression increased the specificity for the prediction of response to 100%. No major responder to therapy had high expression levels for both genes in their peripheral blood. Quantitation of TS and DPD in peripheral blood may be a highly specific analysis to identify a subset of patients who do not respond to neoadjuvant radiochemotherapy and may therefore prevent expensive, noneffective, and potentially harmful therapies in a substantial number of patients with esophageal cancer.

Thymidylate Synthase, Dihydropyrimidine Dehydrogenase, Thymidine Phosphorylase, Orotate Phosphoribosyltransferase mRNA Expression in Lung Cancer Metastatic Lymph Node Samples Obtained by Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration – A Pilot Study

Introduction/BackgroundPyrimidine antimetabolites' target molecules thymidylate synthase (TS) and flopropyrimidine-metabolising enzymes such as dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyltransferase (OPRT) are known as biomarkers of 5-fluorouracil (5-FU) treatment. The purpose of this study was to evaluate the feasibility of mRNA expression analysis for TS, DPD, TP, and OPRT using metastatic lymph node samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in patients with non–small-cell lung cancer (NSCLC). Materials and MethodsMetastatic lymph nodes from 20 patients with NSCLC diagnosed by EBUS-TBNA were analyzed. After pathological diagnosis, additional sections were made and confirmed the existence of tumor cells by hematoxylin and eosin staining. Tumor cells were extracted using laser-captured microdissection and the mRNA levels were measured by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for each marker. ResultsThere were 17 mediastinal and 3 hilar lymph nodes. The histology included 13 cases of adenocarcinoma and 7 cases of squamous cell carcinoma. DPD and TP could be successfully analyzed in all samples and TS and OPRT could be measured in 18 samples. The median values of the relative TS, DPD, TP, and OPRT mRNA expression levels were 10.68, 6.23, 18.32, and 1.77, respectively. ConclusionTS, DPD, TP, and OPRT mRNA expression levels could be analyzed using metastatic lymph node samples obtained by EBUS-TBNA. The result of this pilot study may be applicable for further translational research for the treatment of locally advanced NSCLC.

Thymidylate synthetase and dihydropyrimidine dehydrogenase mRNA levels in esophageal cancer

This study investigated the mRNA levels of thymidylate synthetase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) in esophageal squamous cell carcinoma (ESCC). TYMS and DPYD gene expression was quantified using real-time RT-PCR in 56 patients with ESCC, co-amplified with glyceraldehyde-3-phosphate dehydrogenase as an internal standard. The results were analyzed with reference to the clinicopathological characteristics and the prognosis of the ESCC patients. The TYMS and DPYD expression levels in patients positive with lymphatic invasion were significantly higher compared to those in patients who exhibited negative lymphatic invasion (TYMS P=0.0127, DPYD P=0.0127). Patients were classified into the groups high TYMS/DPYD, high TYMS but low DPYD, low TYMS but high DPYD and low TYMS/DPYD. The highest survival rate was found in the group with low TYMS/DPYD and the lowest survival rate in the group with high TYMS/DPYD (P=0.017). It was concluded that, on the basis of the multivariate analysis, TYMS mRNA expression is a candidate that serves as an independent prognostic factor for ESCC patients.

Thymidylate synthase and dihydropyrimidine dehydrogenase expression levels are associated with response to S-1 plus carboplatin in advanced non-small cell lung cancer

S-1 is an oral fluoropyrimidine derivative that is active against non-small cell lung cancer (NSCLC). Development of S-1 combination chemotherapy for advanced NSCLC is under way. Given the importance of designing therapeutic strategies based on specific tumor biology, we have evaluated the relation between immunohistochemical expression levels of thymidylate synthase (TS), orotate phosphoribosyltransferase (OPRT), or dihydropyrimidine dehydrogenase (DPD) and the response to treatment with S-1 plus carboplatin in patients with advanced NSCLC. Chemotherapy-naïve patients with advanced (stage IIIB or IV) NSCLC, an Eastern Cooperative Oncology Group performance status of 0 or 1, adequate organ function, and archival tumor tissue were assigned to receive S-1–carboplatin (n=22). The predictive or prognostic relevance of the molecular markers was also examined by their evaluation in patients treated with paclitaxel plus carboplatin (n=25). Expression levels of TS, OPRT, or DPD in tumor specimens did not differ significantly between patients treated with S-1–carboplatin and those treated with paclitaxel–carboplatin. A low expression level of TS or of DPD was associated with a better response and longer survival in patients treated with S-1–carboplatin but not in those treated with paclitaxel–carboplatin. Tumor expression levels of TS and DPD are predictive of response to S-1–carboplatin chemotherapy in patients with advanced NSCLC.