Low Dihydropyrimidine Dehydrogenase Research Articles

5-Fluorouracil metabolic pathway genes predict recurrence risk following adjuvant S-1 therapy: Results of an ancillary analysis from a phase III trial of resected biliary tract cancer (JCOG1202A1).

S-1, an oral fluoropyrimidine derivative, is standard adjuvant therapy for resected biliary tract cancer (BTC), based on the results of the JCOG1202, a phase III trial evaluating the survival benefit with adjuvant S-1 following curative resection for BTC compared to surgery alone. This multicenter ancillary study of the JCOG1202 aimed to evaluate the prognostic impact of the 5-fluorouracil (5-FU) metabolic pathway genes including thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). The 5-FU metabolic pathway genes were measured in tumor cells from formalin-fixed paraffin-embedded resected specimens from 183 patients (surgery alone: n = 94; adjuvant S-1: n = 89). We randomly divided them into training (n = 96) and validation sets (n = 87) for evaluating the interaction between gene levels and RFS benefits in the treatment arm. RFS benefits of adjuvant S-1 were observed in the low DPD (HR = 0.440 and 0.748, respectively in the training and validation sets) and the low TP groups (HR = 0.709 and 0.602, respectively). Clinicopathological characteristics were well balanced between low and high DPD populations. More advanced stage tumors were observed in high TP populations as compared to those in low TP populations (p = .0332). The results suggest the RFS benefit of adjuvant S-1 in resected BTC patients with low DPD and low TP gene expressions.

Thymidylate Synthase Inhibition with Capped F10 Is Effective in TP53 Null Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is an aggressive cancer of the myeloid linage characterized by progressive marrow failure and death. Responses to therapy are between 50 and 80% but relapse is common and salvage rates once relapsed are low. The risk of relapse is greatly influenced by specific somatic mutations. One of the highest risk mutations in AML involves loss of the tumor suppressor TP53. Patients with AML harboring loss of TP53 function have a dismal prognosis and there is a clear need for new approaches. To identify novel agents with activity against TP53 null AML, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 in a genetically defined murine AML cell line. Clonal isolates were generated by serial dilution and loss of detectable TP53 was confirmed by Western blotting. A high throughput screen of currently FDA approved agents was performed on these cell lines. When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents either directly targeted thymidylate synthase or its substrates via inhibition of the folate cycle. This finding suggests that thymidylate synthase (TS) is a key enzyme in TP53 null AML cells. To confirm and extend these results, an additional murine AML TP53 null/WT cell line pair was generated. Chemotherapy resistance was confirmed in the TP53 null cell lines when treated with doxorubicin (Figure 1). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that is a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (Figure 1). To extend this result TP53 null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly or doxorubicin 3mg/kg and cytarabine at 100 mg/kg daily for 5 days. CF10 provided a significant survival benefit in the TP53 null AML in this highly aggressive model where doxorubicin and cytarabine did not (p=0.0057, Figure 2). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and containing a GFP reporter. When the mixed population was treated with a titration of doxorubicin there was a highly significant increase in GFP+ cells indicating the involvement of TP53 in doxorubicin mediated cell death. In contrast when cells were treated with a titration of CF10 there was a no significant increase in GFP+ population consistent with CF10 inducing a TP53 independent mechanism of cell death. For CF10 to of use clinically there must be an acceptable therapeutic window. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg to mimic DPD deficiency. Animals were then treated with repeated doses of either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals maintained their weight compared to controls (data not shown). Taken together these data suggest that TS is a viable target in TP53 null AML and CF10 is well tolerated and provides a survival benefit in a highly aggressive, chemotherapy resistant model. Ongoing studies will explore the mechanisms involved in CF10 induced cell death.

Capped F10, a novel DNA directed fluoropyrimidine, in TP53 mutated acute myeloid leukemia (AML).

7046 Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by resistance to treatment and poor outcomes. One of the most devastating mutations in AML involves loss of the tumor suppressor TP53. AML harboring this loss carries a dismal prognosis and there is a clear need for new approaches. Methods: To identify new therapeutics, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 using a murine AML cell line. Clonal isolates were obtained and loss of detectable TP53 was confirmed by Western Blotting. A high throughput screen of currently FDA approved anticancer agents was performed. Results: When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents targeted the folate/thymidylate synthase axis. This suggests that thymidylate synthase (TS) is a vulnerability in TP53 null AML cells. To confirm TS as a target in TP53 null AML an additional murine AML TP53 null/WT cell line pair was generated using CRISPR/CAS9. When these cell lines were treated with doxorubicin the TP53 null cells were significantly more resistant (p≤0.001 at 10nM). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that has been shown to be a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (p = 0.31 at 50pM). To extend this result TP53 WT or null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly. CF10 provided a significant survival benefit in the TP53 null but not WT injected mice consistent with CF10 having activity against TP53 null AML (p = 0.035 vs 0.13). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and a GFP reporter. When the mixed population was treated with the IC90 concentration of 5-fluorouracil there was a highly significant increase in GFP+ cells indicating a TP53 mediated cell death (p = < 0.001). In contrast when cells were treated with the IC90 concentration of CF10 there was a non-significant increase in GFP+ population (p = 0.074) consistent with a mainly TP53 independent mechanism. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg alone or in combination with either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals did not have a significant change in weight compared to controls. Conclusions: Taken together these data suggest that CF10 is a promising new agent for TP53 null AML.

Effects of gene expression in 5-FU metabolic pathways in a phase III trial evaluating adjuvant S-1 therapy compared to surgery alone following curative resection for biliary tract cancer (JCOG1202A1).

732 Background: S-1, an oral fluoropyrimidine derivative, is standard adjuvant therapy in Japanese patients with resected biliary tract cancer (BTC), based on the results of JCOG1202, a phase III trial evaluating adjuvant S-1 following curative resection for BTC compared to surgery alone. The efficacy of 5-FU-based therapy is related to the expression of enzymes involved in 5-FU metabolic pathways, such as thymidylate synthase (TS), thymidine phosphorylase (TP), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD). The aim of this study was to evaluate the impact of messenger RNA (mRNA) levels of the four 5-FU metabolic pathway genes on the outcomes of patients enrolled in JCOG1202. Methods: Tumor cell RNA was isolated from formalin-fixed paraffin-embedded primary BTC specimens resected in 264 of 440 randomized patients in JCOG1202. The four 5-FU metabolic pathway genes were measured in 183 patients (surgery alone: n = 94; adjuvant S-1: n = 89) who were randomly divided to training (n = 96) or validation set (n = 87). The endpoints of interest were the predictive values of the four genes for the efficacy of adjuvant S-1 on overall survival and relapse-free survival (RFS), and we here report the results regarding RFS. Cut-off levels for mRNA expression were selected in the training set which minimized the bootstrap p-values (2,000 samples) of an interaction term of treatment (surgery alone or S-1) and mRNA expression in a Cox regression model. Results: There were no obvious differences in each mRNA level and clinical characteristics between surgery alone and adjuvant S-1 groups. RFS tended to be better with adjuvant S-1 (hazard ratio [HR] = 0.790, [95% confidence interval: 0.524-1.192]) compared to surgery alone, which was maintained in the low DPD population (HR = 0.440 [0.216-0.898] in training set and 0.748 [0.334-1.675] in validation set), in the low TP population (HR = 0.709 [0.388-1.296] and 0.602 [0.287-1.262]), and in the high OPRT population (HR = 0.520 [0.152-1.779] and 0.609 [0.161-2.304]). Conclusions: The efficacy of adjuvant S-1 on RFS was representative in BTC patients with intratumoral gene expressions of low DPD, low TP, and high OPRT, which was disappeared in the population with high DPD, high TP, and low OPRT. These biomarkers might be useful to predict therapeutic benefits with S-1 containing chemotherapy for BTC.

Endogenous metabolic markers for predicting the activity of dihydropyrimidine dehydrogenase.

Five‐fluorouracil (5‐FU) is a chemotherapeutic agent that is mainly metabolized by the rate‐limiting enzyme dihydropyrimidine dehydrogenase (DPD). The DPD enzyme activity deficiency involves a wide range of severities. Previous studies have demonstrated the effect of a DPYD single nucleotide polymorphism on 5‐FU efficacy and highlighted the importance of studying such genes for cancer treatment. Common polymorphisms of DPYD in European ancestry populations are less frequently present in Koreans. DPD is also responsible for the conversion of endogenous uracil (U) into dihydrouracil (DHU). We quantified U and DHU in plasma samples of healthy male Korean subjects, and samples were classified into two groups based on DHU/U ratio. The calculated DHU/U ratios ranged from 0.52 to 7.12, and the two groups were classified into the 10th percentile and 90th percentile for untargeted metabolomics analysis using liquid chromatography‐quantitative time‐of‐flight‐mass spectrometry. A total of 4440 compounds were detected and filtered out based on a coefficient of variation below 30%. Our results revealed that six metabolites differed significantly between the high activity group and low activity group (false discovery rate q‐value < 0.05). Uridine was significantly higher in the low DPD activity group and is a precursor of U involved in pyrimidine metabolism; therefore, we speculated that DPD deficiency can influence uridine levels in plasma. Furthermore, the cutoff values for detecting DPD deficient patients from previous studies were unsuitable for Koreans. Our metabolomics approach is the first study that reported the DHU/U ratio distribution in healthy Korean subjects and identified a new biomarker of DPD deficiency.

The Prognostic and Predictive Value of Dihydropyrimidine Dehydrogenase-Related Indicators in Clinical Outcomes of Chemotherapy in Colorectal Cancer Patients: a Systematic Review and Meta-Analysis.

Colorectal cancer (CRC) is the third most common cancer worldwide. Predictive biomarkers are needed to predict patients' outcomes and to select a chemotherapy regimen. We assessed whether dihydropyrimidine dehydrogenase (DPD)-related indicators can predict CRC patients' outcomes. We searched the studies in PubMed, EmBase, and the Cochrane Library up to March 4, 2018. We mainly analyzed different CRC patients' outcomes according to specific DPD-related indicators. Twenty-five articles were included in the meta-analysis. The results showed that for disease-free survival (DFS), low DPD expression was significantly superior to high expression (I2= 72%; HR: 1.59; 95%CI: 1.21-2.09; p= 0.001). However, this result had a potential publication bias (Begg's test: p= 0.007; Egger's test: p= 0.004). Among patients treated with chemotherapy, a high thymidylate phosphorylase (TP)/DPD ratio was advantageous for DFS (I2= 63.7%; HR: 0.65; 95%CI: 0.46-0.92; p= 0.015), and this result did not have a publication bias. For overall survival (OS), low DPD expression was superior to high expression (I2= 74.4%; HR: 2.11; 95%CI: 1.48-3.00; p< 0.001), although this result had a publication bias (Egger's test: p= 0.003; Begg's test: p= 0.010). There was no difference in OS according to the TP/DPD ratio (I2= 0%; HR: 0.92; 95%CI: 0.75-1.13; p= 0.420). DFS and OS were better in CRC patients with low DPD expression than in those with high DPD expression. However, because of publication bias, more DPD indicator-related studies, especially with negative results, are still needed. Patients with a high TP/DPD ratio have better DFS but not OS.

Value of plasma SN-38 levels and DPD activity in irinotecan-based individualized chemotherapy for advanced colorectal cancer with heterozygous type UGT1A1*6 or UGT1A1*28.

PurposeThe relationship between the pharmacokinetics of irinotecan and outcomes of advanced colorectal cancer is unclear, and few studies have examined individualized irinotecan-based chemotherapy depending on plasma 7-ethyl-10-hydroxy camptothecin (SN-38) levels and dihydropyrimidine dehydrogenase (DPD) activity, particularly for the UGT1A1*6 or UGT1A1*28 heterozygous type.MethodsThis study retrospectively explored the relationship among plasma SN-38 level 1.5 hours after critical enzyme for irinotecan (CPT-11) administration (CSN-38 1.5h), plasma SN-38 level 49 hours after CPT-11 administration (CSN-38 49h), DPD activity, and clinical outcomes for the UGT1A1*6 and UGT1A1*28 heterozygous types.ResultsCSN-38 1.5h and CSN-38 49h of the UGT1A1*6 or UGT1A1*28 heterozygous type were close to those of UGT1A1*6 and UGT1A1*28 wild-types; some of those with relatively high CSN-38 1.5h levels obtained better median progression-free survival (mPFS), whereas others with higher CSN-38 49h concentrations showed a relatively high incidence of adverse reactions possibly because of the decreased activity of DPD.ConclusionIncreasing the dosage of CPT-11 according to CSN-38 1.5h may improve the efficacy in patients with lower CSN-38 1.5h levels. For cases with comparably low DPD activity, advisable primary and subsequent dose adjustment of 5-fluorouracil based on plasma 5-fluorouracil levels may be a practical strategy for reducing the occurrence of adverse reactions for personalized treatment of the UGT1A1*6 or UGT1A1*28 heterozygous type.

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

Dihydropyrimidine dehydrogenase predicts survival and response to interferon-\u03b1 in hepatocellular carcinoma

Metastasis and recurrence contribute to poor prognosis of hepatocellular carcinoma (HCC). Recently, we reported that interferon-α (IFN-α) can suppress metastasis of HCC; however, the underlying mechanism has not been fully described. In this study, we demonstrated that expression of dihydropyrimidine dehydrogenase (DPYD), a pyrimidine catabolic enzyme, was dose-dependently downregulated by IFN-α in HCC tissues from nude mice. Notably, DPYD expression was found to be significantly increased in HCC cell lines with higher metastatic potentials compared with their controls. Moreover, upregulation of DPYD in HCC cells could promote in vitro migration, invasion, and in vivo lung metastasis, and inducing changes characteristic of epithelial-mesenchymal transition (EMT). In contrast, knockdown of DPYD inhibited these processes. Mechanistically, DPYD functioned as a positive regulator of EMT in HCC by targeting the p38/NF-κB/Snail1 pathway. Clinically, tissue microarray analysis showed that high DPYD expression was positively associated with aggressive tumor characteristics, including larger tumor size, tumor recurrence, and advanced tumor node metastasis (TNM) stage, and independently correlated with poorer overall survival times after curative resection. HCC patients with low DPYD expression have better response to IFN-α therapy. Taken together, our findings elucidate that IFN-α could downregulate DPYD expression to inhibit EMT and HCC metastasis, and suggest that DPYD might be a potential prognostic biomarker and a therapeutic target for HCC.

Abstract 3823: High intrinsic DPD activity matters too

Abstract Introduction: Fluoropyrimidines (FP) are of major use in treating cancer. Dihydropyrimidine dehydrogenase (DPD) is the main enzyme responsible for FP catabolism. Low DPD activity screening has proven its predictive clinical value in identifying patients at risk for toxicity. However, high DPD activity may translate to decreased FP activation and efficacy. Patients and methods: One hundred and forty-three patients (monocentric retrospective study) underwent a pretreatment assessment of DPD activity in lymphocytes between 01/01/2004 and 20/04/2016. Included patients were male or female, &amp;gt;18 years, FP-treatment-based. Cut-off for DPD activity was assessed using smoothing spline curves. Results: Median patient follow-up was 30 months [CI95%: 27.3-36.2], mean age 63+/-3 years, females accounted for 53%, 90% of patients had a good performance status (0 or 1). Fifty-eight percent of FP indications (65% 5-FU and 35% capecitabine) were adjuvant or neoadjuvant and 42% were prescribed for local or metastatic recurrences. Tolerance was poor for 43% of patients and 49% needed dose reduction. Objective response (complete and partial) was observed in 50% of patients, stable disease in 38% and progression in 12%. No significant correlation was observed between DPD activity and response to treatment. Mean DPD activity (pmol min−1 mg−1 protein) was 0.21+/-0.1 (quartile: [0.001-0.14-0.20-0.28-0.48]). DPD activity analyzed as a continuous variable was significantly linked to overall survival (OS) (p=0.042) but not with progression-free survival (PFS). The higher the DPD activity, the lower the OS. DPD activity analyzed as a binary variable (cut-off at 0.30) was significantly linked to overall survival (p=0.012) but also with PFS (p=0.016). A Cox regression model for OS and PFS adjusted for age, sex, type of cancer, type of administered FP, and associated surgical or radiotherapy treatment was performed. After adjustment, DPD activity remained significantly linked to OS (p=0.03) and PFS (p=0.021). Conclusions: DPD activity screening could lead to a two-pronged approach: FP dose reduction in the event of low DPD and dose increase for high DPD. Such a strategy needs to be prospectively validated under personalized DPD-based treatment. Note: This abstract was not presented at the meeting. Citation Format: Emmanuel Chamorey, Eric Francois, Marie-Christine Etienne-Grimaldi, Julien Viotti, Frederic Peyrade, Alexandre Bozec, Jean-Marc Ferrero, Renaud Schiappa, Gerard Milano. High intrinsic DPD activity matters too [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3823. doi:10.1158/1538-7445.AM2017-3823

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.

Phase II trial of S-1 plus leucovorin in patients with advanced gastric cancer and clinical prediction by S-1 pharmacogenetic pathway

BackgroundThe first one-arm phase II trial aimed to evaluate and predict efficacy and safety of S-1 plus oral leucovorin (S-1/LV) as first-line chemotherapy for patients with advanced gastric cancer (AGC), using S-1 pharmacogenetic pathway approach.Patients and methodsA total of 39 patients orally took S-1 at conventional dose and LV simultaneously at a dose of 25 mg twice daily for a week, within a 2-week cycle. The primary endpoint was overall response rate (ORR), while the secondary endpoints were progression-free survival (PFS), time to failure (TTF), overall survival (OS), disease control rate (DCR), and adverse events (AEs). Peripheral blood was sampled prospectively for baseline expression of dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase (OPRT), thymidine phosphorylase (TP), and thymidylate synthase (TS), CYP2A6 gene polymorphisms, and 5-FU pharmacokinetics.ResultsThe ORR and DCR were 41.0 and 76.9%. The median PFS, TTF, and OS were 4.13, 3.70, and 11.40 months. Grade 3–4 AEs occurred in only 13 patients, and grade 4 AEs occurred in only 1 of them. High OPRT/TS and peritoneal metastasis (vs. liver metastasis) independently predicted responding. High OPRT/DPD independently predicted grade 3–4 AEs. High AUC0–24h of 5-FU and metastatic/recurrent sites ≤2 (vs. >3) independently predicted prolonged PFS. Low baseline plasmic DPD independently predicted prolonged OS.ConclusionsTwo-week, oral S-1/LV regimen demonstrated promising efficacy and safety as first-line chemotherapy for AGC.ClinicalTrials.gov identifierNCT02090153

Abstract 5028: Reverse translational research for identifying the correlation between EGFR mutation status and DPD protein expression in lung adenocarcinoma

Abstract [Background] Reverse translation research is often described as “From the Bedside to the Bench”, which means taking an understanding gained by observations in a patient population to create hypotheses and confirmation of the hypothesis in the laboratory. According to current chemotherapy treatment for lung adenocarcinoma, empirically, clinicians know that epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) tend to be effective for radiological ground glass nodule- type lung adenocarcinomas. On the other hand, it has been shown that 5-FU sensitivity in patients with non-small cell lung cancer (NSCLC) is associated with EGFR mutation status. From such clinical hints, we identified a relationship between dihydropyrimidine dehydrogenase (DPD), a 5-FU degrading enzyme, and EGFR mutation status. [Methods] We firstly focused on clinicopathologic factors and in vitro correlations between DPD expression and EGFR mutation status. Secondly, we analyzed crosstalk between the EGFR signal cascade and DPD protein expression using EGFR mutated and not mutated cell lines. [Clinicopathological Study] EGFR mutations and messenger RNA (mRNA) levels of DPD were analyzed in 47 resected NSCLC tumors by laser-capture microdissection. EGFR mutation status and the immunohistochemical expression of DPD in 49 resected NSCLC tumors were also examined. As a result, adenocarcinoma in situ showed significantly higher DPD mRNA levels and more EGFR mutation frequency than other histological types (P &amp;lt; 0.05). DPD immunopositive cases were more frequently observed in adenocarcinoma, in females, and in nonsmokers; which were correlated with EGFR mutation status (P &amp;lt; 0.003). [Basic Study] In vitro, EGFR-mutated cell lines (PC9, HCC827; exon19 E746-A750 and H1975; exon21 L858R, T790M, gefitinib resistant) showed higher DPD mRNA and protein expression than wild types (H1437, H1299). The DPD gene (DPYD), and DPD protein expression were known to be regulated by the transcription factor Sp1. In the PC9 study, EGF treatment induced up-regulation of both Sp1 and DPD. Gefitinib, an EGFR-TKI, and mithramycin A, specific Sp-1 inhibitors, suppressed them. However, the phenomena were not observed in EGFR-wild types and EGFR-TKI resistant cell lines. These results suggest DPD is regulated mainly in EGFR signal cascade enhanced lung cancers such as EGFR mutated adenocarcinomas. 5-FU and its derivatives such as uracil-tegafur (UFT) or S-1 might be effective for EGFR wild type adenocarcinomas because of the low DPD expression. [Conclusion] Reverse translational research can provide useful information for clinical treatment. In the present study, we identified crosstalk between the EGFR signal cascade and DPD protein expression, which might affect therapeutic strategy for lung adenocarcinomas. Citation Format: Tomoshi Tsuchiya, Tetsuo Tominaga, Koji Mochinaga, Naoya Yamasaki, Keitaro Matsumoto, Takuro Miyazaki, Go Hatachi, Yuka Kitajima, Takeshi Nagayasu. Reverse translational research for identifying the correlation between EGFR mutation status and DPD protein expression in lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5028.

Abstract 1406: High expression of DPD and ALDH1 are associated with poor prognosis in pancreatic cancer

Abstract Background: Pancreatic cancer is a highly aggressive disease and currently the fourth leading cause for cancer related mortality. The annual incidence of it has been shown to be approximately equal to the annual death rate caused by it because of the propensity for early metastatic spread and because the disease is highly resistant to radiation and chemotherapy. Recently, cancer stem cells may play a crucial role in the development and progression of tumor. According to recent studies, cancer stem cell population in human pancreatic cancers are expressing the cell surface markers CD44+, c-Met, and ALDH1. 5-Fluorouracil (5-FU) is one of the key drugs and widely used in the treatment of pancreatic cancer. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme for 5-FU catabolism in liver, and it was reported that high DPD expression in cancer tissue was associated with 5-FU resistance and poor prognosis. The aim of this study is to examine if the pancreatic cancer stem cells express high DPD as a result acquiring chemoresistance to 5-FU. Methods: A total of 121 patients who underwent curative pancreatectomy for pancreatic cancer from April 2002 to December 2013 were analyzed. Immunohistochemical staining of DPD, ALDH1 and c-Met was performed using paraffin-embedded section of tissues. Expression of them were analyzed in interrelationship each other. Clinical relevance of those markers was confirmed with clinicopathological characteristics that were prospectively collected in our database. Results: Immunohistochemical analysis on clinical sample revealed that DPD expression was high in 55 (45.5%) patients, and high c-Met and ALDH1 expression were seen in 58 (47.9%) and 58 (47.9%) patients retrospectively. ALDH1 activity, DPD expression and sensitivity to 5-FU were examined using pancreatic cancer cell lines in vitro. High DPD expression was significantly associated with high ALDH1 expression not but c-Met, and associated with elderly patients (P = 0.0294) and overall survival (P = 0.0274). Pancreatic cell line with high DPD expression was more resistant to 5-FU than cells with low DPD expression in vitro. Low c-Met expression was significantly associated with high local infiltration (P = 0.0102). ALDH1 expression was significantly associated with relapse free survival (P = 0.0392) and overall survival (P = 0.0358). Conclusion: Our study demonstrated that high DPD expression was significantly associated with specific pancreatic cancer stem cell marker expression, may be involved in resistance to 5-FU treatment in pancreatic cancer. Citation Format: Kota Arima, Hirohisa Okabe, Daisuke Hashimoto, Takayoshi Kaida, Takaaki Higashi, Katsunobu Taki, Hideaki Takeyama, Hidetoshi Nitta, Hiromitsu Hayashi, Akira Chikamoto, Toru Beppu, Hideo Baba. High expression of DPD and ALDH1 are associated with poor prognosis in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1406. doi:10.1158/1538-7445.AM2015-1406

Stage II大腸癌の術後補助化学療法で重篤な副作用を呈したDPD活性低下の1例

Stage II大腸癌の術後補助化学療法で重篤な副作用を呈したDPD活性低下の1例

Expression level of dihydropyrimidine dehydrogenase is associated with clinical outcome in patients with T1G3 bladder cancer treated with Bacillus Calmette-Guerin

BackgroundRecently, it has been shown that 5-fluorouracil (5-FU) with a strong dihydropyrimidine dehydrogenase (DPD) inhibitor elicits a significant response in bladder cancer with a high level of DPD. However, only a few studies investigated the association between the level of the enzyme that regulates the metabolism of 5-FU and prognosis in bladder cancer. Furthermore, to our knowledge, there has also been no such report in T1G3 bladder tumors treated with BCG. Therefore, we evaluated enzymes that regulate the metabolism of 5-FU in T1G3 tumors treated with BCG immunotherapy using the Danenberg tumor profile (DTP) method, a highly accurate measurement of RNA from paraffin-embedded specimens.MethodsThis study included 28 patients with T1G3 bladder cancer, each of whom underwent complete transurethral tumor resection and BCG intravesical instillation at our institution. The median follow-up period was 39 months (range, 3 to 159 months). The DTP method was used to analyze the mRNA expression of 3 enzymes related to 5-FU: DPD, orotate phosphoribosyltransferase (OPRT), and thymidylate synthase (TS).ResultsAmong the 28 patients, 13 developed recurrences (46.4%) and 5 experienced disease progression (17.9%). An elevated DPD mRNA level was significantly associated with recurrence (p = 0.048) and progression (p = 0.045). However, TS and OPRT mRNA levels were not significantly associated with any other clinical features or outcomes. Furthermore, the high DPD group had a significantly lower recurrence-free survival rate than the low DPD group (p = 0.047). Among patients with low DPD, the 2- and 5-year recurrence-free survival rates were 88.9% and 74.1%, respectively; while among patients with high DPD, the corresponding rates were 61.3% and 36.8%, respectively. TS and OPRT were not significantly associated with recurrence-free survival rates.ConclusionDPD is significantly associated with recurrence and progression among T1G3 bladder cancer patients treated with BCG.

Pretherapeutic uracil and dihydrouracil levels in saliva of colorectal cancer patients are associated with toxicity during adjuvant 5-fluorouracil-based chemotherapy.

5-fluorouracil (5-FU) competes with uracil (Ura) as a substrate for dihydropyrimidine dehydrogenase (DPD). Low DPD activity impairs breakdown of Ura to dihydrouracil (UH₂) and is associated with toxicity during 5-FU-based chemotherapy. Calculation of the 5-FU dose is based on body surface area, and new tools are needed to individualize treatment. The aim of study was to measure Ura and UH₂ in saliva of patients with colorectal cancer and relate levels to treatment-induced toxicity. Saliva was collected from 73 patients with stage III colorectal cancer prior to adjuvant 5-FU-based treatment. Ura and UH₂ were analyzed by a column-switching HPLC method. Toxicity was evaluated before each treatment cycle and the highest grade was noted at end of treatment. Toxicity was more common and severe among women compared with men. The Ura and UH₂ concentrations in saliva were 5.0 ± 6.8 and 5.0 ± 4.0 nmol/ml, respectively. The UH₂/Ura ratio was lower in women compared with men (1.2 ± 1.0 and 2.2 ± 2.5, respectively, p = 0.0026). Patients who needed to reduce the drug dose during treatment (or terminate treatment) due to toxicity had a lower ratio (1.3 ± 0.85) compared to patients who completed treatment without dose reduction (4.1 ± 4.3, p < 0.0001). Sampling of saliva is a quick, noninvasive, safe and painless process that gives information about patients Ura and UH₂ levels prior to chemotherapeutical treatment. This information may be useful in order to predict and prevent occurrence of treatment-related toxicities which otherwise may limit drug administration.

Frequency of the DPYD * 2A allele in the Czech population

The pyrimidine analogue 5-fluorouracil (5-FU) is widely used for chemotherapy of many solid tumors, such as colorectal, breast, head/neck, ovarian and skin cancer. More than 80% of administered 5-FU is rapidly detoxified in the liver via a multistep metabolic pathway, involving dihydropyrimidine dehydrogenase (DPD) as the initial and rate-limiting enzyme. Reduced DPD activity can lead to the accumulation of active 5-FU metabolite (FdUMP), which leads to 5-FU toxicity. Individuals with low DPD activity (about 3-5% of patients) cannot effectively inactivate 5-FU and will develop severe to lethal hematological, gastrointestinal or neurological toxicities. Pharmacogenetic variation may contribute to risk of toxicity and/or altered therapeutic benefit. Enzyme DPD has a key function for 5-FU metabolism (degradation and inactivation) and is encoded by DPYD gene. In the majority of cases, a G to A mutation in the splicing recognition sequence of intron 14 (IVS14+1G>A) of the DPD-encoding gene DPYD was shown to be responsible. The mutant allele, designated DPYD*2A, produces a nonfunctional enzyme due to skipping of exon 14. Frequence of DPYD*2A allele in caucasion population is 0,91% and in the deficite of DPD is 52%. It was recommended that screening for DPYD*2A should be routinely carried out prior to start of 5-FU therapy, and that heterozygotes should receive limited dose of 5-FU only, while homozygotes, who are at high risk to develop severe complications, should be treated with alternative therapeutic drugs. 212 males and 210 females (the age of tested patients was 18-69 years) of the Czech population were tested. The material of DNA isolation was obtained oral mucosa by Flocked Swabs or by taking incoagulable peripheral venous blood. DNA was isolated by using QIAmp DNA Minikit or by the semi-automatic isolation DNA QuickGene 810. Mutation IVS14+1 G>A DPYD gene (DPYD * 2 allele) was tested by using certificated method strip assay (PGX-5FU) and by new created method High-Resolution Melting (HRM) (Figure​Figure1).1). Were compared to results of both methods with each other and assessed allele frequency in the Czech population. Figure 1 Analysis of mutation IVS14+1 G>A DPYD gene by methods DPYD * 2A allele was demonstrated in a heterozygous state in 3/422 (0.7%), in the homozygous state was not found. Czech frequency of DPYP * 2A allele 0.36% is 2.5 times lower than the reported frequency 0.91% in the European population. The results of both methods were completely identical. New cheaper method for detection DPYD * 2A was introduced and validated. Low frequency of DPYD * 2A allele in the Czech population assumes the presence of other mutations in the DPYD gene and in other genes affecting the metabolism of 5-FU (eg . TYMS, MTHFR). Genetic test is recommended prior to cancer treatment by 5-FU, because are suitable the lower doses in the heterozygotes and choose other chemotherapy for the homozygotes with risk of toxic reaction.

Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers

Background:Genomic rearrangements at the fragile site FRA1E may disrupt the dihydropyrimidine dehydrogenase gene (DPYD) which is involved in 5-fluorouracil (5-FU) catabolism. In triple-negative breast cancer (TNBC), a subtype of breast cancer frequently deficient in DNA repair, we have investigated the susceptibility to acquire copy number variations (CNVs) in DPYD and evaluated their impact on standard adjuvant treatment.Methods:DPYD CNVs were analysed in 106 TNBC tumour specimens using multiplex ligation-dependent probe amplification (MLPA) analysis. Dihydropyrimidine dehydrogenase (DPD) expression was determined by immunohistochemistry in 146 tumour tissues.Results:In TNBC, we detected 43 (41%) tumour specimens with genomic deletions and/or duplications within DPYD which were associated with higher histological grade (P=0.006) and with rearrangements in the DNA repair gene BRCA1 (P=0.007). Immunohistochemical analysis revealed low, moderate and high DPD expression in 64%, 29% and 7% of all TNBCs, and in 40%, 53% and 7% of TNBCs with DPYD CNVs, respectively. Irrespective of DPD protein levels, the presence of CNVs was significantly related to longer time to progression in patients who had received 5-FU- and/or anthracycline-based polychemotherapy (hazard ratio=0.26 (95% CI: 0.07–0.91), log-rank P=0.023; adjusted for tumour stage: P=0.037).Conclusion:Genomic rearrangements in DPYD, rather than aberrant DPD protein levels, reflect a distinct tumour profile associated with prolonged time to progression upon first-line chemotherapy in TNBC.