Precision fluoropyrimidines dosing in a compound heterozygous variant carrier of the DPYD gene: a case report.

BACKGROUND: Fluoropyrimidines (FPs) form still nowadays the backbone of chemotherapic schemes in colorectal cancer (CRC). Inter-patient variability of the toxicity profile of FPs may be partially accounted for by variable expression of dihydropyrimidine dehydrogenase (DPD). DPD rate activity is genetically determined by its extremely polymorphic coding gene DPYD. In spite of pharmacogenetic guideline-directed-dosing of FPs based regimens treating carrier of multiple variants of DPYD gene remains still challenging. CASE PRESENTATION: We present a case of a 48-year-old Caucasian man, compound heterozygous variant carrier of the DPYD gene (HapB3 and c.2194G>A) who had a diagnosis of adenocarcinoma of the left colon and was safely treated with a pharmacogenetic-guided 25% dose reduction of the standard CAP adjuvant treatment. Compound heterozygosis may have been responsible for an earlier over exposure to CAP resulting into low-grade toxicity with an anticipated median time to toxicity of the c.2194G>A variant to the 4th vs. 6th cycles. Some haplotypes of DPYD variants may have an advantage in terms of survival compared to wild-type patients. Our patient may also have benefitted from compound heterozygosis, as shown by no evidence of disease (NED) at 6-month follow-up. CONCLUSION: Pharmacogenetic-guided dosing of DPYD intermediate metabolizer compound heterozygous HapB3 and c.2194G>A variant carries should be managed by a multidisciplinary team with a dose reduction ranging from 25 to 50% to maintain effectiveness and close clinical monitoring for early detection of ADRs.

Discrepancies between dihydropyrimidine dehydrogenase phenotyping and genotyping: What are the explanatory factors?

AIM: Dihydropyrimidine dehydrogenase (DPD) deficiency can be detected by phenotyping (measurement of plasma uracil [U], with U ≥ 16 µg/L defining a partial deficiency) and/or by genotyping (screening for the four most frequent DPYD variants). We aimed to determine the proportion of discrepancies between phenotypic and genotypic approaches and to identify possible explanatory factors. METHODS: Data from patients who underwent both phenotyping and genotyping were retrospectively collected. Complementary genetic analyses (genotyping of the variant c.557A>G and DPYD sequencing) were performed for patients with U ≥ 16 µg/L without any common variants. The characteristics of patients classified according to the congruence of the phenotyping and genotyping approaches were compared (Kruskal-Wallis test), and determinants of U levels were studied in the whole cohort (linear model). RESULTS: Among the 712 included patients, phenotyping and genotyping were discordant for 12.5%, with 63 (8.8%) having U ≥ 16 µg/L in the absence of a common variant. Complementary genetic investigations marginally reduced the percentage of discrepancies to 12.1%: Among the nine additional identified variants, only the c.557A>G variant, carried by three patients, had been previously reported to be associated with DPD deficiency. Liver dysfunction could explain certain discordances, as ASAT, ALP, GGT and bilirubin levels were significantly elevated, with more frequent liver metastases in patients with U ≥ 16 µg/L and the absence of a DPYD variant. The impact of cytolysis was confirmed, as ASAT levels were independently associated with increased U (p < 0.001). CONCLUSION: The frequent discordances between DPD phenotyping and genotyping approaches highlight the need to perform these two approaches to screen for all DPD deficiencies.

Energy decomposition and waterswapping analysis to investigate the SNP associated DPD mediated 5-FU resistance.

5-fluorouracil is an essential component of systemic chemotherapy for colon, breast, head, and neck cancer patients. However, tumoral overexpression of the dihydropyrimidine dehydrogenase has rendered 5-FU clinically ineffective by inactivating it to 5'-6'-dihydro fluorouracil. The responses to 5-FU in terms of efficacy and toxicity greatly differ depending upon the population group, because of variability in the DPD activity levels. In the current study, key active site amino acids involved in the 5-FU inactivation were investigated by modelling the 3D structure of human DPD in a complex with 5-FU. The identified amino acids were analyzed for their possible missense mutations available in dbSNP database. Out of 12 missense SNPs, four were validated either by sequencing in the 1000 Genomes project or frequency/genotype data. The recorded validated missense SNPs were further considered to analyze the effect of their respective alterations on 5-FU binding. Overall findings suggested that population bearing the Glu611Val DPD mutation (rs762523739) is highly vulnerable to 5-FU resistance. From the docking, electrostatic complementarity, dynamics, and energy decomposition analyses it was found that the above mutation showed superior scores than the wild DPD -5FU complex. Therefore, prescribing prodrug NUC-3373 or DPD inhibitors (Gimeracil/3-Cyano-2,6-Dihydroxypyridines) as adjuvant therapy may overcome the 5-FU resistance.

Assessment of the Clinical Utility of Pretreatment DPYD Testing for Patients Receiving Fluoropyrimidine Chemotherapy.

PURPOSE: Patients who carry pathogenic variants in DPYD have higher systemic fluoropyrimidine (FP) concentrations and greater risk of severe and fatal FP toxicity. Pretreatment DPYD testing and DPYD-guided FP dosing to reduce toxicity and health care costs is recommended by European clinical oncology guidelines and has been adopted across Europe, but has not been recommended or adopted in the United States. The cochairs of the National Comprehensive Cancer Network Guidelines for colon cancer treatment explained their concerns with recommending pretreatment DPYD testing, particularly the risk that reduced FP doses in DPYD carriers may reduce treatment efficacy. METHODS: This special article uses previously published frameworks for assessing the clinical utility of cancer biomarker tests, including for germline indicators of toxicity risk, to assess the clinical utility of pretreatment DPYD testing, with a particular focus on the risk of reducing treatment efficacy. RESULTS: There is no direct evidence of efficacy reduction, and the available indirect evidence demonstrates that DPYD-guided FP dosing results in similar systemic FP exposure and toxicity compared with standard dosing in noncarriers, and is well calibrated to the maximum tolerated dose, strongly suggesting there is minimal risk of efficacy reduction. CONCLUSION: This article should serve as a call to action for clinicians and clinical guidelines committees in the United States to re-evaluate the clinical utility of pretreatment DPYD testing. If clinical utility has not been demonstrated, further dialogue is needed to clarify what additional evidence is needed and which of the available study designs, also described within this article, would be appropriate. Clinical guideline recommendations for pretreatment DPYD testing would increase clinical adoption and ensure that all patients receive maximally safe and effective FP treatment.

Cost-effectiveness of DPYD Genotyping Prior to Fluoropyrimidine-based Adjuvant Chemotherapy for Colon Cancer.

BACKGROUND: Adjuvant fluoropyrimidine-based chemotherapy substantially reduces recurrence and mortality after resection of stage 3 colon cancer. While standard doses of 5-fluorouracil and capecitabine are safe for most patients, the risk of severe toxicity is increased for the approximately 6% of patients with dihydropyimidine dehydrogenase (DPD) deficiency caused by pathogenic DPYD gene variants. Pre-treatment screening for pathogenic DPYD gene variants reduces severe toxicity but has not been widely adopted in the United States. METHODS: We conducted a cost-effectiveness analysis of DPYD genotyping prior to fluoropyrimidine-based adjuvant chemotherapy for stage 3 colon cancer, covering the c.1129-5923C>G (HapB3), c.1679T>G (*13), c.1905+1G>A (*2A), and c.2846A>T gene variants. We used a Markov model with a 5-year horizon, taking a United States healthcare perspective. Simulated patients with pathogenic DPYD gene variants received reduced-dose fluoropyrimidine chemotherapy. The primary outcome was the incremental cost-effectiveness ratio (ICER) for DPYD genotyping. RESULTS: Compared with no screening for DPD deficiency, DPYD genotyping increased per-patient costs by $78 and improved survival by 0.0038 quality-adjusted life years (QALYs), leading to an ICER of $20,506/QALY. In 1-way sensitivity analyses, The ICER exceeded $50,000 per QALY when the cost of the DPYD genotyping assay was greater than $286. In probabilistic sensitivity analysis using a willingness-to-pay threshold of $50,000/QALY DPYD genotyping was preferred to no screening in 96.2% of iterations. CONCLUSION: Among patients receiving adjuvant chemotherapy for stage 3 colon cancer, screening for DPD deficiency with DPYD genotyping is a cost-effective strategy for preventing infrequent but severe and sometimes fatal toxicities of fluoropyrimidine chemotherapy.

DPYD polymorphisms c.496A>G, c.2194G>A and c.85T>C and risk of severe adverse drug reactions in patients treated with fluoropyrimidine-based protocols.

AIMS: Cancer patients with reduced dihydropyrimidine dehydrogenase (DPD) activity are at increased risk of severe fluoropyrimidine (FP)-related adverse events (AE). Guidelines recommend FP dosing adjusted to genotype-predicted DPD activity based on four DPYD variants (rs3918290, rs55886062, rs67376798 and rs56038477). We evaluated the relationship between three further DPYD polymorphisms: c.496A>G (rs2297595), *6 c.2194G>A (rs1801160) and *9A c.85T>C (rs1801265) and the risk of severe AEs. METHODS: Consecutive FP-treated adult patients were genotyped for "standard" and tested DPYD variants, and for UGT1A1*28 if irinotecan was included, and were monitored for the occurrence of grade ≥3 (National Cancer Institute Common Terminology Criteria) vs. grade 0-2 AEs. For each of the tested polymorphisms, variant allele carriers were matched to respective wild type controls (optimal full matching combined with exact matching, in respect to: age, sex, type of cancer, type of FP, DPYD activity score, use of irinotecan/UGT1A1, adjuvant therapy, radiotherapy, biological therapy and genotype on the remaining two tested polymorphisms). RESULTS: Of the 503 included patients (82.3% colorectal cancer), 283 (56.3%) developed grade ≥3 AEs, mostly diarrhoea and neutropenia. Odds of grade ≥3 AEs were higher in c.496A>G variant carriers (n = 127) than in controls (n = 376) [OR = 5.20 (95% CI 1.88-14.3), Bayesian OR = 5.24 (95% CrI 3.06-9.12)]. Odds tended to be higher in c.2194G>A variant carriers (n = 58) than in controls (n = 432) [OR = 1.88 (0.95-3.73), Bayesian OR = 1.90 (1.03-3.56)]. c.85T>C did not appear associated with grade ≥3 AEs (206 variant carriers vs. 284 controls). CONCLUSION: DPYD c.496A>G and possibly c.2194G>A variants might need to be considered for inclusion in the DPYD genotyping panel.

Predictive value of clinical toxicities of chemotherapy with fluoropyrimidines and oxaliplatin in colorectal cancer by DPYD and GSTP1 gene polymorphisms.

BACKGROUND: Fluoropyrimidines and platinum are still widely used for colorectal cancer (CRC) management. Several studies have reported that mutations of dihydropyrimidine dehydrogenase (DPYD) and glutathione S-transferase pi-1 (GSTP1) polymorphisms are related to chemotherapy-related adverse events. In the present study, we purposed to assess the impact of DPYD and GSTP1 variants on the toxicity of adjuvant chemotherapy risk among the Hakka population, minimize adverse events, and to maximize therapy outcome for individualized treatment. METHODS: Genotyping was examined in 104 patients diagnosed with CRC cases and receiving fluoropyrimidine and platinum drug-based chemotherapy regimen by direct sequencing of DPYD and GSTP1 polymorphisms. Three DPYD variants including *2A, *5A, *9A, and GSTP1 c.313A>G were analyzed and clinical outcomes were assessed. RESULTS: The data suggest that the incidence of DPYD*5A, DPYD*9A, and GSTP1 c.313A>G variants were 38.4%, 24%, and 32.7%, respectively. DPYD*2A variant was not found. A total of 23 patients (22.1%) suffered severe vomiting and 19 patients (18.3%) suffered severe anemia. DPYD*5A polymorphism was found significantly associated with grade 3/4 ulceration (p = 0.001). GSTP1 was determined to be an independent risk factor for severe vomiting and skin ulceration (p = 0.042 and p = 0.018, respectively). Patients with GSTP1 c. 313A>G mutant type contributed to a higher risk for grade severe toxicity compared with wild genotype (p = 0.027). Nevertheless, no significant difference was found between patients with DPYD*2A, *5A, and *9A for chemotherapeutic toxicity. CONCLUSIONS: The results demonstrated that GSTP1 polymorphisms were useful predictors of severe events. Screening of single-nucleotide polymorphisms of GSTP1 in colorectal cancer patients before chemotherapy may help to realize personalized therapy.

Dihydropyrimidine Dehydrogenase Testing prior to Treatment with 5-Fluorouracil, Capecitabine, and Tegafur: A Consensus Paper.

BACKGROUND: 5-Fluorouracil (FU) is one of the most commonly used cytostatic drugs in the systemic treatment of cancer. Treatment with FU may cause severe or life-threatening side effects and the treatment-related mortality rate is 0.2-1.0%. SUMMARY: Among other risk factors associated with increased toxicity, a genetic deficiency in dihydropyrimidine dehydrogenase (DPD), an enzyme responsible for the metabolism of FU, is well known. This is due to variants in the DPD gene (DPYD). Up to 9% of European patients carry a DPD gene variant that decreases enzyme activity, and DPD is completely lacking in approximately 0.5% of patients. Here we describe the clinical and genetic background and summarize recommendations for the genetic testing and tailoring of treatment with 5-FU derivatives. The statement was developed as a consensus statement organized by the German Society for Hematology and Medical Oncology in cooperation with 13 medical associations from Austria, Germany, and Switzerland. Key Messages: (i) Patients should be tested for the 4 most common genetic DPYD variants before treatment with drugs containing FU. (ii) Testing forms the basis for a differentiated, risk-adapted algorithm with recommendations for treatment with FU-containing drugs. (iii) Testing may optionally be supplemented by therapeutic drug monitoring.

New DPYD variants causing DPD deficiency in patients treated with fluoropyrimidine.

PURPOSE: Several clinical guidelines recommend genetic screening of DPYD, including coverage of the variants c.1905 + 1G>A(DPYD*2A), c.1679T>G(DPYD*13), c.2846A>T, and c.1129-5923C>G, before initiating treatment with fluoropyrimidines. However, this screening is often inadequate at predicting the occurrence of severe fluoropyrimidine-induced toxicity in patients. METHODS: Using a complementary approach combining whole DPYD exome sequencing and in silico and structural analysis, as well as phenotyping of DPD by measuring uracilemia (U), dihydrouracilemia (UH2), and the UH2/U ratio in plasma, we were able to characterize and interpret DPYD variants in 28 patients with severe fluoropyrimidine-induced toxicity after negative screening. RESULTS: Twenty-five out of 28 patients (90%) had at least 1 variant in the DPYD coding sequence, and 42% of the variants (6/14) were classified as potentially deleterious by at least 2 of the following algorithms: SIFT, Poly-Phen-2, and DPYD varifier. We identified two very rare deleterious mutations, namely, c.2087G>A (p.R696H) and c.2324T>G (p.L775W). We were able to demonstrate partial DPD deficiency, as measured by the UH2/U ratio in a patient carrying the variant p.L775W for the first time. CONCLUSION: Whole exon sequencing of DPYD in patients with suspicion of partial DPD deficiency can help to identify rare or new variants that lead to enzyme inactivation. Combining different techniques can yield abundant information without increasing workload and cost burden, thus making it a useful approach for implementation in patient care.

Phase I pharmacological study of continuous chronomodulated capecitabine treatment.

PURPOSE: Capecitabine is an oral pre-pro-drug of the anti-cancer drug 5-fluorouracil (5-FU). The biological activity of the 5-FU degrading enzyme, dihydropyrimidine dehydrogenase (DPD), and the target enzyme thymidylate synthase (TS), are subject to circadian rhythmicity in healthy volunteers. The aim of this study was to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), safety, pharmacokinetics (PK) and pharmacodynamics (PD) of capecitabine therapy adapted to this circadian rhythm (chronomodulated therapy). METHODS: Patients aged ≥18 years with advanced solid tumours potentially benefitting from capecitabine therapy were enrolled. A classical dose escalation 3 + 3 design was applied. Capecitabine was administered daily without interruptions. The daily dose was divided in morning and evening doses that were administered at 9:00 h and 24:00 h, respectively. The ratio of the morning to the evening dose was 3:5 (morning: evening). PK and PD were examined on treatment days 7 and 8. RESULTS: A total of 25 patients were enrolled. The MTD of continuous chronomodulated capecitabine therapy was established at 750/1250 mg/m2/day, and was generally well tolerated. Circadian rhythmicity in the plasma PK of capecitabine, dFCR, dFUR and 5-FU was not demonstrated. TS activity was induced and DPD activity demonstrated circadian rhythmicity during capecitabine treatment. CONCLUSION: The MTD of continuous chronomodulated capecitabine treatment allows for a 20% higher dose intensity compared to the approved regimen (1250 mg/m2 bi-daily on day 1-14 of every 21-day cycle). Chronomodulated treatment with capecitabine is promising and could lead to improved tolerability and efficacy of capecitabine.

A case report of a severe fluoropyrimidine-related toxicity due to an uncommon DPYD variant.

INTRODUCTION: Fluoropyrimidines such as 5-fluorouracil (5-FU) and its orally active prodrug, capecitabine, are widely used in the treatment of gastrointestinal cancer, including colorectal cancer. Dihydropyrimidine dehydrogenase (DPD) plays an important role in the 5-FU metabolism. Dihydropyrimidine dehydrogenase gene (DPYD) is a highly polymorphic gene with several hundreds of reported genetic variants and DPD activity levels vary considerably among individuals, with different 5-FU-related efficacy and toxicity. About 5% of the population is deficient in DPD enzyme activity. The most well studied DPYD variant is the IVS14+1G>A, also known as DPYD *2A. In this report, we present a case of a patient with a double heterozygote DPYD variant (DPYD activity score: 0,5 according to Clinical Pharmacogenetics Implementation Consortium) who experienced a severe fluoropyrimidine-related toxicity resolved without any consequence. PATIENT CONCERNS: A 46-years-old Caucasian man with diagnosis of left colon adenocarcinoma underwent left hemicolectomy on July 2017: pT3 G3 N1c M0. According to the disease stage, he started an adjuvant therapy with XELOX using capecitabine at 50% of total dose, because of his DPYD IVS14+1G>A variant, detected before the treatment. DIAGNOSIS: After few days, despite of this dose reduction, he experienced life-threatening adverse events such as mucositis G3, diarrhea G3, neutropenia G4, thrombocytopenia G4, and hyperbilirubinemia G3 according to Common Terminology Criteria for Adverse Events v 5.0. INTERVENTIONS: As first, we set up an intensive rehydration therapy, antibiotic and antifungal prophylaxis, Granulocyte-Colony Stimulating Factors, and supportive blood transfusions. Additional genetic tests revealed a double heterozygote variant of DPYD gene (DPYD IVS14+1G>A and 2846A>T) which is a very rare situation and only 3 cases are described in literature, all of them concluded with patient's death. OUTCOMES: After 3 weeks of intensive therapy, the patient was fully recovered. Furthermore, all the whole-body CT scans performed since discharge from the hospital until now, have confirmed no evidence of disease. CONCLUSIONS: Recent studies demonstrated that screening strategy for the most common DPYD variants allowed for avoiding toxicities and saving money. This report underlines the importance of genotyping DPYD before treatment and emphasizes the role of genotype-guided dose individualization.

Long noncoding RNA LINC00261 induces chemosensitization to 5-fluorouracil by mediating methylation-dependent repression of DPYD in human esophageal cancer.

Approximately 85% of a single administered dose of 5-fluorouracil (5-FU) will be degraded by dihydropyrimidine dehydrogenase (DYPD). Studies have highlighted a link between the complete or partial loss of DYPD function and clinical responses to 5-FU; however, the underlying molecular basis of DPD deficiency remains poorly understood. Hence, the aim of the present study was to evaluate the prevailing hypothesis which suggests that overexpression of LINC00261 possesses the ability to modulate the methylation-dependent repression of DPYD, ultimately resulting in an elevation of the sensitivity of human esophageal cancer cells to 5-FU. LINC00261 levels were initially quantified, followed by analysis of DYPD methylation within the cancerous tissues collected from 75 patients diagnosed with esophageal cancer undergoing 5-FU-based adjuvant chemotherapy. In an attempt to determine the levels of LINC00261 related to the esophageal cancer cell resistance to 5-FU and to identify the interaction between the levels of LINC00261 and methylation of the DYPD promoter, esophageal cancer cells TE-1 and -5 were prepared, in which LINC00261 and the 5-FU-resistant TE-1 and -5 cells were overexpressed. The levels of LINC00261 were reduced among the cancerous tissues obtained from patients exhibiting resistance to 5-FU. Overexpression of LINC00261 was determined to dramatically inhibit proliferation and resistance to apoptosis among 5-FU-resistant TE-1 and -5 cells, whereas silencing of LINC00261 was determined to enhance proliferation and resistance to apoptosis among the TE-1 and -5 cells. DPYD, a confirmed target of LINC00261, displayed a greater incidence of DNA methylation among patient's sensitive to 5-FU. A key finding revealed that overexpressed LINC00261 could increase the methylation of the DPYD promoter through the recruitment of DNA methyltransferase (DNMT), which, in turn, acts to decrease DPYD activity in 5-FU-resistant TE-1 cells, whereas a reversible change was recorded once the demethylation reagent 5-aza-2'-deoxyctidine was employed to treat the 5-FU-resistant TE-1 cells. Taken together, the results of the study provided evidence emphasizing the distinct antitumor ability of LINC00261 in cases of esophageal cancer, which was manifested by overexpression of LINC00261 detected to increase the sensitivity of human esophageal cancer cells to 5-FU by mediating methylation-dependent repression of DPYD. Our study highlighted the potential of LINC00261 as a novel target capable of improving the chemotherapeutic response and survival of patients with esophageal cancer.-Lin, K., Jiang, H., Zhuang, S.-S., Qin, Y.-S., Qiu, G.-D., She, Y.-Q., Zheng, J.-T., Chen, C., Fang, L., Zhang, S.-Y. Long noncoding RNA LINC00261 induces chemosensitization to 5-fluorouracil by mediating methylation-dependent repression of DPYD in human esophageal cancer.

Early disease relapse in a patient with colorectal cancer who harbors genetic variants of DPYD, TYMS, MTHFR and DHFR after treatment with 5-fluorouracil-based chemotherapy.

Background Early relapse in colorectal cancer (CRC) after curative resection is mainly attributed to the key determinants such as tumor histology, stage, lymphovascular invasion, and the response to chemotherapy. Case presentation Interindividual variability in the efficacy of adjuvant chemotherapy between patients receiving the same treatment may be ascribed to the patients' genetic profile. In this report, we highlight a clinical case of a patient with stage II CRC who relapsed within a short period after starting adjuvant chemotherapy and was later found to have multiple genetic polymorphisms in the DPYD, TYMS, MTHFR, and DHFR genes. Conclusions Based on the clinical data of the patient and the key role of these genes in 5-fluorouracil pathway, we hypothesize that these variants may contribute to the drug response and early relapse in CRC.

[5-fluorouracil therapeutic drug monitoring: Update and recommendations of the STP-PT group of the SFPT and the GPCO-Unicancer]. / Suivi thérapeutique pharmacologique du 5-fluorouracile : mise au point et recommandations du groupe STP-PT de la SFPT et du GPCO-Unicancer.

Despite being 60-years old now, 5-FU remains the backbone of numerous regimen to treat a variety of solid tumors such as breast, head-and-neck and digestive cancers either in neo-adjuvant, adjuvant or metastatic settings. Standard 5-FU usually claims 15-40% of severe toxicities and up to 1% of toxic-death. Numerous studies show a stiff relationship between 5-FU exposure and toxicity or efficacy. In addition, 5-FU pharmacokinetics is highly variable between patients. Indeed, 80% of the 5-FU dose is catabolized in the liver by dihydropyrimidine dehydrogenase (DPD) into inactive compounds. It is now well established that DPD deficiency could lead to severe toxicities and, thus, require dose reduction in deficient patients. However, despite dosage adaptation based on DPD status, some patients may still experience under- or over-exposure, leading to inefficacy or major toxicity. The "Suivi thérapeutique pharmacologique et personnalisation des traitements" (STP-PT) group of the "Société française de pharmacologie et de thérapeutique" (SFPT) and the "Groupe de pharmacologie clinique oncologique" (GPCO)-Unicancer, based on the latest and most up-to-date literature data, recommend the implementation of 5-FU Therapeutic Drug Monitoring in order to ensure an adequate 5-FU exposure.

The association of polymorphisms in folate-metabolizing genes with response to adjuvant chemotherapy of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the major health issues worldwide. 5-Fluorouracil (5-FU) is a cornerstone of chemotherapy for CRC and the major targets of 5-FU are folate-metabolizing enzymes. METHODS: A total of 103 CRC patients with complete clinical data were included in this prospective cohort study. Genotyping was performed using polymerase chain reaction (PCR) followed by sequencing. Using Kaplan-Meier curves, log-rank tests, and Cox proportional hazard models, we evaluated associations between functional polymorphisms in four genes MTHFR (1298A>C and 677C>T), DPYD (496A>G and 85T>C), DHFR 19 bp del, and MTR (2756 A>G) with disease-free survival (DFS). RESULTS: The minor allele frequencies of MTHFR 1298A>C, MTHFR 677C>T, DPYD 496A>G, DPYD 85T>C, DHFR 19 bp del, and MTR 2756 A>G were 0.364, 0.214, 0.116, 0.209, 0.383, and 0.097, respectively. CRC patients carrying the homozygous GG genotype in DPYD 496A>G had 4.36 times shorter DFS than wild-type AA carriers, (DFSGG vs AA: 8.0 ± 4 vs 69.0 ± 10 months; HR 4.36, 95% CI 1.04-18; p = 0.04). Moreover, female carriers of homozygous CC genotype of DPYD 85T>C had shorter DFS compared to either heterozygous or wild-type genotypes, and were 12.7 times shorter than wild-type TT carriers (DFSCC vs TT: 5.0 ± 1.5 vs 42.0 ± 7.6 months; HR 12.7, 95% CI 2.2-71.4; p = 0.004). However, there were no significant associations with the other studied polymorphisms. CONCLUSION: Genetic polymorphism in DPYD seems to be associated with DFS in CRC patients receiving an adjuvant regimen of 5-FU/capecitabine-based chemotherapy. Further studies are needed to verify these findings.

Expression of dihydropyrimidine dehydrogenase (DPD) and hENT1 predicts survival in pancreatic cancer.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) tumour expression may provide added value to human equilibrative nucleoside transporter-1 (hENT1) tumour expression in predicting survival following pyrimidine-based adjuvant chemotherapy. METHODS: DPD and hENT1 immunohistochemistry and scoring was completed on tumour cores from 238 patients with pancreatic cancer in the ESPAC-3(v2) trial, randomised to either postoperative gemcitabine or 5-fluorouracil/folinic acid (5FU/FA). RESULTS: DPD tumour expression was associated with reduced overall survival (hazard ratio, HR = 1.73 [95% confidence interval, CI = 1.21-2.49], p = 0.003). This was significant in the 5FU/FA arm (HR = 2.07 [95% CI = 1.22-3.53], p = 0.007), but not in the gemcitabine arm (HR = 1.47 [0.91-3.37], p = 0.119). High hENT1 tumour expression was associated with increased survival in gemcitabine treated (HR = 0.56 [0.38-0.82], p = 0.003) but not in 5FU/FA treated patients (HR = 1.19 [0.80-1.78], p = 0.390). In patients with low hENT1 tumour expression, high DPD tumour expression was associated with a worse median [95% CI] survival in the 5FU/FA arm (9.7 [5.3-30.4] vs 29.2 [19.5-41.9] months, p = 0.002) but not in the gemcitabine arm (14.0 [9.1-15.7] vs. 18.0 [7.6-15.3] months, p = 1.000). The interaction of treatment arm and DPD expression was not significant (p = 0.303), but the interaction of treatment arm and hENT1 expression was (p = 0.009). CONCLUSION: DPD tumour expression was a negative prognostic biomarker. Together with tumour expression of hENT1, DPD tumour expression defined patient subgroups that might benefit from either postoperative 5FU/FA or gemcitabine.

Predictors of Hand-Foot Syndrome and Pyridoxine for Prevention of Capecitabine-Induced Hand-Foot Syndrome: A Randomized Clinical Trial.

IMPORTANCE: Hand-foot syndrome (HFS) is a common adverse effect of capecitabine treatment. OBJECTIVE: To compare the incidence and time to onset of grade 2 or greater HFS in patients receiving pyridoxine vs placebo and to identify biomarkers predictive of HFS. DESIGN, SETTING, AND PARTICIPANTS: This single-center, randomized double-blind, placebo-controlled phase 3 trial conducted at National Cancer Centre Singapore assessed whether oral pyridoxine could prevent the onset of grade 2 or higher HFS in 210 patients scheduled to receive single-agent capecitabine chemotherapy for breast, colorectal, and other cancers. INTERVENTIONS: Patients were randomized to receive concurrent pyridoxine (200 mg) or placebo daily for a maximum of 8 cycles of capecitabine, with stratification by sex and use in adjuvant or neoadjuvant vs palliative setting. Patients were withdrawn from the study on development of grade 2 or higher HFS or cessation of capecitabine. MAIN OUTCOMES AND MEASURES: Primary end point was the incidence of grade 2 or higher HFS in patients receiving pyridoxine. Secondary end points included the time to onset (days) of grade 2 or higher HFS and identification of biomarkers predictive of HFS, including baseline folate and vitamin B12 levels, as well as genetic polymorphisms with genome-wide arrays. RESULTS: In this cohort of 210 patients (median [range] age, 58 [26-82] years; 162 women) grade 2 or higher HFS occurred in 33 patients (31.4%) in the pyridoxine arm vs 39 patients (37.1%) in the placebo arm (P = .38). The median time to onset of grade 2 or higher HFS was not reached in both arms. In univariate analysis, the starting dose of capecitabine (odds ratio [OR], 1.99; 95% CI, 1.32-3.00; P = .001), serum folate levels (OR, 1.27; 95% CI, 1.10-1.47; P = .001), and red blood cell folate levels (OR, 1.25; 95% CI, 1.08-1.44; P = .003) were associated with increased risk of grade 2 or higher HFS. In multivariate analyses, serum folate (OR, 1.30; 95% CI, 1.12-1.52; P < .001) and red blood cell folate (OR, 1.28; 95% CI, 1.10-1.49; P = .001) were the only significant predictors of grade 2 or higher HFS. Grade 2 or higher HFS was associated with 300 DNA variants at genome-wide significance (P < 5 × 10-8), including a novel DPYD variant (rs75267292; P = 1.57 × 10-10), and variants in the MACF1 (rs183324967, P = 4.80 × 10-11; rs148221738, P = 5.73 × 10-10) and SPRY2 (rs117876855, P < 1.01 × 10-8; rs139544515, P = 1.30 × 10-8) genes involved in wound healing. CONCLUSIONS AND RELEVANCE: Pyridoxine did not significantly prevent or delay the onset of grade 2 or higher HFS. Serum and red blood cell folate levels are independent predictors of HFS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00486213.

Suppression of ID1 expression in colon cancer cells increases sensitivity to 5-fluorouracil.

Adjuvant chemotherapy with 5-fluorouracil remains the basic treatment for patients with advanced colorectal carcinoma. The major obstacle in successful treatment is the ability of CRC cells to acquire chemoresistance. Here we examined the impact of ID1 silencing on the sensitivity of CRC cells to 5-FU. To suppress ID1 expression in HT-29 and HCT-116 cells the cells were transduced with a lentiviral vector carrying the ID1 silencing sequence. Cells with silenced ID1 showed altered expression of epithelial and mesenchymal markers and exhibited increased proliferation rate compared to the parental cells. HCT-116 cells with suppressed ID1 became sensitized to 5-FU and this was not observed in HT-29 cells. Silencing ID1 resulted in altered expression of genes encoding enzymes metabolizing 5-FU. HT-29 cells with suppressed ID1 had significantly reduced mRNA level for thymidine phosphorylase, uridine-cytydine kinase 2 and dihydropyrimidine dehydrogenase. ID1 suppression in HCT-116 cells resulted in an increase of mRNA level for thymidine phosphorylase, thymidine kinase and uridine-cytydine kinase 2 with concurrent drop of dihydropyrimidine dehydrogenase and thymidylate synthetase mRNA levels. In conclusion, ID1 expression impacts the sensitivity of colon cancer cells to 5-FU and may be considered as a potential predictive marker in CRC treatment.

Low-dose hyperthermia enhances the antitumor effects of chemotherapy in squamous cell carcinoma.

Esophageal squamous cell carcinoma is a highly aggressive neoplasm and the sixth leading cause of global cancer-related death; the 5-year survival rate for esophageal cancer is only about 20%-25% for all stages. Therefore, improving the therapeutic effect is important. This study assessed whether low-dose hyperthermia (LDH) enhances the antitumor effects of chemotherapy. The antitumor effect of chemotherapy with/without LDH in the squamous cell carcinoma cell line SCCVII was evaluated. A comprehensive analysis was performed with real-time polymerase chain reaction (PCR) to study the hyperthermia-induced changes in the gene expression of SCCVII cell lines. In addition, the cytotoxic and apoptotic changes in the cells treated with LDH combined with/without 5-fluorouracil (5-FU) were measured. LDH combined with 5-FU (10 nM) strongly inhibited the cell growth of SCCVII, with flow cytometry showing an increased population of apoptotic cells. PCR showed that LDH promoted a 25.22-fold increase of p53 mRNA and 18.08-fold increase of Bax mRNA in vitro. MDR1 expression was decreased to 28.7% after LDH. This treatment can result in much higher efficacy of antitumor drugs. After LDH, the expressions of TS decreased to 12.06%, OPRT increased by 4.17-fold, and DPD did not change (1.03-fold). This transformations will induce susceptibility to 5-FU. LDH may be a useful enhancer of chemotherapy drugs for squamous cell carcinoma.

Evaluation of 5-fluorouracil degradation rate and Pharmacogenetic profiling to predict toxicity following adjuvant Capecitabine.

BACKGROUND: On account of the lack of predictive biomarkers of toxicity, we investigated whether polymorphisms of genes involved in fluoropyrimidine metabolism and 5-fluorouracil (5-FU) degradation rate were associated with outcomes of adjuvant capecitabine in patients with early stage gastrointestinal cancers. METHODS: Genotyping of DPYD GIVS14A, MTHFR C677T and A1298C SNPs were performed by pyro-sequencing technology. PCR analysis was used for genotyping TYMS-TSER. We also evaluated the 5-FU degradation rate, which determines the amount of drug consumed by PBMC in a time unit. Association of these variables with clinical outcome was evaluated using multivariate logistic regression analysis. RESULTS: One hundred forty-two patients with early stage colon (39%), rectal (28%), stomach (20%) and pancreatic (13%) cancer, treated with adjuvant capecitabine, were included in this retrospective analysis. Seventy and 20% of the patients suffered from at least one G1-4 and G3-4 adverse events, respectively. According to the 5-FU degradation rate, three and 13 patients were assigned as poor (<0.86 ng/mL/106 cells/min) and ultra-rapid (>2.1 ng/mL/106 cells/min) metabolizers, respectively. At a multivariate logistic regression analysis, an altered 5-FU degradation rate (values <0.86 or >2.10 ng/mL/106 cells/min) was associated with grade 3-4 adverse events (OR = 2.09, 95% CI: 1.14-3.82, P = 0.01). No correlation was reported between toxicity and gene polymorphisms except for hand-foot syndrome that was more frequent in the MTHFR 1298CC homozygous variant genotype (OR = 2.03, 95% CI 1.04-3.96, P = 0.03). CONCLUSIONS: 5-FU degradation rate may be regarded as possible predictive biomarker of capecitabine toxicity in early stage gastrointestinal cancer.