DPYD Variants Research Articles

DPYD genotype-guided dose individualisation of fluoropyrimidine therapy in patients with cancer: a prospective safety analysis.

Fluoropyrimidine treatment can result in severe toxicity in up to 30% of patients and is often the result of reduced activity of the key metabolic enzyme dihydropyrimidine dehydrogenase (DPD), mostly caused by genetic variants in the gene encoding DPD (DPYD). We assessed the effect of prospective screening for the four most relevant DPYD variants (DPYD*2A [rs3918290, c.1905+1G>A, IVS14+1G>A], c.2846A>T [rs67376798, D949V], c.1679T>G [rs55886062, DPYD*13, I560S], and c.1236G>A [rs56038477, E412E, in haplotype B3]) on patient safety and subsequent DPYD genotype-guided dose individualisation in daily clinical care. In this prospective, multicentre, safety analysis in 17 hospitals in the Netherlands, the study population consisted of adult patients (≥18 years) with cancer who were intended to start on a fluoropyrimidine-based anticancer therapy (capecitabine or fluorouracil as single agent or in combination with other chemotherapeutic agents or radiotherapy). Patients with all tumour types for which fluoropyrimidine-based therapy was considered in their best interest were eligible. We did prospective genotyping for DPYD*2A, c.2846A>T, c.1679T>G, and c.1236G>A. Heterozygous DPYD variant allele carriers received an initial dose reduction of 25% (c.2846A>T and c.1236G>A) or 50% (DPYD*2A and c.1679T>G), and DPYD wild-type patients were treated according to the current standard of care. The primary endpoint of the study was the frequency of severe (National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03 grade ≥3) overall fluoropyrimidine-related toxicity across the entire treatment duration. We compared toxicity incidence between DPYD variant allele carriers and DPYD wild-type patients on an intention-to-treat basis, and relative risks (RRs) for severe toxicity were compared between the current study and a historical cohort of DPYD variant allele carriers treated with full dose fluoropyrimidine-based therapy (derived from a previously published meta-analysis). This trial is registered with ClinicalTrials.gov, number NCT02324452, and is complete. Between April 30, 2015, and Dec 21, 2017, we enrolled 1181 patients. 78 patients were considered non-evaluable, because they were retrospectively identified as not meeting inclusion criteria, did not start fluoropyrimidine-based treatment, or were homozygous or compound heterozygous DPYD variant allele carriers. Of 1103 evaluable patients, 85 (8%) were heterozygous DPYD variant allele carriers, and 1018 (92%) were DPYD wild-type patients. Overall, fluoropyrimidine-related severe toxicity was higher in DPYD variant carriers (33 [39%] of 85 patients) than in wild-type patients (231 [23%] of 1018 patients; p=0·0013). The RR for severe fluoropyrimidine-related toxicity was 1·31 (95% CI 0·63-2·73) for genotype-guided dosing compared with 2·87 (2·14-3·86) in the historical cohort for DPYD*2A carriers, no toxicity compared with 4·30 (2·10-8·80) in c.1679T>G carriers, 2·00 (1·19-3·34) compared with 3·11 (2·25-4·28) for c.2846A>T carriers, and 1·69 (1·18-2·42) compared with 1·72 (1·22-2·42) for c.1236G>A carriers. Prospective DPYD genotyping was feasible in routine clinical practice, and DPYD genotype-based dose reductions improved patient safety of fluoropyrimidine treatment. For DPYD*2A and c.1679T>G carriers, a 50% initial dose reduction was adequate. For c.1236G>A and c.2846A>T carriers, a larger dose reduction of 50% (instead of 25%) requires investigation. Since fluoropyrimidines are among the most commonly used anticancer agents, these findings suggest that implementation of DPYD genotype-guided individualised dosing should be a new standard of care. Dutch Cancer Society.

Dose modification for safe treatment of a compound complex heterozygous DPYD variant carrier with 5-fluorouracil.

Dose modification for safe treatment of a compound complex heterozygous DPYD variant carrier with 5-fluorouracil.

74P - Common and rare DPYD variants are predictive for 5FU/capecitabine (5FU) toxicity: The MRC COIN and COIN-B trials

Rare genetic variants in DPYP increase toxicity and screening for them prevents serious complications by upfront reduction in 5FU dose; however, most patients with severe toxicities do not have a rare mutation. We have previously shown that 2 common DPYD variants were associated with toxicity in patients with advanced colorectal cancer treated on COIN & COIN-B (abstract 3509, ASCO 2013): Cys29Arg [rs1801265] (Minor Allele Frequency (MAF) 0.21) and Val732Ile [rs1801160] (MAF 0.04). We have now genotyped 4 rare variants using the same cohort.

561P - Standard fluoropyrimidine dosages in chemoradiation therapy result in an increased risk of severe toxicity in DPYD variant allele carriers

Abstract Background Prospective DPYD genotyping prevents severe fluoropyrimidine (FP)-induced toxicity by decreasing dosages in DPYD variant allele carriers. FP dosages in chemoradiation therapy (CRT) are lower than those in other FP-containing regimens. Pharmacogenetic guidelines do not distinguish between regimens, leaving physicians in doubt to apply dose reductions. Our aim was to investigate severe toxicity in DPYD variant allele carriers receiving CRT. Methods Medical records of 828 patients who received FP-based CRT were reviewed from three centres. Severe (grade ≥III) toxicity in DPYD variant allele carriers receiving upfront FP dose reductions according to pharmacogenetic dosing guidelines and DPYD variant allele carriers not receiving FP dose reductions was compared with DPYD wild-type patients receiving standard dose of FPs in CRT. Results DPYD variant allele carriers treated with standard dosages (N = 34) showed an increased risk of severe gastrointestinal (adjusted OR = 2.58, confidence interval [CI] = 1.02–6.53, P = 0.045) or severe haematological (adjusted OR = 4.19, CI = 1.32–13.25, P = 0.015) toxicity compared with wild-type patients (N = 771). DPYD variant allele carriers who received dose reductions (N = 22) showed a comparable frequency of severe gastrointestinal toxicity compared with wild-type patients, but more (not statistically significant) severe haematological toxicity. Hospitalisations for all DPYD variant allele carriers were comparable, independent of dose adjustments; however, the mean duration of hospitalisation was significantly shorter in the dose reduction group (P = 0.010). Conclusions Standard FP dosages in CRT resulted in an increased risk of severe toxicity in DPYD variant allele carriers. We advise to apply FP dose reductions according to current guidelines in DPYD variant allele carriers starting CRT.

Integration of Germline Pharmacogenetics Into a Tumor Sequencing Program.

Evidence-based guidelines inform treatment decisions for patients for whom germline genetic information is available. Our real-time tumor sequencing program, which makes precision treatment decisions for patients with cancer, produces matched germline information, providing a unique opportunity to efficiently implement pharmacogenetics and benefit patients. The germline genetic database from the Michigan Oncology Sequencing (MI-Oncoseq) program was searched for 21 clinically actionable polymorphisms in five cancer-relevant genes: TPMT, DPYD, CYP2C19, CYP3A5, and UGT1A1. Residual germ line DNA was sent to an external Clinical Laboratory Improvement Amendments-approved laboratory for confirmatory genotyping. The medical records of MI-Oncoseq patients with actionable phenotypes were searched for receipt of relevant drugs and to determine whether having genetic information at the time of treatment would have led to a treatment recommendation. All nine variants in TPMT, DPYD, and CYP2C19 that were detected in MI-Oncoseq were confirmed by external genotyping. Genotype determinations could not be made for CYP3A5*3, UGT1A1*28, or UGT1A1*80. On the basis of retrospective assessment of 115 adult and pediatric patient records, 4.3% (n = 5) had a potentially clinically actionable phenotype for TPMT, DPYD, or CYP2C19 and received a relevant medication. After accounting for differences in adult and pediatric recommendations, three of these patients could have received a treatment recommendation at the time of prescribing. Germline genotype determinations for TPMT, DPYD, and CYP2C19 can be used to make evidence-based treatment recommendations in MI-Oncoseq patients. Although the proportion of patients for whom recommendations can be made is small, this added value to MI-Oncoseq and patient care comes at no additional genotyping cost. Pharmacogenetic assessment should be integrated into tumor sequencing programs that genotype matched germline DNA; however, the complexity and additional cost of implementing pharmacogenetics remain challenging.

Abstract 3894: DPYD-Varifier, a computational model to identify 5-FU toxicity-associated DPYD variants

Abstract 5-Fluorouracil (5-FU) is a cornerstone therapy for the treatment of several aggressive tumors. However, nearly 1/3 of patients receiving 5-FU experience severe (grade 3+) adverse 5-FU toxicity. Genetic variations in dihydropyrimidine dehydrogenase (DPD; DPYD gene) are major contributors of 5-FU toxicity. With continued progress in next-generation sequencing technologies, the number of reported genetic variants is substantially increasing; to date, there are more than 450 reported missense variants in the DPYD gene. Only a handful of those variants, including *2A, I560S, D949V, and rs75017182, have been established as predictors of toxicity. Because most of the remaining variants are unstudied, mainly due to their rare occurrence and unequal racial distribution, it is challenging to conduct statistically powered clinical association studies. Therefore, in vitro and computational methods to identify potential toxicity-associated variants have major appeal. The primary goal of the current study was to develop a highly accurate gene-specific in silico classifier to predict deleterious DPYD variants. For this purpose, using a recombinant cellular model, we characterized 69 missense DPYD variants reported in online databases. We then utilized DPD activity data from the 69 variants in combination with data from 87 previously characterized variants (see Offer, SM et al. Cancer Research 2013 & 2014) to develop the DPYD variant classifier model (DPYD-Varifier). DPD protein-specific biochemical and structural features and amino acid conservation scores were integrated into the model's random forest classifier-based algorithm. DPYD-Varifier showed improved accuracy (85% accurate) compared to other in silico tools. Additionally, the model provided mechanistic insights into alterations of DPD function conferred by DPYD variants. In general, deleterious variants were located in closer proximity to important binding/coordinating sites of the DPD protein. Additionally, DPYD-Varifier allowed us to estimate the cumulative allele frequency of all reported deleterious variants in various geographical/racial groups, which can substantially add to our knowledge of DPD deficiency and 5-FU toxicity observed in these populations. In conclusion, the integration of in vitro and accurate computational data from our study permitted classification of relevant DPYD variants, which could be useful to more effectively individualize 5-FU therapy in the future. Citation Format: Shikshya Shrestha, Cheng Zhang, Calvin R. Jerde, Hu Li, Steven M. Offer, Robert B. Diasio. DPYD-Varifier, a computational model to identify 5-FU toxicity-associated DPYD variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3894.

P-213 - An evaluation of the clinical utility of a panel of variants in DPYD and ENOSF1 for predicting common capecitabine related toxicities

P-213 - An evaluation of the clinical utility of a panel of variants in DPYD and ENOSF1 for predicting common capecitabine related toxicities

Germline pharmacogenomics of DPYD*9A (c.85T>C) variant in patients with gastrointestinal malignancies treated with fluoropyrimidines.

The correlation between DPYD*9A (c.85T>C) genotype and dihydropyrimidine dehydrogenase (DPD) deficiency clinical phenotype is controversial. Reference laboratories either did not perform DPYD*9A genotyping or have stopped DPYD*9A genotyping and limited genotyping to high-risk variants (DPYD*2A, DPYD*13 and DPYD*9B) only. This study explored DPYD*9A genotype and clinical phenotype correlation in patients with gastrointestinal (GI) malignancies treated with fluoropyrimidines. Between 2011 and 2017, 67 patients with GI malignancies were genotyped for DPYD variants. Fluoropyrimidines-associated toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0). Fisher's exact test was used for statistical analysis. DPYD variants were identified in 17 out of 67 (25%) patients. One patient was homozygous for DPYD*9A variant and one patient was double heterozygous for DPYD*9A and DPYD*9B variants. In patients with identified DPYD variants, 13/17 (76%) patients had DPYD*9A variant, 3/17 (18%) patients had DPYD*2A variant and 2/17 (12%) patient had DPYD*9B variant. Only patients genotyped prior to 2015 were genotyped for DPYD*9A variant (N=28). Of those, 13/28 patients (46%) had DPYD*9A variant. Grade 3-4 diarrhea was associated with DPYD*9A variant in patients treated with full dose fluoropyrimidines (P=0.0055). In our cohort, DPYD*9A variant was the most common diagnosed variant. The correlation between DPYD*9A genotype and DPD deficiency in clinical phenotype was noticeable in patients who received full dose fluoropyrimidines as they all experienced grade 3-4 toxicities (diarrhea).

Functional Characterization of 21 Allelic Variants of Dihydropyrimidine Dehydrogenase Identified in 1070 Japanese Individuals.

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2), encoded by the DPYD gene, is the rate-limiting enzyme in the degradation pathway of endogenous pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil (5-FU). DPD catalyzes the reduction of uracil, thymine, and 5-FU. In Caucasians, DPYD mutations, including DPYD*2A, DPYD*13, c.2846A>T, and c.1129-5923C>G/hapB3, are known to contribute to interindividual variations in the toxicity of 5-FU; however, none of these DPYD polymorphisms has been identified in the Asian population. Recently, 21 DPYD allelic variants, including some novel single-nucleotide variants (SNVs), were identified in 1070 healthy Japanese individuals by analyzing their whole-genome sequences (WGSs), but the functional alterations caused by these variants remain unknown. In this study, in vitro analysis was performed on 22 DPD allelic variants by transiently expressing wild-type DPD and 21 DPD variants in 293FT cells and characterizing their enzymatic activities using 5-FU as a substrate. DPD expression levels and dimeric forms were determined using immunoblotting and blue-native PAGE, respectively. Additionally, the values of three kinetic parameters-the Michaelis constant (Km ), maximum velocity (Vmax ), and intrinsic clearance (CLint = Vmax/Km )-were determined for the reduction of 5-FU. Eleven variants exhibited significantly decreased intrinsic clearance compared with wild-type DPD. Moreover, the band patterns observed in the immunoblots of blue-native gels indicated that DPD dimerization is required for enzymatic activity in DPD. Thus, the detection of rare DPYD variants might facilitate severe adverse effect prediction of 5-FU-based chemotherapy in the Japanese population.

Gene-Specific Variant Classifier (DPYD-Varifier) to Identify Deleterious Alleles of Dihydropyrimidine Dehydrogenase.

Deleterious variants in dihydropyrimidine dehydrogenase (DPD, DPYD gene) can be highly predictive of clinical toxicity to the widely prescribed chemotherapeutic 5-fluorouracil (5-FU). However, there are very limited data pertaining to the functional consequences of the >450 reported no-synonymous DPYD variants. We developed a DPYD-specific variant classifier (DPYD-Varifier) using machine learning and in vitro functional data for 156 missense DPYD variants. The developed model showed 85% accuracy and outperformed other in silico prediction tools. An examination of feature importance within the model provided additional insight into functional aspects of the DPD protein relevant to 5-FU toxicity. In the absence of clinical data for unstudied variants, prediction tools like DPYD-Varifier have great potential to individualize medicine and improve the clinical decision-making process.

Germline pharmacogenomics of DPYD*9A (c.85T>C) variant in patients with gastrointestinal malignancies treated with fluoropyrimidines.

598 Background: The correlation between DPYD*9A (c.85T > C) genotype and dihydropyrimidine dehydrogenase (DPD) deficiency clinical phenotype is controversial. Reference laboratories either did not perform DPYD*9A genotyping or have stopped DPYD*9A genotyping and limited genotyping to high-risk variants (DPYD*2A, DPYD*13 and DPYD*9B) only. This study explored DPYD*9A genotype and clinical phenotype correlation in patients with gastrointestinal (GI) malignancies treated with fluoropyrimidines. Methods: Between 2011 and 2017, 67 patients with GI malignancies were genotyped for DPYD variants. Fluoropyrimidines-associated toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0). Fisher’s exact test was used for statistical analysis. Results: DPYD variants were identified in 17 out of 67 (25%) patients. One patient was homozygous for DPYD*9A variant and one patient was double heterozygous for DPYD*9A and DPYD*9B variants. 13/17 (76%) patients had DPYD*9A variant, 3/17 (18%) patients had DPYD*2A variant and 2/17 (12%) patient had DPYD*9B variant. Only patients genotyped prior to 2015 were genotyped for DPYD*9A variant (N = 28). Of those, 13/28 patients (46%) had DPYD*9A variant. Grade 3-4 diarrhea was associated with DPYD*9A variant in patients treated with full dose fluoropyrimidines (P = 0.0055). Conclusions: In our cohort, DPYD*9A variant was the most common diagnosed variant. The correlation between DPYD*9A genotype and DPD deficiency clinical phenotype was noticeable in patients who received full dose fluoropyrimidines as they all experienced grade 3-4 toxicities (diarrhea).

DPYD and UGT1A1 genotyping to predict adverse events during first-line FOLFIRI or FOLFOXIRI plus bevacizumab in metastatic colorectal cancer.

Our study addresses the issue of the clinical reliability of three candidate DPYD and one UGT single nucleotide polymorphisms in predicting 5-fluorouracil- and irinotecan-related adverse events. To this purpose, we took advantage of a large cohort of metastatic colorectal cancer patients treated with first-line 5-fluorouracil- and irinotecan-based chemotherapy regimens (i.e., FOLFIRI or FOLFOXIRI) plus bevacizumab in the randomized clinical trial TRIBE by GONO (clinicaltrials.gov: NCT00719797), in which adverse events were carefully and prospectively collected at each treatment cycle. Here we show that patients bearing DPYD c.1905+1G/A and c.2846A/T genotypes, together with UGT1A1*28 variant carriers, have an increased risk of experiencing clinically relevant toxicities, including hematological AEs and stomatitis. No carrier of the DPYD c.1679T>G minor allele was identified. Present results support the preemptive screening of mentioned DPYD and UGT1A1 variants to identify patients at risk of clinically relevant 5-fluoruracil- and irinotecan-related AEs, in order to improve treatments’ safety through a “genotype-guided” approach.

Pharmacogenetic landscape of DPYD variants in south Asian populations by integration of genome-scale data.

Adverse drug reactions to 5-Fluorouracil(5-FU) is frequent and largely attributable to genetic variations in the DPYD gene, a rate limiting enzyme that clears 5-FU. The study aims at understanding the pharmacogenetic landscape of DPYD variants in south Asian populations. Systematic analysis of population scale genome wide datasets of over 3000 south Asians was performed. Independent evaluation was performed in a small cohort of patients. Our analysis revealed significant differences in the the allelic distribution of variants in different ethnicities. This is the first and largest genetic map the DPYD variants associated with adverse drug reaction to 5-FU in south Asian population. Our study highlights ethnic differences in allelic frequencies.

Dihydropyrimidine dehydrogenase pharmacogenetics for predicting fluoropyrimidine-related toxicity in the randomised, phase III adjuvant TOSCA trial in high-risk colon cancer patients

Background:Dihydropyrimidine dehydrogenase (DPD) catabolises ∼85% of the administered dose of fluoropyrimidines. Functional DPYD gene variants cause reduced/abrogated DPD activity. DPYD variants analysis may help for defining individual patients’ risk of fluoropyrimidine-related severe toxicity.Methods:The TOSCA Italian randomised trial enrolled colon cancer patients for 3 or 6 months of either FOLFOX-4 or XELOX adjuvant chemotherapy. In an ancillary pharmacogenetic study, 10 DPYD variants (*2A rs3918290 G>A, *13 rs55886062 T>G, rs67376798 A>T, *4 rs1801158 G>A, *5 rs1801159 A>G, *6 rs1801160 G>A, *9A rs1801265 T>C, rs2297595 A>G, rs17376848 T>C, and rs75017182 C>G), were retrospectively tested for associations with ⩾grade 3 fluoropyrimidine-related adverse events (FAEs). An association analysis and a time-to-toxicity (TTT) analysis were planned. To adjust for multiple testing, the Benjamini and Hochberg’s False Discovery Rate (FDR) procedure was used.Results:FAEs occurred in 194 out of 508 assessable patients (38.2%). In the association analysis, FAEs occurred more frequently in *6 rs1801160 A allele carriers (FDR=0.0083). At multivariate TTT analysis, significant associations were found for *6 rs1801160 A allele carriers (FDR<0.0001), *2A rs3918290 A allele carriers (FDR<0.0001), and rs2297595 GG genotype carriers (FDR=0.0014). Neutropenia was the most common FAEs (28.5%). *6 rs1801160 (FDR<0.0001), and *2A rs3918290 (FDR=0.0004) variant alleles were significantly associated with time to neutropenia.Conclusions:This study adds evidence on the role of DPYD pharmacogenetics for safety of patients undergoing fluoropyrimidine-based chemotherapy.

Predictors of Hand-Foot Syndrome and Pyridoxine for Prevention of Capecitabine–Induced Hand-Foot Syndrome

ImportanceHand-foot syndrome (HFS) is a common adverse effect of capecitabine treatment.ObjectiveTo 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 ParticipantsThis 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.InterventionsPatients 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 MeasuresPrimary 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.ResultsIn 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 RelevancePyridoxine 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 Registrationclinicaltrials.gov Identifier: NCT00486213

Abstract 4069: Trimethylation and acetylation of Histone H3K27 modulates 5-fluorouracil response by regulating DPYD expression

Abstract The antimetabolite 5-fluorouracil (5-FU) is one of the most widely used chemotherapy drugs. Dihydropyrimidine dehydrogenase (DPD) initiates the catabolic degradation of 5-FU to inactive metabolites and is widely accepted as a major determinant of 5-FU response and toxicity. While genetic variants in DPYD have been shown to variants may affect 5-FU metabolism, they are rare events and do not completely explain the reported variability in DPD function or the resultant differences in treatment response. Here, we report that DPYD expression is epigenetically regulated by histone modification at relevant promoter and enhancer regions. Using both chemical inhibitors and genetic approaches, we show that that H3K27 tri-methylation (H3K27me3) at the DPYD promoter is regulated by the epigenetic modifiers Ezh2 and UTX. Promoter H3K27me3 suppresses DPYD expression by inhibiting the binding of the transcription factor PU.1 bindingto the promoter and ,increases sensitivity leading to increased resistance to 5-FU. In a cohort of healthy volunteers, H3K27me3 levels were inversely correlated with An enhancer that regulates DPYD expression was also identified. Modifying the H3K27 status of the DPYD promoter or enhancer modulates DPYD gene expression. Deletions and mutations on the DPYD promoter or enhancer abolish their transcription regulation function. Enrichment of H3K27me3 at the DPYD promoter was negatively correlated with both DPYD expression and DPD enzyme activity in peripheral blood mononuclear cellsspecimens from healthy volunteers. Lastly, tumor expression data suggests that DPYD repression by Ezh2 is a strong predictor ofs poor survival in 5-FU treated cancers. By using a combination of in silico prediction and in vitro reporter assays we also identified a functional enhancer region for DPYD that is also likely to be clinically relevant to 5-FU treatment outcome. Collectively, the presentthese findings suggest that a previously uncharacterized mechanisms regulates DPD expression and may likely contribute to tumor resistance to 5-FU. Citation Format: Rentian Wu, Qian Nie, Erin E. Tapper, Calvin R. Jerde, Steven M. Offer, Robert B. Diasio. Trimethylation and acetylation of Histone H3K27 modulates 5-fluorouracil response by regulating DPYD expression [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 4069. doi:10.1158/1538-7445.AM2017-4069

Quantitative Contribution of rs75017182 to Dihydropyrimidine Dehydrogenase mRNA Splicing and Enzyme Activity

Dihydropyrimidine dehydrogenase (DPD; DPYD gene) variants have emerged as reliable predictors of adverse toxicity to the chemotherapy agent 5-fluorouracil (5-FU). The intronic DPYD variant rs75017182 has been recently suggested to promote alternative splicing of DPYD. However, both the extent of alternative splicing and the true contribution of rs75017182 to DPD function remain unclear. In the present study we quantified alternative splicing and DPD enzyme activity in rs75017182 carriers utilizing healthy volunteer specimens from the Mayo Clinic Biobank. Although the alternatively spliced transcript was uniquely detected in rs75017182 carriers, canonically spliced DPYD levels were only reduced by 30% (P = 2.8 × 10-6 ) relative to controls. Similarly, DPD enzyme function was reduced by 35% (P = 0.025). Carriers of the well-studied toxicity-associated variant rs67376798 displayed similar reductions in DPD activity (31% reduction). The modest effects on splicing and function suggest that rs75017182 may have clinical utility as a predictor of 5-FU toxicity similar to rs67376798.

New advances in DPYD genotype and risk of severe toxicity under capecitabine.

BackgroundDeficiency in dihydropyrimidine dehydrogenase (DPD) enzyme is the main cause of severe and lethal fluoropyrimidine-related toxicity. Various approaches have been developed for DPD-deficiency screening, including DPYD genotyping and phenotyping. The goal of this prospective observational study was to perform exhaustive exome DPYD sequencing and to examine relationships between DPYD variants and toxicity in advanced breast cancer patients receiving capecitabine.MethodsTwo-hundred forty-three patients were analysed (88.5% capecitabine monotherapy). Grade 3 and grade 4 capecitabine-related digestive and/or neurologic and/or hemato-toxicities were observed in 10.3% and 2.1% of patients, respectively. DPYD exome, along with flanking intronic regions 3’UTR and 5’UTR, were sequenced on MiSeq Illumina. DPD phenotype was assessed by pre-treatment plasma uracil (U) and dihydrouracil (UH2) measurement.ResultsAmong the 48 SNPs identified, 19 were located in coding regions, including 3 novel variations, each observed in a single patient (among which, F100L and A26T, both pathogenic in silico). Combined analysis of deleterious variants *2A, I560S (*13) and D949V showed significant association with grade 3–4 toxicity (sensitivity 16.7%, positive predictive value (PPV) 71.4%, relative risk (RR) 6.7, p<0.001) but not with grade 4 toxicity. Considering additional deleterious coding variants D342G, S492L, R592W and F100L increased the sensitivity to 26.7% for grade 3–4 toxicity (PPV 72.7%, RR 7.6, p<0.001), and was significantly associated with grade 4 toxicity (sensitivity 60%, PPV 27.3%, RR 31.4, p = 0.001), suggesting the clinical relevance of extended targeted DPYD genotyping. As compared to extended genotype, combining genotyping (7 variants) and phenotyping (U>16 ng/ml) did not substantially increase the sensitivity, while impairing PPV and RR.ConclusionsExploring an extended set of deleterious DPYD variants improves the performance of DPYD genotyping for predicting both grade 3–4 and grade 4 toxicities (digestive and/or neurologic and/or hematotoxicities) related to capecitabine, as compared to conventional genotyping restricted to consensual variants *2A, *13 and D949V.

Pretreatment serum uracil concentration as a predictor of severe and fatal fluoropyrimidine-associated toxicity

Background:We investigated the predictive value of dihydropyrimidine dehydrogenase (DPD) phenotype, measured as pretreatment serum uracil and dihydrouracil concentrations, for severe as well as fatal fluoropyrimidine-associated toxicity in 550 patients treated previously with fluoropyrimidines during a prospective multicenter study.Methods:Pretreatment serum concentrations of uracil and dihydrouracil were measured using a validated LC-MS/MS method. The primary endpoint of this analysis was global (any) severe fluoropyrimidine-associated toxicity, that is, grade ⩾3 toxicity according to the NCI CTC-AE v3.0, occurring during the first cycle of treatment. The predictive value of uracil and the uracil/dihydrouracil ratio for early severe fluoropyrimidine-associated toxicity were compared. Pharmacogenetic variants in DPYD (c.2846A>T, c.1679T>G, c.1129-5923C>G, and c.1601G>A) and TYMS (TYMS 5′-UTR VNTR and TYMS 3′-UTR 6-bp ins/del) were measured and tested for associations with severe fluoropyrimidine-associated toxicity to compare predictive value with DPD phenotype. The Benjamini-Hochberg false discovery rate method was used to control for type I errors at level q<0.050 (corresponding to P<0.010).Results:Uracil was superior to the dihydrouracil/uracil ratio as a predictor of severe toxicity. High pretreatment uracil concentrations (>16 ng ml−1) were strongly associated with global severe toxicity (OR 5.3, P=0.009), severe gastrointestinal toxicity (OR 33.7, P<0.0001), toxicity-related hospitalisation (OR 16.9, P<0.0001), as well as fatal treatment-related toxicity (OR 44.8, P=0.001). None of the DPYD variants alone, or TYMS variants alone, were associated with severe toxicity.Conclusions:High pretreatment uracil concentration was strongly predictive of severe, including fatal, fluoropyrimidine-associated toxicity, and is a highly promising phenotypic marker to identify patients at risk of severe fluoropyrimidine-associated toxicity.

Identification of new SNPs associated with severe toxicity to capecitabine

Predicting individual risk of chemotherapy-induced severe adverse reaction is a critical issue when selecting the best treatment for cancer patients. SNPs have been identified in genes involved in the pharmacodynamics of fluoropyrimidines, and guidelines even recommend genotyping some DPYD variants in order to estimate the risk of toxicity. However, the predictive value of this approach remains insufficient, thus limiting its clinical implementation. The aim of the present study was to identify new genetic variants by selecting a group of tag SNPs in genes associated with the pharmacodynamics of fluoropyrimidines (CDA, DPYD, ENOSF1, CES1, TYMS, SLC22A7, TYMP, and UMPS). For this purpose, 23 selected SNPs were genotyped on an OpenArray™ platform in a cohort of 301 colorectal cancer patients receiving capecitabine-based chemotherapy. Univariate and multivariate statistical analysis by logistic regression revealed 10 SNPs associated with severe adverse reactions to capecitabine (P<0.05): rs1048977, rs12726436, and rs2072671 in CDA; rs12119882 in DPYD; rs2853741 in TYMS; rs699517 in TYMS/ENOSF1; rs2270860 and rs4149178 in SLC22A7; and rs2279199 and rs4678145 in UMPS. Except for rs2072671, no association had previously been reported between these SNPs and the risk of capecitabine-induced toxicity. The use of tag SNPs to find new polymorphisms related to adverse reactions to capecitabine was successful. These new variants could increase the predictive power of currently available tests and thus prevent severe adverse reactions to capecitabine.