Germline pharmacogenomics in patients with dihydropyrimidine dehydrogenase (DPD) deficiency.

BackgroundPretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended or required prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD-deficient patients remains elusive.MethodsAmong a nationwide cohort of 5886 phenotyped patients with cancer who were screened for DPD deficiency over a 3 years period, we assessed the characteristics of both DPD phenotypes and DPYD genotypes in a subgroup of 3680 patients who had completed the two tests. The extent to which defective allelic variants of DPYD predict DPD activity as estimated by the plasma concentrations of uracil [U] and its product dihydrouracil [UH2] was evaluated.ResultsWhen [U] was used to monitor DPD activity, 6.8% of the patients were classified as having DPD deficiency ([U] > 16 ng/ml), while the [UH2]:[U] ratio identified 11.5% of the patients as having DPD deficiency (UH2]:[U] < 10). [U] classified two patients (0.05%) with complete DPD deficiency (> 150 ng/ml), and [UH2]:[U] < 1 identified three patients (0.08%) with a complete DPD deficiency. A defective DPYD variant was present in 4.5% of the patients, and two patients (0.05%) carrying 2 defective variants of DPYD were predicted to have low metabolism. The mutation status of DPYD displayed a very low positive predictive value in identifying individuals with DPD deficiency, although a higher predictive value was observed when [UH2]:[U] was used to measure DPD activity. Whole exon sequencing of the DPYD gene in 111 patients with DPD deficiency and a “wild-type” genotype (based on the four most common variants) identified seven heterozygous carriers of a defective allelic variant.ConclusionsFrequent genetic DPYD variants have low performances in predicting partial DPD deficiency when evaluated by [U] alone, and [UH2]:[U] might better reflect the impact of genetic variants on DPD activity. A clinical trial comparing toxicity rates after dose adjustment according to the results of genotyping or phenotyping testing to detect DPD deficiency will provide critical information on the best strategy to identify DPD deficiency.

Dihydropyrimidine dehydrogenase (DPD) deficiency, a known pharmacogenetic syndrome associated with 5-fluorouracil (5-FU) toxicity, has been detected in 3% to 5% of the population. Genotypic studies have identified >32 sequence variants in the DPYD gene; however, in a number of cases, sequence variants could not explain the molecular basis of DPD deficiency. Recent studies in cell lines indicate that hypermethylation of the DPYD promoter might down-regulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency. DPD deficiency was identified phenotypically by both enzyme assay and uracil breath test, and genotypically by denaturing high-performance liquid chromatography. The methylation status was evaluated in PCR products (209 bp) of bisulfite-modified DPYD promoter, using a novel denaturing high-performance liquid chromatography method that distinguishes between methylated and unmethylated alleles. Clinical samples included five volunteers with normal DPD enzyme activity, five DPD-deficient volunteers, and five DPD-deficient cancer patients with a history of 5-FU toxicity. No evidence of methylation was detected in samples from volunteers with normal DPD. Methylation was detected in five of five DPD-deficient volunteers and in three of five of the DPD-deficient cancer patient samples. Of note, one of the two samples from patients with DPD-deficient cancer with no evidence of methylation had the mutation DPYD*2A, whereas the other had DPYD*13. Methylation of the DPYD promoter region is associated with down-regulation of DPD activity in clinical samples and should be considered as a potentially important regulatory mechanism of DPD activity and basis for 5-FU toxicity in cancer patients.

To the editor, With interest we read the article by Dr Cubero and colleagues, in which they evaluated the safety of tegafur-uracil (UFT®) in five cases with partial dihydropyrimidine dehydrogenase (DPD) deficiency [Cubero et al. 2012]. Based on our previous experience [Deenen et al. 2010], however, we would like to express our concern about their conclusion that UFT is a safe alternative for the treatment of patients with partial DPD deficiency. Cubero and colleagues make the erroneous and unproven statement that the presence of uracil in UFT creates an artificial DPD deficiency, and that the DPD activity in patients with normal DPD activity would then be similarly as low as in DPD-deficient patients. This assumption, however, is incorrect. As uracil is a competitive inhibitor of DPD, it competes with 5-fluorouracil (5-FU) for DPD-mediated metabolism. This does not mean that the activity of DPD is depleted, as suggested by Cubero and colleagues, in contrast, its activity is fully utilized, as well as for the metabolism of uracil, as for the metabolism of 5-FU. We would like to caution that treating patients with partial DPD deficiency with the standard dose of UFT may unnecessarily lead to severe, potentially lethal toxicity. Unlike the cases described by Cubero and colleagues, we could previously describe four cases presenting with comparable severe toxicity profiles upon treatment with UFT as had previously occurred during treatment with capecitabine or 5-FU. In all subjects an underlying partial DPD deficiency was identified by genotype and phenotype analyses [Deenen et al. 2010]. Furthermore, there are several pharmacological lines of argument that support our clinical observation, i.e. that the standard dose of UFT is not safe in (partial) DPD-deficient patients. First, pharmacokinetic studies have shown that DPD remains essential for the metabolism of UFT, with significantly longer half-lives of 5-FU after administration of UFT compared with 5-FU administered intravenously [Ho et al. 1998]. This is due to the presence of uracil in UFT. Since DPD-deficient patients already have longer half-lives of 5-FU than other patients [Mattison et al. 2006], presence of uracil increases its half-life even further. This in turn leads to prolonged and elevated circulating levels of 5-FU, with a subsequently increased risk of 5-FU-induced severe toxicity. Another argument underscoring the importance of normal DPD function in the safe application of UFT, is the experience with S-1. S-1 is another drug combination of tegafur, consisting of tegafur, 5-chloro-2,4-dihydroxypyridine (CDHP) and potassium oxonate in a molar ratio of 1:0.4:1. CDHP inhibits DPD 200-fold more potently than does uracil [Shirasaka et al. 1996a, 1996b]. Even after administration of S-1, the primary 5-FU metabolite formed by DPD is observed in significant concentrations in plasma [Kim et al. 2007]. Thus, DPD remains an essential detoxification enzyme of 5-FU, even when its activity is strongly inhibited. The ultimate proof of theory is the occurrence of 18 treatment-related deaths in patients with cancer and herpes zoster given UFT plus the antiviral drug sorivudine [Pharmaceutical Affairs Bureau, 1994]. Subsequent studies in rats showed that a metabolite of sorivudine, (E)-5-(2-bromovinyl)uracil, instantly and irreversibly inactivates DPD by covalent binding, which has been identified as the underlying mechanism of these toxic deaths [Ogura et al. 1998; Okuda et al. 1998]. It is for these arguments that the Summary of Product Characteristics of UFT notes a known DPD deficiency as a contra-indication [Merck Serono, 2011]. The fact that the patients described by Cubero and colleagues did not develop significant toxicity might be due to patient selection, the slightly decreased dose intensity of 90%, or despite their DPYD*2A genotype a DPD enzyme activity within the (lower) range of normal. We are not aware of this, because DPD enzyme activity was not determined in these patients. In summary, we would like to state that standard-dose UFT is not a safe treatment in (partial) DPD-deficient patients. Instead, dose reductions of on average 50% of either capecitabine, 5-FU or UFT with careful monitoring of safety and further dose titration are proposed as the standard of care [Deenen et al. 2011].

Dihydropyrimidine dehydrogenase (DPD) deficiency is prevalent in 3-5% of the Caucasian population; however, the frequency of this pharmacogenetic syndrome in the Indian population and other racial and ethnic groups remains to be elucidated. We describe an Indian patient who presented to clinic for the treatment of gastric adenocarcinoma with 5-flurouracil (5-FU) therapy who subsequently was diagnosed with DPD deficiency by using the peripheral blood mononuclear cell (PBMC) DPD radioassay. This observation prompted us to examine the data generated from healthy (cancer-free) Indian subjects who were enrolled in a large population study to determine the sensitivity and specificity of the uracil breath test (UraBT) in the detection of DPD deficiency. Thirteen Indian subjects performed the UraBT. UraBT results were confirmed by PBMC DPD radioassay. The Indian cancer patient demonstrated reduced DPD activity (0.11 nmol/min/mg protein) and severe 5-FU toxicities commonly associated with DPD deficiency. Of the 13 Indian subjects [ten men and three women; mean age, 26 years (range: 21-31 years)] enrolled in the UraBT, 12 Indian subjects demonstrated UraBT breath profiles and PBMC DPD activity within the normal range; one Indian subject demonstrated a reduced breath profile and partial DPD deficiency. DPD deficiency is a pharmacogenetic syndrome which is also present in the Indian population. If undiagnosed, the DPD deficiency can lead to death. Future epidemiological studies would be helpful to determine the prevalence of DPD deficiency among racial and ethnic groups, allowing for the optimization of 5-FU chemotherapy.

Treating patients with dihydropyrimidine dehydrogenase (DPD) deficiency with fluoropyrimidine chemotherapy since the onset of routine prospective testing—The experience of a large oncology center in the United Kingdom

2003 Background: Although DPD deficiency is a well established cause of severe FU-related toxicities, relationships between the depth of the deficiency and the intensity of the toxicity is still poorly documented. We analyzed DPD activity in a large population of patients with FU-related toxicities. Methods: Blood lymphocytes from 144 consecutive cancer patients having developed FU toxicities were collected from different French institutions between January 1993 and July 2004 (53 men, 91 women; mean age 57, extremes 31–94). DPD activity was measured with a radioenzymatic HPLC assay. The IVS14+1G>A mutation was analyzed in 102 patients (RFLP assay). Results: Grade 3–4 toxicity (WHO classification) was 64% for mucositis, 58% for neutropenia, 42% for thrombopenia, 19% for diarrhea and 14% for neurotoxicity. Toxicity led to patient death in 9 cases (8 women, 1 man). DPD activity ranged from 8 to 504 pmol/min/mg (mean 200, median 188, N=144). Nine patients had an activity < 50 pmol/min/mg (severe deficiency) and 19 had an activity between 50 and 100 pmol/min/mg (partial deficiency), thus accounting for a total of 19% deficient patients. The relative risk of developing grade 3–4 toxicity in DPD deficient patients relative to non-deficient patients was 7.69 for neurotoxicity (Fisher’s Exact test, p< 0.001), 2.93 for diarrhea (p<0.001), 1.73 for thrombopenia (p=0.01), 1.63 for mucositis (p<0.001) and 1.59 for neutropenia (p=0.005). The lower the DPD activity, the higher the mucositis, neutropenia or diarrhea grading (Spearman rank correlations, p< 0.03). Toxic deaths were significantly related to low DPD activity (Mann-Whitney p=0.002), with 7 patients out of 9 exhibiting a DPD deficiency. The DPYD mutation (wt/mut) was detected in only 2 patients; both exhibited a low DPD activity (44 and 142 pmol/min/mg) and developed grade 4 mucositis, neutropenia, thrombopenia and grade 3 neurotoxicity without toxic death. Conclusions: 1- Patients with normal DPD activity may develop FU-related toxicities. 2- The intensity of the FU toxicity is related to the severity of the DPD deficiency. No significant financial relationships to disclose.

1618 Background: DPD deficiency is the most important risk factor for developing fluoropyrimidine-related adverse events. Genetic variants causing DPD deficiency are found in 6-8% of Caucasian patients. However, there is limited information on their prevalence in underrepresented ethnic groups, such as Hispanics and Latinos, and testing for these variants is not routinely recommended in Latin America. Our goal was to assess the allele frequency of clinically actionable dihydropyrimidine dehydrogenase ( DPYD) risk variants defined by the Clinical Pharmacogenetics Implementation Consortium (CPIC) and the European Medicines Agency (EMA) among admixed Mexican patients with GI malignancies. Methods: Patients with recently diagnosed GI cancer candidates for fluropyrimidine therapy were recruited from a single institution in Mexico City. After providing informed consent, a blood sample and clinical characteristics were collected. We utilized the Illumina Infinium Global Screening Array (GSA)-to genotype 34 DPYD variants, six of which are known to lead to an increased risk of fluoropyrimidine toxicity and are considered clinically actionable. Results: Two hundred and eight patients with a mean age of 62 years (SD 13.2) were included. 47% were female. The most common type of cancer was colorectal (38%) followed by pancreas (22%) and biliary tract (18%). DNA samples from 192 patients passed quality control, of which 156 (62%) received fluoropyrimidines during follow-up. Only 2 patients (1%) were heterozygous for actionable DPYD intermediate metabolizer risk variant alleles: one with c.2846A &gt; T ( rs67376798 , D949V) and one with c.1129–5923C &gt; G [ rs75017182; HapB3 SNP c.1236G &gt; A; rs56038477]. No patients were found to have other CPIC-listed DPYD risk variants . Additionally, we investigated the allele frequencies of other 30 DPYD variants and observed low-frequency variation (between 0.260 and 0.0032) in rs56038477, rs1801160, rs17376848, rs1801159, rs1801158, rs45589337, rs2297595, rs200562975, and rs1801265. Several of these may be related to decreased DPYD activity and warrant further analysis regarding their impact on adverse drug reactions. Conclusions: In contrast with reports from Caucasic populations, we found a very low allele frequency of DPYD actionable variants. Our findings highlight the limitation of current pharmacogenomic testing recommendations and panels, which may not be appropriate for admixed ethnic populations such as Hispanics/Latinos due to disparities in representation. There is a need to study the role of other DPYD variants in larger patient samples to understand their role in the toxicity risk of admixed populations in Mexico and Latin America, to explore the use of novel techniques such as Next Generation Sequencing, and to investigate the effect of other related genes on toxicity risk.

Is Capecitabine Safe in Patients with Gastrointestinal Cancer and Dihydropyrimidine Dehydrogenase Deficiency?

e13134 Background: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the metabolism of 5-fluorouracil (5-FU). Patients with a partial or complete DPD deficiency are at risk to develop severe toxicity after 5-FU administration. Uracil (U) is degraded in dihydrouracil (DHU) in a similar way as 5-FU. An oral uracil test dose might be useful to determine the systemic DPD activity by measuring uracil and its metabolite dihydrouracil in plasma. DPD deficiency is hypothesized to result in higher uracil levels and a reduced turnover of uracil into dihydrouracil. Methods: Uracil (500 mg/m2) was administered to 11 healthy volunteers with normal DPD activity (≥ 6 nmol/mg/hour) and 1 patient with colorectal carcinoma with a novel DPYD mutation and decreased DPD activity (4.6 nmol/mg/hour). DPD activity was measured in PBMC, as determined as described earlier. Blood samples were taken at t= 15, 30, 45, 60, 80, 100, 120, 150, 180, and 220 or 240 min after oral uracil intake. U and DHU pla...

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype. Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity. In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug. Sequence analysis in a patient with complete DPD deficiency, previously shown to be heterozygous for the delta C1897 frame-shift mutation, revealed the presence of a novel missense mutation, R235W. Expression of this novel mutation and previously identified missense mutations C29R and R886H in Escherichia coli showed that both C29R and R235W lead to a mutant DPD protein without significant residual enzymatic activity. The R886H mutation, however, resulted in about 25% residual enzymatic activity and is unlikely to be responsible for the DPD-deficient phenotype. We show that the E. coli expression system is a valuable tool for examining DPD enzymatic variants. In addition, two new patients who were both heterozygous for the C29R mutation and the common splice donor site mutation were identified. Only one of these patients showed convulsive disorders during childhood, whereas the other showed no clinical phenotype, further illustrating the lack of correlation between genotype and phenotype in DPD deficiency.

Dihydropyrimidine dehydrogenase (DPD) deficiency is critical in the predisposition to 5-fluorouracil dose-related toxicity. We recently characterized the phenotypic [2-(13)C]uracil breath test (UraBT) with 96% specificity and 100% sensitivity for identification of DPD deficiency. In the present study, we characterize the relationships among UraBT-associated breath (13)CO(2) metabolite formation, plasma [2-(13)C]dihydrouracil formation, [2-(13)C]uracil clearance, and DPD activity. An aqueous solution of [2-(13)C]uracil (6 mg/kg) was orally administered to 23 healthy volunteers and 8 cancer patients. Subsequently, breath (13)CO(2) concentrations and plasma [2-(13)C]dihydrouracil and [2-(13)C]uracil concentrations were determined over 180 minutes using IR spectroscopy and liquid chromatography-tandem mass spectrometry, respectively. Pharmacokinetic variables were determined using noncompartmental methods. Peripheral blood mononuclear cell (PBMC) DPD activity was measured using the DPD radioassay. The UraBT identified 19 subjects with normal activity, 11 subjects with partial DPD deficiency, and 1 subject with profound DPD deficiency with PBMC DPD activity within the corresponding previously established ranges. UraBT breath (13)CO(2) DOB(50) significantly correlated with PBMC DPD activity (r(p) = 0.78), plasma [2-(13)C]uracil area under the curve (r(p) = -0.73), [2-(13)C]dihydrouracil appearance rate (r(p) = 0.76), and proportion of [2-(13)C]uracil metabolized to [2-(13)C]dihydrouracil (r(p) = 0.77; all Ps < 0.05). UraBT breath (13)CO(2) pharmacokinetics parallel plasma [2-(13)C]uracil and [2-(13)C]dihydrouracil pharmacokinetics and are an accurate measure of interindividual variation in DPD activity. These pharmacokinetic data further support the future use of the UraBT as a screening test to identify DPD deficiency before 5-fluorouracil-based therapy.

AimsTreatment with dihydropyrimidines poses a significant risk of serious adverse reactions for patients with dihydropyrimidine dehydrogenase (DPD) deficiency. This study seeks to analyse the correlation between DPD deficiency and plasmatic...

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).

Specific language impairment (SLI) is a complex neurodevelopmental disorder defined as an unexpected failure to develop normal language abilities for no obvious reason. Copy number variants (CNVs) are an important source of variation in the susceptibility to neuropsychiatric disorders. Therefore, a CNV study within SLI families was performed to investigate the role of structural variants in SLI. Among the identified CNVs, we focused on CNVs on chromosome 15q11-q13, recurrently observed in neuropsychiatric conditions, and a homozygous exonic microdeletion in ZNF277. Since this microdeletion falls within the AUTS1 locus, a region linked to autism spectrum disorders (ASD), we investigated a potential role of ZNF277 in SLI and ASD. Frequency data and expression analysis of the ZNF277 microdeletion suggested that this variant may contribute to the risk of language impairments in a complex manner, that is independent of the autism risk previously described in this region. Moreover, we identified an affected individual with a dihydropyrimidine dehydrogenase (DPD) deficiency, caused by compound heterozygosity of two deleterious variants in the gene DPYD. Since DPYD represents a good candidate gene for both SLI and ASD, we investigated its involvement in the susceptibility to these two disorders, focusing on the splicing variant rs3918290, the most common mutation in the DPD deficiency. We observed a higher frequency of rs3918290 in SLI cases (1.2%), compared to controls (~0.6%), while no difference was observed in a large ASD cohort. DPYD mutation screening in 4 SLI and 7 ASD families carrying the splicing variant identified six known missense changes and a novel variant in the promoter region. These data suggest that the combined effect of the mutations identified in affected individuals may lead to an altered DPD activity and that rare variants in DPYD might contribute to a minority of cases, in conjunction with other genetic or non-genetic factors.

712 Background: Capecitabine is an integral part of treatment of gastrointestinal cancers. Dihydropyrimidine dehydrogenase (DPD) enzyme is rate limiting in the metabolism of capecitabine, deficiency of which leads to myelsuppression, mucositis, diarrhea, hand foot syndrome (HFS) and rarely, death. Data regarding the toxicity of capecitabine in patients with DPD deficiency in the Indian context is scarce. Methods: 506 patients were treated with capecitabine containing regimens with a dose range of 1250 mg/m2/day to 2000 mg/m2/day during the period from June 2013 to May 2015 in the Gastrointestinal Medical Oncology Unit of our institution. Patients with Grade 3/4 toxicities requiring in-patient care (life threatening complications) were planned for DPD activity testing by Peripheral Blood PCR sequencing. Results: 27 patients developed Gr 3/4 toxicities during cycle 1 of capecitabine. It included mucositis in 22 (81.5 %), diarrhea in 25 (92.6%), HFS in 10 (37%) and myelosuppression in 4 (14.8%). 19 were found to be DPD deficient with 5 patients negative for DPD mutation. 3 patients did not do the DPD analysis as advised. Homozygous mutations were seen in 9 (33.3%) and heterozygous mutations in 10 (27%) of patients. More than one mutation was seen in 10 patients (37%). The relative frequencies of mutation were Exon 14 (44.4%), Exon 2 (25.9%), Exon 13 (25.9%), Exon 6 (11.1%) and Exon 18 (7.4 %). Post Cycle 1 of capecitabine, the drug was stopped in 5 patients (18.5%), regimen changed in 2 (7.4%) and dose reduction by 50% of the drug was done in the remaining patients. Despite dose reduction and change in therapy during Cycle 2, patients still had Grade 3/4 toxicities including mucositis in 7(25.9%), diarrhoea in 10 (37%), HFS in 7 (25.9 %) and myelosuppression in 6 (22%) of patients. Conclusions: Capecitabine can also lead to severe toxicities in DPD-deficient patients. Dose reduction of capecitabine in DPD deficient patients may not completely ameliorate the future risk of life – threatening complications. Screening for DPD deficiency prior to administration of capecitabine in toxicity prone nutritionally deficient Indian patients should be further evaluated based on this data.