Dihydropyrimidine Dehydrogenase Deficiency Research Articles

FDA Approval Summary: Capecitabine Labeling Update under Project Renewal.

On December 14, 2022, the U.S. Food and Drug Administration (FDA) approved revisions to the United States Prescribing Information (USPI) for capecitabine that revised existing indications and dosage regimens, added new indications and their recommended dosage regimens, revised safety information, updated the description of the risk of capecitabine in patients with dihydropyrimidine dehydrogenase (DPD) deficiency, and edited other sections of the USPI to conform with FDA's current labeling guidance. These supplements were reviewed and approved under Project Renewal, a public health initiative established by the FDA's Oncology Center of Excellence that aims to update the prescribing information of certain older oncology drugs to ensure information is clinically meaningful and scientifically up to date. This article summarizes the FDA approach that supported revisions to the capecitabine USPI within the context of Project Renewal.

Pharmacogenetics of DPYD and treatment-related mortality on fluoropyrimidine chemotherapy for cancer patients: a meta-analysis and trial sequential analysis

BackgroundFluoropyrimidines are chemotherapy drugs utilized to treat a variety of solid tumors. These drugs predominantly rely on the enzyme dihydropyrimidine dehydrogenase (DPD), which is encoded by the DPYD gene, for their metabolism. Genetic mutations affecting this gene can cause DPYD deficiency, disrupting pyrimidine metabolism and increasing the risk of toxicity in cancer patients treated with 5-fluorouracil. The severity and type of toxic reactions are influenced by genetic and demographic factors and, in certain instances, can result in patient mortality. Among the more than 50 identified variants of DPYD, only a subset has clinical significance, leading to the production of enzymes that are either non-functional or impaired. The study aims to examine treatment-related mortality in cancer patients undergoing fluoropyrimidine chemotherapy, comparing those with and without DPD deficiency.MethodsThe meta-analysis selected and evaluated 9685 studies from Pubmed, Cochrane, Embase and Web of Science databases. Only studies examining the main DPYD variants (DPYD*2A, DPYD p.D949V, DPYD*13 and DPYD HapB3) were included. Statistical Analysis was performed using R, version 4.2.3. Data were examined using the Mantel-Haenszel method and 95% CIs. Heterogeneity was assessed with I2 statistics.ResultsThere were 36 prospective and retrospective studies included, accounting for 16,005 patients. Most studies assessed colorectal cancer, representing 86.49% of patients. Other gastrointestinal cancers were evaluated by 11 studies, breast cancer by nine studies and head and neck cancers by five studies. Four DPYD variants were identified as predictors of severe fluoropyrimidines toxicity in literature review: DPYD*2A (rs3918290), DPYD p.D949V (rs67376798), DPYD*13 (rs55886062) and DPYD Hap23 (rs56038477). All 36 studies assessed the DPYD*2A variant, while 20 assessed DPYD p.D949V, 7 assessed DPYD*13, and 9 assessed DPYDHap23. Among the 587 patients who tested positive for at least one DPYD variant, 13 died from fluoropyrimidine toxicity. Conversely, in the non-carrier group there were 14 treatment-related deaths. Carriers of DPYD variants was found to be significantly correlated with treatment-related mortality (OR = 34.86, 95% CI 13.96–87.05; p < 0.05).ConclusionsThis study improves our comprehension of how the DPYD gene impacts cancer patients receiving fluoropyrimidine chemotherapy. Identifying mutations associated with dihydropyrimidine dehydrogenase deficiency may help predict the likelihood of serious side effects and fatalities. This knowledge can be applied to adjust medication doses before starting treatment, thus reducing the occurrence of these critical outcomes.Graphical abstract

Irinotecan plus raltitrexed as second-line treatment in locally advanced or metastatic colorectal cancer patients: a prospective open-label, single-arm, multi-center, phase II study

BackgroundColorectal cancer is the third most common cancer and the second leading cause of cancer death. There are limited therapeutic options for the treatment of locally advanced or metastatic colorectal cancers which fail first-line chemotherapy. Phase I/II studies showed that the combined application of the raltitrexed and irinotecan has significant synergistic effect and acceptable toxicity. However, most of these previous studies have relatively small sample size.MethodsThis is a prospective open-label, single-arm, multi-center, Phase II trial. Brief inclusion criteria: patients were aged 18 to 75 years with locally advanced or metastatic colorectal cancer after failure of 5-FU and oxaliplatin therapy. Enrolled patients received raltitrexed (3 mg/m2, d1) and irinotecan (180 mg/m2, d1) each 21-day cycle until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival, and the secondary endpoints were disease control rate, objective response rate, overall survival and safety.ResultsA total of 108 patients were enrolled between September 2016 and May 2020. The median age was 61 years, ECOG 1 score accounts for 67.6%, the rest were ECOG 0. A total of 502 cycles were completed, with an average of 4.6 cycles and a median of 4 cycles. 108 patients were evaluated, with an objective response rate of 17.6%, and disease control rate of 76.9%. The median follow-up time was 27 months (range:3.1–61.0 m) at data cut-off on March 2023. Median progression-free survival was 4.9 months (95% CI 4.1–5.7) and median overall survival was 13.1 months (95% CI 12.2–15.5). The most common adverse events that were elevated are alanine aminotransferase increased, aspartate aminotransferase increased, fatigue, diarrhoea, neutrocytopenia, thrombocytopenia, hypohemoglobin, and leukocytopenia. Most of the adverse events were Grade I/II, which were relieved after symptomatic treatment, and there were no treatment-related cardiotoxicities and deaths.ConclusionsThe combination of raltitrexed and irinotecan as second-line treatment for mCRC could be a reliable option after failure of standard 5-Fu-first-line chemotherapy in locally advanced or metastatic colorectal cancers, especially for patients with 5-FU intolerance (cardiac events or DPD deficiency patients).Trial registrationClinicalTrials.gov identifier: NCT03053167, registration date was 14/2/2017.

1822: Impact of DPD deficiency on outcomes for rectal cancer patients receiving concurrent chemo-radiation

1822: Impact of DPD deficiency on outcomes for rectal cancer patients receiving concurrent chemo-radiation

Strategies for DPYD testing prior to fluoropyrimidine chemotherapy in the US.

Patients with dihydropyrimidine dehydrogenase (DPD) deficiency are at high risk for severe and fatal toxicity from fluoropyrimidine (FP) chemotherapy. Pre-treatment DPYD testing is standard of care in many countries, but not the United States (US). This survey assessed pre-treatment DPYD testing approaches in the US to identify best practices for broader adoption. From August to October 2023, a 22-item QualtricsXM survey was sent to institutions and clinicians known to conduct pre-treatment DPYD testing and broadly distributed through relevant organizations and social networks. Responses were analyzed using descriptive analysis. Responses from 24 unique US sites that have implemented pre-treatment DPYD testing or have a detailed implementation plan in place were analyzed. Only 33% of sites ordered DPYD testing for all FP-treated patients; at the remaining sites, patients were tested depending on disease characteristics or clinician preference. Almost 50% of sites depend on individual clinicians to remember to order testing without the assistance of electronic alerts or workflow reminders. DPYD testing was most often conducted by commercial laboratories that tested for at least the four or five DPYD variants considered clinically actionable. Approximately 90% of sites reported receiving results within 10days of ordering. Implementing DPYD testing into routine clinical practice is feasible and requires a coordinated effort among the healthcare team. These results will be used to develop best practices for the clinical adoption of DPYD testing to prevent severe and fatal toxicity in cancer patients receiving FP chemotherapy.

Rapid determination of uracil in biological fluids at mercury thin film electrode for early detection of potential 5-fluorouracil toxicity due to dihydropyrimidine dehydrogenase deficiency

Determination of plasma uracil was reported as a method for evaluation of Dihydropyrimidine dehydrogenase (DPD) activity that is highly demanded to ensure the safe administration of 5-fluorouracil (5-FU)-based therapies to cancer patients. This work reports the development of a simple electroanalytical method based on adsorptive stripping square wave voltammetry (AdSWV) at mercury film-coated glassy carbon electrode (MF/GCE) for the highly sensitive determination of uracil in biological fluids that can be used for diagnosis of decreased DPD activity. Due to the formation of the HgII–Uracil complex at the electrode surface, the accuracy of the measurement was not affected by the complicated matrices in biological fluids including human serum, plasma, and urine. The high sensitivity of the developed method results in a low limit of detection (≈1.3 nM) in human plasma samples, falling below the practical cut-off level of 15 ng mL−1 (≈0.14 μM). This threshold concentration is crucial for predicting 5-FU toxicity, as reported in buffer, and ≤1.15% in biological samples), and accuracy (recovery percentage close to 100%).

Can we identify patients carrying targeted deleterious DPYD variants with plasma uracil and dihydrouracil? A GPCO-RNPGx retrospective analysis.

Dihydropyrimidine dehydrogenase (DPD) deficiency is the main cause of severe fluoropyrimidine-related toxicities. The best strategy for identifying DPD-deficient patients is still not defined. The EMA recommends targeted DPYD genotyping or uracilemia (U)testing. We analyzed the concordance between both approaches. This study included 19,376 consecutive French patients with pre-treatment plasma U, UH2 and targeted DPYD genotyping (*2A, *13, D949V, *7) analyzed at Eurofins Biomnis (2015-2022). Mean U was 9.9±10.1 ng/mL (median 8.7, range 1.6-856). According to French recommendations, 7.3 % of patients were partially deficient (U 16-150 ng/mL) and 0.02 % completely deficient (U≥150 ng/mL). DPYD variant frequencies were *2A: 0.83 %, *13: 0.17 %, D949V: 1.16 %, *7: 0.05 % (2 homozygous patients with U at 22 and 856 ng/mL). Variant carriers exhibited higher U (median 13.8 vs. 8.6 ng/mL), and lower UH2/U (median 7.2 vs. 11.8) and UH2/U2 (median 0.54 vs. 1.37) relative to wild-type patients (p<0.00001). Sixty-six% of variant carriers exhibited uracilemia <16 ng/mL, challenging correct identification of DPD deficiency based on U. The sensitivity (% patients with a deficient phenotype among variant carriers) of U threshold at 16 ng/mL was 34 %. The best discriminant marker for identifying variant carriers was UH2/U2. UH2/U2<0.942 (29.7 % of patients) showed enhanced sensitivity (81 %) in identifying deleterious genotypes across different variants compared to 16 ng/mL U. These results reaffirm the poor concordance between DPD phenotyping and genotyping, suggesting that both approaches may be complementary and that targeted DPYD genotyping is not sufficiently reliable to identify all patients with complete deficiency.

Tolerance and safety of adjuvant metronomic capecitabine in Indian patients with non-metastatic triple negative breast cancer (TNBC): An institutional experience.

e12530 Background: Triple-negative breast cancer (TNBC) subtype has a high risk of recurrence and overall poorer survival after standard treatment. SYSUCC-001 study presented in ASCO 2020 reported an increased disease-free survival after one year of metronomic capecitabine in operable TNBC patients after completion of local therapy. As per our clinical experience in metastatic breast cancer, many Indian patients are not able to tolerate capecitabine for longer duration. Hence, we wanted to assess the tolerance and safety of one year of daily dosing of capecitabine in our patients with Non-Metastatic TNBC. Methods: We performed a retrospective analysis of non-metastatic TNBC patients receiving adjuvant capecitabine 650 mg /m2 twice daily continuously for one year who took treatment from June 2020 to August 2022. The data extracted included patient socio-demographics, capecitabine information, side effects, and capecitabine treatment changes (dose reductions and dose interruptions). Statistical analysis was conducted using SPSS, version 24. Results: We analyzed 90 patients, out of which majority (62%) were stage II. Median age of the cohort was 48 years. Germline data was available for 64 patients, out which 8 patients were BRCA 1 positive and 8 patients had a variant of undetermined significance. Median duration of capecitabine in our patients was 230 days (IQR: 10 - 360). Twenty-six (28.8%) patients underwent dose reduction and 15 (16.6%) had reported grade 3 toxicity. Mean dose of capecitabine received was 535 mg / m2 twice daily. Treatment was interrupted due to toxicity in 20 (22%) of patients. The most common capecitabine-induced toxicity was hand foot syndrome, which was also the most common reason for dose reduction and treatment interruption. Dihydropyrimidine dehydrogenase (DPD) mutation status was known for 38 patients out of which only one (2.6%) was found to have DPD deficiency. Incidence of grade 3 toxicity was observed to be less in patients with age less than 50 years, ECOG performance status 0-1 and absence of comorbidities. Two-year disease free survival (DFS) and overall survival (OS) was estimated to be 79.3% and 80% respectively. Most common site of metastases was reported to be brain followed by lung. Conclusions: Continuous metronomic dosing of adjuvant capecitabine for 1 year is fairly well tolerated in Indian patients with non-metastatic TNBC, but may still require dose reduction in some patients due to toxicity.

527P Systematic detection of dihydropyrimidine dehydrogenase (DPD) deficiency using UH2/U ratio in patients with digestive cancers in central Tunisia: Toxicity profile

527P Systematic detection of dihydropyrimidine dehydrogenase (DPD) deficiency using UH2/U ratio in patients with digestive cancers in central Tunisia: Toxicity profile

Unveiling Discrepant and Rare Dihydropyrimidine Dehydrogenase (DPYD) Results Using an In-House Genotyping Test: A Case Series.

Fluoropyrimidine chemotherapy is a primary component of many solid tumor treatment regimens, particularly those for gastrointestinal malignancies. Approximately one-third of patients receiving fluoropyrimidine-based chemotherapies experience serious adverse effects. This risk is substantially higher in patients carrying DPYD genetic variants, which cause reduced fluoropyrimidine metabolism and inactivation (ie, dihydropyridine dehydrogenase [DPD] deficiency). Despite the known relationship between DPD deficiency and severe toxicity risk, including drug-related fatalities, pretreatment DPYD testing is not standard of care in the United States. We developed an in-house DPYD genotyping test that detects 5 clinically actionable variants associated with DPD deficiency, and genotyped 827 patients receiving fluoropyrimidines, of which 49 (6%) were identified as heterozygous carriers. We highlight 3 unique cases: (1) a patient with a false-negative result from a commercial laboratory that only tested for the c.1905 + 1G>A (*2A) variant, (2) a White patient in whom the c.557A>G variant (typically observed in people of African ancestry) was detected, and (3) a patient with the rare c.1679T>G (*13) variant. Lastly, we evaluated which DPYD variants are detected by commercial laboratories offering DPYD genotyping in the United States and found 6 of 13 (46%) did not test for all 5 variants included on our panel. We estimated that 20.4% to 81.6% of DPYD heterozygous carriers identified on our panel would have had a false-negative result if tested by 1 of these 6 laboratories. The sensitivity and negative predictive value of the diagnostic tests from these laboratories ranged from 18.4% to 79.6% and 95.1% to 98.7%, respectively. These cases underscore the importance of comprehensive DPYD genotyping to accurately identify patients with DPD deficiency who may require lower fluoropyrimidine doses to mitigate severe toxicities and hospitalizations. Clinicians should be aware of test limitations and variability in variant detection by commercial laboratories, and seek assistance by pharmacogenetic experts or available resources for test selection and result interpretation.

Lethal Capecitabine Toxicity in Patients With Complete Dihydropyrimidine Dehydrogenase Deficiency Due to Ultra-Rare DPYD Variants.

A DPD deficiency should be considered in case of severe toxicity even in the absence of common risk variants in DPYD.

Design and Implementation of an Opt-Out, End-to-End, Preemptive DPYD Testing Program for Patients Planned for a Systemic Fluoropyrimidine.

Several allelic variants of the gene DPYD encoding dihydropyrimidine dehydrogenase (DPD) are associated with impaired metabolism of the systemic fluoropyrimidine fluorouracil (5FU) and its oral prodrug, capecitabine, which elevates the risk for severe toxicity. Following a patient death related to capecitabine toxicity in which DPD deficiency was suspected, a multidisciplinary advisory panel was convened to develop an institution-wide approach to future patients planned for a systemic fluoropyrimidine. The panel selected an opt-out testing strategy which focused on developing reliable processes to collect and report test results and targeted education. An electronic health record-based automated reminder was designed to activate when a 5FU- or capecitabine-containing chemotherapy regimen was ordered for a patient without prior exposure to either agent and without a prior DPYD sequencing test result. DPYD testing was standardized across all sites of care, and a closed loop reporting system for abnormal test results was created. Before implementation, targeted education was provided to providers, pharmacists, and nurses, and a failure mode and effects analysis was performed. Program rollout was staged over a 6-month period. At 10 months, the rate of preemptive testing increased from a baseline of 26% to a sustained rate of >90%. In the six network sites, the testing rate increased from 9% to 96%. A total of 1,043 patients have been tested preemptively; allelic variants have been identified in 43 (4.1%). Among 25 evaluable patients, dose reduction or change to a non-fluoropyrimidine-based regimen was accomplished in 96%. Preemptive DPYD testing is feasible, and high rates of testing can be achieved using an opt-out, reminder-based program. We provide the details of the implementation and encourage others to emulate it.

Assessing the Hepatotoxic Effects of Fluoropyrimidine Chemotherapy in Male Iraqi Colorectal Cancer Patients.

Colorectal cancer (CRC) is one the most frequently occurring cancer types among various populations. Fluoropyrimidine is the backbone of first-line chemotherapy, the oral capecitabine, or intravenous 5-fluorouracil (5-FU) in various combinations and schedules the chemotherapy regime in the treatment of a wide variety of gastrointestinal cancers. The enzyme dihydropyrimidine dehydrogenase (DPD) functions as the rate-limiting step in the metabolism of fluoropyrimidine chemotherapies, and patients with complete or partial DPD deficiency are at increased risk of severe and fatal toxicity during treatment with fluorouracil. This study aimed to examine thechemotoxicity of the 5-FU drug on hepatocytes in male Iraqi CRC patients. This research is a cross-sectional study conducted between November 2022 and April 2023. The study included 80 male participants who had undergone surgical intervention for stage III CRC under the care of the Misan Health Directorate, Misan Center for Tumors Treatment, located in Misan, Iraq. Based on their subsequent surgical treatment, the participants were divided into two groups. The first group, comprising 45 males aged between 41 and 71 years, experienced a relapse despite receiving adjuvant therapy, which involved a singular cycle of fluoropyrimidine-based chemotherapy (5-FU). The second group consisted of 35 male patients with CRC, aged between 40 and 57 years, who did not experience a relapse post-adjuvant therapy. Their adjuvant therapy involved a single round of fluoropyrimidine-based chemotherapy with 5-FU. Relapse in patients was determined by assessing the white blood cell count(WBC). Liver enzymes were significantly increased after 5-FU treatment, while the concentration of albumin was significantly decreased. The findings of our study clearly indicate that 5-FU induced hepatic injury, lowering the hepatocytefunction with elevated levels of hepatic enzymes and low concentration of albumin in the blood, which is an important predictive marker of chemotherapy toxicity.

A novel large intragenic DPYD deletion causing dihydropyrimidine dehydrogenase deficiency: a case report

BackgroundDihydropyrimidine dehydrogenase (DPD), is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines. Deleterious variants in the DPYD gene cause DPD deficiency, a rare autosomal recessive disorder. The clinical spectrum of affected individuals is wide ranging from asymptomatic to severely affected patients presenting with intellectual disability, motor retardation, developmental delay and seizures. DPD is also important as the main enzyme in the catabolism of 5-fluorouracil (5-FU) which is extensively used as a chemotherapeutic agent. Even in the absence of clinical symptoms, individuals with either complete or partial DPD deficiency face a high risk of severe and even fatal fluoropyrimidine-associated toxicity. The identification of causative genetic variants in DPYD is therefore gaining increasing attention due to their potential use as predictive markers of fluoropyrimidine toxicity.MethodsA male infant patient displaying biochemical features of DPD deficiency was investigated by clinical exome sequencing. Bioinformatics tools were used for data analysis and results were confirmed by MLPA and Sanger sequencing.ResultsA novel intragenic deletion of 71.2 kb in the DPYD gene was identified in homozygosity. The deletion, DPYD(NM_000110.4):c.850 + 23455_1128 + 8811del, eliminates exons 9 and 10 and may have resulted from a non-homologous end-joining event, as suggested by in silico analysis.ConclusionsThe study expands the spectrum of DPYD variants associated with DPD deficiency. Furthermore, it raises the concern that patients at risk for fluoropyrimidine toxicity due to DPYD deletions could be missed during pre-treatment genetic testing for the currently recommended single nucleotide polymorphisms.

Complete DPYD genotyping combined with dihydropyrimidine dehydrogenase phenotyping to prevent fluoropyrimidine toxicity: A retrospective study.

In April 2019, French authorities mandated dihydropyrimidine dehydrogenase (DPD) screening, specifically testing uracilemia, to mitigate the risk of toxicity associated with fluoropyrimidine-based chemotherapy. However, this subject is still of debate as there is no consensus on a standardized DPD deficiency screening test. We conducted a real-life retrospective study with the aim of assessing the impact of DPD screening on the occurrence of severe toxicity and exploring the potential benefits of complete genotyping using next-generation sequencing. All adult patients consecutively treated with 5-fluorouracil (5-FU) or its oral prodrug at six cancer centers between March 2018 and February 2019 were considered for inclusion. Dihydropyrimidine dehydrogenase deficiency screening included gene encoding DPD (DPYD) genotyping using complete genome sequencing and DPD phenotyping (uracilemia or dihydrouracilemia/uracilemia ratio) or both tests. Associations between each DPD screening method and (i) severe (grade ≥3) early toxicity and (ii) fluoropyrimidine dose reduction in the second chemotherapy cycle were evaluated using multivariable logistic regression analysis. Furthermore, we assessed the concordance between DPD genotype and phenotype using Cohen's kappa. A total of 551 patients were included. Most patients were tested for DPD deficiency (86%) including DPYD genotyping only (6%), DPD phenotyping only (8%), or both (72%). Complete DPD deficiency was not detected in the study population. Severe early toxicity events were observed in 73 patients (13%), with two patients (0.30%) presenting grade 5 toxicity. Despite the numerically higher toxicity rate in untested patients, the occurrence of severe toxicity was not significantly associated with the DPD screening method (p = 0.69). Concordance between the DPD genotype and phenotype was weak (Cohen's kappa of 0.14). Due to insufficient numbers, our study was not able to demonstrate any added value of DPYD genotyping using complete genome sequencing to prevent 5-FU toxicity. The optimal strategy for DPD screening before fluoropyrimidine-based chemotherapy requires further clinical evaluation.

Association of plasma uracil concentration with 5-FU pharmacokinetics, dose-limiting toxicity, and DPYD genotype in patients with gastrointestinal cancer.

751 Background: Plasma uracil concentration &gt;16 ng/mL is used in some countries as a screening test for DPD deficiency. We conducted this exploratory study to evaluate the association of uracil concentration with 5-FU exposure, toxicity, and DPYD genotype in patients without known DPD deficiency. The main hypothesis was that lower uracil concentration would be predictive of subtherapeutic 5-FU drug exposure (5-FU AUC &lt; 20 mg*h/L) in patients treated with infusional 5-FU. Methods: The study prospectively enrolled patients with GI cancer who were receiving initial chemotherapy with a regimen containing a 46-hour 5-FU infusion initially dosed at 2400 mg/m2. Study candidates were screened for the DPYD *2A, *13 and c.2846A&gt;T gene variants; variant carriers were excluded. Participants gave pretreatment fasting morning blood samples for determination of plasma uracil concentration and DPYD whole-gene sequencing. Plasma samples were collected at hour 45 of 5-FU infusion in chemotherapy cycles 1 and 2 for determination of 5-FU exposure, measured as the 5-FU AUC. We estimated a linear mixed-effects model to test the association of uracil concentration with 5-FU AUC, adjusting for cycle, sex, creatinine, and 5-FU infusion dose (in mg/m2). Results: There were 29 evaluable participants with a median age of 64 years (range 41-81); nine (31%) were female. The most common cancer sites were colorectal (n = 15) and pancreatic (10). The median (IQR) plasma uracil concentration was 10.4 ng/mL (7.2, 12.6). Data for 5-FU AUC were available for 19 subjects in cycle 1 (median = 22.0 mg*h/L) and 27 subjects in cycle 2 (median = 19.3 mg*h/L). In the mixed-effects model, male sex was significantly associated with 5-FU AUC (beta = -12.0 [95% CI -17.2, -6.7], p &lt;0.001) but plasma uracil concentration was not (beta = 0.35 [-0.16, 0.86], p=0.16). Holding other covariates constant, male sex was associated with a 29% decrease in 5-FU AUC (vs female) and plasma uracil concentration in the lowest quartile was associated with a 12% decrease in 5-FU AUC (vs highest quartile). Three of 29 participants (10%, all male) had uracil concentrations of &gt;16 ng/mL, suggestive of DPD deficiency. The subject with the highest uracil concentration (23.1 ng/mL) had severe toxicity in cycle 1 (neutropenia, mucositis) requiring 5-FU dose reduction; whole gene sequencing identified a rare DPYD missense variant in this subject (c.2185G&gt;A [p.A729T]). The other two subjects with uracil concentration &gt;16 ng/mL did not have culprit DPYD gene variants and tolerated infusional 5-FU at 2400 mg/m2 without early dose-limiting toxicity. Conclusions: Our findings suggest that male sex is more important than low pretreatment plasma uracil concentration as a predictor of risk for subtherapeutic 5-FU drug exposure. This result is consistent with prior evidence that men experience less 5-FU associated toxicity than women.

Dihydropyrimidine dehydrogenase gene variants for predicting grade 4-5 fluoropyrimidine-induced toxicity: FUSAFE individual patient data meta-analysis.

Dihydropyrimidine dehydrogenase (DPD) deficiency is the main known cause of life-threatening fluoropyrimidine (FP)-induced toxicities. We conducted a meta-analysis on individual patient data to assess the contribution of deleterious DPYD variants *2A/D949V/*13/HapB3 (recommended by EMA) and clinical factors, for predicting G4-5 toxicity. Study eligibility criteria included recruitment of Caucasian patients without DPD-based FP-dose adjustment. Main endpoint was 12-week haematological or digestive G4-5 toxicity. The value of DPYD variants *2A/p.D949V/*13 merged, HapB3, and MIR27A rs895819 was evaluated using multivariable logistic models (AUC). Among 25 eligible studies, complete clinical variables and primary endpoint were available in 15 studies (8733 patients). Twelve-week G4-5 toxicity prevalence was 7.3% (641 events). The clinical model included age, sex, body mass index, schedule of FP-administration, concomitant anticancer drugs. Adding *2A/p.D949V/*13 variants (at least one allele, prevalence 2.2%, OR 9.5 [95%CI 6.7-13.5]) significantly improved the model (p < 0.0001). The addition of HapB3 (prevalence 4.0%, 98.6% heterozygous), in spite of significant association with toxicity (OR 1.8 [95%CI 1.2-2.7]), did not improve the model. MIR27A rs895819 was not associated with toxicity, irrespective of DPYD variants. FUSAFE meta-analysis highlights the major relevance of DPYD *2A/p.D949V/*13 combined with clinical variables to identify patients at risk of very severe FP-related toxicity.

Pharmacogenetic Variants Can Influence Optical Medication Use.

Single Nucleotide Polymorphisms (SNPs) are used as drug susceptibility biomarkers in metabolic diseases. Alterations in the gene encoding triggers the enzyme flavin monooxygenase 3 (FMO3), involved in the Sulindac metabolization, which also is responsible for the inherited metabolic disorder. Trimethylaminuria (TMAu, OMIM: 602079). DPYD gene variants are associated with the enzyme dihydropyrimidine dehydrogenase deficiency (DPD; OMIM: 274270). This autosomal recessive metabolic disorder, ultimately leads to the inability to metabolize fluoropyrimidines, which causes severe toxicity in individuals treated with these drugs. Variants in genes responsible for the expression of enzymes that encode transporters or receptors involved in the metabolization pathways of certain drugs may condition the individuals response to certain drugs, compromising the therapeutic response and clinical prognosis. Thus the sequencing and identification of variants become relevant, not only gain knowledge on effects of these variants' on disease causality but also in terms of its side effects resulting from the coding enzymes responsible for drug metabolization. It was found that patients with the c.472G>A (p.Glu158Lys) and c.923A>G (p.Glu308Gly) polymorphisms, in homozygosity, in FMO3 gene did not develop polyps, thus have a protective effect in the treatment of Familial Adenomatous Polyposis (PAF). However, in the case of the DPYD gene, c.1905+1G>A (IVS14+1G>A), c.1679T>G (p.Ile560Ser), c.2846A>T (p.Asp949Val) e c.1236G>A/HapB3 variants can be lethal in cancer patients indicated for fluoropyrimidine-based chemotherapy. Knowledge on the drug mechanisms will affect the therapeutic response of patients treated with a given drug. Thus, pharmacogenetics is an essential tool in personalized medicine, since molecular studies allows the clinician to predict the probability of efficacy and toxicity of certain drugs, resulting higher efficiency in individualizing treatment and also improving the safety of the patient. From a personalized medicine perspective, the study of the characteristics of the drug and its metabolization site, the genes involved in the encoding of enzymes responsible for its metabolization will be of great interest.

Risk of Toxicity From Topical 5-Fluorouracil Treatment in Patients Carrying DPYD Variant Alleles.

Patients carrying DPYD variant alleles have increased risk of severe toxicity from systemic fluoropyrimidine chemotherapy. There is a paucity of data regarding risk of toxicity from topical 5-fluorouracil (5-FU) treatment in these patients, leading to inconsistent guideline recommendations for pretreatment testing and topical 5-FU dosing. The objective of this retrospective cohort study was to investigate whether DPYD variant allele carriers have increased risk of toxicity from topical 5-FU. Treatment and toxicity data were retrospectively abstracted from the electronic medical records. Genotypes for the five DPYD variants that are associated with increased toxicity from systemic fluoropyrimidine chemotherapy (DPYD*2A, DPYD*13, DPYD p.D949V, DPYD HapB3, and DPYD p.Y186C) were collected from a genetic data repository. Incidence of grade 3+ (primary end point) and 1+ (secondary end point) toxicity was compared between DPYD variant carriers vs. wild-type patients using Fisher's exact tests. The analysis included 201 patients, 7% (14/201) of whom carried a single DPYD variant allele. No patients carried two variant alleles or experienced grade 3+ toxicity. DPYD variant allele carriers did not have a significantly higher risk of grade 1+ toxicity (21.4% vs. 10.2%, odds ratio = 2.40, 95% confidence interval: 0.10-2.53, P = 0.19). Given the low toxicity risk in patients carrying a single DPYD variant allele, there is limited potential clinical benefit of DPYD genetic testing prior to topical 5-FU. However, the risk of severe toxicity in patients with complete DPD deficiency remains unknown and topical 5-FU treatment should be avoided in these patients.

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

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