Development and Validation of an NGS Assay for the Detection Of Clinically Actionable Genetic Variants in DPYD and UGT1A1

ACMG Annual Clinical Genetics Meeting 2023 PRESENTATION
Authors Robert Huether, Kyung Choi, Ian Zavitz, Yan Yang, Christopher Vlangos, Rachel Star, Francisco M. De La Vega

Introduction: Dihydropyrimidine dehydrogenase (DPYD) and UDP-glucuronosyltransferase isoform 1A1 (UGT1A1) are enzymes that metabolize many clinically important chemotherapeutic drugs including 5-fluorouracil (5-FU), irinotecan, sacituzumab, and belinostat, which are used in treatment of colorectal, breast, pancreatic, lung or lymphoma cancers.  The DPYD gene encodes for the rate limiting enzyme in the metabolism of cytotoxic 5-FU. UGT1A1 catalyzes glucuronylation of SN-38, the active compound of irinotecan and sacituzumab govitecan. Genetic polymorphism of DPYD and UGT1A1 associated with loss or decreased function (poor metabolizer phenotype) elevates the plasma concentration of 5-FU or SN-38 and increases the risk of severe adverse events in the patients receiving the chemotherapeutic drugs. Identification of the patients carrying the poor metabolizer variants enables clinicians to optimize the drug therapy based on the patient’s genotype.  DPYD or UGT1A1 genotypes are commonly determined via dedicated tests using Sanger sequencing, real time PCR, or short tandem repeat analysis.  Matched tumor/normal genomic profiling by Next Generation Sequencing (NGS) for cancer therapy decision support has increased in adoption, which provides an ideal opportunity to identify the germline genetic variants associated with chemotherapy-induced adverse events. Here, we report the validation of an NGS assay which includes a novel repeat polymorphism calling algorithm for the detection of DPYD and UGT1A1 genetic variants from Tempus xT, a NGS paired tumor/normal 648-gene assay for cancer therapy decision support.

Methods: The method identifies 7 common and 3 underserved population variants in DPYD or UGT1A1: DPYD*2a, DPYD*13, DPYDHapB3, DPYDc.557A>G, DPYDc.2846A>T, UGT1A1*6, UGT1A1*27, UGT1A1*28, UGT1A1*36, and UGT1A1*37. For the variant calling of the UGT1A1 repeat diplotypes, we implemented a novel calling algorithm that is resilient to stutter created by DNA polymerase in repeats. The calls of the TA repeat polymorphisms were orthogonally confirmed by a CLIA/CAP lab using a fragment analysis by capillary electrophoresis method and the SNVs were orthogonally confirmed using Sanger sequencing.  

Results: Analytical validation was performed using DNA from 50 cell lines from the GetRM cell line repository and 21 patient samples orthogonally confirmed by another NGS assay and sanger sequencing. In total 128 clinical samples (98 positive, 30 reference controls) were evaluated and showed a concordance rate of 99.4% (198/199 samples).  The single discordant result was a GetRM cell line sample with a homozygous call at the UGT1A1*28 locus.   The NGS call was heterozygous at this site and was confirmed by short tandem repeat assay.  The discordance is potentially due to limitations in the array platform used to initially genotype a complex region in the GetRM sample. 

Conclusions: In conclusion, our method allows for clinical DPYD and UGT1A1 genotyping from NGS data collected for tumor profiling, enabling clinicians to consider potential adverse drug reactions (loss or decrease function of DPYD/UGT1A1) simultaneously with therapy selection for cancer patients.