Pharmacogenetic assays for personalized cancer treatment

Pharmacogenomics is increasingly utilized in various areas of medicine for personalization of drug selection and dosing. Nevertheless, the cost of large pharmacogenomic panels may be prohibitive for some practices due to reimbursement and utility limitations. Smaller, cancer treatment-specific pharmacogenetic panels that are currently available as services provide a limited number of gene targets and do not include important HLA alleles associated in published studies with adverse effects for specific cancer drugs (note that this work does not establish diagnostic capability). We evaluated the feasibility of developing a targeted pharmacogenetic test for cancer related drugs, which includes HLA along with other critical gene targets not captured by commonly available panels. The number of individual qPCR reactions in a given reaction mix is known as the plex level. Standard genotyping assays are designed as 2-plex mixtures, which are provided as an array of multiple reactions per sample present on a single qPCR plate. Increasing the qPCR plex level reduces the number of reactions required per sample, potentially reducing operational cost, reducing operator error rates and improving turn-around time for pharmacogenetic testing. We evaluated the feasibility of implementing maximal multiplexing in our targeted pharmacogenetic panel. We have designed 50 individual qPCR assays and optimized multiplex mixes into fewer than 8 individual reactions. Our assay targets include single nucleotide polymorphisms (SNPs) in HLA-A, HLA-B, HLA-DRB1, HLA-DQA1, UGT1A1, DPYD and CYP2C9. Based on multiple publications, certain polymorphisms in these genes are associated with increased risk of adverse effects for specific cancer drugs. Our new data analysis method allows derivation of genotyping results from up to 9-plex qPCR reactions performed on a standard qPCR instrument. Assays were tested using a combination of synthetic DNAs and human DNA samples, from Terasaki collection of well-characterized DNA samples and the Coriell repository. Our multiplex reactions included assays for detection of HLA and non-HLA alleles in single reactions using a combination of standard qPCR dyes (i.e; ABY, JUN, etc.) as well as FRET and long-stoke shift dyes. Future development of such pharmacogenetic panels may contribute to further advancement in precision medicine.