UBC Theses and Dissertations

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UBC Theses and Dissertations

Novel ultra-sensitive digital PCR assays for screening and detection of rare missense mutations in (proto)-oncogenes Bidshahri, Arezoo (Roza)


Somatic mutations can lead to cancer, often by altering the activity of kinases within signaling pathways that control cell growth and proliferation. Targeted cancer therapeutics are designed and used to regulate these aberrant signaling pathways in cases where somatic mutations within kinase genes predict a positive patient response to those treatments. For example, the V600E mutation in BRAF, the gene coding for the BRAF serine threonine kinase, predicts the effectiveness of vemurafenib in treating metastatic melanoma, while the mutational status of codons G12/G13 in the KRAS gene predicts likely colorectal cancer patient response to the monoclonal antibody (mAb) cetuximab.¹-³ However, FDA approved assays currently used to detect missense mutations in BRAF V600 and KRAS G12/G13 are not capable of detecting clinically actionable mutations at mutational frequencies low enough to permit their robust application to early disease detection or minimal residual disease monitoring. Moreover, detection of all clinically actionable missense mutations is not certain or generally achieved, in part due to limitations to assay specificities and the inability to unequivocally discriminate missense mutations from synonymous germline sequence variations. This thesis addresses that limitation through the development and validation of a novel platform for creating highly sensitive assays against all possible missense mutations in an oncogenic hotspot codon or adjacent set of hotspot codons that ameliorates the known limitations to current FDA-approved assays. The platform is designed to enable development of assays against all possible missense mutations in oncogenic hotspots and, if required, unequivocally differentiate them from synonymous germline alleles. It utilizes droplet digital PCR (ddPCR) technology and chimeric wild-type specific LNA/DNA probes to create a novel “WT-negative” screening paradigm. The platform is applied to the creation of two new assays of potential clinical use in cancer diagnostics and theranostics. The first provides a reliable and sensitive screening and detection of all known clinically actionable mutations in BRAF V600, and the second achieves the same for KRAS G12/G13. Both assays show complete diagnostic accuracy when applied to formalin-fixed paraffin-embedded (FFPE) tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.

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