Development of a novel liquid biopsy test to assist targeted MSI-H cancer treatment

Abstract The goal of this SBIR project is to develop an innovative SA PCPE PCR technology that can simultaneously genotype multiple mutated mononucleotide STRs in a vast background of wild type DNAwhich will be employed to develop an assay for liquid biopsy of high frequency microsatellite instabilityMSI HcancerThe hallmark of MSI H is extensive instability in short tandem repeatSTRwhich alters the repeat numberExamination of five mononucleotide STRs is currently recommended for the MSI classificationTargeted cancer treatment is emerging as an effective approach to cancer care because it allows doctors to select the right treatment for the right patient at the right time based on a genetic understanding of tumorBecause MSI H has different profiles than microsatellite stableMSStumorstheir treatment differs from each otherRecentlyFDA approved the first two drugs that are tailored to MSI H treatmentFor exampleFDA granted an accelerated approval of Keytruda to a treatment for patients with MSI HUntil thenFDA approved cancer treatments based on where in the body the cancer started for examplelung or breast cancersKeytruda is the first approved drug based on a tumorandapos s biomarkerMSI Hwithout regard to the tumorandapos s original locationLiquid biopsy is a method enabling doctors to discover a range of information about a tumor through a blood sampleand detecting mutations in cell free DNAcfDNAis emerging as the method of choice of liquid biopsyTargeted treatment is one of its most important application areasBecause of its values to targeted cancer treatmentvarious liquid biopsy assays as the accompany test for targeted treatment have been or are being developedFor exampleFDA has approved liquid biopsy tests for targeted EGFR treatmentClearlyliquid biopsy will be equally valuable to targeted MSI H treatmentHoweverno liquid biopsy test is available for targeted MSI H cancer treatment because of the lack of technologies that can detect the biomarker of MSI H in a sensitivespecificand cost effective mannerHereinwe propose a novel method termed SA PCPE PCR to address this unmet needThis method solves two of the fundamental problems plaguing the detection of mutated STRs in cfDNAFirstit enriches multiple mutated STRs simultaneously prior to PCRThis solves the problem of PCR slippagesrendering it possible to genotype mutated STRs in a vast background of wild type DNA using fragment analysisSecondit manipulates fragment size of amplicons by introducing size tagscreating a sufficient difference in length of the ampliconsThis solves the size constraint problems imposed by cfDNAmaking it possible to genotype multiple STRs based on the unique size of each ampliconA SA PCPE PCR assay consists of three stepsFirsta DNA sample is subjected to SA PCPEwhich preferentially produces amplifiablelongextension products from mutant allelesenriching mutants prior to PCRwhile introducing size tags to these extension productsSecondmultiplexed PCR is performed with extension products as templatesbut only long extension products are amplifiedFinallysize adjusted amplicons are fragment analyzed by a conventional DNA sequencer to identify mutated STRsIn this studywe will first develop a SA PCPE PCR assay to detect five mutated mononucleotide STRs simultaneously in a large background of wild type DNAThenwe will assess the detection limit and reproducibility of the assayFinallywe will assess the clinical detection sensitivity and specificity of the assayClearlysuccess of this project can provide a liquid biopsy assay that may transform the landscape of personalized MSI H cancer medicine Narrative The goal of this SBIR project is to develop an innovative technology and the associated assay for liquid biopsy of MSI H cancer