Custom assays for personalized, longitudinal monitoring of circulating tumor DNA using PIPSenSeq

PROJECT SUMMARYABSTRACT Targeted DNA sequencing of cell free tumor DNAcirculating tumor DNActDNAshed into a patient s blood holds great promise for detectiondiagnosisand monitoring of cancerGiven that all tumor cells have the potential to shed ctDNAtargeted assays using modern next generation sequencing methods and digital PCR can provide insight into the entirety of a patient s tumor burdenctDNA is therefore an attractive biomarker for diagnosis and monitoring of cancer patients via non invasive peripheral blood drawSuch assayshoweverrequire ultra sensitive sequencing fidelityflexible target multiplexingand uniform target amplification for efficientquantitativeand cost effective testingIn additionseveral challenges arise in the implementation of assays to identify and monitor tumor specific mutations extracted from bloodFirstthe mutant DNA from cancer cells is rare compared to the background of normal DNArequiring detection sensitivities belowfor some clinical applicationsSecondcell free DNA extracted from plasma is limited in massandltngml sampleand typically degraded to very short fragment lengthsrequiring efficient capture and amplification of the targets of interestThirdcancer diagnosis and monitoring may require surveillance of a multitude of tumor or patient specific mutationsrequiring multiplex amplification for efficient sample utilizationIn collaboration with our academic partners at Boston University and the University of California San Franciscowe have developed a novel next generation sequencing sample preparation platform that addresses these critical challengesPIPSenSeqPre templated Instant Partitions for Sensitive SequencingThis approach takes advantage of molecular indexing for consensus read sequencing in combination with single molecule amplification in Poisson distributed nanoscale partitionsFurthermorePIPSenSeq provides a simplerapid library preparation that does not require complexexpensive instrumentation or microfluidic consumablesIn this proposal we will develop PIPSenSeq as a commercial ready platform for cancer monitoringand demonstrate clinical utility in a study ofhead and neck squamous cell carcinoma patientsThese patients will be monitored post operatively for tumor recurrence using personalized PIPSenSeq panels on longitudinally collected ctDNA samples PROJECT NARRATIVE Circulating tumor DNActDNAobtained via a blood drawis increasingly seen as a clinically valuable biomarker for cancer detection and diagnosisThe objective of this proposal is to develop and demonstrate the clinical utility of a new technology for personalized cancer detection and longitudinal monitoringThis proposal combines the core innovation of our companypre templated instant partitionswith a validated multiplex PCR assay from our collaborator to create a novel technology that overcomes the critical barriers to flexiblemultiplexed PCR based ctDNA tests for cancer monitoring