While massive parallel sequencing technology is quite mature, a problematic aspect is that plasma tumor markers are measured relative to total DNA, and total DNA levels vary with biologic factors like inflammation and with pre-analytic interferences such as leukocyte lysis during blood collection and handling ex vivo.
This research aims to develop a novel strategy to quantify tumor markers in ?copies per mL of plasma?, thus harmonizing the massive parallel sequencing assay with gold standard values generated by quantitative PCR. First, we will spike plasma with synthetic DNAs (called ?EndoGenus Spikes?) which are then targeted for enrichment during library preparation and are quantified using informatic scripts after massive parallel sequencing. By normalizing levels of each tumor marker to the fractional recovery of spiked DNAs, numerical values are reportable in units of ?copies per mL of plasma?, which we will show reflect clonal abundance. This new capability for absolute quantification of clonal abundance is likely to benefit basic scientists studying tumor heterogeneity and clonal evolution, and is likely to benefit patients and healthcare providers who seek more informative ways to monitor tumor burden, to evaluate the impact of medical interventions, and to find emerging drug resistance clones so that alternate therapy may be considered in a timely fashion. At the conclusion of this study, we will have developed and validated the ?EndoGenus Toolkit?, comprised of synthetic DNAs, reagents to enrich for them during library preparation, and bioinformatic scripts to convert tumor marker levels from fractions to absolute concentrations. We will show that applying the ?Toolkit? to mock plasma specimens yields sensitive, specific, linear and reproducible sequencing results for multiple tumor markers. In blood from active cancer subjects, we will show that the ?Toolkit? helps overcome pre-analytic problems associated with blood storage. These tools should facilitate future clinical trials aimed at setting numeric thresholds for changing patient management.
A novel technology called the ?EndoGenus Toolkit? permits absolute quantification of multiple DNA tumor markers in plasma using a method that overcomes certain biologic and technical interferences that hamper current massive parallel sequencing technology. The ?Toolkit? relies on co-amplification of synthetic DNAs spiked into plasma before library preparation, and informatic scripts to normalize read counts of each tumor marker that is detected after DNA sequencing. Importantly, tumor marker levels are expressed in ?copies per mL of plasma? to facilitate harmonization with units of measurement in quantitative PCR.
