Molecular markers are essential for cancer diagnosis, clinical trial enrollment, and some surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform due to time-consuming DNA extraction, targeted amplification, and library preparation times. In this work, we present a proof-of-principle approach for sub-1 hour targeted variant detection using real-time DNA sequencers. By modifying existing protocols, optimizing for diagnostic time-to-result, we demonstrate confirmation of a hot-spot mutation from tumor tissue in ~52 minutes. To further reduce time, we explore rapid, targeted Loop-mediated Isothermal Amplification (LAMP) and design a bioinformatics tool—LAMPrey—to process sequenced LAMP product. LAMPrey’s concatemer aware alignment algorithm is designed to maximize recovery of diagnostically relevant information leading to a more rapid detection versus standard read alignment approaches. Using LAMPrey, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes.

Molecular markers are becoming increasingly important for cancer diagnosis, proper clinical trial enrollment, and even surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform. In this work, we present Threshold Sequencing, a methodology for designing protocols for targeted variant detection on real-time sequencers with a minimal time to result. Threshold Sequencing analytically identifies a time-optimal threshold to stop target amplification and begin sequencing. To further reduce diagnostic time, we explore targeted Loop-mediated Isothermal Amplification (LAMP) and design a LAMP-specific bioinformatics tool--LAMPrey--to process sequenced LAMP product. LAMPrey's concatemer aware alignment algorithm is designed to maximize recovery of diagnostically relevant information leading to a more rapid detection versus standard read alignment approaches. Coupled with time-optimized DNA extraction and library preparation, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes.

3575 Background: Patients (pts) treated with checkpoint inhibitors (CPI) may uncommonly experience accelerated progression in their tumor burden when compared to their rate of progression prior to receiving CPI. This hyperprogression has been varyingly defined and no biomarker has yet been identified. Methods: We reviewed the database from the Tumor Response Assessment Core (TRAC) at University of Michigan to identify these patients. Hyperprogression was defined as increase in tumor burden per specific immune RECIST criteria by at least 40% from baseline on the first follow-up scan with a minimum increase of 10 mm, and at least 2 times rate of growth than observed prior to start of CPI therapy. Results: Out of 741 pts who underwent baseline and 1st follow-up assessment enrolled on 118 trials, 302 (34.4%) pts received immunotherapy alone or in combination with chemotherapy/targeted agents across 49 trials. Of them, 15 pts (5%) with 5 females (33%) and median age of 63 years (range, 44 -72) met criteria for hyperprogression. The primary cancers included lung (5), colorectal (2), renal (2), biliary (1), pancreatic (1), esophageal (1), bladder (1), small bowel (1), and melanoma (1). The median time to hyperprogression was 67 (range 42-110) days, and the mean survival was 7.9 months from trial enrollment. We did not identify any clinical factor or specific CPI therapy that associated with hyperprogression. Exploratory biomarker analysis of genomic (gene panel assay) and immune subsets of tissue microenvironment (multiplex staining) is underway. Conclusions: This is the largest cohort investigated for hyperprogression across multiple cancers in literature. The rate of hyperprogression observed is less than previously reported in literature, and physicians need to be aware of this possibility while administering CPI to their patients.