Background: While detection of circulating tumor DNA (ctDNA) is associated with poor cancer prognosis, the clinical utility for guiding treatment decisions is unresolved. Patients with minimal residual disease (MRD) often have less than one genome equivalent of ctDNA per 10 mL blood. Consequently, it is stochastic whether a 10 mL sample contains ctDNA from a particular genomic locus. Consequently, the sensitivity of ctDNA detection methods targeting a limited number of tumor loci is heavily affected by sampling bias. To overcome this challenge, we developed MRDetect; a whole genome sequencing (WGS) approach, which detects ctDNA using the patient-specific cumulative signal from tens of thousands of mutations throughout the genome. Recently, we showed how MRDetect found ctDNA fractions down to 10-4. Here, we performed a validation study to confirm the prognostic impact of MRDetect.
Aim: Validation of MRDetect for sensitive ctDNA detection to monitor residual disease in stage III colorectal cancer (CRC) patients treated with curative intent.
Methods: From a large, uniform cohort of stage III CRC patients n = 146), we had plasma samples collected every third month (n = 938, median = 9 per patient) and a median follow-up of 34 months. For each patient, a genome-wide mutational signature was established by WGS of tumor and matched normal DNA. Enhanced by an AI-based error suppression model, this signature was used to detect ctDNA in 1-2 mL plasma samples using WGS (20x coverage). We used de-novo point mutation and copy number variation analysis to investigate cancer evolution after treatment. To evaluate the reproducibility of MRDetect, aliquot samples (n = 2x190 samples) from 5 recurrence and 10 non-recurrence patients were processed and sequenced at two independent laboratories. Outcome measures: ctDNA status, tumor fraction, false positive rate, Time To ctDNA Recurrence (TTcR), and Time To radiological Recurrence (TTrR).
Results: Analysis of paired samples showed great reproducibility with high agreement between both ctDNA status calls (Cohens Kappa = 0.81) and the estimated tumor fractions (r2 = 0.99). MRDetect revealed post-operative ctDNA in all recurrence patients (5/5) with detected tumor fractions down to 2 x 10-4. Median TTcR was 0.9 month (range 0.5 - 7.3 months) while median TTrR was 12.8 months (range 11.3 - 31.1 months). The false positive rate was 1% (1/100), assessed in longitudinal samples from the 10 non-relapsing patients. Tumor evolution dynamics in plasma samples revealed novel amplification and deletions, which were absent in the primary tissue but confirmed in metachronous metastases. We will present results from the full cohort at AACR 2022.
Conclusion: MRDetect detects ctDNA with high sensitivity and specificity and enables effective postoperative assessment of MRD, cancer evolution dynamics and early relapse detection.
Citation Format: Amanda Frydendahl, Thomas Reinert, Jesper Nors, Sunil Deochand, Dillon Maloney, Noah Friedman, Tomer Lauterman, Danielle Afterman, Imane Bourzgui, Nidhi Ramaraj, Zohar Donenhirsh, Ronel Veksler, Ravi Kandasamy, Iman Tavassoly, Jonathan Rosenfeld, Anders Husted Andersen, Uffe S. Løve, Per V. Andersen, Ole Thorlacius-Ussing, Lene Hjerrild Iversen, Kåre Andersson Gotschalck, Boris Oklander, Asaf Zviran, Claus Lindbjerg Andersen. Sensitive detection of circulating tumor DNA by whole genome sequencing: Validation of MRDetect using serial blood samples from stage III colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1959.