Sensitive detection of minimal residual disease: methods and application to patients treated for early-stage breast cancer

Parsons HA, Rhoades J, Reed S, Gydush G, Ram P, Exman P, Xiong K, Lo CC, Li T, Fleharty M, Kirkner G, Rotem D, Cohen O, Yu F, Fitarelli-Kiehl M, Leong KW, Hughes ME, Rosenberg SM, Collins LC, Miller KD, Blumenstiel B, Trippa L, Cibulskis C, Neuberg DS, DeFelice M, Freeman SS, Lennon NJ, Wagle N, Ha G, Stover DG, Choudhury AD, Getz G, Winer EP, Meyerson M, Lin NU, Krop I, Love JC, Makrigiorgos GM, Patridge AH, Mayer EL, Golub TR, Adalsteinsson V.
Clinical Cancer Research 26, 2556-2564 (2020).


Purpose: Existing cell-free DNA (cfDNA) methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. We developed a test for tracking hundreds of patient-specific mutations to detect MRD with a 1000-fold lower error rate than conventional sequencing. Experimental Design: We compared the sensitivity of our approach to digital droplet PCR (ddPCR) in a dilution series, then retrospectively identified two cohorts of patients who had undergone prospective plasma sampling and clinical data collection: 16 patients with ER+/HER2- metastatic breast cancer (MBC) sampled within six months following metastatic diagnosis and 142 patients with stage 0-III breast cancer who received curative-intent treatment with most sampled at surgery and one year post-op. We performed whole-exome sequencing of tumors and designed individualized MRD tests, which we applied to serial cfDNA samples.

Results: Our approach was 100-fold more sensitive than ddPCR when tracking 488 mutations, but most patients had fewer identifiable tumor mutations to track in cfDNA (median 57, range 2-346). Clinical sensitivity was 81% (n=13/16) in newly diagnosed MBC, 23% (n=7/30) at post-op and 19% (n=6/32) at one year in early-stage disease, and highest in patients with the most tumor mutations available to track. MRD detection at one year was strongly associated with distant recurrence (HR=20.8 [95%CI: 7.3-58.9]). Median lead time from first positive sample to recurrence was 18.9 months (range: 3.4-39.2 months).

Conclusions: Tracking large numbers of individualized tumor mutations in cfDNA can improve MRD detection, but its sensitivity is driven by the number of tumor mutations available to track.