Abstract 3616: Low-pass whole-genome sequencing of single circulating tumor cells for detection of tumor mutation burden and chromosomal instability across multiple cancer types
Authors: Rodriguez Angel , Lee Jerry , Sutton Ramsay , Jiles Rhett , Vansant Gordon , Wang Yipeng , Landers Mark , Dittamore Ryan 
Published: August 2018

Background: Recent studies show tumor mutation burden (TMB) is a biomarker for patient responses to immune checkpoint inhibitors whereas chromosomal instability (CIN) is a marker for PARP inhibitors drug response. Assessment of TMB and CIN in bulk tumor samples is well explored but limited by sample accessibility and tumor heterogeneity. Analysis of ctDNA is challenging for TMB and CIN, especially for patients who harbor subclonal genomic alterations, limiting its clinical utility. Epic Sciences' CTC platform employs a non-enrichment based approach to provide insight into subclonal heterogeneity. Here we present a single cell genomics assay for detection of TMB and CIN from individual CTCs by low pass whole genome sequencing.

Methods: Contrived samples were prepared by spiking prostate cancer cell lines LNCaP, PC3 and VCaP into healthy donor blood. Red blood cells were lysed, nucleated cells deposited onto glass slides and immunofluorescently stained (DAPI, CK, CD45 and androgen receptor). Identified cancer cells were individually isolated from the slides, lysed, whole-genome amplified (WGA), shotgun library prepared, and low pass whole genome sequenced to ~ 0.1X coverage. Data were analyzed for TMB and large-scale transitions (LST, a surrogate of CIN). Microsatellite instability (MSI) was measured by the Qiagen Type-It Microsatellite PCR kit as per manufacturer's protocol on the control cell lines. 1,047 CTCs from 108 prostate, breast, colorectal, bladder and lung cancer patients were evaluated for clinical feasibility.

Results: TMB scores from LNCaP (average 181) were significantly higher than PC3 (127), VCaP (132), and healthy donor WBC (94). MSI confirmed LNCaP was MSI-H (high) with INDELs detected in 3 of 4 microsatellite sites, whereas PC3 was MSI-L (low) and VCaP was MSS (stable). Although with lower MSI, PC3 (average 33) and VCaP (33) had higher LST scores vs. LNCaP (11). A wide range of TMB (21-480) and LST scores (0-67) was observed in patient samples. TMB cutoff of 175 was set based on cell line TMB scores, and an LST cutoff of 10 was set based on WBC data. Across CTCs from all cancer types and stages, 117 (11.2%) CTCs were TMB-H/LST-L (H: high; L: low), 479 (45.7%) CTCs were TMB-L/LST-H, 31 (3.0%) CTCs were TMB-H/LST-H, and 420 (40.1%) CTCs were TMB-L/LST-L. A significant lower incidence of TMB-H/LST-H CTCs (3.0%) was observed by Fisher's exact test (p&lt;0.0001).

Conclusions: These data demonstrate the feasibility of detecting TMB and CIN simultaneously at the single cell level using the Epic Sciences CTC Platform. Inter- and intrapatient heterogeneity was observed in this large patient cohort. Although MSI and HRD are were mutually exclusive driver events in single cells, patients often had both subclonal populations. Studies are ongoing to investigate the potential correlations of TMB with checkpoint inhibitor and CIN with PARPi response.

Citation Format: Angel Rodriguez, Jerry Lee, Ramsay Sutton, Rhett Jiles, Gordon Vansant, Yipeng Wang, Mark Landers, Ryan Dittamore. Low-pass whole-genome sequencing of single circulating tumor cells for detection of tumor mutation burden and chromosomal instability across multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3616.