Abstract A061: Tumor mutation burden (TMB), microsatellite instability (MSI), and chromosomal instability (CIN) analysis using low pass whole genome sequencing of single circulating tumor cell (CTC)
Authors: Rodriguez Angel , Lee Jerry , Sutton Ramsay , Jiles Rhett , Wang Yipeng , Landers Mark , Dittamore Ryan 
Published: August 2018

Background: Genomic instability (GI) is a hallmark of cancer often associated with poor patient outcome. TMB, MSI, and CIN represent the majority of GI in metastatic patients. Recent studies show that TMB and MSI are emerging immune checkpoint inhibitor drug sensitivity biomarkers and CIN is a sensitivity marker for PARP inhibitors. Assessment of genomic instability in bulk tumor samples is well explored, but it is limited by sample availability and tumor heterogeneity. Analysis of ctDNA is feasible for TMB and MSI analysis but not CIN, and it also suffers in sensitivity and specificity in patients who harbor subclonal GI, limiting the clinical utility of these assays to detect early clonal alterations. The Epic Sciences CTC platform is a non-enrichment-based approach for the detection and characterization of rare tumor cells in clinical blood samples, and could provide insight into subclonal heterogeneity. Here we present downstream single cell GI assay(s) for the detection of TMB, MSI and CIN from individual CTCs using next-generation sequencing and PCR. Methods: Contrived samples were prepared by spiking three prostate cancer cell lines, LNCaP, PC3, and VCaP, into healthy donor blood. Red blood cells were lysed, all nucleated cells deposited onto slides, slides immunofluorescence stained (DAPI, CK, CD45, and Androgen Receptor), and identified cancer cells individually picked up from the slides. Each recovered cell was lysed, whole genome amplified (WGA), shotgun library prepared, and low pass whole genome sequenced using Illumina NextSeq 500. Data were analyzed for TMB scores (# of INDELs per Mbp) and large-scale transitions (LSTs, a surrogate of CIN). MSI was measured using Qiagen Type-It microsatellite PCR kit for four sites (BAT26, BAT25, D2S123, and D5S346). Samples from metastatic castration resistant prostate cancer (mCRPC) patients were included to evaluate clinical feasibility. Results: TMB scores for LNCaP (average 652, coefficient of variation 15%) were significantly higher than PC3 (558, 0.9%), VCaP (548, 1.1%), and WBC from healthy donor (540, 7.6%) with p&lt;0.01. MSI assay confirmed that LNCaP is the only MSI-H cell with insertions/deletions found in 3 (BAT26, BAT25, and D5S346) of 4 microsatellite sites. No MMR deficiency was found in PC3 and VCaP. LST analysis shows PC3 (average 33) and VCaP (33) have much higher LST scores than LNCaP (11). A wide range of TMB (375-861) and LST scores (0-70) are observed inter- and intra-mCRPC patient samples. Conclusions: The data shown here demonstrate the feasibility of detecting three types of genomic instabilities at the single-cell level using the Epic Sciences CTC Platform. Inter- and intra-patient heterogeneity is observed in the small patient cohort. Additionally, the results confirm that MSI and HRD are likely mutually exclusive driver events driving tumor selection in mCRPC. Further studies are ongoing to investigate the correlation of GI markers with PARPi and I/O checkpoint inhibitor responses.

Citation Format: Angel Rodriguez, Jerry Lee, Ramsay Sutton, Rhett Jiles, Yipeng Wang, Mark Landers, Ryan Dittamore. Tumor mutation burden (TMB), microsatellite instability (MSI), and chromosomal instability (CIN) analysis using low pass whole genome sequencing of single circulating tumor cell (CTC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A061.