Abstract 1549: Molecular profiling of circulating tumor cells as a surrogate for distant metastasis in stage IV breast cancer
Authors: Ring Alexander , Forte Victoria , Barrak Dany , Porras Tania , Punj Vasu , Carrasco Steven , Yu Min , Tripathy Debu , Lang Julie 
Published: July 2016

Background: Metastasis is responsible for virtually all breast cancer related deaths. While circulating tumor cells (CTCs) have been shown to be prognostic in metastatic breast cancer (MBC), their use as a biomarker to date has been limited. While initial treatment recommendations are based on primary tumor biology, ASCO guidelines call for biopsy of a metastatic site to guide decision making for systemic therapy. The ANGLE Parsortix system is a microfluidics device that separates CTCs based on size and deformability, without the need for cell-surface marker selection. We hypothesize that the ANGLE Parsortix system permits isolation and gene expression profiling of pure CTCs and allows comparison of the biopsied metastatic site, thereby acting as a surrogate for macrometastases.

Methods: We are currently enrolling metastatic breast cancer patients to a prospective, observational clinical study in which CTCs are enumerated and captured from 10-20 mL peripheral blood (PB) via the ANGLE Parsortix system. CTCs, peripheral blood (PB), and metastatic sites were profiled with RNA Seq via the Illumina HiSeq (primary predictor). We received fresh frozen tissue biopsies from the following metastatic sites: skin, brain, pleural effusion, pericardial effusion, breast, cerebrospinal fluid and bone tissue. Bioinformatics analysis was then performed. NanoString PAM50 and real-time polymerase chain reaction will be used as validation studies.

Results: To date we have successfully isolated and molecularly profiled CTCs and metastatic tissue (MT) from 9/9 stage IV breast cancer patients. Final bioinformatics analysis on the last five patients is underway. Principal component analysis demonstrated clustering of MT and CTCs, with clear separation from PB. These results demonstrated high purity of CTCs, eliminating the need for subtraction of PB background signal, and demonstrated common gene expression between CTCs and MT. Differential gene expression analysis (FDR p&lt;0.05) identified a 214 gene-expression signature that statistically significantly correlated (R&gt;0.98) between CTCs and MT. Gene Set Enrichment Analysis (GSEA) analysis of 214-gene signature identified biological pathways related to metastasis in CTCs and MT. Individual intra-patient analysis confirmed gene-expression correlation between CTCs and MT, but not PB.

Conclusion and outlook: Cell surface marker independent CTC isolation is feasible for RNA Seq analysis without background subtraction. Furthermore, RNA Seq of CTCs can identify gene expression signatures that correlate with distant macrometastatic sites. The gene-expression patterns revealed biological relevant information that could be used as biomarkers or identify potential therapeutic targets.

Citation Format: Alexander Ring, Victoria Forte, Dany Barrak, Tania Porras, Vasu Punj, Steven Carrasco, Min Yu, Debu Tripathy, Julie Lang. Molecular profiling of circulating tumor cells as a surrogate for distant metastasis in stage IV breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1549.