Gene expression analysis to detect disseminated tumor cells in the bone marrow of triple-negative breast cancer patients predicts metastatic relapse.

PURPOSE: Disseminated tumor cells (DTCs) in the BM of breast cancer patients predict early disease relapse, but the molecular heterogeneity of these cells is less well characterized. Expression of a 46-gene panel was used to detect DTCs and classify patient BM samples to determine whether a composite set of biomarkers could better predict metastatic relapse. METHODS: Using a high-throughput qRT-PCR assay platform, BM specimens collected from 70 breast cancer patients prior to neoadjuvant therapy were analyzed for the expression of 46 gene transcripts. Gene expression was scored positive (detectable) relative to a reference pool of 16 healthy female control BM specimens. To validate findings from a subset of 28 triple-negative breast cancer (TNBC) patients in the initial 70 patient cohort, an independent set of pre-therapeutic BM specimens from 16 TNBC patients was analyzed. RESULTS: Expression of each of the 46 gene transcripts was highly variable between patients. Individual gene expression was detected in 0-84% of BM specimens analyzed and all but two patient BM specimens expressed at least one transcript. Among a subset of 28 patients with TNBC, positivity of one or more of eight transcripts correlated with time to distant relapse (p = 0.03). In an independent set of 16 triple-negative patient BM samples, detection of five of these same eight gene transcripts also correlated with time to distant relapse (p = 0.03) with a positive predictive value of 89%. CONCLUSIONS: We identified a set of gene transcripts whose detection in the BM of TNBC patients, prior to any treatment intervention, predicts time to first distant relapse, thus identifying a TNBC patient population which requires additional treatment intervention. Because these genes are presumably expressed in populations of DTCs and many encode proteins that are known therapeutic targets (e.g., ERBB2), these results also suggest a potential approach for targeted DTC therapy to mitigate distant metastases in TNBC.

A microfluidic-based filtration system to enrich for bone marrow disseminated tumor cells from breast cancer patients.

Disseminated tumors cells (DTCs) present in the bone marrow (BM) are believed to be the progenitors of distant metastatic spread, a major cause of mortality in breast cancer patients. To better understand the behavior and therapeutic vulnerabilities of these rare cell populations, unbiased methods for selective cell enrichment are required. In this study, we have evaluated a microfluidic-based filtration system (ParsortixR, Angle PLC), previously demonstrated for use in circulating tumor cell (CTC) capture, to capture BM DTCs. Performance using BM samples was also compared directly to enrichment of CTCs in the peripheral blood (PB) from both metastatic and non-metastatic breast cancer patients. Although the non-specific capture of BM immune cells was significant, the device could routinely achieve significant cytoreduction of BM and PB WBCs and at least 1,000-fold enrichment of DTCs, based on labeled tumor cell spike-in experiments. Detection of previously characterized DTC-associated gene expression biomarkers was greatly enhanced by the enrichment method, as demonstrated by droplet digital PCR assay. Cells eluted from the device were viable and suitable for single cell RNA sequencing experiments. DTCs in enriched BM samples comprised up to 5% of the total cell population, allowing for effective single cell and population-based transcriptional profiling of these rare cells. Use of the Parsortix instrument will be an effective approach to enrich for rare BM DTCs in order to better understand their diverse molecular phenotypes and develop approaches to eradicate these cells to prevent distant disease development in breast cancer patients.