Abstract P2-01-04: The whole transcriptional landscape of circulating tumor cells compared to metastases in stage IV breast cancer
Authors: Ring A , Porras T , Campo D , Kaur P , Forte VA , Tripathy D , Lu J , Zada G , Wagle N , Wecsler JS , Lang JE 
Published: February 2018

Background: Metastatic breast cancer (MBC) and the circulating cells (CTCs) leading to macrometastasis are inherently different than primary breast cancer, evolving under the selection pressure of systemic therapy. A better understanding of the tumor biology of CTCs compared to metastasis may shed light on treatment opportunities.

Methods: We performed whole transcriptome sequencing (RNA Seq) on fresh metastatic tumor biopsies (mets), CTCs, and peripheral blood (PB) from 21 newly diagnosed MBC patients. CTCs were harvested using the ANGLE Parsortix to isolate cells based on size and deformability. Data were analyzed for differential expression, pathways, single nucleotide variants (SNV), fusions, intrinsic subtype, and a CTC-mets shared gene signature was validated using data from The Cancer Genome Atlas (TCGA). Detailed clinical-pathological and treatment data was evaluated.

Results: CTCs as a group showed much stronger gene expression of oncogenes, stem cell genes, keratins and mesenchymal markers than did mets from the same patients. Matched patient comparisons for 66 potentially clinically actionable genes for 8/9 pathways showed no significant difference in gene expression targets between CTCs and mets on ANOVA, although fold-change did vary. Eight SNVs in the ESR1 gene (n=5 patients) and 5 SNVs in the HER2 gene (n=2 patients) were shared between CTCs and distant metastases.

Differential gene expression analysis identified a signature of 8870 genes that were statistically significantly correlated between CTCs and mets (FDR adjusted p&lt;0.05). Ingenuity pathway analysis was applied to the list of genes shared between CTCs and mets, with analysis of canonical pathways and upstream regulators revealing numerous oncogenes and breast cancer related genes. The top upstream regulators of CTCs-mets were beta-estradiol, progesterone, FOXA1, HNRPA2B1 and HNF1A. The top 50 genes of this CTC-mets shared signature were prognostic of worse overall survival in the TCGA breast cancer dataset (p&lt;0.001), which included 817 patients with a median follow-up of 59.5 months. Second time-point data for n=5 patients with subsequent PB draws 6 months after baseline is currently pending. Intrinsic subtyping of mets by either NanoString assays or RNA Seq were not concordant with intrinsic subtyping of CTCs by RNA Seq.

Four of 21 CTC samples showed strong whole transcriptome RPKM correlation with PB (R2)&gt;0.9, however, 3/21 CTC samples showed strong whole transcriptome RPKM correlation with mets (R2)&gt;0.8. The remainder showed low correlation with both. Coverage was 91.4X for CTCs, 140.2X for mets and 138.5X for PB.

Conclusions: We present the transcriptomic landscape of CTCs with comparison to metastases and peripheral blood all acquired prior to treatment of newly diagnosed Stage IV breast cancer. Multiple genes, including oncogenes and stem cell genes, were found with higher expression in CTCs versus metastases. When focusing on 66 known potentially clinically actionable genes in breast cancer, CTCs did not show significantly different patterns of expression than mets in terms of up-regulation versus down-regulation compared to PB. RNA Seq of CTCs may be utilized to identify molecular alterations that are potentially clinically actionable.

Citation Format: Ring A, Porras T, Campo D, Kaur P, Forte VA, Tripathy D, Lu J, Zada G, Wagle N, Wecsler JS, Lang JE. The whole transcriptional landscape of circulating tumor cells compared to metastases in stage IV breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-04.