Dissection of major cancer gene variants in subsets of circulating tumor cells in advanced breast cancer

Abstract

Enumeration of circulating tumor cells (CTCs) may reflect the metastatic potential of breast cancer (BC). By using the DEPArray, we investigated CTCs with respect to their epithelial-to-mesenchymal transition phenotype and compared their genomic heterogeneity with tissue biopsies. Seventeen stage IV BC patients were enrolled. Pre-enriched CTC suspensions were stained with fluorescent-labeled antibodies to epithelial (E) and mesenchymal (M) markers. CTC samples were processed by DEPArray system and clustered in relation to their markers. DNA from CTCs, as well as from primary tumor samples, was sequenced by next generation sequencing to assess the mutational state of 50 major cancer-related genes. We identified four different CTC subsets that harbored different gene variants. The most heterogenous CTC subsets included the M+/E- phenotype, which, however, expressed only 7 repeatedly mutated genes, while in the M-/E+ subset multiple mutations affected only 2 out of 50 genes. When matching all gene variants among CTC subsets, a small number of mutations was shared by only 4 genes, namely ATM, FGFR3, PIK3CA, and TP53 that, however, were absent in primary tumors. Our results postulate that the detected mutations in all CTC subsets may be considered as genomic markers of metastatic dissemination to be investigated during early stages of BC.

Figure 1

Representative images of CTCs isolated by DEPArray. The upper panel shows spiking experiments performed by using MDA-MB231 and MCF-7 BC cell lines predominantly expressing mesenchymal (M: N-cadherin/CD146/CD44; PE, red) and epithelial markers (E: E-cadherin/EPCAM; FITC, green), respectively. The lower part of the figure shows four different CTC sub-populations from the same blood sample (patient #242), showing variable expression of M and/or E markers as well as negative blood and endothelial markers (CD45/CD31/CD34; APC, purple). Nuclei are stained by Hoechst 33342 (blue). The first line shows a CTC expressing only M markers (M+/E−). Line 2 represents a CTC lacking both E and M markers (M−/E−). The APC fluorescence detectable near M−/E− CTC was due to non-nucleated blood components, namely erythrocytes and platelets that in several instances bind CTCs. Line 3 shows a CTC expressing both M and E markers (M+/E+), whereas line four depicts a CTC with E phenotype (M−/E+). Two lymphocytes derived from the CD45+ fraction of the same blood sample, are also shown as control for blood cell markers (purple), with or without CD44 expression (red).

Figure 2

Distribution of CTC subsets. Magnitude of EMT-related CTCs from metastatic BC patients, grouped as A (treatment-naïve) or B (pre-treated). The upper section depicts percent values of these cohorts with significant differences regarding both M+/E− and M−/E− subsets. The lower part shows the extent of all CTC subsets in each patient depicting intra- and inter-patient heterogeneity, even within the same group of patients. The numbers in brackets are the recovered CTCs from each patient (range: 7–106). Abbreviations: CTCs: circulating tumor cells; M+/E−: CTCs expressing only M markers; M−/E−: CTCs negative for both M and E markers; M+/E+: CTCs expressing both M and E markers; M−/E+: CTCs expressing only E markers.

Figure 3

Percent of pathogenic variants recurring in single genes in EMT-related CTC subsets. Genes showing a unique variant were included in ‘other genes’ box. By contrast, the recurrence of multiple variants in single genes is expressed as percent values. As shown, a restricted number of genes, namely 7 in M+/E− and only 2 in M−/E+ subset, out of 50 genes of the Cancer Hotspot panel, harbored repeated mutations by NGS. The most mutated genes in all CTC subsets included PIK3CA, TP53, FGFR3, and ATM, whereas PTEN also expressed multiple variants although only in the M+/E− CTCs to further support high genomic heterogeneity in this subset. Numbers refer to variants and sequenced CTCs. Abbreviations: CTCs: circulating tumor cells; M+/E−: CTCs expressing mesenchymal markers only; M−/E−: CTCs which do not express either mesenchymal or epithelial markers; M+/E+: CTCs expressing both mesenchymal and epithelial markers; M−/E+: CTCs expressing epithelial markers only; #: number.

Figure 4

Venn diagram depicting both shared and exclusive pathogenic variants in CTC subsets. Numbers of the variants shared between the subsets are differently colored while those of the exclusive mutations are highlighted in black (left). Shared variants involved only 4 genes (ATM, FGFR3, TP53, and PIK3CA), whereas the subset-specific mutations were at higher number of genes in each CTC subpopulation (right).