Co-expression of MET and CD47 is a novel prognosticator for survival of luminal breast cancer patients

Abstract

Although luminal-type primary breast cancer can be efficiently treated, development of metastatic disease remains a significant clinical problem. We have previously shown that luminal-type circulating tumor cells (CTCs) co-expressing the tyrosine-kinase MET and CD47, a ligand involved in cancer cell evasion from macrophage scavenging, are able to initiate metastasis in xenografts. Here, we investigated the clinical relevance of MET-CD47 co-expression in 255 hormone receptor positive breast tumors by immunohistochemistry and found a 10.3- year mean overall-survival difference between MET-CD47 double-positive and double-negative patients (p<0.001) MET-CD47 co-expression defined a novel independent prognosticator for overall-survival by multivariate analysis (Cox proportional hazards model: HR: 4.1, p<0.002) and CD47 expression alone or in combination with MET was strongly associated with lymph node metastasis. Furthermore, flow cytometric analysis of metastatic patient blood revealed consistent presence of MET+CD47+ CTCs (range 0.8 - 33.3% of CTCs) and their frequency was associated with increased metastatic spread. Finally, primary uncultured CTCs with high MET+CD47+ content showed an enhanced capacity to initiate metastasis in mice. Detection and targeting of MET and CD47 may thus provide a rational basis for risk stratification and treatment of patients with luminal-type breast cancer.

Figure 1. Analysis of MET and CD47 expression in luminal-type breast cancer by tissue microarray analysis

(A) Flow-chart of the tissue microarray study. (B) Examples of MET (top) or CD47 (bottom) staining of luminal-type breast tumors by immunohistochemistry. (C) Expression of CD47 (left) or MET (right) on serial sections of a hormone receptor positive primary breast tumor. Arrows show examples of double-positive tumor cells. (D-F) Kaplan-Meier survival analyses of hormone receptor positive breast cancer patients based on (E) MET expression (F), CD47 expression and (G) CD47-MET co- expression. Log-rank tests were used to probe for significance. Abbreviations: HR: hazard ratio; CI: confidence interval.

Figure 2. Association between MET⁺CD47⁺ CTCs and metastatic spread

Association between flow cytometry-determined (A) MET⁺CD47⁺ CTCs (n = 8, p = 0.03, unpaired T-test)) or (B) bulk CTCs (n = 8, p > 0.99, unpaired T-test) and number of metastatic sites in patients. Low and high groups of patients were defined according to the median (13 for MET⁺CD47⁺ CTCs and 196 for bulk CTCs, see supplementary Table 2). Each dot represents data for one patient. CTCs are defined here as PI^-CD45^-EPCAM⁺ cells by flow cytometry. Data for MET⁺CD47⁺ CTCs and bulk CTCs are calculated based on datasets reported in [41]. Abbreviations: CTC: circulating tumor cell.

Figure 3. Analysis of CTC-induced bone metastasis by immunohistopathology

(A-H) Expression of human Ki67, CK7, ER, PR, HER2, MET and CD47 in bone metastasis induced by CTCs derived from patient 8 (see Table 5), as inidicated. Abbreviations: CK7: cytokeratin 7; ER: estrogen receptor; PR: progesterone receptor.