Detection of Bcl-2 and apoptosis in circulating tumor cells during treatment of metastatic breast cancer

Abstract

11016 Background: Induction of apoptosis is a common effect of many new and existing drugs, and Bcl-2 expression is associated with resistance to apoptosis. CTCs are a minimally invasive blood test and are a source of tumor cells that can be obtained serially during therapy. Monitoring Bcl-2, apoptosis, and other markers in CTCs may be useful endpoints in trials of new and targeted agents. The goal of this pilot study is to estimate Bcl-2 expression and apoptosis in CTCs from pts being treated for metastatic breast cancer (MBC). Methods: Whole blood was collected from pts initiating therapy for MBC at baseline, post-treatment (24, 48, or 72 hours), and 3–4 weeks. Samples were split into 3 × 7.5ml fractions. CTCs were isolated and enumerated using the CellTracks and CellSpotter systems. CTCs were defined as DAPI+, cytokeratin+, CD45-, with a cellular morphology. Apoptosis defined by positive staining with MAb M30. Bcl-2 status defined by staining with MAb Bcl-2–100. Results: 39/81 patients (49%) had ≥5 CTCs at baseline. 25% had ≥5 CTCs at 3–4 weeks. 35% of pts with CTCs had positive Bcl-2 staining in 91–100% of their CTCs (highest decile). Other pts were equally distributed across the remaining Bcl-2 deciles. At baseline, apoptosis correlates with CTC number. In samples with 1–4 CTCs, 80% of cells were apoptotic. For 5–49 CTCs, 48% were apoptotic; for 50–99, 34% were apoptotic; for 100–500, 28% were apoptotic; and for >500, 22% were apoptotic. Bcl-2 staining was inversely correlated with apoptosis staining. Pts were risk stratified by changes in CTC number after one cycle of therapy, and apoptosis increased with improving prognostic groups. One pt had markedly elevated CTCs of >20,000. The cells were remarkably 100% Bcl-2 positive and M30 negative (1% staining). Conclusions: These data confirm the prevalence of CTCs seen in the pivotal trial of CTCs in MBC (Cristofanilli NEJM 2004) and provide novel observations for Bcl-2 expression and apoptosis in CTCs. Higher Bcl-2 expression appears to correlate with decreased apoptosis. High fractions of apoptotic cells correlate with lower numbers of CTCs, which is associated with a better prognosis. Analysis of the post-treatment (24, 48, or 72 hour) samples and correlation of markers with progression free survival are ongoing and will be presented. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Immunicon Immunicon Immunicon Immunicon