Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients

Abstract

IntroductionCirculating tumor cells (CTCs) are commonly isolated from the blood by targeting the epithelial cell adhesion molecule (EpCAM) through positive selection. However, EpCAM can be downregulated during metastatic progression, or it can be initially not present. We designed the present prospective trial to characterize CTCs as well as other circulating cell populations in blood samples from women with metastatic breast cancer without EpCAM-dependent enrichment and/or isolation technology.MethodsA total of 32 patients with metastatic breast cancer were enrolled, and blood samples were processed using a previously described negative depletion immunomagnetic methodology. Samples from healthy volunteers were run as controls (n = 5). Multistep sequential labeling was performed to label and fix cell-surface markers followed by permeabilization for cytokeratins (CK) 8, 18 and 19. Multiparametric flow cytometry (FCM) analysis was conducted using a BD LSR II flow cytometer or a BD FACSAria II or FACSAria III cell sorter. Immunocytochemical staining on postenrichment specimens for DAPI, EpCAM, CD45, CK, epidermal growth factor receptor and vimentin was performed. Expression of these markers was visualized using confocal microscopy (CM).ResultsCD45-negative/CK-positive (CD45− CK+) populations with EpCAM + and EpCAM − expression were identified with both FCM and CM from the negatively enriched patient samples. In addition, EpCAM + and EpCAM − populations that were CK + and coexpressing the pan-hematopoietic marker CD45 were also noted. There were more CK + EpCAM − events/ml than CK + EpCAM + events/ml in both the CD45− and CD45+ fractions (both statistically significant at P ≤ 0.0005). The number of CK + CD45− and CK + CD45+ events per milliliter in blood samples (regardless of EpCAM status) was higher in patient samples than in normal control samples (P ≤ 0.0005 and P ≤ 0.026, respectively). Further, a significant fraction of the CK + CD45+ events also expressed CD68, a marker associated with tumor-associated macrophages. Higher levels of CD45-CK + EpCAM − were associated with worse overall survival (P = 0.0292).ConclusionsMetastatic breast cancer patients have atypical cells that are CK + EpCAM − circulating in their blood. Because a substantial number of these patients do not have EpCAM + CTCs, additional studies are needed to evaluate the role of EpCAM − circulating cells as a prognostic and predictive marker.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells

“…In all, 84% of the samples (16 of 19) were positive for panCK(ϩ)/AR-V7(Ϫ) CTCs, 68% of samples (13 of 19) were positive for panCK(ϩ)/AR-V7(ϩ) CTCs, and 32% of samples (6 of 19) were positive for panCK(Ϫ)/AR-V7(ϩ) CTCs. In rare cases, single cells were detected showing staining for both, panCK and CD45, similar to the results of others (30 ). These cells were not counted as CTCs.…”

Pooled Analysis of the Prognostic Relevance of Circulating Tumor Cells in Primary Breast Cancer

“…Therefore, EpCAM-based CTC detection methods such as the CellSearch system might miss circulating cancer cells that are particularly likely to initiate distant organ metastasis (34,35). EpCAM-negative CTCs have been detected in the blood of patients with different subtypes of MBC patients (36), but it has been argued that the problem of detecting only EpCAM-positive CTCs and missing EpCAM-negative circulating cancer cells might apply particularly to some basal-like tumors, as these are characterized by low or even nonexistent EpCAM expression (37). However, EpCAM-positive CTCs are commonly found also in triple-negative (basal-like) breast cancer, and the presence of EpCAM-positive CTCs as detected using the CellSearch system has been identified as a prognostic marker for disease-free and overall survival in patients with both early (this study) and metastatic triple-negative breast cancer (2,6,38).…”

Circulating tumor cell technologies

“…These studies support the need for additional molecular characterization of circulating epithelial cells. Multiple groups have also reported the occurrence of a CKþ/CD45þ or 'double positive' cell phenotype in the bloodstream of metastatic cancer patients (Yoon et al, 2013;Lustberg et al, 2014;Riethdorf et al, 2007;Sheng et al, 2014;Stott et al, 2010). Both the identity and the significance of these obfuscating cells have received much debate with no resolution.…”