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.

Purpose Triple negative breast cancers (TNBC) frequently have high epidermal growth factor receptor (EGFR) expression and are sensitive to DNA-damaging agents. Improved therapies are needed for this aggressive malignancy. Patients and methods We performed a phase I trial of bendamustine and erlotinib, an EGFR tyrosine kinase inhibitor, in patients with metastatic TNBC, ECOG performance status ≤2, and ≤1 prior chemotherapy for metastatic disease. Each 28-day cycle included intravenous bendamustine on days 1, 2 and oral erlotinib on days 5–21 with dose escalation according to a 3 + 3 phase I study design. Dose-limiting toxicity (DLT) was determined by toxicities related to study therapy observed during cycle 1. Results Eleven patients were treated, 5 on dose level 1 and 6 on dose level 2. One patient had DLT on dose level 2. However, cumulative toxicities were observed, including grade 3/4 lymphopenia in 91 % (95 % CI 0.59–0.998) with progressively decreased CD4 counts and grade ≥3 infections in 36 % (95 % CI 0.11–0.69) of patients. Conclusions Combination therapy with bendamustine and erlotinib causes excessive toxicity with severe, prolonged lymphopenia, depressed CD4 counts, and opportunistic infections and should not be pursued further. Future trials of bendamustine combinations in TNBC patients should account for potential cumulative lymphocyte toxicity necessitating patient monitoring during and after treatment.

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