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Introduction: Circulating 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., Methods: A 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)., Results: CD45-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)., Conclusions: Metastatic 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.

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.

The term gray zone lymphoma has been applied to tumors that demonstrate transitional morphologic and immunophenotypic features between classical Hodgkin's lymphoma and diffuse large B-cell lymphoma, especially primary mediastinal large B-cell lymphoma. Histopathological and genetic data are limited for these unusual cases. We analyzed cases of gray zone lymphoma (n=27), mediastinal composite lymphoma (n=3) and mediastinal synchronous/metachronous lymphoma (n=3) by morphology, immunophenotyping and fluorescence in situ hybridization. Mediastinal involvement was assured in 24/33 patients (73%). The patient cohort showed a male predominance (M:F ratio; 20:13) and a median age of 32 years (range, 16-91 years). Patients with mediastinal disease were significantly younger (median age: 29.5 years) than patients presenting without evident mediastinal disease (median age: 55 years). Gains including amplifications in 2p16.1 (REL/BCL11A locus) were observed in 33% of all patients, whereas alterations affecting the JAK2/PDL2 locus in 9p24.1 were present in 55%. Further studies revealed rearrangement of the CIITA locus at 16p13.13 in 8/30 cases (27%) and 7/26 cases (27%) demonstrated gains of 8q24 (MYC). Genetic aberrations involving 2p16.1, 9p24.1 and 8q24 showed a higher incidence in cases with evident mediastinal involvement. However, this was not statistically significant when compared with cases without known mediastinal involvement. Twelve of the 27 cases of gray zone lymphoma were morphologically more reminiscent of classical Hodgkin's lymphoma, whereas the other gray zone lymphomas presented with morphological features more closely resembling large B-cell lymphoma. Both morphological groups of gray zone lymphoma were similarly positive for Cyclin E (75 and 93%) and p63 (50 and 53%, respectively) expression. These findings further support a close relationship between gray zone lymphoma, classical Hodgkin's lymphoma and primary mediastinal large B-cell lymphoma, and suggest that some cases of gray zone lymphoma without mediastinal disease may share similar genetic alterations.