Scientists use two different methods to visualize rare CTCs, double positive cells in mBC

New research from The Ohio State University utilizes two different methods to visualize circulating tumor cells (CTCs) as well as other unusual circulating cells with both epithelial and hematopoietic characteristics in metastatic breast cancer (mBC). Results of the data were presented during a poster session at the American Association for Cancer Research Annual Meeting 2012 in Chicago, Ill.

The research, supported in part by pilot funding from the Ohio State Center for Clinical and Translational Science (CCTS), was conducted by a team of scientists with The Ohio State University Comprehensive Cancer Center - Arthur G. James Cancer Hospital and Richard J. Solove Research Institute using flow cytometry and immunocytochemistry to analyze enriched blood samples from patients with mBC.

"By utilizing a negative depletion process we were able to identify rare CTCs that are often overlooked by current methods of study," said Maryam B. Lustberg, MD, MPH, study investigator and assistant professor of internal medicine at The Ohio State University College of Medicine. "While more information is needed to understand the clinical significance of these cells, with further research it's possible these atypical cells could influence prognoses and treatments in the future."

A CTC is currently defined as a nucleated cell that is positive for epithelial cell adhesion activating molecule (EpCAM), is cytokeratin positive and is CD45 negative. These cells are identified by a trained specialist using fluorescently stained slides also known as EpCAM enrichment technology. During this process only EpCAM positive CTCs are identifiable.

"Some of the most aggressive forms of cancer, including triple negative breast cancer (TNBC), downregulate EpCAM and have high numbers of EpCAM negative CTCs, cells that would be missed by the most commonly utilized technologies," said Lustberg. "The results of this study support the theory that there is a heterogeneous population of circulating cells in the blood of cancer patients which may hold clues regarding the metastatic process."

During the study, researchers also identified abnormal double positive cells, a little understood type of cell which has only been found in cancer patients. The double positive cells tested positive for both markers CD45 and cytokeratin, implying the cells have both cancer and normal blood characteristics, a combination that was previously thought impossible.

While the double positive cells have been observed anecdotally in past studies, researchers dismissed them as artifacts of the testing process. This new research suggests the characteristics of these double positive cells may have more meaning than previously thought.

"The presence of these double positive cells points to an unusual mutation taking place in the blood of cancer patients," said Jeffrey Chalmers, PhD, study investigator, professor in Ohio State's Department of Chemical and Biomolecular Engineering and director of the Cytometry Shared Resource at the Center for Scientific Review. "Our team is currently working with researchers in the field of cell biology and immunology to further characterize the cells and understand their implications in the field of cancer research."

Patients with mBC who were older than 18 and had two or less prior lines of systemic therapy were eligible to enroll in the study. Participants included 40 women, between the ages of 28 and 78, who were either estrogen receptor positive (52%) or had TNBC (47%). Sixty percent of participants had undergone two prior lines of chemotherapy and all had bone and organ metastases. Healthy volunteers without a known diagnosis of malignancy were also enrolled in the study.

Follow-up studies are currently underway to characterize the CTCs and double positive cells identified by researchers.