Genomic Profiling of Viable and Proliferative Micrometastatic Cells from Early-Stage Breast Cancer Patients

Abstract

Purpose: Metastases in distant organs are the major cause of death for cancer patients, and bone marrow is a prominent homing organ for early disseminated cancer cells. However, it remains still unclear which of these cells evolve into overt metastases. We therefore established a new approach based on the analysis of viable and proliferating cancer cells by single-cell comparative genomic hybridization.Experimental Design: The bone marrow of early-stage breast tumor patients (pN 0 M 0 ) was screened for tumor cells by immunostaining. By applying special short-term culturing, we selected for viable and proliferative tumor cells. The short-term culturing allowed us to evaluate the proliferative potential of micrometastatic cells, which we had previously shown to represent an independent prognostic marker. We assessed genomic changes in single disseminated cancer cells by single-cell comparative genomic hybridization.Results: We found that these viable disseminated cancer cells already had a plethora of copy number changes in their genome. All of these cells showed chromosomal copy number changes with a substantial intercellular heterogeneity and differences to the matching primary tumors.Conclusions: The established experimental strategy might pave the way for the identification of metastatic stem cells in cancer patients. Our preliminary results support the new concept that early disseminated cancer cells evolve independently from their primary tumor.

Sequential application of interphase-FISH and CGH to single cells

“…2,3,[18][19][20] The high quality of our ratio profiles is probably a reason why the accuracy rate was generally in the range of close to 100%. Therefore, we could use our single-cell CGHapproach already for monitoring minimal residual disease albeit without FISH, 13 and we can now apply our strategy for diagnostic applications (own unpublished data).…”

“…These cells may have no or a various number of copy number changes. 13,21,22 A preceding interphase-FISH analysis offers the opportunity for the sophisticated selection of cells for further analysis based on certain interphase-FISH patterns.…”

“…Cells were isolated by the PALM MicroBeam System (PALM Microlaser Technologies, Bernried, Germany; [12][13][14]. Isolated cells were lifted into the cap of a 200 ml Eppendorf tube containing 4.5 ml digestion-mix.…”

A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation

“…However, molecular analysis has provided enough evidence to assume that cytokeratin-positive DTCs are malignant cells. [38][39][40][41][42] Nevertheless, several investigators use >2 categories to describe immunostained cells, including apoptotic cells, probable hematopoietic cells, or probable tumor cells. 24,25,37 The clinical significance of such multiple categories is under investigation.…”

Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing

“…In patients with non-metastatic breast cancer, the majority of identified cells displayed either a normal euploid profile or an aberrant DNA copy number landscape seemingly unrelated to the primary tumor [23,24], suggesting parallel evolution. Additionally, copy number aberrations (CNAs) detected among DTCs from the same nonmetastatic patient were generally non-recurrent [23,25].…”