Thomas F. Tucker

Circulating Tumor Cells in Patients with Breast Cancer Dormancy

Purpose: The purpose of this study was to test the hypothesis that circulating tumor cells (CTCs) are present in patients many years after mastectomy without evidence of disease and that these CTCs are shed from persisting tumor in patients with breast cancer dormancy. Experimental Design: We searched for CTCs in 36 dormancy candidate patients and 26 age-matched controls using stringent criteria for cytomorphology, immunophenotype, and aneusomy. Results: Thirteen of 36 dormancy candidates, 7 to 22 years after mastectomy and without evidence of clinical disease, had CTCs, usually on more than one occasion. Only 1 of 26 controls had a possible CTC (no aneusomy). The statistical difference of these two distributions was significant (exact P = 0.0043). The CTCs in patients whose primary breast cancer was just removed had a half-life measured in 1 to 2.4 hours. Conclusions: The CTCs that are dying must be replenished every few hours by replicating tumor cells somewhere in the tissues. Hence, there appears to be a balance between tumor replication and cell death for as long as 22 years in dormancy candidates. We conclude that this is one mechanism underlying tumor dormancy.

Methods for isolating circulating epithelial cells and criteria for their classification as carcinoma cells

Novel assay methods developed for the isolation and characterization of circulating tumor cells (CTC) of epithelial origin offer the potential of markers for the non-invasive gathering of clinical information relevant to the diagnosis, evolution and treatment of carcinoma. Of the numerous techniques currently used to analyze CTC, slide-based assays are perhaps the most common. While traditional combined immunocytochemical/brightfield microscopy systems continue to be the most frequently employed, fluorescence-based analysis is gaining in importance. This is partly because fluorescence microscopy analysis of slide-mounted CTC can provide simultaneously cytogenetic as well as morphologic and multiple phenotypic information. In particular, fluorescence microscopy analysis of slide-mounted CTC can accurately determine genetic changes at the chromosomal level in patients with recurrent disease. More importantly, by identifying genetic aberrations in CTC, it becomes possible to choose those patients most likely to benefit from a given treatment. The potential of this technique has already been demonstrated by employing fluorescence in situ hybridization (FISH) methods to measure expression of the HER2/neu gene in tissue from patients with breast carcinoma for the specific purpose of identifying those patients most likely to respond to Trastuzumab targeted therapy. Here, we review the major methodologies used in the preparation and analysis of the slide-based assays.

Patterns of aneusomy for three chromosomes in individual cells from breast cancer tumors.

Multi-color fluorescence in situ hybridization (FISH) can determine the changes in the copy numbers of several chromosomes simultaneously and can therefore be used to identify aneusomic patterns in individual cells. Aneusomic patterns may be useful for determining the malignant nature of rare epithelial cells in the blood of cancer patients. Touch preparations from 74 primary breast tumors were evaluated for aneusomy of chromosomes 1, 8 and 17 by tri-color-FISH. In the first part of the analysis, percentages of aneusomy for individual chromosomes and their combinations were determined. In the second part of the analysis, aneusomic patterns for these three chromosomes were analyzed in individual tumor cells and compared to aneusomic patterns observed in leukocytes and in individual cells from benign and normal breast tissue to determine aneusomic patterns indicative of malignancy. Ninety-two percentage of the primary breast carcinomas showed aneusomy for one or more enumerator probes. Comparison with benign breast tissue identified six aneusomic patterns in individual carcinoma cells indicative for malignancy by statistical analysis and not observed in leukocytes. Hence, certain patterns of aneusomy in individual cells involving chromosomes 1, 8 and 17 are indicative of malignancy in individual breast tumor cells and may be useful for determining malignancy of rare epithelial cells in the blood of breast cancer patients.

Induction of B cell tumor dormancy by anti-idiotypic antibodies

Long-term dormancy of murine B-cell lymphomas can be experimentally induced by immunizing the host with the idiotype expressed on the tumor. Interaction of the cells with anti-idiotype antibodies is sufficient to induce and maintain the dormant state. The growth of lymphoma cells interacting with anti-idiotype antibodies is arrested and they undergo dramatic changes in their morphology, cell-cycle status and oncogene expression. Regrowth of a tumor after long-term dormancy results from the emergence of a tumor cell variant that no longer responds to the antibodies with growth inhibition. These data demonstrate the feasibility of reversing a malignant phenotype of cells by specific growth arrest signals and suggest new approaches for therapeutic intervention in cancer.

Role of Antibody Signaling in Inducing Tumor Dormancy

Cancer dormancy is a well-recognized clinical phenomenon in which tumor cells are present, but the tumor burden does not increase for long periods of time1–3. However, tumor cells can regrow many years later. In breast cancer, there is a steady rate of recurrence 10 to 20 years after removal of the primary tumorl3,4 and the recurrent tumor frequently grows at a rapid rate(5). A particularly pertinent example is the low grade (follicular) form of non-Hodgkin’s lymphoma (NHL) in which long-term remissions are common but, eventually, virtually all die of a recurrence. Levy and Miller(5) have treated such patients with monoclonal anti-idiotype (Id) and have achieved remissions in a high proportion of patients. Relapses, many caused by Id-negative variants, are frequent indicating that the antibody (Ab) was particularly effective in inducing dormancy in cells bearing the corresponding idiotope but that hypermutation of VH and VL genes eventually allow some tumor cells from the original clone to escape(5–7).

Failure of vaccination with idiotypic protein or DNA, (+/-IL-2), the depletion of regulatory T cells, or the blockade of CTLA-4 to prolong dormancy in mice with BCL1 lymphoma

Immunization of mice with the idiotype (Id) immunoglobulin from the murine B cell lymphoma, BCL1, before inoculating tumor cells can induce tumor dormancy. In this model, the tumor cells grow for a short period of time and then regress. The mice live for months or years with approximately 1 million tumor cells in their spleens. Some mice relapse due to decreases in the anti-Id antibody titers or the development of mutations in the residual tumor cells which render them refractory to negative signaling by the anti-Id antibody. In this study we determined whether we could eliminate the residual dormant cells by using a DNA vaccine against the Id or by immunomodulation of T-cell subsets in vivo. Our results demonstrate that dormancy can be maintained by further immunizations with either the BCL1 Id protein or DNA vaccine encoding its single-chain Fv fragment. We also found that a cytotoxic T-cell response was not induced by either in vivo administration of vaccine alone or by the vaccine plus interleukin-2. In addition the injection of anti-cytotoxic T-lymphocyte-associate antigen did not prolong dormancy. Finally, the in vivo administration of anti-CD25 to deplete regulatory T cells did not prolong dormancy. Dormancy in this model is dependent primarily upon anti-Id antibodies, our results suggest that other strategies to target residual dormant BCL1 cells are warranted. They also suggest that the elimination of dormant tumor may represent a greater challenge than the elimination of primary tumors.