A. Description of the Research: Among breast cancer patients, 20% will die of recurrent disease. A reason for recurrence has been discovered: a small fraction of the cancer cells in a personís body, which are found in the tumor nodules as well as the peripheral blood, resemble embryonic stem cells in the genes that they express, in their capability to produce progeny and in their resistance to radiation therapy and chemotherapy. The peripheral blood stem cells may be the most aggressive. The failure to rcognize the presence of these ìcancer stem cellsî has led to targeting therapies to the majority of cells which are not contributing to recurrence. Now that we know about the presence of the tumor stem cell, we need to access large enough numbers of these cancer stem cells to identify markers in these cancer stem cells to develop tests for prognosis, therapy selection and therapy development.
B. Background: Cancer cells which exhibit overexpression of the Polycomb group (PcG) gene BMI-1 are present in the peripheral blood and solid tumors of recurrent disease breast cancer patients. The Polycomb group is a family of transcription factors important in the regulation of the differentiation of the embryonic stem cell (2), the presence of which was found to be predictive of adverse prognosis in carcinoma of the breast by the Glinsy lab. These cells form spheroids in serum free medium, and the cell in these spheroids displayed expression of stem cell gene signatures. They form tumors when injected sc at low dose (100 cells) into NODXSCID mice.
C. Proposed Work: Isolation of the peripheral blood breast cancer stem cells provides a method of sampling the cancer cell stem cell fraction from the entire body in order to identify new targets to which we can direct therapy to destroy these cells. Unfortunately, the number of these cells in peripheral blood is very low (5/cc). Our laboratory has FDA approval for 2 pheresis procedures from each of 12 recurrent breast cancer patients who are participants in a cancer vaccine trial sponsored by our laboratry at [REDACTED], which will generate the numbers of peripheral blood cancer stem cells necessary to test for correlations between individual gene expression signatures and assays of therapy resistance, tumorigenicity and metastasis.
D. How the Research will Contribute to Development of New Cancer Stem Cell Diagnostic Tools: The pheresis collections will allow us to compare the gene expression signatures of peripheral blood cancer stem cells with that already determined for human and mouse embryonic stem cells, and to test for correlations between these signatures and the tumorigenicity, chemotherapy resistance and motility assays to be carried out on these peripheral blood cancer cells. These studies may therefore lead to new technolgy for assays for prognosis, therapy selection and therapy development for breast cancer.
Statement of Benefit to California:
Gene expression profiling is one way to study the complexity of genetic change in each patientís population of peripheral blood breast cancer stem cells. What has been learned so far is that some of the stem cell genes expressed in breast cancer cells isolated from solid tumors and peripheral blood are involved in transcriptional regulatory systems (Polycomb Pathway) that direct the partitioning of embryonic stem cells between the stem cell compartment and differentiating populations (4). Importantly, may of the stem cell genetic signatures discovered so far have been shown to be useful in assigning breast cancer patients to good or poor prognosis categories (2). Moreover, studies have established that there are cancer cells circulating in the peripheral blood of breast cancer patients in relapse. It is important now to access sufficient number of these peripheral blood cancer cells to study their properties and to link particular gene expression signatures with the transformed properties of these cells.Insufficient numbers of cells have been available for carrying out this type of analysis. We will have available to us purified populations of cancer cells from two pheresis collections/patient from 12 patients with relapse breast cancer. These collections will contain 130,000 breast cancer cells/collection generated by processing 2.5 intravascular fluid volumes/patient. These patients will be in early relapse and participants in a cancer vaccine trial from the [REDACTED] lab. The cells will be collected efore and after vaccination. Permission has been given to the [REDACTED] lab by the FDA to open this phase I trial upon which the collections of peripheral blood cancer cells depends.
The goal of the proposed grant is to test for correlations between these gene expression signatures in the peripheral blood breast cancer cells and their transformed properties as determined by the following functional studies: chemotherapy resistance, migration (a surrogate of metastasis), tumor formation, and self renewal (as studied by serial passage in NODXSCID mice). The completion of this work will make it possible to identify gene expression signatures and markers that are predictive of chemotherap resistance, tumor formation, metastasis, and self-renewal in the cancer stem cells. These markers will then be available for incorporation in tests that could be used for prognosis, therapy selection and therapy target identification. These discoveries could be the first clinically relevant and important applications of the CIRM initiative. We will make all information from these studies available at the time of publication or within 6 months of the end of the two year grant period to everyone in the fied. Thus, this work will have a global impact on the management of breast cancer patients in California, and may create jobs and develop capitol for the health science industry in California.
The goal of this proposal is to identify new biomarkers for breast cancer stem cells. The applicant will purify circulating cancer cells, which have stem cell properties and are a feature of recurrent breast cancer, from patients participating in a vaccine trial. Isolated cells will be analyzed by gene expression profiling, and expression patterns will be correlated with the results of functional assays. Information from this analysis may allow the recognition of aggressive cancer phenotypes that can guide treatment options
The proposed research has a high potential impact given the prevalence of breast cancer. Gene expression profiles of breast cancer stem cells could prove to be important prognostic indicators of chemotherapy resistance, tumor formation, metastasis and self-renewal. This information could be valuable in guiding therapeutic decisions. However, reviewers felt that the study was largely derivative of previous work, would not break new ground, and was unlikely to provide significant new information.
The applicant outlined a straightforward strategy for enrichment and analysis of circulating cancer cells. The proposal builds on an existing IND and clinical trial as a source of patient material. Details regarding possible pitfalls and alternative strategies were clearly outlined. However, key feasibility issues diminished reviewer enthusiasm. In particular, the proposed number of patients (12) was viewed as too small to provide statistically meaningful data, and it was unclear that the cell enrichment techniques would yield cell populations of adequate purity and number to allow significant analysis. Sufficient preliminary data or consideration of cell numbers was lacking. Additionally, the inclusion of data accrual from prostate cancer patients and information about the vaccination strategy and about chromatin changes in prostate cancer were largely irrelevant or peripheral to the goals and feasibility of the proposal.
The PI has a long history and many accomplishments in the cancer field and in the development of cancer vaccines. Other members of the research team appear highly experienced. However, neither the applicant nor collaborators have published research on stem cells.
Overall, reviewers viewed the identification of new biomarkers for breast cancer to be an important goal, but were unconvinced that the study as proposed would contribute significantly to this effort.