Metastasis to distal organs causes the majority of cancer deaths. Circulating tumor cells (CTCs) originate from a primary tumor and initiate a metastasis cascade that ultimately results in metastatic tumors appearing at distal sites. See Sun et al. (2011) Circulating tumor cells: advances in detection methods, biological issues, and clinical relevance, J. Cancer Res. Clin. Oncol. 137:1151-1173. Several genetic, morphological, immunological and physiological tests may be used to identify CTCs. See Id., and Man, et al. (2011) Currently used markers for CTC isolation—advantages, limitations and impact on cancer prognosis, J. Clin. Exper. Pathol. 1:1. Because the number of CTCs in peripheral blood even in patients with advanced metastatic disease, is extremely low compared to the number of normal blood cells (one in a billion), positive selection is a commonly used method for enumeration and isolation of CTCs. CTCs may be captured by using an antibody directed against the epithelial cell adhesion molecule (EpCAM) expressed within the cell membrane of many CTCs. Following selection, the CTCs are ultimately identified by immunostaining using a combination of reagents targeting live cells, leukocyte-specific markers and tumor-specific markers. For example, the FDA-approved CELLSEARCH® CTC test (Veridex, LLC, Raritan, N.J.) detects CTCs using anti-EpCAM monoclonal antibodies poised on paramagnetic beads, followed by CTC identification using DAPI, cytokeratins (CK) and CD45. See Hayes, D. F. and Smerage, J. (2008) Is there a role for circulating tumor cells in the management of breast cancer? Clin. Cancer Res., 14:3646-3650; Cristofanilli, M., et al. (2004) Circulating tumor cells, disease progression, and survival in mestatic breast cancer. N. Engl. J. Med., 351:781-791. However, reports have suggested that this approach does not capture cells with low or no expression of EpCAM; and the assay shows poor sensitivity and specificity for metastatic cases. See Lu, J. et al. (2010) Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients. Intl. J. of Cancer, 126:669-683; Sieuwerts, A. M. et al. (2009) Anti-epithelial adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells. J Natl Cancer Inst, 101:61-66. However, cells with low or no EpCAM expression are also likely to be highly clinically significant. It has been suggested that tumor cells expressing invasive phenotypes down-regulate and lose their epithelial antigens (including EpCAM) in a process called the epithelial-to-mesenchymal transition (EMT). Mego, M. et al. (2010) Molecular mechanisms of metastasis in breast cancer-clinical applications. Nat. Rev. Clin. Oncol., 7:693-701; Brabletz, T. et al. (2005) Invasion and metastasis in colorectal cancer: Epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and Beta-catenin. Cell Tissues Organs, 179:56-65; Ralmondi, C. et al. (2011) Epithelial-mesenchynmal transition and stemness features in circulating tumor cells from breast cancer patients. Breast Cancer Res, 130:449-455. It has also been suggested that CTCs contain sub-populations with a continuum of phenotypes besides the epithelial one. Therefore, attempts to capture all CTC sub-populations (especially invasive ones that have little if any EpCAM expression) by targeting EpCAM alone may be ineffectual. Sabile, A. et al. (1999) Efficiency of Ber-EP4 antibody for isolating circulating epithelial tumor cells before RT-PCR detection. Am. J. Clin. Pathol., 112:171-178; Thurm, H., et al. (2003) Rare expression of epithelial cell adhesion molecule on residual micrometastatic breast cancer cells after adjuvant chemotherapy. Clin. Cancer Res., 9:2598-2604. This is further substantiated by the observation that one gram of an epithelial-based tumor can release up to 10° cells per day. Butler, T. P. et al. (1975) Quantitation of cell shedding into efferent blood of mammary adenocarcinoma. Cancer Res., 35, 512-516. While clearly not all of these cells possess the ability to initiate metastasis, some rare and elusive sub-populations do.
It may be possible to select CTC sub-populations using a combination of antibodies, e.g., CD45 antibody combined with antibodies for various tumor makers, e.g., HER2 or estrogen receptor. Exemplary tests are offered by BioCept, Inc. (San Diego, Cal.). However, the antibody cocktails typically used in such tests are generated using immortalized cells lines that may not truly recapitulate the continuum of changes occurring in CTCs released from patient tumors. Pecot, C. V. et al. (2011) A novel platform for detection of CK+ and CK− CTCs. Cancer Discovery, 1(7):580-586. Accordingly, there is a need for a method able to effectively detect and target rare invasive sub-populations of CTCs present in patient samples.
USPTO Backgrounds
USPTO Backgrounds