Symposium 1:

-Search and establishment of biomarkers for cancer as predictive Factors-

Molecular and functional characterization of circulating tumor cells as diagnostic and therapeutic targets

Klaus Pantel, MD

Institute of Tumor Biology, Center of Experimental Medicine,
University Medical Center Hamburg-Eppendorf, Germany

Metastatic relapse of carcinoma patients is mainly due to clinically occult micrometastases present at primary diagnosis, but undetectable even by high-resolution imaging technologies. Frequently, traditional prognostic factors are insuffi cient to predict metastasis and treatment decisions are mainly based on statistical risk parameters. Highly sensitive and specific cytometric and molecular methods enable now the detection of disseminated tumor cells (DTC) in bone marrow (BM) and circulating tumor cells (CTC) in peripheral blood of breast carcinoma patients. The presence of DTC has independent prognostic impact for patients with primary breast cancer with regard to metastatic relapse and overall survival (1) and DTC may even contribute to local relapse (2).
Interestingly, bone marrow seems to be a common homing organ for cells derived from various epithelial tumors including breast, prostate, lung and colon cancer (3); (4). This surprising fi nding is consistent with recent results obtained in mouse models (5),, supporting the hypothesis that BM might be an important reservoir for metastatic cells from where they can re-circulate into various organs and may be even back to the primary site (6); (7); (8). DTC may have adapted to the special environmental conditions in the BM and may survive in so called “bone marrow niches” over decades. This hypothesis has important clinical implications for the design of future clinical trials with drugs that are able to specifi cally block the interaction between tumor cells and the bone marrow microenvironement (e.g. bisphosphonate or antibodies to RANK ligand).
However, a signifi cant fraction of DTC remain over years in a “dormant” stage, and little is known about the conditions required for the persistence of dormancy or the escape from the dormant phase into the active phase of metastasis formation (9). Transition from a dormant into a dynamic phase may be caused by genetic changes within the disseminated tumor cells (i.e. acquisition of growth factor receptor Her-2/ neu amplifi cation (10); (11) but also by the infl uence of the surrounding bone marrow microenvironment.
Furthermore, BM has a particular capability to host stem cells, which may also contribute to keep DTC in a stem cell-like state. This assumption is also supported by the fact that most DTC in BM and blood are in a non-proliferating state and survive systemic chemotherapy (12); (13)). Moreover, most DTC in breast cancer patients showed a breast stem cell phenotype (CD44+/CD24- or MUC1-/CK19+) (14); (15)). Moreover, epidermal growth factor (EGF) and fi broblast growth factor-2 (FGF-2) – two known stem cell factors – were relevant for the in vitro growth of DTC obtained from BM of cancer patients (Solakoglu et al., PNAS 2002). Nevertheless, strong direct evidence that some of the few DTC or CTC detected in the BM or blood samples have cancer stem cell properties is still missing. Future studies including xenotransplantation of DTC/CTC into immunodefi cient mice need to demonstrate that these cells are the actual founder cells of overt metastasis.
BM analyses are not well accepted by the medical community for the clinical management patients in breast cancer and other solid tumors. Therefore, most current research efforts are directed to evaluate the clinical utility of CTC detection (16). Because of the high variability of results obtained by different cytometric and molecular approaches, standardization of current technologies is urgently required (17); (18). While the prognostic signifi cance of CTC could be demonstrated for metastatic breast cancer patients (Hayes & Smerage, CCR 2008), studies on the impact of CTC in primary breast cancer patients are still ongoing but the intermediate results are so far promising (9). Moreover, encouraging results on monitoring CTC during primary systemic or adjuvant chemotherapy in breast cancer patients were obtained in recent studies.
Further characterization of CTC is pivotal to understand the biology of tumor cell dissemination (19). The molecular characterization of CTC with special emphasis on potential cancer stem cell features and therapeutically relevant targets such as HER2 (19) might improve individual risk assessment and stratifi cation of patients for targeted therapies. The HER2 proto-oncogene is currently the most predominant biological target for systemic therapy with remarkable results of clinical trials using a humanized monoclonal antibody (trastuzumab) in breast cancer. The detection of HER2-positive DTC/CTC might enable a “real-time” assessment of the HER2 status during the clinical course of disease. Several groups reported a striking discrepancy between the detection of HER2-positive DTC/CTC and the HER2 score of the corresponding primary tumor, suggesting that a small subclone of HER2-overexpressing cancer cells easily missed by routine primary tumor analysis may have the potential to disseminate (9). The detection of Her2-positive DTC and CTC was correlated to an unfavourable clinical outcome in breast and oesophageal cancer and HER2 gene amplifi cation can be acquired during tumor progression of the cancer (10); (19); (20). Thus, the assessment of the HER2 status on DTC and CTC might add important information for the clinical management of cancer patients.
The characterization of DTC/CTC will contribute to more “tailored” and personalized anti-metastatic therapies. At present, the success or failure of anti-cancer therapies is only assessed retrospectively by the absence or presence of overt metastases during the post-operative follow up period. Real-time monitoring of peripheral blood (i.e., during and after systemic adjuvant therapy) for CTC might provide unique information for the clinical management of the individual cancer patient and allow an early change in therapy years before the appearance of overt metastases signals incurability (20). Future clinical trials will show whether the assessment and monitoring of therapeutic targets (e.g., EGF-R, HER2 or VEGF) on CTC (and probably DTC) might become an important diagnostic tool for cancer patients undergoing targeted therapies and may provide new insights into the selection of tumour cells under biological therapies.

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