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Symposium 34: Determinants of Metastasis

Bone marrow: Homing organ for dormant micrometastatic cancer cells

University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany

Abstract

SY34-2

Although many different assays have been developed over the past ten years to detect micrometastatic tumor cells, the two major approaches used involve either immunocytochemical staining or polymerase chain reaction (PCR) analysis. If these assays are sensitive and specific enough, they can detect a single metastatic cell in the background of millions of normal cells. For epithelial tumors, cytokeratins have become the best marker for the immunocytochemical detection of micrometastatic tumor cells¹. Bone marrow (BM), which can be easily collected from the iliac crest, is the most important site for detecting micrometastatic epithelial tumor cells, which are present in BM samples of 20-40% of patients with carcinomas at various primary sites; even in the absence of lymph node metastases (stage N0) or clinical signs of overt distant metastases (stage M0) ^2–4. BM samples can also be monitored for the presence of micrometastatic tumor cells after primary surgical treatment, to detect tumor recurrence^5,6. Interestingly, the presence of disseminated tumor cells in the BM is not only useful in predicting the development of skeletal metastases, but also in predicting the development of metastases in other distant organs, such as lung or liver. This is even true for tumors that rarely show skeletal metastases, such as colon cancer ³. In fact disseminated cancer cells have also been found in the BM of patients with head and neck cancer that did not have lymph node metastases, although the clinical significance of these cells is not clear ⁷. So BM might be an important reservoir that allows for disseminated epithelial tumor cells to adapt and disseminate into other organs. An alternative explanation is that the presence of occult cancer cells in the BM might only reflect the general propensity of these cells to disseminate and to survive in organs, rather than just in the BM. Several studies have documented the persistence of dormant micrometastatic tumor cells in BM of cancer patients without clinical signs of overt metastases^5,6,8–12. These findings are direct proof of the concept of dormancy. Moreover, the persistence of these dormant cells is an indicator for subsequent metastatic relapse. Heiss et al. previously showed a close association between post-operative persistence of tumor cells in BM and metastatic relapse in patients with gastric cancer ⁶. More recent data in breast cancer published by two independent groups demonstrated that micrometastatic tumor cells in BM three years after initial diagnosis in disease-free breast cancer patients predicted unfavourable clinical outcome ^11,12. Persistence of tumor cells in BM was an independent prognostic factor. These findings indicate that adjuvant systemic treatment may be insufficient to eliminate micrometastatic cells and that at least some of the surviving micrometastatic cells can escape dormancy. It will be now a major challenge to unravel the mechanisms underlying this capacity. One approach to achieve this task might be the further characterization of micrometastatic cells. Anti-cytokeratin antibodies, which are used to identify disseminated tumor cells in BM, can be used in combination with antibodies against other tumor-associated antigens to profile these cells. Using such immunocytochemical procedures, it has been possible to identify a number of tumor-associated characteristics of the cytokeratin-positive cells found in BM¹ (see Fig. 1, in Reference 1). Phenotyping of disseminated tumor cells in BM of early stage patients has also yielded additional prognostic information. In patients with gastric cancer, the presence of micrometastatic cells that express the urokinase-type plasminogen activator receptor (uPAR) correlates with an unfavourable prognosis ⁶. Similar observations have been made in patients with breast cancer; Her2/neu overexpression by tumor cells that have disseminated to the BM predicts poor clinical outcome ¹³. So uPAR and Her2/neu might be important for the survival and growth of disseminated tumor cells. Recently, Jonathan Uhr's group presented evidence that the genotype of persistent circulating tumor cells may change towards a Her2-amplified genotype¹⁴, which might be in line with our previous findings on the selection of a Her2-positive phenotype in BM micrometastases^13,15. Recent technical developments have made it possible to examine the genome of disseminated tumor cells. A combination of immunocytochemistry and fluorescence in situ hybridization (FISH) have shown that BM contains proliferating micrometastatic cells with various numeric chromosomal aberrations found of malignant origin¹⁶. The viability and proliferative capacity of these cells has also been monitored in these models, and correlated with clinical outcome^16,17. By developing a new procedure for whole genome amplification and subsequent comparative genomic hybridisation (CGH) of single immunostained cells, it was found that cytokeratin-positive cells in BM of epithelial breast cancer patients without clinical signs of overt metastases (stage M0) are genetically heterogeneous¹⁸. This heterogeneity was strikingly reduced with the emergence of clinically evident metastasis (stage M1). The stage at which individual cells leave the primary tumor is unclear. In patients with early stage invasive breast cancer, the cytokeratin-positive cells isolated from the BM had few features in common with those found in their respective primary tumors¹⁹. A provocative interpretation of this surprising finding is that the disseminated tumor cells separated from the primary lesion at a very early stage. This hypothesis is also supported by the finding that only few disseminated tumor cells in these patients had TP53 mutations, which are associated with the later stages of tumourigenesis^18,20. Disseminated tumor cells might therefore evolve independently into overt metastases, driven by the specific selective pressures of the BM environment. Functional analysis of micrometastatic cells remains a challenge, because even after short-term culture their numbers are still small (on average between 1,000 and 10,000 cells). Permanent cell lines have therefore been established, and show gene expression and genomic characteristics that are typical of epithelial tumor cells in situ ^21,22. These could therefore serve as models for functional studies on dormancy of micrometastatic cells. The detection and characterization of micrometastatic tumor cells in cancer patients has provided important new information about the cascade of metastatic events. This information may also have important implications for cancer prognosis and for therapy.