Abstract A106: Mechanisms underlying extra-medullary myeloma development
Authors: Mishima Yuji , Roccaro Aldo , Moschetta Michele , Terui Yasuhito , Hatake Kiyohiko , Ghobrial Irene M. 
Published: May 2016

Extramedullary disease (EMD) involvement in Multiple Myeloma (MM) is associated with poor prognosis and resistance to therapeutic agents. However, molecular alterations that lead to extramedullary metastasis of malignant plasma cells have not been identified. In this study, we developed MM subclones that have tropism to liver EMD sites by performing repetitive in vivo selection in xenograft mice using 2 MM cell lines. Whole body imaging showed that liver-prone clones infiltrated into various EMD site including liver, developing macroscopic EMD tumors, whereas control BM-prone subclones developed little or no macroscopic EMD. Interestingly no significant differences were observed in BM involvement between these subclones. In vitro characterization study showed that cell proliferation rates of these sub-clones were almost identical, while the transwell migration assay revealed that EMD-prone cells had higher chemotaxis index towards serum containing media compared to BM-prone clones. Flow-cytemetry analysis was performed, showing several chemokine receptors were upregulated in EMD-clones compared to BM clones. Among them, CXCR4 was remarkably upregulated in both two MM cell lines. Immunohistochemistry of clinical samples further demonstrated a significant difference in protein expression of CXCR4 in EMD sites compared to BM sites of involvement in the same patients, suggesting that CXCR4 upregulation in mice model is clinical relevant. Gain-of function studies using stable cell lines with high or low CXCR4 expression in a competitive bone metastasis model confirmed that high CXCR4 expressing cell lines were more “fit” for dissemination and engraftment in the liver.

For further understanding of the participation of CXCR4 in EMD development, we investigated characteristics of these subclones in hypoxic condition. CXCR4 expression of both EMD and BM-prone subclones were upregulated in hypoxic condition (0.5% O2), becoming almost same level, despite EMD-prone clones had higher CXCR4 expression in normoxia. In normoxic condition, EMD-prone cells exhibited higher chemotaxis toward primary stellate cells, which is thought to be a major source of SDF-1 in liver, compared to BM-prone clones, while no difference was observed in chemotaxis towards BM mesenchymal stem cells at hypoxic condition. These results may suggest possible explanation of EMD tropism of the cells constitutively expressing CXCR4. As BM is highly hypoxic milieu, myeloma cells, in general express higher levels of CXCR4 in BM, but once they migrate outside the BM, the cells decrease CXCR4 expression. The EMD-prone cells remain expressing higher levels of CXCR4 even outside BM and may infiltrate into extrmedullary tissues in response to SDF-1 density gradient.

Together, these studies identify novel molecular alterations that are present in MM subclones that metastasize to EMD sites indicating a potential therapeutic role for CXCR4 inhibitors in the treatment of patients with extramedullary myeloma.

Citation Format: Yuji Mishima, Aldo Roccaro, Michele Moschetta, Yasuhito Terui, Kiyohiko Hatake, Irene M. Ghobrial. Mechanisms underlying extra-medullary myeloma development. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A106.