Multiple Myeloma and Bone Marrow Microenvironment Immunohistochemical Study of the Expression of 15 Proteins Related to Myeloma Bone Disease

Objective: Neoplastic milieu is an integral part of all malignant diseases including multiple myeloma and plays variable role in their development, retention/adhesivity, resistency or sensitivity to therapeutic approach, homing and also paraneoplastic manifestations. Relatively genetically stable milieu may play an important role in new specific molecular therapeutic approaches and therefore should be contextually studied with neoplastic cells as complex neoplastic tissues. The expressions of 15 proteins with close relation to the development of myeloma bone disease (MBD) were analysed in consecutive multiple myeloma specimens.

Methods: Bone marrow trephine biopsy specimens (n=57) with multiple myeloma were included in our prospective study. FFPE tissues were processed in app. 5microm sections and placed on charged slides. The indirect immunohistochemical staining was applicated after antigen retrieval and commercial primary antibodies were used for the detection of observed proteins. Standard secondary antibody and ABC method were included in visualisation. We analysed the expressions of MIP1alfa, Annexin A2, TRAP, DKK-1, RANK, RANKL, OPG, Sclerostin, Activin A, NFkappaB proteins (p50, p52, p65), p62 (sequestosome 1), MMP9 and RUNX2.

Results: Bone marrow multiple myeloma specimens showed variable positivity of MIP1alfa in 60% (cut-off point 20%), Annexin A2 in 42% (myeloma cells, cut-off point 30%) and in 74% (stromal cells, cut-off point 5%), TRAP in 28% (cut-off point 5%), DKK-1 in 23% (cut-off point 30%), RANK in 53% (cut-off point 30%), RANKL in 70%, OPG in 39% (cut-off point 5%), Sclerostin in 95% (cut-off point 90%), Activin A in 35% (cut-off point 30%), cytoplasmic positivity of p50 in 5% (cut-off point 10%), p52 in 86% (cut-off point 10%), p62 in 91% (cut-off point 10%), p65 in 89% (cut-off point 10%), positivity of MMP9 in 22% (cut-off point 30%) and positivity of RUNX2 in 56% (cut-off point 30%).

Conclusion: Our study showed variable expression of proteins related to MBD in multiple myeloma and its bone marrow microenvironment that imply biological heterogeneity, different development and stromal plasticity in this complex hemato-oncological disease. The exact and contextual knowledge of the engaged signaling pathways may suggest more specific or tailored therapeutic approaches (e.g. anti-RANKL, anti-DKK-1, anti-Sclerostin, anti-Activin A).

Supported by the grant NT 14393.