Assessment of Signalling Pathways in Myeloma Bone Disease Using Selected Parameters of Bone Marow Microenvironment
Authors: Minarik Jiri , Flodr Patrik , Pika Tomas , Bacovsky Jaroslav , Latalova Pavla , Pusciznova Petra , Scudla Vlastimil 
Published: October 2019

Abstract
Aim: Myeloma bone disease (MBD) is present in 80-90% patients with multiple myeloma (MM). Up to now, three signalling pathways have been described in the pathogenesis of MBD – receptor activator of nuclear factor kappa B and its ligand (RANK/RANKL) pathway, macrophage inflammatory proteins (MIP) pathway, and wingless type (Wnt) pathway. Moreover, several cytokines and parameters of bone microenvironment have been shown to interfere with bone homeostasis in MM. The aim of our study was to assess the activity of selected parameters in bone marrow of patients with MM and monoclonal gammopathy of undetermined significance (MGUS) in order to define the principal processess occuring within MBD.
Materials and methods: We designed a prospective study aimed at signalling in MBD. Formaline-fixed, parafin-embedded diagnostic tissue of patients with MM and MGUS has been processed in routine tissue sections (app. 5 um) and placed on plus-charged slides. After the antigen retrieval (Table 1) indirect immunohistochemical evaluation has been processed with the use of commercial available primary antibody for particular detected protein (according to manufactor´s manual) in optimalised dilution. For the visualisation secondary antibody has been applicated with the use of the standard method avidin-biotin (ABC). Following parameters have been evaluated: RANK on myeloma and mononuclear stromal cells, RANKL and osteoprotegerin (OPG) on stromal cells, MIP-1α in plasma cells (both membrane and cytoplasm), sclerostin, MMP 9 and DKK-1 in the cytoplasm of plasma cells, Annexin A2 in plasma and stromal cells, tartrate resistant acid phosphatase (TRAP) in the cytoplasm of osteoclasts and precursor cells, Activin A in the nucleus of plasma cells, p50, p52, p62, p65, in nucleus and cytoplasm of plasma cells.
Results: Activity of RANK varied between 0-100% in plasma cells with 0% activity in stromal cells. Positivity of RANKL was found on endosteum of stromal cells in 12/17 patients (71%). The activity of OPG on stromal cells was in all patients under 10%. Assessment of MIP-1α revealed 100% positivity in 9/17 patients (53%), in 13 patients (76%) the activity was more than 50%. The activity of sclerostin reached 90-100% in all patients. The levels of DKK reached in 3 patients more than 60%, in the rest it was under 10%. The levels of Annexin A2 in stromal cells were low, in 16/17 patients below 20%. In plasma cells, higher activity (above 60%) was found in 4 patients (24%). Activity of p50 above 70% was found in cytoplasm of 2 patients only (12%). The levels of p52 varied between 1-90%, majority of the patients (53%) having more than 80% activity. Similar results were found within the assessment of p65. The levels of p62 were with high activity above 70% in 16/17 patients. The activity of MMP-9 was in 9/17 patients above 70%, the rest of patients had the activity of MMP-9 under 20%.
Conclusions: Patientswith monoclonal gammopathies displayed significant activation of all three signalling pathways of MBD. There were however, differences in the involvement of each individual pathway as well as in the levels of other cytokines participating on bone homeostasis, suggesting different mechanisms of the cascade occurring in patients with similar skeletal involvement.
With support of the grant NT14393.
Abstract 5679. Table 1 Primary antibodies Antibodies clone Antigen retrieval Dilution Source Anti- MIP1alfa C-16 MW 1:200 Santa Cruz Anti- RANK 9A725 MW 1:100 Santa Cruz Anti- RANKL N-19 MW 1:100 Santa Cruz Anti- Ann A2 Polyclonal MW 1:1000 Abcam Anti- TRAP Polyclonal MW 1:1000 GeneTex Anti- Act A Polyclonal MW 1:50 Sigma-Aldrich Anti- OPG N-20 MW 1:50 Santa Cruz Anti- p50 E-10 MW 1:50 Santa Cruz Anti- p52 C-5 MW 1:100 Santa Cruz Anti- p65 F-6 MW 1:100 Santa Cruz Anti- p62 SQSTM1 (ab56416) MW and methanol unblocking 1:50 Abcam Anti- Sclerotisin Polyclonal MW 1:100 Abcam Anti- MMP9 Polyclonal MW 1:50 Abcam Anti- Dkk-1 H-120 MW 1:100 Santa Cruz
Abbreviations: MW – microwave oven

Disclosures
No relevant conflicts of interest to declare.