In Vitro Osteoclastogenesis and T-Cell RANKL Expression In Multiple Myeloma-Bone Disease At Diagnosis and In The Setting Of Frontline Treatment
Authors: Oranger Angela , Rizzi Rita , Brunetti Giacomina , Daraia Bruna , Gigante Isabella , Mongelli Teresa , Mori Giorgio , Germano Candida , Curci Paola , Grano Maria , Specchia Giorgina , Colucci Silvia 
Published: November 2019

Abstract

Introduction
Multiple myeloma (MM)-bone disease occurs in 70% to 80% of patients at MM diagnosis, and up to 90% at relapse; skeletal related events cause high morbidity and mortality. MM-bone disease is mostly consisting of lytic lesions arising as a consequence of an unbalanced bone remodelling due to activation of osteoclasts (OCs), and inactivation of osteoblasts. Osteoclastogenesis may be under immunoregulation by T-cells through the production of receptor activator of NF-kB ligand (RANKL), that is the OC differentiating molecule.

Aim
By means of an in vitro osteoclastogenesis model derived from peripheral blood mononuclear cells (PBMCs) of patients with MM-lytic bone disease, we purposed to evaluate OC formation and T-cell RANKL expression at diagnosis, and in the setting of frontline therapy.

Methods
PB was taken by venipuncture from 18 patients (7 men and 11 women), aged from 63 to 84, diagnosed as having symptomatic MM by International Myeloma Working Group (IMWG) criteria. In all of them, lytic lesions were demonstrated by skeleton standard radiography, and in some cases by nuclear magnetic resonance (NMR) too. On the basis of patients’ age and fitness, initial standard therapy consisting of proteasome inhibitor (Bortezomib) associated with Melphalan and Prednisone (VMP) was administered for nine cycles. The therapy response was assessed according to IMWG criteria. The controls included PB collected from 20 healthy donors, matched for age and sex with the patients. Controls and patients gave their written informed consent to the study, that was approved by the local Ethical Committee and performed according to the Declaration of Helsinki. PB was taken from the patients at diagnosis, and following the fourth and the ninth VMP cycles, respectively. OCs were obtained from unfractionated PBMC cultures either stimulated or not with the exogenous pro-osteoclastogenic cytokines [macrophage-colony stimulating factor (M-CSF) and RANKL]. Mature OCs were identified as multinucleated tartrate-resistant acid phosphatase (TRAP) positive cells. Freshly isolated T-cells from patients’ and controls’ PBMCs were lysed and analyzed by Western Blotting to evaluate RANKL expression.

Results
Spontaneous osteoclastogenesis was demonstrated in the cultures derived from PBMCs isolated at diagnosis. With particular regard to the patients achieving a deep treatment response, at the end of the culture period we found that PBMCs formed few small-sized OCs, when cultured in the absence of M-CSF and RANKL. Moreover, fresh T-cells purified after four and nine VMP cycles expressed progressively lower RANKL levels than fresh T-cells purified at diagnosis. Otherwise the cultures derived from non-responsive patients’ PBMCs, collected during and after treatment respectively, showed in vitro spontaneous formation of numerous and large OCs, overlapping the results at diagnosis. Fresh T-cells purified during or after treatment expressed comparable or even higher RANKL levels respect to diagnosis.

Conclusions
These findings show that in vitro spontaneous osteoclastogenesis and T-cell RANKL expression may be correlated with the response to treatment in patients with MM-lytic bone disease. However, further studies will be necessary to confirm these data, and to better translate them from a biologic to a clinical point of view.

Disclosures:
No relevant conflicts of interest to declare.