Identification of Phenotype Profile Related to the Extramedullary Involvement in Multiple Myeloma Relapse
Authors: Rihova Lucie , Vsianska Pavla , Bezdekova Renata , Adam Zdenek , Penka Miroslav , Jelinek Tomas , Sevcikova Sabina , Pour Ludek , Hájek Roman 
Published: November 2019

Abstract
Background: Multiple myeloma (MM) is characterised by proliferation of malignant plasma cells (PCs) usually in bone marrow (BM). The mechanisms of extramedullary (EM) spread in MM are poorly understood. It probably involves possible changes in expression of adhesive molecules. An increase incidence of aggressive and mostly resistant EM relapses was reported in the era of new treatment approaches. Finding a marker allowing predict a risk of EM relapse could help to improve treatment strategies and prolong patient life.
Aim: Phenotype profile of PCs in BM and tumor tissue (TU) samples of EM relapses and comparison with "intramedullary" (IM) relapses of MM.
Material and methods: There were analysed 40 patients with confirmed EM relaps and 17 patients with IM relaps (without EM spread). Analysis of 57 BM and 34 TU was done by polychromatic flow cytometry (FC). Phenotype of CD38+CD138+ PC was analysed using CD19, CD27, CD28, CD44, CD56, CD81, CD117 and nestin. PCs were considered positive for given marker when its expression on PCs was higher than 25 %.
Results: There were detected CD38+CD138+ PCs in 95.0 % (38/40) of EM-BM samples (due to sample dilution), in 100 % IM-BM samples and in 88.2 % (30/34) of analysed TU samples (probably acquired not from tumor site). PC infiltration was statistically significantly higher in TU then in EM-BM [median of infiltration 51.7 % (range 0.0-98.9) vs. 1.5 % (0.01-68.9); p<0.001]; IM-BM has higher PC infiltration then EM-BM [5.7 % (0.7-66.1) vs. 1.5 % (0.0-68.9); p<0.05].
Although TU PCs have relatively similar phenotype like EM-BM PCs, there was found significant increase in median of expression of CD81 on abnormal PCs (A-PCs) [2.6 % (0.0-96.9) in EM-BM vs. 11.2 % (0.0-99.2) in TU; p<0.02] and also in number of CD81+ cases (positivity) [20 % (4/20) of EM-BM vs. 41.2 % (7/17) of TU]. Moreover, no presence of CD19+ normal PCs was found in TU even if mixture of CD19+ normal and CD19- A-PCs was detected in EM-BM. TU PCs we almost negative for C27 and CD117 (median of expression 2.6 and 0.1 %; positivity 17.9 and 14.3 %; respectively); slightly positive for CD28 (median of expression 2.6 %; positivity 33.3 %) and usually positive for CD56 and nestin (median of expression 97.8 and 45.7 %; positivity 69.0 and 57.9 %; respectively). There was found significant increase of CD44 and nestin expression in EM-BM when compared to IM-BM [median of expression 91.8 (3.4-99.9) vs. 25.6 (1.4-99.8) %; p<0.04 and 52.7 (0-100) vs. 0.2 (0-99.4) %; p<0.01; respectively], other markers were expressed similarly.
Conclusion: Almost all EM PCs (BM and/or TU) usually express nestin, a marker of stem/progenitor cells, and CD44, a glycoprotein involved in cell-cell interactions, cell adhesion and migration. Adhesion molecule CD56, which loss is considered as a marker of extramedullary spread, was not changed in different MM cases (EM vs. IM). Flow cytometry analysis of EM PCs allowed to identify a phenotype profile of A-PCs (CD19-CD27-CD56+CD81+CD117-CD44+nestin+) related to possible extramedullary involvement in MM.

Disclosures
Hájek: TAKEDA: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Novartis: Research Funding.