A Maturation Index Defines Newly Diagnosed Multiple Myeloma Patients with Advanced Immunophenotypic and Molecular Differentiation Profiles Associated with Poor Prognosis
Authors: Martello Marina , Termini Rosalinda , Borsi Enrica , Solli Vincenza , Poletti Andrea , Cifarelli Luca , Agboyi Lakpo , Armuzzi Silvia , Pantani Lucia , Rocchi Serena , Mancuso Katia , Zamagni Elena , Tacchetti Paola , Arpinati Mario , Chirumbolo Gabriella , Orlando Antonella , Marzocchi Giulia , Testoni Nicoletta , Zinzani Pier Luigi Luigi , Cavo Michele , Terragna Carolina 
Published: November 2019

Introduction
The differentiation status plasticity of Multiple Myeloma (MM) plasma cells (PCs) is an adaptive strategy that might confer a specific fitness to tumour cells, enabling their interaction with an evolving microenvironment. Therefore, proliferation rates of MM clones are not constant, and the immunophenotypic profile of the most skilled MM PCs might be the expression of specific genetic and genomic programs, that emerge under the therapeutic pressure and promote tumour development. However, the genomic background that supports any diverse plasma cell differentiation phenotypes has not yet been inferred.
Aim
To correlate the genetic and genomic background with the immunophenotypic profile of MM clones at diagnosis, in order to stratify patients (pts) according to a Maturation Index, and ultimately to evaluate the impact of this stratification on the disease outcome.
Patients and Methods
117 newly diagnosed MM (NDMM) pts, mostly treated up-front with a proteasome inhibitor (PI) -based treatment, were included in the study. For each pts, both neoplastic PCs and CD19+ B cells compartments were characterized by 8-color multi-parameter flow cytometry analysis, combining CD138-PE, CD38-PE-Cy7, CD56-APC-Cy7, CD20-APC, CD19-FITC, CD27-APC, CD45-PerCp, CD28-APC, CD117-FITC, CD28-APC, CD81-APC, IgK-APC and IgL-FITC. In a subgroup of pts, both a SNP array profile for copy number alterations (CNAs) and a 25-genes targeted mutational panel were assessed in CD138+ PCs. A custom ddPCR assay was also employed to evaluate through a 10-Hh gene signature the self-renewal status of PCs.
Results
In order to define a Maturation Index, pts were evaluated at different levels of heterogeneity for: a) differential expression of CD19/CD81 markers; b) level of chromosomal instability (CIN) and point mutations; d) self-renewal status.
Based on the co-expression of CD19 and CD81 markers, pts were stratified in 3 different subgroups, recapitulating a progressive PC maturation process: the most immature, which included pts with CD19+/CD81+ PCs (18/117 = 15%), the intermediate, including CD19-/CD81+ PCs (42/117 = 36%) and the most mature, including CD19-/CD81- PCs (57/117 = 49%). The advanced PC differentiation status characterizing this latter subgroup was further confirmed by the high expression of CD28 and CD44, and the reduced expression of CD20, CD27 and CD45, commonly associated with less mature stages of the disease (p<.05). Accordingly, a reduced CD138-/low/CD19+/CD20-/CD38high plasmablastic population was observed, reflecting a more quiescent immature reservoir pool in these more differentiated PCs (5,1% vs. 0,5%; p<.05).
CIN was defined according both to the total CNAs count and to the number of chromosomes' breakpoints (BPs). Pts with an advanced differentiation status displayed a higher number of total CNAs and BPs (median tot. CNAs = 550 vs. 105, and median BPs nr. = 18.5 vs. 8 in CD19-81- and CD19+81+ pts, respectively, p<.05). Interestingly, 16q deletion (CYLD, WWOX, FANCA) and 17p deletion (TP53) were the most recurrent abnormalities in mature PCs (p<.05). Genomic instability was also confirmed by a higher incidence of clonal pathogenic mutations in critical genes (e.g. NRAS, KRAS, TP53). Interestingly, the application of a 10 Hh-genes signature, resuming the Hedgehog pathway, demonstrated that PCs with more advanced differentiation status displayed a substantial overexpression of all the genes, indicating a more proliferative, aggressive and, possibly, persistent phenotype.
Finally, a more mature PC clone was significantly related to a higher prevalence of unfavourable features at baseline (e.g. ≥ 3 PET lesions, ≥ 100 k/l ratio, ISS III disease stage p<.05), ultimately resulting in shorter progression-free (HR=3.5; CI:1.3-9.4; p= 0.088) and overall survival (median not reached, p = 0.005).
Conclusions
A Maturation Index defined NDMM pts with an advanced differentiation status both at immunophenotypic and molecular level, finding lastly associated with a prevalence of poor prognostic features. Chromosomal instability, together with cellular phenotypic plasticity, represents an important, yet poorly defined, mechanism by which MM clones accelerate their own evolution and survival.
Acknowledgements: AIRC_IG2014-15839, RF-2016-02362532.

Disclosures
Mancuso: Celgene: Honoraria, Speakers Bureau; Amgen: Honoraria; Takeda: Honoraria; Janssen: Honoraria. Zamagni:Sanofi: Honoraria, Other: Advisory Board, Speakers Bureau; Celgene Corporation: Honoraria, Other: Advisory board, Speakers Bureau; Janssen: Honoraria, Other: Advisory board, Speakers Bureau; Amgen: Honoraria, Other: Advisory board, Speakers Bureau; BMS: Honoraria, Other: Advisory Board, Speakers Bureau; Takeda: Honoraria, Speakers Bureau. Tacchetti:Oncopeptides: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; BMS: Honoraria; Celgene: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Janssen: Honoraria. Zinzani:CELGENE: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PORTOLA: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ROCHE: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GILEAD: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; JANSSEN-CILAG: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SANDOZ: Membership on an entity's Board of Directors or advisory committees; IMMUNE DESIGN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSAPHARMA: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; KYOWA KIRIN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SANOFI: Consultancy; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELLTRION: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VERASTEM: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.