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509 The Pathogenesis of Multiple Myeloma (MM) Is Preceded By Mutated Lymphopoiesis and B Cell Oligoclonality That Persist in Patients with Negative Minimal Residual Disease (MRD)

Program: Oral and Poster Abstracts
Type: Oral
Session: 651. Myeloma: Biology and Pathophysiology, excluding Therapy: Pathogenesis and Bone Marrow Microenvironment
Hematology Disease Topics & Pathways:
Diseases, multiple myeloma, Biological Processes, Technology and Procedures, Plasma Cell Disorders, Lymphoid Malignancies, Clinically relevant, genomics, genetic profiling, hematopoiesis, flow cytometry, RNA sequencing
Sunday, December 8, 2019: 5:30 PM
Hall E2, Level 2 (Orange County Convention Center)

Sara Rodríguez, PhD1*, Noemi Puig, MD, PhD2*, Katerina Gemenetzi3*, Ibai Goicoechea, PhD4, Cirino Botta, MD5, Andreas Agathangelidis, PhD3*, Rafael Valdés-Mas6*, Juan José Garcés4*, María Teresa Cedena7*, José J Pérez8*, Leire Burgos4*, Maria Jose Calasanz, PhD, BSc9,10*, Diego Alignani, PhD4*, Amaia Vilas-Zornoza, PhD4*, Erika Lorenzo-Vivas, PhD4*, Irene Aires2*, Idoia Rodriguez4*, Sarvide Sarai4*, Felipe Prosper, MD4,10,11*, Jose Angel Martinez-Climent, MD, PhD4,11,12*, Alberto Orfao, MD, PhD13, Ramón García-Sanz, MD, PhD2*, Joaquin Martinez-Lopez, MD, PhD14*, Juan-José Lahuerta, MD, PhD15*, Laura Rosinol Dachs16*, Joan Bladé, MD16*, Maria-Victoria Mateos2, Anastasia Hadzidimitriou, PhD3*, Jesus San-Miguel, MD, PhD4,10,11 and Bruno Paiva, PhD4,11,17,18*

1Centro de Investigación Médica Aplicada, University of Navarra, Clínica Universidad de Navarra, PAMPLONA, Spain
2Departamento de Hematología, Hospital Universitario de Salamanca (HUSAL), IBSAL, IBMCC (USAL-CSIC), CIBERONC, Salamanca, Spain
3Institute of Applied Biosciences (INAB), Centre for Research and Technology Hellas (CERTH), Thessaloniki, Greece
4Centro de Investigación Médica Aplicada, University of Navarra, Clínica Universidad de Navarra, Pamplona, Spain
5DMSC, Magna Graecia University of Catanzaro, Catanzaro, Italy
6Dreamgenics, Oviedo, ESP
7Hospital Universitario 12 de Octubre, Madrid, Spain
8Hospital Universitario de Salamanca (IBSAL). Centro de Investigación del Cáncer (IBMCC-CSIC). Universidad de Salamanca., Salamanca, Spain
9Scientific co-Director of CIMA LAB Diagnostics, CIMA Lab Diagnostics, University of Navarra, Pamplona, Spain
10Centro de Investigación Biomédica en Red de Cáncer, CIBERONC, Pamplona, Spain
11Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain
12Centro de Investigación Biomédica en Red de Cancer, CIBERONC, Pamplona, Spain
13Cancer Research Centre (IBMCC, USAL-CSIC), Department of Medicine and Cytometry Service (NUCLEUS), University of Salamanca and IBSAL, Salamanca, Spain
14Hematology Department Hospital 12 de Octubre, Complutense University, H12O-CNIO Clinical research unit, CIBERONC, Madrid, Spain
15Hospital 12 de Octubre, CIBERONC, Madrid, Spain
16Hospital Clínica de Barcelona - Servicio de Onco-Hematología, Barcelona, Spain
17CIMA Lab Diagnostics, University of Navarra, Pamplona, Spain
18Centro de Investigación Biomédica en Red de Cáncer, CIBERONC, Madrid, Spain

In MM patients relapsing after MRD-negativity, the disease could reemerge from immature cells or from undetectable MRD. However, it remains unknown if immature cells have the same genetic background as MM plasma cells (PCs), as well as the amount of MRD that persists below the limit of detection (LOD) of next-generation techniques.

To obtain further insight, we compared the biological landscape of MM PCs at diagnosis to that of CD34 progenitors, B cells and normal PCs isolated from patients with negative MRD by next-generation flow (NGF) after treatment.

We performed whole-exome sequencing (WES, mean depth: 90x) with the 10XGenomics Exome Solution for low DNA-input as well as deep NGS of B-cell receptor immunoglobulin (BcR IG) gene rearrangements (mean, 69,975 sequences), in a total of 68 cell-samples isolated from the bone marrow (BM) of 7 MM patients with MRD-negativity by EuroFlow NGF after induction with VRD and auto-transplant (GEM2012MENOS65 trial). Patients with negative MRD were intentionally selected to avoid contamination with MM PCs during sorting of CD34 progenitors, B-cell precursors, mature B cells and normal PCs after induction and transplant. We investigated in these populations the presence of somatic mutations and clonotypic BcR Ig rearrangements detectable in MM PCs sorted at diagnosis, using peripheral blood T cells as germline control. We also performed WES in matched diagnostic MM PCs and MRD cells persisting after VRD induction in 14 cases as control. In another 6 patients with untreated MM, we performed single-cell RNA and BcR IG sequencing (scRNA/BcRIGseq) of total BM B cells and PCs (n=16,380) to investigate before treatment, if the clonotypic BcR IG sequence of MM PCs was detectable in other B cell stages defined by their molecular phenotype. We used multidimensional flow cytometry (MFC) to investigate the frequency of B cell clonality in BM samples from a larger series of 195 newly-diagnosed MM patients, prospectively enrolled in the GEM-CLARIDEX trial.

Somatic mutations present in diagnostic MM PCs were detectable in the lymphopoiesis of 5/7 patients achieving MRD-negativity after treatment. In one case, out of 55 mutations present in diagnostic MM PCs, a single mutation in PCSK1N (VAF: 0.30) was detectable in normal PCs. In the other four patients, a total of 85 mutations were present in MM PCs and up to 10 (median VAF, 0.16) were found all the way from CD34 progenitors into B-cell precursors, mature B cells and normal PCs, but not in T cells. Of note, most mutations were reproducibly detected in each cell type after induction and after transplant. All somatic mutations shared by MM PCs and normal cells were non-recurrent, and genes recurrently mutated in MM (eg. ACTG1, ATM, DIS3, FAM46C, KRAS, LTB, MAX, TRAF3) were found in MM PCs but never in normal cells. Copy number alterations (CNA) were found only in MM PCs. By contrast, up to 513/827 (62%) mutations and 48/67 (72%) CNA were detectable in matched diagnostic MM PCs and persistent MRD cells, indicating that the few somatic variants present in normal cells were unlikely related to contaminating MRD below NGF’s LOD. Accordingly, MM clonotypic BcR IG rearrangements were detectable in normal PCs (4/7patients) and in immature B cells (5/7 patients) but at much lower frequencies (mean of 0.02% in both). Of note, 9 additional clonotypes (mean 8.4%) were found in MM PCs of 5/7 patients (range, 1-3). scRNR/BcRIGseq unveiled that clonotypic cells were confined mostly but not entirely within PC clusters, and that in 1 patient another clonotype was detectable in mature B cells. Accordingly, using MFC we found in a larger series that 25/195 (13%) of newly-diagnosed MM patients display B-cell clonality (median of 0.7% BM clonal B cells, range 0.02%-6.3%).

In conclusion, we show for the first time that MM patients bear somatic mutations in CD34 progenitors that specifically differentiate into the B cell lineage, likely before the disease onset. Because diagnostic, MRD (and relapse) MM PCs display great genetic similarity, these results suggest that undetectable MRD <10-6 rather than normal cells with a few non-recurrent mutations are responsible for relapses after MRD-negativity. This study also challenges our understanding of myelomagenesis and clonal heterogeneity, and proposes that mutated lymphopoiesis may increase risk of developing B cell and PC oligoclonality, which precedes secondary driver mutations or CNA leading to the expansion of MM PCs.

Disclosures: Puig: The Binding Site: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Martinez-Lopez: BMS: Honoraria, Other: Advisory boards; Janssen: Honoraria, Other: Advisory boards and Non-Financial Support ; Amgen: Honoraria, Other: Non-Financial Support ; Celgene: Honoraria, Other: Advisory boards and Non-Financial Support ; Incyte: Honoraria, Other: Advisory boards; Novartis: Honoraria, Other: Advisory boards; VIVIA Biotech: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria. Lahuerta: Takeda, Amgen, Celgene and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosinol Dachs: Janssen, Celgene, Amgen and Takeda: Honoraria. Bladé: Jansen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. Mateos: EDO: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees. San-Miguel: Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

*signifies non-member of ASH