Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells
Authors: Rodríguez Sara , Botta Cirino , Celay Jon , Goicoechea Ibai , Garcia-Barchino Maria J , Valdés-Mas Rafael , Carrasco Yolanda , Alameda Daniel , Garcés Juan José , Larrayoz Marta , Santos Susana , Alignani Diego , Vilas-Zornoza Amaia , Lorenzo-Vivas Erika , Pérez Ruiz Cristina , Garate Sonia , Sarai Sarvide , Gascue Amaia , Reinhardt Christian , Sanchez-Garcia Isidro , Larrayoz Maria Jose , Calasanz Maria Jose , Prosper Felipe , Motta Marina , Anastasia Antonella , Sacco Antonio , Gentile Massimo , Duarte Sara , Vitoria Helena , Geraldes Catarina , Paiva Artur , Roccaro Aldo M. , San-Miguel Jesús , Martinez-Climent Jose Angel , Paiva Bruno 
Published: November 2019

Background: Although MYD88 L265P is highly frequent in WM, by itself is insufficient to explain disease progression since most cases with IgM MGUS also have mutated MYD88. In fact, the percentage of MYD88 L265P in CD19+ cells isolated from WM patients is typically &lt;100%, which questions if this mutation initiates the formation of B-cell clones. Furthermore, a few WM patients have detectable MYD88 L265P in total bone marrow (BM) cells and not in CD19+ selected B cells, raising the possibility that other hematopoietic cells carry the MYD88 mutation. However, no one has investigated if the pathogenesis of WM is related to somatic mutations occurring at the hematopoietic stem cell level, similarly to what has been shown in CLL or hairy cell leukemia.
Aim: Define the cellular origin of WM by comparing the genetic landscape of WM cells to that of CD34 progenitors, B cell precursors and residual normal B cells.
Methods: We used multidimensional FACSorting to isolate a total of 43 cell subsets from BM aspirates of 8 WM patients: CD34+ progenitors, B cell precursors, residual normal B cells (if detectable), WM B cells, plasma cells (PCs) and T cells (germline control). Whole-exome sequencing (WES, mean depth 74x) was performed with the 10XGenomics Exome Solution for low DNA-input due to very low numbers of some cell types. We also performed single-cell RNA and B-cell receptor sequencing (scRNA/BCRseq) in total BM B cells and PCs (n=32,720) from 3 IgM MGUS and 2 WM patients. Accordingly, the clonotypic BCR detected in WM cells was unbiasedly investigated in all B cell maturation stages defined according to their molecular phenotype. In parallel, MYD88p.L252P (orthologous position of the human L265P mutation) transgenic mice were crossed with conditional Sca1Cre, Mb1Cre, and Cγ1Cre mice to selectively induce in vivo expression of MYD88 mutation in CD34 progenitors, B cell precursors and germinal center B cells, respectively. Upon immunization, mice from each cohort were necropsied at 5, 10 and 15 months of age and screened for the presence of hematological disease.
Results: All 8 WM patients showed MYD88 L265P and 3 had mutated CXCR4. Notably, we found MYD88 L265P in B cell precursors from 1/8 cases and in residual normal B cells from 3/8 patients, which were confirmed by ASO-PCR. In addition, CXCR4 was simultaneously mutated in B cell precursors and WM B cells from one patient. Overall, CD34+ progenitors, B-cell precursors and residual normal B cells shared a median of 1 (range, 0-4; mean VAF, 0.16), 2 (range, 1-5; mean VAF, 0.14), and 4 (range, 1-13; mean VAF, 0.26) non-synonymous mutations with WM B cells. Some mutations were found all the way from CD34+ progenitors to WM B cells and PCs. Interestingly, concordance between the mutational landscape of WM B cells and PCs was &lt;100% (median of 85%, range: 25%-100%), suggesting that not all WB B cells differentiate into PCs.
A median of 7 (range, 2-19; mean VAF, 0.39) mutations were unique to WM B cells. Accordingly, many clonal mutations in WM B cells were undetectable in normal cells. Thus, the few somatic mutations observed in patients' lymphopoiesis could not result from contamination during FACSorting since in such cases, all clonal mutations would be detectable in normal cells. Of note, while somatic mutations were systematically detected in normal cells from all patients, no copy number alterations (CNA) present in WM cells were detectable in normal cells. scRNA/BCRseq unveiled that clonotypic cells were confined mostly within mature B cell and PC clusters in IgM MGUS, whereas a fraction of clonotypic cells from WM patients showed a transcriptional profile overlapping with that of B cell precursors.
In mice, induced expression of mutated MYD88 led to a moderate increase in the number of B220+CD138+ plasmablasts and B220-CD138+ PCs in lymphoid tissues and BM, but no signs of clonality or hematological disease. Interestingly, such increment was more evident in mice with activation of mutated MYD88 in CD34+ progenitors and B-cell precursors vs mice with MYD88 L252P induced in germinal center B cells.
Conclusions: We show for the first time that WM patients have somatic mutations, including MYD88 L265P and in CXCR4, at the B cell progenitor level. Taken together, this study suggests that in some patients, WM could develop from B cell clones carrying MYD88 L265P rather than it being the initiating event, and that other mutations or CNA are required for the expansion of B cells and PCs with the WM phenotype.

Disclosures
Roccaro: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: 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.