Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma

Multiple myeloma, a plasma cell malignancy, is the second most common blood cancer. Despite extensive research, disease heterogeneity is poorly characterized, hampering efforts for early diagnosis and improved treatments. Here, we apply single cell RNA sequencing to study the heterogeneity of 40 individuals along the multiple myeloma progression spectrum, including 11 healthy controls, demonstrating high interindividual variability that can be explained by expression of known multiple myeloma drivers and additional putative factors. We identify extensive subclonal structures for 10 of 29 individuals with multiple myeloma. In asymptomatic individuals with early disease and in those with minimal residual disease post-treatment, we detect rare tumor plasma cells with molecular characteristics similar to those of active myeloma, with possible implications for personalized therapies. Single cell analysis of rare circulating tumor cells allows for accurate liquid biopsy and detection of malignant plasma cells, which reflect bone marrow disease. Our work establishes single cell RNA sequencing for dissecting blood malignancies and devising detailed molecular characterization of tumor cells in symptomatic and asymptomatic patients

Early Dynamics and Depth of Response in Multiple Myeloma Patients Treated With BCMA CAR-T Cells

Chimeric antigen receptor T-cell (CAR-T) therapy targeted against B-cell maturation antigen (BCMA) in multiple myeloma (MM) has produced rapid responses but many eventually relapse. In light of this new treatment, novel predictors of progression-free survival (PFS) are needed. We performed a single institution analysis of 54 BCMA-CAR-T patients. We analyzed patient's overall response rate (ORR) by the IMWG criteria, involved serum-free light chains (iFLC), and minimal residual disease testing by next-generation sequencing (MRD-NGS). Between patients who achieved a ≤SD and those who achieved a ≥PR, PFS differed significantly (p < 0.0001); though there was no difference between patients who achieved a ≥CR vs. VGPR/PR (p = 0.2). In contrast, patients who achieved a nonelevated iFLC at 15 days (p < 0.0001, HR = 6.8; 95% CI, 2.7-17.3) or 30 days (p < 0.001, HR = 16.7; 95% CI, 3.9-71.7) had a prolonged PFS compared with those with an elevated iFLC. Patients achieving MRD-NGS less than the detectable limit at a sensitivity of 10-6 had a better PFS than those with detectable disease at 1 month (p = 0.02) and 3 months (p = 0.02). In conclusion, achieving a nonelevated iFLC and an undetectable MRD-NGS quickly were factors that were strongly associated with improved PFS. Further studies are needed to confirm the role of these markers in MM patients receiving CAR-T therapies

Mytype: A Capture Based Sequencing Approach to Detect Somatic Mutations, Copy Number Changes and IGH Translocations in Multiple Myeloma

At diagnosis, Multiple Myeloma (MM) is traditionally classified into two clinical and prognostic subgroups groups on the basis of initiating cytogenetic abnormalities: IGH translocations and hyperdiploidy. Currently, these events are clinically ascertained by Fluorescent In-Situ Hybridization (FISH). In recent years, comprehensive genome profiling studies have shown that MM pathogenesis is defined by a spectrum of acquired somatic lesions, many of which are biologically and clinically relevant. To this effect, targeted gene sequencing approaches are becoming routine in the upfront diagnostic settings. Here we present myTYPE, a MM-specific targeted next generation sequencing panel to identify germline and somatic substitutions, indels, Copy Number Aberrations (CNA) and IGH translocations. Methods A multiplex bait panel was designed to capture the exons of 120 genes implicated in MM pathogenesis, entire IGH locus as well as genome wide representation of single nucleotide polymorphisms (SNPs) (1 in 3Mb) to enable detection of arm level copy number events and recurrent focal events. These 120 genes were selected on the basis of 1) frequently mutated and driver genes in MM 2) genes in important signaling pathways, e.g the NFKB pathway 3) treatment targets and candidate genes for drug resistance, e.g. cereblon.To validate the efficacy of the assay, 16 constitutional bone marrow samples and 18 tumor samples were sequenced using myTYPE. For validation, 6/18 tumor/normal pairs sequenced using myTYPE were subject to WGS and remaining 12/18 tumor samples were subject to FISH. After sequencing, we obtained an overall median target coverage of 815x. Results After alignment, substitutions and indels were called using Caveman, Pindel and Strelka. CNAs were identified using Facets and IGH translocations were identified using Delly along with a modified version of BRASS. Below is a description of the genomic abnormalities captured by the myTYPE assay. SNVs and Indels For the 6 tumor/normal pairs sequenced using myTYPE and WGS, we obtained a total of 21 (median = 3) non-synonymous mutations using myTYPE. When limiting the WGS calls to myTYPE targets, we recovered 20/21 non-synonymous mutations identified by myTYPE. These involved SNVs and indels in key MM related drivers including NRAS, KRAS, FAM46C and TP53 among others. For the mutations identified by both myTYPE and WGS, there was a high correlation between the variant VAFs, R2 = 0.99 and as expected is better in capturing subclonal mutations. IGH rearrangements and Copy Number Aberrations (CNA) Next we compared myTYPE and WGS results for recurrent CNAs in MM. We specifically looked at deletions of 1p, 13p, 16q, 17p and gains of 1q, 11q and found a 100% concordance of these aberrations identified by both assays. The remaining 12 samples sequenced using myTYPE also had orthogonal FISH. myTYPE identified a total of 7 IGH rearrangements, 4 of which are also reported by FISH. Three additional t(11;14) translocations were uniquely identified by myTYPE in cases that remained clinically uncharacterized. FISH was also used to probe deletions in 17q, 13q, 1p and 1q gain. All aberrations identified by FISH were also identified in myType. Additionally, 13q- in four samples and 1p- in one sample were uniquely identified by myTYPE. Conclusion In summary, we present a targeted assay capable of identifying somatic mutations, CNAs and IGH translocations of prognostic and diagnostic relevance in MM. When compared to conventional assays currently used in clinical practice, myTYPE identified at least one disease defining alterations in all samples screened. Evaluation of sensitivity and specificity will require larger clinical cohorts. Importantly, myTYPE enables comprehensive profiling, large sample multiplexing and short turn around times which renders it as an optimal assay for utilisation in the upfront clinical setting

Distinct bone marrow blood vessels differentially regulate haematopoiesis

Bone marrow (BM) endothelial cells (BMECs) form a network of blood vessels (BVs) which regulate both leukocyte trafficking and hematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles and if these events occur at the same vascular site. We found that BM stem cell maintenance and leukocyte trafficking are regulated by distinct BV types with different permeability properties. Less permeable arterial BVs maintain HSCs in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the BM. A functional consequence of high BVs permeability is that exposure to blood plasma increases BM HSPC ROS levels, augmenting their migration capacity while compromising their long term repopulation and survival potential. These findings may have relevance for clinical hematopoietic stem cell transplantation and mobilization protocols.Stem Cell and Regenerative Biolog

Timing the initiation of multiple myeloma

International audienceThe evolution and progression of multiple myeloma and its precursors over time is poorly understood. Here, we investigate the landscape and timing of mutational processes shaping multiple myeloma evolution in a large cohort of 89 whole genomes and 973 exomes. We identify eight processes, including a mutational signature caused by exposure to melphalan. Reconstructing the chronological activity of each mutational signature, we estimate that the initial transformation of a germinal center B-cell usually occurred during the first 2 nd -3 rd decades of life. We define four main patterns of activation-induced deaminase (AID) and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutagenesis over time, including a subset of patients with evidence of prolonged AID activity during the pre-malignant phase, indicating antigen-responsiveness and germinal center reentry. Our findings provide a framework to study the etiology of multiple myeloma and explore strategies for prevention and early detection