Timing the initiation of multiple myeloma

The 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 2nd-3rd 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

Mytype targeted next generation sequencing assay for detection of igh translocations and copy number alterations in multiple myeloma

Background: Multiple myeloma is a genetically complex disease where early hits include chromosome 14 (IGH) translocations or hyperdiploidy. Later events are additional copy number alterations (CNAs), e.g. gain 1q, deletion 13q or 17p as well as somatic mutations. Currently, translocations and CNAs are commonly assessed via fluorescent in situ hybridization (FISH) in the clinical setting. Recently, targeted DNA assays have been evaluated in myeloma cell lines and showed correlation with conventional methods. Aims: To interrogate genomic aberrations in primary samples from patients with multiple myeloma and we developed a targeted next generation sequencing panel, called myTYPE, and compared detection rates to those from FISH. Methods: In the myTYPE assay, baits were designed to capture the entire IGH locus where the vast majority of the chromosome 14 breakpoints occur, genome wide single nucleotide polymorphisms (SNPs) for hyperdiploidy and other CNAs, as well as exons of 120 frequently mutated somatic genes in multiple myeloma. To validate the capture of IGH translocations and CNAs using myTYPE, 46 samples from 22 patients with multiple myeloma as well as bone marrow samples from 16 healthy individuals were analyzed. All samples were sequenced using 126 bp paired end reads using Illumina HiSeq with a mean target depth of ~600x. All patient samples contained a high percentage of plasma cells. After sequencing, CNAs and translocations were identified using validated bioinformatic algorithms such as CNVkit, Brass and Delly. Results: We found that the targeted sequencing assay myTYPE was equally good or better for detecting IGH translocations and CNAs compared to FISH. Using myTYPE, a higher number of t(4;14) and t(11;14) and equally many t(14;16) were captured using myTYPE in relation to FISH. (Table 1) Hyper- diploidy was detected in 14% and 22% with FISH and myTYPE, respectively. Greater numbers of 1q gains and 13/13q deletions were found using myTYPE while the number of 17p deletions were similar. Since myTYPE covers genome-wide SNPs, this assay was able to detect additional CNAs such as 6q deletion, 8p deletion, 16q gain, and trisomy 8 that were not targeted by FISH. In 4 multiple myeloma patients enrolled on a study protocol allowing for sev- eral parallel bone marrow/extramedullary disease biopsies in the same patient, the same IGH translocations and CNAs were detected across all sites of extramedullary disease except in two samples where a 6q deletion and 8p deletion were not detected in one sample each. This is likely explained by clonal heterogeneity, i.e. that the CNA was not present since we have no rea- son to suspect sequencing failure in these samples. Summary and Conclusions: In this analysis, based on the targeted DNA capture assay myTYPE used head-to-head with FISH on primary samples from patients with plasma cell myeloma, we found that the sensitivity for detecting IGH translocations and CNAs was higher using the targeted sequencing approach as compared to FISH. Furthermore, using myTYPE, we found additional CNAs not covered by FISH, as FISH detects only the probe-specific alterations. Translocations and CNAs can be detected with high sensitivity also using whole genome sequencing, however, targeted sequencing is less complex and more cost-effective. In summary, targeted sequencing using myTYPE is an effective and sensitive approach to assess IGH translocations and chromosomal gains and losses in multiple myeloma and there are ongoing efforts to implement sequencing-based assays to replace FISH in the clinical setting

A niche-dependent myeloid transcriptome signature defines dormant myeloma cells

The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.Weng Hua Khoo … Khatora S. Opperman, Kate Vandyke … Justine R. Clark … Duncan R. Hewett … Andrew C. W. Zannettino … et al

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

A niche-dependent myeloid transcriptome signature defines dormant myeloma cells

© 2019 by The American Society of Hematology. The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells

Dissecting intratumour heterogeneity of nodal B-cell lymphomas at the transcriptional, genetic and drug-response levels

Tumour heterogeneity encompasses both the malignant cells and their microenvironment. While heterogeneity between individual patients is known to affect the efficacy of cancer therapy, most personalized treatment approaches do not account for intratumour heterogeneity. We addressed this issue by studying the heterogeneity of nodal B-cell lymphomas by single-cell RNA-sequencing and transcriptome-informed flow cytometry. We identified transcriptionally distinct malignant subpopulations and compared their drug-response and genomic profiles. Malignant subpopulations from the same patient responded strikingly differently to anti-cancer drugs ex vivo, which recapitulated subpopulation-specific drug sensitivity during in vivo treatment. Infiltrating T cells represented the majority of non-malignant cells, whose gene-expression signatures were similar across all donors, whereas the frequencies of T-cell subsets varied significantly between the donors. Our data provide insights into the heterogeneity of nodal B-cell lymphomas and highlight the relevance of intratumour heterogeneity for personalized cancer therapy. Roider et al. combine scRNA-seq and transcriptome-informed flow cytometry, and uncover transcriptionally different malignant subclones with distinct drug responses and T-cell profiles in B-cell non-Hodgkin lymphoma