Comprehensive detection of recurring genomic abnormalities : a targeted sequencing approach for multiple myeloma

Recent genomic research efforts in multiple myeloma have revealed clinically relevant molecular subgroups beyond conventional cytogenetic classifications. Implementing these advances in clinical trial design and in routine patient care requires a new generation of molecular diagnostic tools. Here, we present a custom capture next-generation sequencing (NGS) panel designed to identify rearrangements involving the IGH locus, arm level, and focal copy number aberrations, as well as frequently mutated genes in multiple myeloma in a single assay. We sequenced 154 patients with plasma cell disorders and performed a head-to-head comparison with the results from conventional clinical assays, i.e., fluorescent in situ hybridization (FISH) and single-nucleotide polymorphism (SNP) microarray. Our custom capture NGS panel had high sensitivity (>99%) and specificity (>99%) for detection of IGH translocations and relevant chromosomal gains and losses in multiple myeloma. In addition, the assay was able to capture novel genomic markers associated with poor outcome such as bi-allelic events involving TP53. In summary, we show that a multiple myeloma designed custom capture NGS panel can detect IGH translocations and CNAs with very high concordance in relation to FISH and SNP microarrays and importantly captures the most relevant and recurrent somatic mutations in multiple myeloma rendering this approach highly suitable for clinical application in the modern era

Integrative analysis of the genomic and transcriptomic landscape of double-refractory multiple myeloma

In multiple myeloma, novel treatments with proteasome inhibitors (PIs) and immunomodulatory agents (IMiDs) have prolonged survival but the disease remains incurable. At relapse, next-generation sequencing has shown occasional mutations of drug targets but has failed to identify unifying features that underlie chemotherapy resistance. We studied 42 patients refractory to both PIs and IMiDs. Whole-exome sequencing was performed in 40 patients, and RNA sequencing (RNA-seq) was performed in 27. We found more mutations than were reported at diagnosis and more subclonal mutations, which implies ongoing evolution of the genome of myeloma cells during treatment. The mutational landscape was different from that described in published studies on samples taken at diagnosis. The TP53 pathway was the most frequently inactivated (in 45% of patients). Conversely, point mutations of genes associated with resistance to IMiDs were rare and were always subclonal. Refractory patients were uniquely characterized by having a mutational signature linked to exposure to alkylating agents, whose role in chemotherapy resistance and disease progression remains to be elucidated. RNA-seq analysis showed that treatment or mutations had no influence on clustering, which was instead influenced by karyotypic events. We describe a cluster with both amp(1q) and del(13) characterized by CCND2 upregulation and also overexpression of MCL1, which represents a novel target for experimental treatments. Overall, high-risk features were found in 65% of patients. However, only amp(1q) predicted survival. Gene mutations of IMiD and PI targets are not a preferred mode of drug resistance in myeloma. Chemotherapy resistance of the bulk tumor population is likely attained through differential, yet converging evolution of subclones that are overall variable from patient to patient and within the same patient

A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

Acute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase inhibitors has demonstrated that targeting of prevalent FMS-related receptor tyrosine kinase 3 (FLT3) gain-of-function mutations can provide significant survival benefits for patients, although the efficacy of FLT3 inhibitors in eliminating FLT3-mutated clones is variable. We identified a T cell receptor (TCR) reactive to the recurrent D835Y driver mutation in the FLT3 tyrosine kinase domain (TCRFLT3D/Y). TCRFLT3D/Y-redirected T cells selectively eliminated primary human AML cells harboring the FLT3D835Y mutation in vitro and in vivo. TCRFLT3D/Y cells rejected both CD34+ and CD34− AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34+ AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo. Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Moving from Cancer Burden to Cancer Genomics for Smoldering Myeloma : a Review

Importance: All patients who develop multiple myeloma have a preceding asymptomatic expansion of clonal plasma cells, clinically recognized as monoclonal gammopathy of undetermined significance or smoldering multiple myeloma (SMM). During the past decade, significant progress has been made in the classification and risk stratification of SMM. Observations: This review summarizes current clinical challenges and discusses available models for risk stratification in the context of SMM. Owing to several novel, more effective, and less toxic drugs, these aspects are becoming increasingly important to identify patients eligible for early treatment. However, all proposed criteria were built around indirect markers of disease burden and therefore are generally able to identify a fraction of patients with SMM in whom transformation to multiple myeloma and genomic subclonal diversification are already happening. In contrast, next-generation sequencing approaches have the potential to identify myeloma precursor disease that will progress even before the major clonal expansion and progression, providing a potential base for more effective treatment and better precision regarding the optimal timing of treatment initiation. Conclusions and Relevance: Owing to modern technologies, in the near future, prognostic models derived from genomic signatures independent of the disease burden will allow better identification of the optimal timing to treat a plasma cell clonal disorder at the very early stages, when the chances of eradication are higher

Early Relapse Risk in Newly Diagnosed Multiple Myeloma Patients Characterized by Next-Generation Sequencing

Purpose: Duration of first remission is important for the survival of patients with multiple myeloma. Experimental design: From the CoMMpass study (NCT01454297), 926 patients with newly diagnosed multiple myeloma, characterized by next-generation sequencing, were analyzed to evaluate those who experienced early progressive disease (PD; time to progression, TTP 6418 months). Results: After a median follow-up of 39 months, early PD was detected in 191/926 (20.6%) patients, 228/926 (24.6%) patients had late PD (TTP >18 months), while 507/926 (54.8%) did not have PD at the current follow-up. Compared with patients with late PD, patients with early PD had a lower at least very good partial response rate (47% vs. 82%, P < 0.001) and more frequently acquired double refractoriness to immunomodulatory drugs (IMiD) and proteasome inhibitors (PI; 21% vs. 8%, P < 0.001). Patients with early PD were at higher risk of death compared with patients with late PD and no PD (HR, 3.65; 95% CI, 2.7-4.93; P < 0.001), showing a dismal median overall survival (32.8 months). In a multivariate logistic regression model, independent factors increasing the early PD risk were TP53 mutation (OR, 3.78, P < 0.001), high lactate dehydrogenase levels (OR, 3.15, P = 0.006), \u3bb-chain translocation (OR, 2.25, P = 0.033), and IGLL5 mutation (OR, 2.15, P = 0.007). Carfilzomib-based induction (OR, 0.15, P = 0.014), autologous stem-cell transplantation (OR, 0.27, P < 0.001), and continuous therapy with PIs and IMiDs (OR, 0.34, P = 0.024) mitigated the risk of early PD. Conclusions: Early PD identifies a high-risk multiple myeloma population. Further research is needed to better identify baseline features predicting early PD and the optimal treatment approaches for patients at risk

Integrative analysis of the genomic and transcriptomic landscape of double-refractory multiple myeloma

In multiple myeloma, novel treatments with proteasome inhibitors (PIs) and immunomodulatory agents (IMiDs) have prolonged survival but the disease remains incurable. At relapse, next-generation sequencing has shown occasional mutations of drug targets but has failed to identify unifying features that underlie chemotherapy resistance. We studied 42 patients refractory to both PIs and IMiDs. Whole-exome sequencing was performed in 40 patients, and RNA sequencing (RNA-seq) was performed in 27. We found more mutations than were reported at diagnosis and more subclonal mutations, which implies ongoing evolution of the genome of myeloma cells during treatment. The mutational landscape was different from that described in published studies on samples taken at diagnosis. The TP53 pathway was the most frequently inactivated (in 45% of patients). Conversely, point mutations of genes associated with resistance to IMiDs were rare and were always subclonal. Refractory patients were uniquely characterized by having a mutational signature linked to exposure to alkylating agents, whose role in chemotherapy resistance and disease progression remains to be elucidated. RNA-seq analysis showed that treatment or mutations had no influence on clustering, which was instead influenced by karyotypic events. We describe a cluster with both amp(1q) and del(13) characterized by CCND2 upregulation and also overexpression of MCL1, which represents a novel target for experimental treatments. Overall, high-risk features were found in 65% of patients. However, only amp(1q) predicted survival. Gene mutations of IMiD and PI targets are not a preferred mode of drug resistance in myeloma. Chemotherapy resistance of the bulk tumor population is likely attained through differential, yet converging evolution of subclones that are overall variable from patient to patient and within the same patient. © 2020 by The American Society of Hematology

Integrative analysis of the genomic and transcriptomic landscape of double-refractory multiple myeloma

In multiple myeloma, novel treatments with proteasome inhibitors (PIs) and immunomodulatory agents (IMiDs) have prolonged survival but the disease remains incurable. At relapse, next-generation sequencing has shown occasional mutations of drug targets but has failed to identify unifying features that underlie chemotherapy resistance. We studied 42 patients refractory to both PIs and IMiDs. Whole-exome sequencing was performed in 40 patients, and RNA sequencing (RNA-seq) was performed in 27. We found more mutations than were reported at diagnosis and more subclonal mutations, which implies ongoing evolution of the genome of myeloma cells during treatment. The mutational landscape was different from that described in published studies on samples taken at diagnosis. The TP53 pathway was the most frequently inactivated (in 45% of patients). Conversely, point mutations of genes associated with resistance to IMiDs were rare and were always subclonal. Refractory patients were uniquely characterized by having a mutational signature linked to exposure to alkylating agents, whose role in chemotherapy resistance and disease progression remains to be elucidated. RNA-seq analysis showed that treatment or mutations had no influence on clustering, which was instead influenced by karyotypic events. We describe a cluster with both amp(1q) and del(13) characterized by CCND2 upregulation and also overexpression of MCL1, which represents a novel target for experimental treatments. Overall, high-risk features were found in 65% of patients. However, only amp(1q) predicted survival. Gene mutations of IMiD and PI targets are not a preferred mode of drug resistance in myeloma. Chemotherapy resistance of the bulk tumor population is likely attained through differential, yet converging evolution of subclones that are overall variable from patient to patient and within the same patient