Blood Advances, 2021
Tazemetostat represents the first epigenetic therapy approved for the treatment of follicular lymphoma (FL). It inhibits the activity of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase, the first of a multitude of epigenetic regulators that have been identified as recurrently mutated in FL and germinal center diffuse large B-cell lymphoma. In this review, we discuss the initial discovery and ongoing exploration of the functional role of EZH2 mutations in lymphomagenesis. We also explore the path from the preclinical development of tazemetostat to its approval for the treatment of relapsed FL, and potential future therapeutic applications. We discuss the clinical data that led to the approval of tazemetostat and ongoing research into the function of EZH2 and of tazemetostat in lymphomas that derive from the germinal center, which could increase the applicability of this drug in the future.
The Journal of pathology, 2019
Despite being the most common childhood bone tumor, the genomic characterization of osteosarcoma remains incomplete. In particular, very few osteosarcoma metastases have been sequenced to date, critical to better understand mechanisms of progression and evolution in this tumor. We performed an integrated whole genome and exome sequencing analysis of paired primary and metastatic pediatric osteosarcoma specimens to identify recurrent genomic alterations. Sequencing of 13 osteosarcoma patients including 13 primary, 10 metastatic, and 3 locally recurring tumors revealed a highly heterogeneous mutational landscape, including cases of hypermutation and microsatellite instability positivity, but with virtually no recurrent alterations except for mutations involving the tumor suppressor genes RB1 and TP53. At the germline level, we detected alterations in multiple cancer related genes in the majority of the cohort, including those potentially disrupting DNA damage response pathways. Metastases retained only a minimal number of short variants from their corresponding primary tumors, while copy number alterations showed higher conservation. One recurrently amplified gene, KDR, was highly expressed in advanced cases and associated with poor prognosis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/THs) include a group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcomes after standard chemoimmunotherapy. We recently described a gene expression signature that identifies 27% of germinal center B-cell DLBCLs (GCB-DLBCLs) as having a double-hit-like expression pattern (DHITsig) and inferior outcomes; however, only half of these cases have both MYC and BCL2 translocations identifiable using standard breakapart fluorescence in situ hybridization (FISH). Here, 20 DHITsig+ GCB-DLBCLs apparently lacking MYC and/or BCL2 rearrangements underwent whole-genome sequencing. This revealed 6 tumors with MYC or BCL2 rearrangements that were cryptic to breakapart FISH. Copy-number analysis identified 3 tumors with MYC and 6 tumors with MIR17HG gains or amplifications, both of which may contribute to dysregulation of MYC and its downstream pathways. Focal deletions of the PVT1 promoter were observed exclusively among DHITsig+ tumors lacking MYC translocations; this may also contribute to MYC overexpression. These results highlight that FISH fails to identify all HGBL-DH/THs, while revealing a range of other genetic mechanisms potentially underlying MYC dysregulation in DHITsig+ DLBCL, suggesting that gene expression profiling is more sensitive for identifying the biology underlying poor outcomes in GCB-DLBCL.
Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as , , and , we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.
The Journal of molecular diagnostics : JMD, 2019
Recurrent activating point mutations in KRAS are critical drivers in pancreatic cancer and have been attributed to resistance to anti-epidermal growth factor receptor therapy in colorectal cancer. Although KRAS genotyping provides limited clinical utility in the diagnosis and management of pancreatic cancer patients at present, inferences about the fractional abundance of KRAS mutations may inform on tumor purity in traditionally challenging clinical specimens and their potential use in precision medicine. KRAS genetic testing has indeed become an essential tool to guide treatment decisions in colorectal cancer, but an unmet need for methods standardization exists. Here, we present a unique droplet digital PCR method that enables the simultaneous detection and quantification of KRAS exon 2, 3, and 4 point mutations and copy number alterations. We have validated 13 mutations (G12S, G12R, G12D, G12A, G12V, G12C, G13D, G60V, Q61H, Q61L, A146V, A146T, and A146P) and focal KRAS amplifications by conducting this assay in a cohort of 100 DNA samples extracted from fresh frozen tumor biopsies, formaldehyde-fixed, paraffin-embedded tissue, and liquid biopsy specimens. Despite its modest lower limit of detection (approximately 1%), this assay will be a rapid cost-effective means to infer the purity of biopsy specimens carrying KRAS mutations and can be used in noninvasive serial monitoring of circulating tumor DNA to evaluate clinical response and/or detect early signs of relapse.
Methods in molecular biology (Clifton, N.J.), 2019
Liquid biopsies are rapidly emerging as powerful tools for the early detection of cancer, noninvasive genomic profiling of localized or metastatic tumors, prompt detection of treatment resistance-associated mutations, and monitoring of therapeutic response and minimal residual disease in patients during clinical follow-up. Growing evidence strongly supports the utility of circulating tumor DNA (ctDNA) as a biomarker for the stratification and clinical management of lymphoma patients. However, ctDNA is diluted by variable amounts of cell-free DNA (cfDNA) shed by nonneoplastic cells causing a background signal of wild-type DNA that limits the sensitivity of methods that rely on DNA sequencing. Here, we describe an error suppression method for single-molecule counting that relies on targeted sequencing of cfDNA libraries constructed with semi-degenerate barcode adapters. Custom pools of biotinylated DNA baits for target enrichment can be designed to specifically track somatic mutations in one patient, survey mutation hotspots with diagnostic and prognostic value or be comprised of comprehensive gene panels with broad patient coverage in lymphoma. Such methods are amenable to track ctDNA levels during longitudinal liquid biopsy testing with high specificity and sensitivity and characterize, in real time, the genetic profiles of tumors without the need of standard invasive biopsies. The analysis of ultra-deep sequencing data according to the bioinformatics pipelines also described in this chapter affords to harness lower limits of detection for ctDNA below 0.1%.
Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 2019
High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression.
Nature communications, 2018
Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.
Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.
Nature genetics, 2010
Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells. Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-kappaB pathway that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified. Here we report recurrent somatic mutations affecting the polycomb-group oncogene EZH2, which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27). After the recent discovery of mutations in KDM6A (UTX), which encodes the histone H3K27me3 demethylase UTX, in several cancer types, EZH2 is the second histone methyltransferase gene found to be mutated in cancer. These mutations, which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641), occur in 21.7% of GCB DLBCLs and 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro.