Application of the LymphGen classification tool to 928 clinically and genetically-characterised cases of diffuse large B cell lymphoma (DLBCL).

We recently published results of targeted sequencing applied to 928 unselected cases of DLBCL registered in the Haematological Malignancy Research Network (HMRN) registry (1). Clustering allowed us to resolve five genomic subtypes. These subtypes shared considerable overlap with those proposed in two independent genomic studies(2, 3), suggesting the potential to use genetics to stratify patients by both risk and biology. In the original studies, clustering techniques were applied to sample cohorts to reveal molecular substructure, but left open the challenge of how to classify an individual patient. This was addressed by the LymphGen classification tool (4). LymphGen assigns an individual case to one of six molecular subtypes. The tool accommodates data from exome or targeted sequencing, either with or without copy number variant (CNV) data. Separate gene expression data allows classification of a seventh, MYC-driven subtype defined by a double hit (DHL) or molecular high-grade (MHG) gene expression signature(5-7).HR was funded by a studentship from the Medical Research Council. DH was supported by a Clinician Scientist Fellowship from the Medical Research Council (MR/M008584/1). The Hodson laboratory receives core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute and core funding from the CRUK Cambridge Cancer Centre. HMRN is supported by BCUK 15037 and CRUK 18362

Mutation screening using formalin-fixed paraffin-embedded tissues: a stratified approach according to DNA quality.

DNA samples from formalin-fixed paraffin-embedded tissues are highly degraded with variable quality, and this imposes a big challenge for targeted sequencing due to false positives, largely caused by PCR errors and cytosine deamination. To eliminate false positives, a common practice is to validate the detected variants by Sanger sequencing or perform targeted sequencing in duplicate. Technically, PCR errors could be removed by molecular barcoding of template DNA prior to amplification as in the HaloPlexHS design. Nonetheless, it is uncertain to what extent variants detected using this approach should be further validated. Here, we addressed this question by correlating variant reproducibility with DNA quality using HaloPlexHS target enrichment and Illumina HiSeq4000, together with an in-house validated variant calling algorithm. The overall sequencing coverage, as shown by analyses of 70 genes in 266 cases of large B-cell lymphoma, was excellent (98%) in DNA samples amenable for PCR of ≥400 bp, but suboptimal (92%) and poor (80%) in those amenable for PCR of 300 bp and 200 bp respectively. By mutation analysis in duplicate in 93 cases, we demonstrated that 20 alternative allele depth (AAD) was an optimal cut-off value for separating reproducible from non-reproducible variants in DNA samples amenable for PCR of ≥300 bp, with 97% sensitivity and 100% specificity. By cross validation with a previously established targeted sequencing protocol by Fluidigm-PCR and Illumina MiSeq, the HaloPlexHS protocol was shown to be highly sensitive and specific in mutation screening. To conclude, we proposed a stratified approach for mutation screening by HaloplexHS and Illumina HiSeq4000 according to DNA quality. DNA samples with good quality (≥400 bp) are amenable for mutation analysis with a single replicate, with only variants at 15-20 AAD requiring for further validation, while those with suboptimal quality (300 bp) are better analysed in duplicate with reproducible variants at >15 AAD regarded as true genetic changes

Targeted sequencing in DLBCL, molecular subtypes, and outcomes: a Haematological Malignancy Research Network report.

Based on the profile of genetic alterations occurring in tumor samples from selected diffuse large B-cell lymphoma (DLBCL) patients, 2 recent whole-exome sequencing studies proposed partially overlapping classification systems. Using clustering techniques applied to targeted sequencing data derived from a large unselected population-based patient cohort with full clinical follow-up (n = 928), we investigated whether molecular subtypes can be robustly identified using methods potentially applicable in routine clinical practice. DNA extracted from DLBCL tumors diagnosed in patients residing in a catchment population of ∼4 million (14 centers) were sequenced with a targeted 293-gene hematological-malignancy panel. Bernoulli mixture-model clustering was applied and the resulting subtypes analyzed in relation to their clinical characteristics and outcomes. Five molecular subtypes were resolved, termed MYD88, BCL2, SOCS1/SGK1, TET2/SGK1, and NOTCH2, along with an unclassified group. The subtypes characterized by genetic alterations of BCL2, NOTCH2, and MYD88 recapitulated recent studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. Our findings confirm the existence of molecular subtypes of DLBCL, providing evidence that genomic tests have prognostic significance in non-selected DLBCL patients. The identification of both good and poor risk subtypes in patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) clearly show the clinical value of the approach, confirming the need for a consensus classification.Bloodwise (grant number 15037) funded the majority of this study. Genetic sequencing was funded by 14M Genomics, a start-up company that ceased trading February 2016. DJH was funded by a clinician scientist fellowship from the MRC and receives core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute. Some of the analysis in this study was performed on the “Viking” high performance computing cluster at the University of York.1

DDX3X loss is an adverse prognostic marker in diffuse large B-cell lymphoma and is associated with chemoresistance in aggressive non-Hodgkin lymphoma subtypes.

Funder: addenbrooke's charitable trust, cambridge university hospital

Molecular subclusters of follicular lymphoma: a report from the UK's Haematological Malignancy Research Network

Follicular lymphoma (FL) is morphologically and clinically diverse, with mutations in epigenetic regulators alongside t(14;18) identified as disease-initiating events. Identification of additional mutational entities confirms this cancer’s heterogeneity, but whether mutational data can be resolved into mechanistically distinct subsets remains an open question. Targeted sequencing was applied to an unselected population-based FL cohort (n = 548) with full clinical follow-up (n = 538), which included 96 diffuse large B-cell lymphoma (DLBCL) transformations. We investigated whether molecular subclusters of FL can be identified and whether mutational data provide predictive information relating to transformation. DNA extracted from FL samples was sequenced with a 293-gene panel representing genes frequently mutated in DLBCL and FL. Three clusters were resolved using mutational data alone, independent of translocation status: FL_aSHM, with high burden of aberrant somatic hypermutation (aSHM) targets; FL_STAT6, with high STAT6 & CREBBP mutation and low aSHM; and FL_Com, with the absence of features of other subtypes and enriched KMT2D mutation. Analysis of mutation signatures demonstrated differential enrichment of predicted mutation signatures between subgroups and a dominant preference in the FL_aSHM subgroup for G(C>T)T and G(C>T)C transitions consistent with previously defined aSHM-like patterns. Of transformed cases with paired samples, 17 of 26 had evidence of branching evolution. Poorer overall survival (OS) in the aSHM group (P = .04) was associated with older age; however, overall tumor genetics provided limited information to predict individual patient risk. Our approach identifies 3 molecular subclusters of FL linked to differences in underlying mechanistic pathways. These clusters, which may be further resolved by the inclusion of translocation status and wider mutation profiles, have implications for understanding pathogenesis as well as improving treatment strategies in the future

Prognostic Significance of MYC Rearrangement and Translocation Partner in Diffuse Large B-Cell Lymphoma : A Study by the Lunenburg Lymphoma Biomarker Consortium

PURPOSE: MYC rearrangement (MYC-R) occurs in approximately 10% of diffuse large B-cell lymphomas (DLBCLs) and has been associated with poor prognosis in many studies. The impact of MYC-R on prognosis may be influenced by the MYC partner gene (immunoglobulin [IG] or a non-IG gene). We evaluated a large cohort of patients through the Lunenburg Lymphoma Biomarker Consortium to validate the prognostic significance of MYC-R (single-, double-, and triple-hit status) in DLBCL within the context of the MYC partner gene. METHODS: The study cohort included patients with histologically confirmed DLBCL morphology derived from large prospective trials and patient registries in Europe and North America who were uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone therapy or the like. Fluorescence in situ hybridization for the MYC, BCL2, BCL6, and IG heavy and light chain loci was used, and results were correlated with clinical outcomes. RESULTS: A total of 5,117 patients were identified of whom 2,383 (47%) had biopsy material available to assess for MYC-R. MYC-R was present in 264 (11%) of 2,383 patients and was associated with a significantly shorter progression-free and overall survival, with a strong time-dependent effect within the first 24 months after diagnosis. The adverse prognostic impact of MYC-R was only evident in patients with a concurrent rearrangement of BCL2 and/or BCL6 and an IG partner (hazard ratio, 2.4; 95% CI, 1.6 to 3.6; P < .001). CONCLUSION: The negative prognostic impact of MYC-R in DLBCL is largely observed in patients with MYC double hit/triple-hit disease in which MYC is translocated to an IG partner, and this effect is restricted to the first 2 years after diagnosis. Our results suggest that diagnostic strategies should be adopted to identify this high-risk cohort, and risk-adjusted therapeutic approaches should be refined further

Molecular High-Grade B-Cell Lymphoma: Defining a Poor-Risk Group That Requires Different Approaches to Therapy.

PURPOSE: Biologic heterogeneity is a feature of diffuse large B-cell lymphoma (DLBCL), and the existence of a subgroup with poor prognosis and phenotypic proximity to Burkitt lymphoma is well known. Conventional cytogenetics identifies some patients with rearrangements of MYC and BCL2 and/or BCL6 (double-hit lymphomas) who are increasingly treated with more intensive chemotherapy, but a more biologically coherent and clinically useful definition of this group is required. PATIENTS AND METHODS: We defined a molecular high-grade (MHG) group by applying a gene expression-based classifier to 928 patients with DLBCL from a clinical trial that investigated the addition of bortezomib to standard rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy. The prognostic significance of MHG was compared with existing biomarkers. We performed targeted sequencing of 70 genes in 400 patients and explored molecular pathology using gene expression signature databases. Findings were validated in an independent data set. RESULTS: The MHG group comprised 83 patients (9%), with 75 in the cell-of-origin germinal center B-cell-like group. MYC rearranged and double-hit groups were strongly over-represented in MHG but comprised only one half of the total. Gene expression analysis revealed a proliferative phenotype with a relationship to centroblasts. Progression-free survival rate at 36 months after R-CHOP in the MHG group was 37% (95% CI, 24% to 55%) compared with 72% (95% CI, 68% to 77%) for others, and an analysis of treatment effects suggested a possible positive effect of bortezomib. Double-hit lymphomas lacking the MHG signature showed no evidence of worse outcome than other germinal center B-cell-like cases. CONCLUSION: MHG defines a biologically coherent high-grade B-cell lymphoma group with distinct molecular features and clinical outcomes that effectively doubles the size of the poor-prognosis, double-hit group. Patients with MHG may benefit from intensified chemotherapy or novel targeted therapies.Supported by Bloodwise grant number 15002: Precision Medicine for Aggressive Lymphoma. The Randomized Evaluation of Molecular-Guided Therapy for DLBCL With Bortezomib (REMoDL-B) trial was endorsed by Cancer Research UK, reference number CRUKE/10/024, and Janssen-Cillag provided funding. A.S. is partly funded by the National Institute for Health Research Oxford Biomedical Research Centre. D.R.W. acknowledges UK Medical Research Council grant MR/L01629X/1 for infrastructure support

Distinct genetic changes reveal evolutionary history and heterogeneous molecular grade of DLBCL with MYC / BCL2 double-hit

Abstract: Using a Burkitt lymphoma-like gene expression signature, we recently defined a high-risk molecular high-grade (MHG) group mainly within germinal centre B-cell like diffuse large B-cell lymphomas (GCB-DLBCL), which was enriched for MYC/BCL2 double-hit (MYC/BCL2-DH). The genetic basis underlying MHG-DLBCL and their aggressive clinical behaviour remain unknown. We investigated 697 cases of DLBCL, particularly those with MYC/BCL2-DH (n = 62) by targeted sequencing and gene expression profiling. We showed that DLBCL with MYC/BCL2-DH, and those with BCL2 translocation, harbour the characteristic mutation signatures that are associated with follicular lymphoma and its high-grade transformation. We identified frequent MYC hotspot mutations that affect the phosphorylation site (T58) and its adjacent amino acids, which are important for MYC protein degradation. These MYC mutations were seen in a subset of cases with MYC translocation, but predominantly in those of MHG. The mutations were more frequent in double-hit lymphomas with IG as the MYC translocation partner, and were associated with higher MYC protein expression and poor patient survival. DLBCL with MYC/BCL2-DH and those with BCL2 translocation alone are most likely derived from follicular lymphoma or its precursor lesion, and acquisition of MYC pathogenic mutations may augment MYC function, resulting in aggressive clinical behaviour

Longitudinal expression profiling identifies a poor risk subset of patients with ABC-type Diffuse Large B Cell Lymphoma

Despite the effectiveness of immuno-chemotherapy, 40\cell lymphoma (DLBCL) experience relapse or refractory disease. Longitudinal studies have previously focused on the mutational landscape of relapse but fell short of providing a consistent relapse-specific genetic signature. In our study, we have focussed attention on the changes in gene expression profile accompanying DLBCL relapse using archival paired diagnostic/relapse specimens from 38 de novo DLBCL patients. Cell of origin remained stable from diagnosis to relapse in 80\ with only a single patient showing COO switching from ABC to GCB. Analysis of the transcriptomic changes that occur following relapse suggest ABC and GCB relapses are mediated via different mechanisms. We developed a 30-gene discriminator for ABC-DLBCLs derived from relapse-associated genes, that defined clinically distinct high and low risk subgroups in ABC-DLBCLs at diagnosis in datasets comprising both population-based and clinical trial cohorts. This signature also identified a population of \lt;60-year-old patients with superior PFS and OS treated with Ibrutinib-R-CHOP as part of the PHOENIX trial. Altogether this new signature adds to the existing toolkit of putative genetic predictors now available in DLBCL that can be readily assessed as part of prospective clinical trials