Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven lymphomagenesis.

DDX3X is a ubiquitously expressed RNA helicase involved in multiple stages of RNA biogenesis. DDX3X is frequently mutated in Burkitt lymphoma, but the functional basis for this is unknown. Here, we show that loss-of-function DDX3X mutations are also enriched in MYC-translocated diffuse large B cell lymphoma and reveal functional cooperation between mutant DDX3X and MYC. DDX3X promotes the translation of mRNA encoding components of the core translational machinery, thereby driving global protein synthesis. Loss-of-function DDX3X mutations moderate MYC-driven global protein synthesis, thereby buffering MYC-induced proteotoxic stress during early lymphomagenesis. Established lymphoma cells restore full protein synthetic capacity by aberrant expression of DDX3Y, a Y chromosome homolog, the expression of which is normally restricted to the testis. These findings show that DDX3X loss of function can buffer MYC-driven proteotoxic stress and highlight the capacity of male B cell lymphomas to then compensate for this loss by ectopic DDX3Y expression.

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.

Comparative analysis of gene expression platforms for cell-of-origin classification of diffuse large B-cell lymphoma shows high concordance.

Cell-of-origin subclassification of diffuse large B cell lymphoma (DLBCL) into activated B cell-like (ABC), germinal centre B cell-like (GCB) and unclassified (UNC) or type III by gene expression profiling is recommended in the latest update of the World Health Organization's classification of lymphoid neoplasms. There is, however, no accepted gold standard method or dataset for this classification. Here, we compare classification results using gene expression data for 68 formalin-fixed paraffin-embedded DLBCL samples measured on four different gene expression platforms (Illumina wG-DASLTM arrays, Affymetrix PrimeView arrays, Illumina TrueSeq RNA sequencing and the HTG EdgeSeq DLBCL Cell of Origin Assay EU using an established platform agnostic classification algorithm (DAC) and the classifier native to the HTG platform, which is CE marked for in vitro diagnostic use (CE-IVD). Classification methods and platforms show a high level of concordance, with agreement in at least 80% of cases and rising to much higher levels for classifications of high confidence. Our results demonstrate that cell-of-origin classification by gene expression profiling on different platforms is robust, and that the use of the confidence value alongside the classification result is important in clinical applications.

Gene-expression profiling of bortezomib added to standard chemoimmunotherapy for diffuse large B-cell lymphoma (REMoDL-B): an open-label, randomised, phase 3 trial.

BACKGROUND: Biologically distinct subtypes of diffuse large B-cell lymphoma can be identified using gene-expression analysis to determine their cell of origin, corresponding to germinal centre or activated B cell. We aimed to investigate whether adding bortezomib to standard therapy could improve outcomes in patients with these subtypes. METHODS: In a randomised evaluation of molecular guided therapy for diffuse large B-cell lymphoma with bortezomib (REMoDL-B), an open-label, adaptive, randomised controlled, phase 3 superiority trial, participants were recruited from 107 cancer centres in the UK (n=94) and Switzerland (n=13). Eligible patients had previously untreated, histologically confirmed diffuse large B-cell lymphoma with sufficient diagnostic material from initial biopsies for gene-expression profiling and pathology review; were aged 18 years or older; had ECOG performance status of 2 or less; had bulky stage I or stage II-IV disease requiring full-course chemotherapy; had measurable disease; and had cardiac, lung, renal, and liver function sufficient to tolerate chemotherapy. Patients initially received one 21-day cycle of standard rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP; rituximab 375 mg/m2, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, and vincristine 1·4 mg/m2 [to a maximum of 2 mg total dose] intravenously on day 1 of the cycle, and prednisolone 100 mg orally once daily on days 1-5). During this time, we did gene-expression profiling using whole genome cDNA-mediated annealing, selection, extension, and ligation assay of tissue from routine diagnostic biopsy samples to determine the cell-of-origin subtype of each participant (germinal centre B cell, activated B cell, or unclassified). Patients were then centrally randomly assigned (1:1) via a web-based system, with block randomisation stratified by international prognostic index score and cell-of-origin subtype, to continue R-CHOP alone (R-CHOP group; control), or with bortezomib (RB-CHOP group; experimental; 1·3 mg/m2 intravenously or 1·6 mg/m2 subcutaneously) on days 1 and 8 for cycles two to six. If RNA extracted from the diagnostic tissues was of insufficient quality or quantity, participants were given R-CHOP as per the control group. The primary endpoint was 30-month progression-free survival, for the germinal centre and activated B-cell population. The primary analysis was on the modified intention-to-treat population of activated and germinal centre B-cell population. Safety was assessed in all participants who were given at least one dose of study drug. We report the progression-free survival and safety outcomes for patients in the follow-up phase after the required number of events occurred. This study was registered at ClinicalTrials.gov, number NCT01324596, and recruitment and treatment has completed for all participants, with long-term follow-up ongoing. FINDINGS: Between June 2, 2011, and June 10, 2015, 1128 eligible patients were registered, of whom 918 (81%) were randomly assigned to receive treatment (n=459 to R-CHOP, n=459 to RB-CHOP), comprising 244 (26·6%) with activated B-cell disease, 475 (51·7%) with germinal centre B cell disease, and 199 (21·7%) with unclassified disease. At a median follow-up of 29·7 months (95% CI 29·0-32·0), we saw no evidence for a difference in progression-free survival in the combined germinal centre and activated B-cell population between R-CHOP and RB-CHOP (30-month progression-free survival 70·1%, 95% CI 65·0-74·7 vs 74·3%, 69·3-78·7; hazard ratio 0·86, 95% CI 0·65-1·13; p=0·28). The most common grade 3 or worse adverse event was haematological toxicity, reported in 178 (39·8%) of 447 patients given R-CHOP and 187 (42·1%) of 444 given RB-CHOP. However, RB-CHOP was not associated with increased haematological toxicity and 398 [87·1%] of 459 participants assigned to receive RB-CHOP completed six cycles of treatment. Grade 3 or worse neuropathy occurred in 17 (3·8%) patients given RB-CHOP versus eight (1·8%) given R-CHOP. Serious adverse events occurred in 190 (42·5%) patients given R-CHOP, including five treatment-related deaths, and 223 (50·2%) given RB-CHOP, including four treatment-related deaths. INTERPRETATION: This is the first large-scale study in diffuse large B-cell lymphoma to use real-time molecular characterisation for prospective stratification, randomisation, and subsequent analysis of biologically distinct subgroups of patients. The addition of bortezomib did not improve progression-free survival. FUNDING: Janssen-Cilag, Bloodwise, and Cancer Research UK.

A phase II study to assess the safety and efficacy of the dual mTORC1/2 inhibitor vistusertib in relapsed, refractory DLBCL.

Patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) who are unfit for or relapsed postautologous stem-cell transplantation have poor outcomes. Historically, mTORC1 inhibitors have produced responses in approximately 30% of patients in this setting. mTORC1 inhibitor efficacy may be limited by resistance mechanisms including AKT activation by mTORC2. To date, dual mTORC1/2 inhibitors targeting both the TORC1 and TORC2 complexes have not been investigated in DLBCL. This phase II trial investigated the oral dual mTORC1/2 inhibitor vistusertib in an intermittent dosing schedule of 125 mg b.d. for 2 days per week. Thirty patients received vistusertib and six received vistusertib-rituximab for up to six cycles (28-day cycles). Two partial responses were achieved on monotherapy. Durations of response were 57 and 62 days, respectively, for these patients. 19% had stable disease within six cycles. In the monotherapy arm, the median progression-free survival was1.69 (95% confidence interval [CI] 1.61-2.14) months and median overall survival was 6.58 (95% CI 3.81-not reached) months, respectively. The median duration of response or stable disease across the trial duration was 153 days (95% CI 112-not reached). Tumour responses according to positron emission tomography/computed tomography versus computed tomography were concordant. There were no differences noted in tumour volume response according to cell of origin by either gene expression profiling or immunohistochemistry. Vistusertib ± rituximab was well tolerated; across 36 patients 86% of adverse events were grade (G) 1-2. Common vistusertib-related adverse events were similar to those described with mTORC1 inhibitors: nausea (47% G1-2), diarrhoea (27% G1-2, 6% G3), fatigue (30% G1-2, 3% G3), mucositis (25% G1-2, 6% G3), vomiting (17% G1-2), and dyspepsia (14% G1-2). Dual mTORC1/2 inhibitors do not clearly confer an advantage over mTORC1 inhibitors in relapsed or refractory DLBCL. Potential resistance mechanisms are discussed within.

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 identifies patients with preclinical MDS at high risk of disease progression.

The diagnosis of myelodysplastic syndromes (MDS) remains problematic due to the subjective nature of morphologic assessment. The reported high frequency of somatic mutations and increased structural variants by array-based cytogenetics have provided potential objective markers of disease; however, this has been complicated by reports of similar abnormalities in the healthy population. We aimed to identify distinguishing features between those with early MDS and reported healthy individuals by characterizing 69 patients who, following a nondiagnostic marrow, developed progressive dysplasia or acute myeloid leukemia. Targeted sequencing and array-based cytogenetics identified a driver mutation and/or structural variant in 91% (63/69) of prediagnostic samples with the mutational spectrum mirroring that in the MDS population. When compared with the reported healthy population, the mutations detected had significantly greater median variant allele fraction (40% vs 9% to 10%), and occurred more commonly with additional mutations (≥2 mutations, 64% vs 8%). Furthermore, mutational analysis identified a high-risk group of patients with a shorter time to disease progression and poorer overall survival. The mutational features in our cohort are distinct from those seen in the healthy population and, even in the absence of definitive disease, can predict outcome. Early detection may allow consideration of intervention in poor-risk patients.

N-terminally truncated FOXP1 protein expression and alternate internal FOXP1 promoter usage in normal and malignant B cells.

Strong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and N-terminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5' non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1-Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation.

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity.

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL.

Lymphoid blood cancers, incidence and survival 2005-2023: A report from the UK's Haematological Malignancy Research Network.

BACKGROUND: Population-based information on cancer incidence and outcome are required to inform clinical practice and research; but contemporary data are lacking for many lymphoid cancer subtypes. METHODS: Set within a socio-demographically representative UK population of ∼4 million, data are from an established UK patient cohort (N = 22,414 diagnoses). Information on incidence (crude and age-standardised) and survival (overall and net) is presented for > 40 subtypes. RESULTS: The median diagnostic age was 69.9 years (interquartile range 59.1-78.3), but unlike many other cancers, lymphoid malignancies can be diagnosed at any age; different subtypes dominating at different ages. Males were more likely to be diagnosed than females (age-standardised sex rate ratio: 1.55 (95% Confidence Interval: 1.50,1.59)), and most subtypes had a male predominance, some more than three-fold (e.g. Burkitt lymphoma 3.26 (2.42, 4.40)). Five-year net survival estimates varied hugely, ranging from 97.4% (95% CI: 56.5, 99.9) in patients with hairy cell leukaemia to 31.6% (95% CI: 2.5, 69.8) in those with T-cell prolymphocytic leukaemia. No significant sex difference in survival were observed for the majority of diagnoses; one exception being classical Hodgkin lymphoma, where males had a higher mortality (Excess Mortality Ratio: 1.44 (95% CI: 1.11, 1.87)). An improvement in survival over time was observed for some, but not all, of the major diagnostic groups. CONCLUSIONS: Marked incidence and survival variations by subtype, sex and age confirm the heterogeneity of lymphoid neoplasms and highlight the importance of accurately characterising disease entities. Despite recent improvements, routine cancer registration of lymphoid neoplasms remains challenging and new issues continue to emerge; including the lack of an international consensus on classification and the recording of progressions and transformations. Furthermore, the increasing need for additional molecular and genomic information required for accurate classification is likely to impact negatively on the quality of cancer registration data, especially in low income countries.

Non-IG::MYC in diffuse large B-cell lymphoma confers variable genomic configurations and MYC transactivation potential.

MYC translocation occurs in 8-14% of diffuse large B-cell lymphoma (DLBCL), and may concur with BCL2 and/or BCL6 translocation, known as double-hit (DH) or triple-hit (TH). DLBCL-MYC/BCL2-DH/TH are largely germinal centre B-cell like subtype, but show variable clinical outcome, with IG::MYC fusion significantly associated with inferior survival. While DLBCL-MYC/BCL6-DH are variable in their cell-of-origin subtypes and clinical outcome. Intriguingly, only 40-50% of DLBCL with MYC translocation show high MYC protein expression (>70%). We studied 186 DLBCLs with MYC translocation including 32 MYC/BCL2/BCL6-TH, 75 MYC/BCL2-DH and 26 MYC/BCL6-DH. FISH revealed a MYC/BCL6 fusion in 59% of DLBCL-MYC/BCL2/BCL6-TH and 27% of DLBCL-MYC/BCL6-DH. Targeted NGS showed a similar mutation profile and LymphGen genetic subtype between DLBCL-MYC/BCL2/BCL6-TH and DLBCL-MYC/BCL2-DH, but variable LymphGen subtypes among DLBCL-MYC/BCL6-DH. MYC protein expression is uniformly high in DLBCL with IG::MYC, but variable in those with non-IG::MYC including MYC/BCL6-fusion. Translocation breakpoint analyses of 8 cases by TLC-based NGS showed no obvious genomic configuration that enables MYC transactivation in 3 of the 4 cases with non-IG::MYC, while a typical promoter substitution or IGH super enhancer juxtaposition in the remaining cases. The findings potentially explain variable MYC expression in DLBCL with MYC translocation, and also bear practical implications in its routine assessment.

Differential Efficacy From the Addition of Bortezomib to R-CHOP in Diffuse Large B-Cell Lymphoma According to the Molecular Subgroup in the REMoDL-B Study With a 5-Year Follow-Up.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.The REMoDL-B phase III adaptive trial compared rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) versus R-CHOP + bortezomib (RB-CHOP) in patients with diffuse large B-cell lymphoma (DLBCL), stratified by molecular subtype. Primary analysis at a median follow-up of 30 months found no effect of bortezomib on progression-free survival (PFS) or overall survival (OS). Retrospective analysis using a gene expression-based classifier identified a molecular high-grade (MHG) group with worse outcomes. We present an updated analysis for patients successfully classified by the gene expression profile (GEP). Eligible patients were age older than 18 years with untreated DLBCL, fit enough for full-dose chemotherapy, and with adequate biopsies for GEP. Of 1,077 patients registered, 801 were identified with Activated B-Cell (ABC), Germinal Center B-cell, or MHG lymphoma. At a median follow-up of 64 months, there was no overall benefit of bortezomib on PFS or OS (5-year PFS hazard ratio [HR], 0.81; P = .085; OS HR, 0.86; P = .32). However, improved PFS and OS were seen in ABC lymphomas after RB-CHOP: 5-year OS 67% with R-CHOP versus 80% with RB-CHOP (HR, 0.58; 95% CI, 0.35 to 0.95; P = .032). Five-year PFS was higher in MHG lymphomas: 29% versus 55% (HR, 0.46; 95% CI, 0.26 to 0.84). Patients with ABC and MHG DLBCL may benefit from the addition of bortezomib to R-CHOP in initial therapy.

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% of patients with diffuse large B-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 focused attention on the changes in GEP accompanying DLBCL relapse using archival paired diagnostic/relapse specimens from 38 de novo patients with DLBCL. COO remained stable from diagnosis to relapse in 80% of patients, with only a single patient showing COO switching from activated B-cell-like (ABC) to germinal center B-cell-like (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 <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.

Whole genome expression profiling based on paraffin embedded tissue can be used to classify diffuse large B-cell lymphoma and predict clinical outcome.

This study tested the validity of whole-genome expression profiling (GEP) using RNA from formalin-fixed, paraffin-embedded (FFPE) tissue to sub-classify Diffuse Large B-cell Lymphoma (DLBCL), in a population based cohort of 172 patients. GEP was performed using Illumina Whole Genome cDNA-mediated Annealing, Selection, extension & Ligation, and tumours were classified into germinal centre (GCB), activated B-cell (ABC) and Type-III subtypes. The method was highly reproducible and reliably classified cell lines of known phenotype. GCB and ABC subtypes were each characterized by unique gene expression signatures consistent with previously published data. A significant relationship between subtype and survival was observed, with ABC having the worst clinical outcome and in a multivariate survival model only age and GEP class remained significant. This effect was not seen when tumours were classified by immunohistochemistry. There was a significant association between age and subtype (mean ages ABC - 72·8 years, GC - 68·4 years, Type-III - 64·5 years). Older patients with ABC subtype were also over-represented in patients who died soon after diagnosis. The relationship between prognosis and subtype improved when only patients assigned to the three categories with the highest level of confidence were analysed. This study demonstrates that GEP-based classification of DLBCL can be applied to RNA extracted from routine FFPE samples and has potential for use in stratified medicine trials and clinical practice.

Molecular subclusters of follicular lymphoma: a report from the United Kingdom'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.