Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Molecular features encoded in the ctDNA reveal heterogeneity and predict outcome in high-risk aggressive B-cell lymphoma.

Inadequate molecular and clinical stratification of the patients with high-risk diffuse large B-cell lymphoma (DLBCL) is a clinical challenge hampering the establishment of personalized therapeutic options. We studied the translational significance of liquid biopsy in a uniformly treated trial cohort. Pretreatment circulating tumor DNA (ctDNA) revealed hidden clinical and biological heterogeneity, and high ctDNA burden determined increased risk of relapse and death independently of conventional risk factors. Genomic dissection of pretreatment ctDNA revealed translationally relevant phenotypic, molecular, and prognostic information that extended beyond diagnostic tissue biopsies. During therapy, chemorefractory lymphomas exhibited diverging ctDNA kinetics, whereas end-of-therapy negativity for minimal residual disease (MRD) characterized cured patients and resolved clinical enigmas, including false residual PET positivity. Furthermore, we discovered fragmentation disparities in the cell-free DNA that characterize lymphoma-derived ctDNA and, as a proof-of-concept for their clinical application, used machine learning to show that end-of-therapy fragmentation patterns predict outcome. Altogether, we have discovered novel molecular determinants in the liquid biopsy that can noninvasively guide treatment decisions.

Clinical significance of gene defects in B-cell lymphomas.

Lymphomas are a heterogenous group of malignant diseases. Identification of sub-groups has created pressure for a more detailed diagnosis and individualized treatment. Although the underlying genetic and molecular pathologic factors of the most common B-cell derived lymphomas, i.e. diffuse large B-cell lymphoma and follicular lymphoma, have become more accurate, prognosis is evaluated and treatment options still selected mainly on the basis of clinical variables. In the future, new generation sequencing methods that are becoming more common in clinical practice will allow the assessment of prognosis and treatment on the basis of biologic and genetic risk factors. To achieve this both comprehensive basic research and clinical drug trials taking the pathogenesis of different diseases into consideration are required.

MRD-driven treatment with venetoclax-R2 in mantle cell lymphoma: the Nordic Lymphoma Group MCL7 VALERIA trial.

ABSTRACT: Despite improvements in treatment of mantle cell lymphoma (MCL), most patients eventually relapse. In this multicenter phase 1b/2 trial, we evaluated safety and efficacy of minimal residual disease (MRD)-driven venetoclax, lenalidomide, and rituximab (venetoclax-R2) in relapsed/refractory (R/R) MCL and explored the feasibility of stopping treatment in molecular remission. The primary end point was overall response rate (ORR) at 6 months. After dose escalation, the recommended phase 2 dose was lenalidomide 20 mg daily, days 1 to 21; venetoclax 600 mg daily after ramp-up; and rituximab 375 mg/m2 weekly for 4 weeks, then every 8 weeks. MRD monitoring by RQ-PCR was performed every 3 months. When MRD-negativity in the blood was reached, treatment was continued for another 3 months; if MRD-negativity was then confirmed, treatment was stopped. In total, 59 patients were enrolled, with a median age of 73 years. At 6 months, the ORR was 63% (29 complete remission [CR], 8 partial remission [PR]), and 40% (4 CR, 2 PR) for patients previously failing a Bruton tyrosine kinase (BTK) inhibitor. Median progression-free survival (PFS) was 21 months, with median overall survival of 31 months. TP53 mutation was associated with inferior PFS (P < .01). Overall, 28 patients (48%) discontinued treatment in molecular remission, and 25 remain MRD negative after a median of 17.4 months. Hematological toxicity was frequent, with 52 of 59 (88%) patients with G3-4 neutropenia and 21 of 59 (36%) patients with G3-4 thrombocytopenia. To conclude, MRD-driven venetoclax-R2 is feasible and tolerable and shows efficacy in R/R MCL, also after BTK inhibitor failure. This trial was registered at www.ClinicalTrials.gov as #NCT03505944.

Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-cell Lymphoma.

Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL), are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin-ligase (CRL) with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiological KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the BTB domain of KLHL6 disrupted its localization and heterodimerization, and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL.

Inflammatory and subtype-dependent serum protein signatures predict survival beyond the ctDNA in aggressive B cell lymphomas.

BACKGROUND: Biological heterogeneity of large B cell lymphomas (LBCLs) is poorly captured by current prognostic tools, hampering optimal treatment decisions. METHODS: We dissected the levels of 1,463 serum proteins in a uniformly treated trial cohort of 109 patients with high-risk primary LBCL (ClinicalTrials.gov: NCT01325194) and correlated the profiles with molecular data from tumor tissue and circulating tumor DNA (ctDNA) together with clinical data. FINDINGS: We discovered clinically and biologically relevant associations beyond established clinical estimates and ctDNA. We identified an inflamed serum protein profile, which reflected host response to lymphoma, associated with inflamed and exhausted tumor microenvironment features and high ctDNA burden, and translated to poor outcome. We composed an inflammation score based on the identified inflammatory proteins and used the score to predict survival in an independent LBCL trial cohort (ClinicalTrials.gov: NCT03293173). Furthermore, joint analyses with ctDNA uncovered multiple serum proteins that correlate with tumor burden. We found that SERPINA9, TACI, and TARC complement minimally invasive subtype profiling and that TACI and TARC can be used to evaluate treatment response in a subtype-dependent manner in the liquid biopsy. CONCLUSIONS: Altogether, we discovered distinct serum protein landscapes that dissect the heterogeneity of LBCLs and provide agile, minimally invasive tools for precision oncology. FUNDING: This research was funded by grants from the Research Council of Finland, Finnish Cancer Organizations, Sigrid Juselius Foundation, University of Helsinki, iCAN Digital Precision Cancer Medicine Flagship, Orion Research Foundation sr, and Helsinki University Hospital.

Blood has never been thicker: Cell-free DNA fragmentomics in the liquid biopsy toolbox of B-cell lymphomas.

Liquid biopsies utilizing plasma circulating tumor DNA (ctDNA) are anticipated to revolutionize decision-making in cancer care. In the field of lymphomas, ctDNA-based blood tests represent the forefront of clinically applicable tools to harness decades of genomic research for disease profiling, quantification, and detection. More recently, the discovery of nonrandom fragmentation patterns in cell-free DNA (cfDNA) has opened another avenue of liquid biopsy research beyond mutational interrogation of ctDNA. Through examination of structural features, nucleotide content, and genomic distribution of massive numbers of plasma cfDNA molecules, the study of fragmentomics aims at identifying new tools that augment existing ctDNA-based analyses and discover new ways to profile cancer from blood tests. Indeed, the characterization of aberrant lymphoma ctDNA fragment patterns and harnessing them with powerful machine-learning techniques are expected to unleash the potential of nonmutant molecules for liquid biopsy purposes. In this article, we review cfDNA fragmentomics as an emerging approach in the ctDNA research of B-cell lymphomas. We summarize the biology behind the formation of cfDNA fragment patterns and discuss the preanalytical and technical limitations faced with current methodologies. Then we go through the advances in the field of lymphomas and envision what other noninvasive tools based on fragment characteristics could be explored. Last, we place fragmentomics as one of the facets of ctDNA analyses in emerging multiview and multiomics liquid biopsies. We pay attention to the unknowns in the field of cfDNA fragmentation biology that warrant further mechanistic investigation to provide rational background for the development of these precision oncology tools and understanding of their limitations.

Molecular background delineates outcome of double protein expressor diffuse large B-cell lymphoma.

Concomitant deregulation of MYC and BCL2 comprises clinically significant, yet poorly characterized biological high-risk feature in diffuse large B-cell lymphoma (DLBCL). To interrogate these lymphomas, we analyzed translocations and protein expression of BCL2, BCL6, and MYC; correlated the findings with comprehensive mutational, transcriptomic, and clinical data in 181 patients with primary DLBCL; and validated the key findings in independent data sets. Structural variations of BCL2 were subtype-specific and specifically increased BCL2 expression. Molecular dissection of MYC deregulation revealed associations with other lymphoma drivers, including loss of TP53, and distinctive gene expression profiles. Double protein expression (DPE) arose from heterogeneous molecular backgrounds that exhibited subtype-dependent patterns. In the germinal center B-cell (GCB) DLBCL, concurrent alterations of MYC and BCL2 loci gave rise to the majority of DPE DLBCLs, whereas among the activated B-cell (ABC) DLBCLs, concurrent alterations were infrequent. Clinically, DPE DLBCL defined a prognostic entity, which was independent of the International Prognostic Index (IPI) and cell of origin, and together with the loss of TP53 had a synergistic dismal impact on survival. In the DPE DLBCL, the loss of TP53 was associated with a chemorefractory disease, whereas among the other DLBCLs, no correlation with survival was seen. Importantly, BCL6 translocations identified non-GCB lymphomas with favorable BN2/C1-like survival independent of IPI and concurrent DPE status. Taken together, our findings define molecular characteristics of the DPE in DLBCL, and recognize clinically feasible predictors of outcome. Given the emerging taxonomical significance of BCL2, BCL6, MYC, and TP53, our findings provide further depth and validation to the genomic classification of DLBCL.

Immunogenomic Landscape of Hematological Malignancies.

Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. We integrated over 8,000 transcriptomes and 2,000 samples with multilevel genomics of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival, and validated key findings experimentally. Infiltration of cytotoxic lymphocytes was associated with TP53 and myelodysplasia-related changes in acute myeloid leukemia, and activated B cell-like phenotype and interferon-γ response in lymphoma. CIITA methylation regulating antigen presentation, cancer type-specific immune checkpoints, such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity and predicted survival. Our study provides a resource linking immunology with cancer subtypes and genomics in hematological malignancies.

Patients with high-risk DLBCL benefit from dose-dense immunochemotherapy combined with early systemic CNS prophylaxis.

Survival of patients with high-risk diffuse large B-cell lymphoma (DLBCL) is suboptimal, and the risk of central nervous system (CNS) progression is relatively high. We conducted a phase 2 trial in 139 patients aged 18 to 64 years who had primary DLBCL with an age-adjusted International Prognostic Index (aaIPI) score of 2 to 3 or site-specific risk factors for CNS recurrence. The goal was to assess whether a dose-dense immunochemotherapy with early systemic CNS prophylaxis improves the outcome and reduces the incidence of CNS events. Treatment consisted of 2 courses of high-dose methotrexate in combination with biweekly rituximab (R), cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP-14), followed by 4 courses of R-CHOP-14 with etoposide (R-CHOEP) and 1 course of high-dose cytarabine with R. In addition, liposomal cytarabine was administered intrathecally at courses 1, 3, and 5. Coprimary endpoints were failure-free survival and CNS progression rates. Thirty-six (26%) patients experienced treatment failure. Progression occurred in 23 (16%) patients, including three (2.2%) CNS events. At 5 years of median follow-up, failure-free survival, overall survival, and CNS progression rates were 74%, 83%, and 2.3%, respectively. Treatment reduced the risk of progression compared with our previous trial, in which systemic CNS prophylaxis was given after 6 courses of biweekly R-CHOEP (hazard ratio, 0.49; 95% CI, 0.31-0.77; P = .002) and overcame the adverse impact of an aaIPI score of 3 on survival. In addition, outcome of the patients with BCL2/MYC double-hit lymphomas was comparable to the patients without the rearrangements. The results are encouraging, with a low toxic death rate, low number of CNS events, and favorable survival rates. This trial was registered at www.clinicaltrials.gov as #NCT01325194.

Distinct subtypes of diffuse large B-cell lymphoma defined by hypermutated genes.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease whose personalized clinical management requires robust molecular stratification. Here, we show that somatic hypermutation (SHM) patterns constitute a marker for DLBCL molecular classification. The activity of SHM mutational processes delineated the cell of origin (COO) in DLBCL. Expression of the herein identified 36 SHM target genes stratified DLBCL into four novel SHM subtypes. In a meta-analysis of patients with DLBCL treated with immunochemotherapy, the SHM subtypes were significantly associated with overall survival (1642 patients) and progression-free survival (795 patients). Multivariate analysis of survival indicated that the prognostic impact of the SHM subtypes is independent from the COO classification and the International Prognostic Index. Furthermore, the SHM subtypes had a distinct clinical outcome within each of the COO subtypes, and strikingly, even within unclassified DLBCL. The genetic landscape of the four SHM subtypes indicated unique associations with driver alterations and oncogenic signaling in DLBCL, which suggests a possibility for therapeutic exploitation. These findings provide a biologically driven classification system in DLBCL with potential clinical applications.