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Hotspot mutations in the PEST-domain of NOTCH1 and NOTCH2 are recurrently identified in B cell malignancies. To address how NOTCH-mutations contribute to a dismal prognosis, we have generated isogenic primary human tumor cells from patients with Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL), differing only in their expression of the intracellular domain (ICD) of NOTCH1 or NOTCH2. Our data demonstrate that both NOTCH-paralogs facilitate immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-γ signaling. In addition, NOTCH-activation causes silencing of the entire HLA-class II locus via epigenetic regulation of the transcriptional co-activator CIITA. Notably, while NOTCH1 and NOTCH2 govern similar transcriptional programs, disease-specific differences in their expression levels can favor paralog-specific selection. Importantly, NOTCH-ICD also strongly down-regulates the expression of CD19, possibly limiting the effectiveness of immune-therapies. These NOTCH-mediated immune escape mechanisms are associated with the expansion of exhausted CD8+ T cells in vivo.

The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited and systematic analyses are lacking. Using chronic lymphocytic leukaemia (CLL) as a model disease, we investigated the influence of 17 microenvironmental stimuli on 12 drugs in 192 genetically characterised patient samples. Based on microenvironmental response, we identified four subgroups with distinct clinical outcomes beyond known prognostic markers. Response to multiple microenvironmental stimuli was amplified in trisomy 12 samples. Trisomy 12 was associated with a distinct epigenetic signature. Bromodomain inhibition reversed this epigenetic profile and could be used to target microenvironmental signalling in trisomy 12 CLL. We quantified the impact of microenvironmental stimuli on drug response and their dependence on genetic alterations, identifying interleukin 4 (IL4) and Toll-like receptor (TLR) stimulation as the strongest actuators of drug resistance. IL4 and TLR signalling activity was increased in CLL-infiltrated lymph nodes compared with healthy samples. High IL4 activity correlated with faster disease progression. The publicly available dataset can facilitate the investigation of cell-extrinsic mechanisms of drug resistance and disease progression.

Current therapeutic approaches for chronic lymphocytic leukemia (CLL) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the phosphatidylinositol-3-phosphate/AKT (PI3K/AKT)-signaling pathway may be leveraged to trigger CLL cell death. Though counterintuitive, our data show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SH2-containing-inositol-5′-phosphatase-1 (SHIP1) induces acute cell death in CLL cells. Our mechanistic studies reveal that increased AKT activity upon inhibition of SHIP1 leads to increased mitochondrial respiration and causes excessive accumulation of reactive oxygen species (ROS), resulting in cell death in CLL with immunogenic features. Our results demonstrate that CLL cells critically depend on mechanisms to fine-tune PI3K/AKT activity, allowing sustained proliferation and survival but avoid ROS-induced cell death and suggest transient SHIP1-inhibition as an unexpectedly promising concept for CLL therapy., Current therapeutic approaches in chronic lymphocytic leukemia (CLL) focus on the suppression of PI3K/AKT signaling. Here, the authors show that CLL cells are vulnerable to hyperactivation of the PI3K/AKT signaling pathway and suggest this as a promising concept for CLL therapy.

AIM OF THE STUDY: Chimeric antigen receptor T (CAR-T) cells are a powerful form of immune-cell therapy for patients with relapsed/refractory B-cell lymphoma and acute B lymphoblastic leukaemia. CAR-T cells have been commercially available in Switzerland since 2018. Because of the complexity and costs of this treatment it is critical to review patient outcomes in real-world settings, to examine whether the promising results from pivotal trials can be reproduced and to identify clinical parameters that determine their efficacy. METHODS: Here we present results of a retrospective study analysing outcomes of patients treated with CAR-T cells in a single academic centre in Switzerland during the first two years after commercial approval (BASEC-No. 2020-02271). Cytokine release syndrome (CRS), immune-cell associated neurotoxicity syndrome (ICANS), responses to treatment, ancillary laboratory studies and administrative specifics of CAR-T treatment were examined and are discussed. RESULTS: From October 2018 to August 2020 CAR-T cell therapy was evaluated in 34 patients, mostly with relapsed/refractory aggressive B-cell lymphoma (87% had refractory disease). Thirty-one patients underwent leukapheresis. Three of 31 patients (9.6%) died of rapid disease progression before the CAR-T cell product was delivered, two patients were enrolled into a clinical trial, three patients were not given CAR-T cells for other reasons. Ultimately, 23 patients were infused with a commercial CAR-T cell product and included in this analysis. Fourteen (61%) patients received bridging therapy while waiting for a median of 41 days (range 31–62) for delivery of the CAR-T cell product. Toxicity and severe side effects were rare (CRS >3 in 13%, ICANS > grade 3 in 10% of patients), manageable and resolved completely thereafter. The best overall response rate was 65%, with complete responses in 38% of lymphoma patients. At 12 months postinfusion, 61% of patients were alive and 35% progression free. With a median follow-up of 14 months, 13/23 (56%) patients were alive at the time of writing. CONCLUSION: CAR-T cell therapy proved to be safe and manageable under adequate hospital conditions. Outcomes resemble results from pivotal trials. The majority of patients was heavily pretreated and refractory at the time of CAR-T cell infusion. Patient selection, time point of leukapheresis, bridging strategies and timing of CAR-T cell infusion may be critical to further improve outcomes.

Primary lymphomas of the central nervous system (PCNSL) are mainly diffuse large B-cell lymphomas (DLBCLs) confined to the central nervous system (CNS). Molecular drivers of PCNSL have not been fully elucidated. Here, we profile and compare the whole-genome and transcriptome landscape of 51 CNS lymphomas (CNSL) to 39 follicular lymphoma and 36 DLBCL cases outside the CNS. We find recurrent mutations in JAK-STAT, NFkB, and B-cell receptor signaling pathways, including hallmark mutations in MYD88 L265P (67%) and CD79B (63%), and CDKN2A deletions (83%). PCNSLs exhibit significantly more focal deletions of HLA-D (6p21) locus as a potential mechanism of immune evasion. Mutational signatures correlating with DNA replication and mitosis are significantly enriched in PCNSL. TERT gene expression is significantly higher in PCNSL compared to activated B-cell (ABC)-DLBCL. Transcriptome analysis clearly distinguishes PCNSL and systemic DLBCL into distinct molecular subtypes. Epstein-Barr virus (EBV)+ CNSL cases lack recurrent mutational hotspots apart from IG and HLA-DRB loci. We show that PCNSL can be clearly distinguished from DLBCL, having distinct expression profiles, IG expression and translocation patterns, as well as specific combinations of genetic alterations.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy arising from germinal center or post-germinal center B-cells that retain many of the properties of normal B-cells. Here we show that a subset of DLBCL express the cytokine IL-10 and its receptor. The genetic ablation of IL-10 receptor signaling abrogates the autocrine STAT3 phosphorylation triggered by tumor cell-intrinsic IL-10 expression and impairs growth of DLBCL cell lines in subcutaneous and orthotopic xenotransplantation models. Furthermore, we demonstrate using an immunocompetent Myc-driven model of DLBCL that neutralization of IL-10 signaling reduces tumor growth, which can be attributed to reduced Treg infiltration, stronger intratumoral effector T-cell responses, and restored tumor-specific MHCII expression. The effects of IL-10R neutralization were phenocopied by the genetic ablation of IL-10 signaling in the Treg compartment and could be reversed by MHCII blockade. The BTK inhibitor ibrutinib effectively blocked tumor cell-intrinsic IL-10 expression and tumor growth in this Myc-driven model. Tumors from patients with high IL-10RA expression are infiltrated by higher numbers of Tregs than IL-10RAlow patients. Finally, we show in 16 cases of DLBCL derived from transplant patients on immunosuppressive therapy that IL-10RA expression is less common in this cohort, and Treg infiltration is not observed., OncoImmunology, 10 (1), ISSN:2162-4011, ISSN:2162-402X

Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7–8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

The development of cancer therapies may be improved by the discovery of tumor-specific molecular dependencies. The requisite tools include genetic and chemical perturbations, each with its strengths and limitations. Chemical perturbations can be readily applied to primary cancer samples at large scale, but mechanistic understanding of hits and further pharmaceutical development is often complicated by the fact that a chemical compound has affinities to multiple proteins. To computationally infer specific molecular dependencies of individual cancers from their ex vivo drug sensitivity profiles, we developed a mathematical model that deconvolutes these data using measurements of protein-drug affinity profiles. Through integrating a drug-kinase profiling dataset and several drug response datasets, our method, DepInfeR, correctly identified known protein kinase dependencies, including the EGFR dependence of HER2+ breast cancer cell lines, the FLT3 dependence of acute myeloid leukemia (AML) with FLT3-ITD mutations and the differential dependencies on the B-cell receptor pathway in the two major subtypes of chronic lymphocytic leukemia (CLL). Furthermore, our method uncovered new subgroup-specific dependencies, including a previously unreported dependence of high-risk CLL on Checkpoint kinase 1 (CHEK1). The method also produced a detailed map of the kinase dependencies in a heterogeneous set of 117 CLL samples. The ability to deconvolute polypharmacological phenotypes into underlying causal molecular dependencies should increase the utility of high-throughput drug response assays for functional precision oncology.

Chronic Lymphocytic Leukemia (CLL) has a complex pattern of driver mutations and much of its clinical diversity remains unexplained. We devised a method for simultaneous subgroup discovery across multiple data types and applied it to genomic, transcriptomic, DNA methylation and ex-vivo drug response data from 217 Chronic Lymphocytic Leukemia (CLL) cases. We uncovered a biological axis of heterogeneity strongly associated with clinical behavior and orthogonal to the known biomarkers. We validated its presence and clinical relevance in four independent cohorts (n=547 patients). We find that this axis captures the proliferative drive (PD) of CLL cells, as it associates with lymphocyte doubling rate, global hypomethylation, accumulation of driver aberrations and response to pro-proliferative stimuli. CLL-PD was linked to the activation of mTOR-MYC-oxidative phosphorylation (OXPHOS) through transcriptomic, proteomic and single cell resolution analysis. CLL-PD is a key determinant of disease outcome in CLL. Our multi-table integration approach may be applicable to other tumors whose inter-individual differences are currently unexplained.

The genomic landscape of mantle cell lymphoma (MCL) includes frequent alterations of TP53, ATM, CCND1 and KMT2D. Thus far, the mutational landscape provides little information for treatment stratification. We characterized a cohort of MCL by targeted next generation sequencing and discovered SAMHD1 as a novel recurrently mutated gene (8.5% of investigated cases, 4/47 samples). Furthermore, we provide evidence of in vitro resistance of SAMHD1 mutated patient-derived MCL cells to cytarabine and fludarabine.

Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

In chronic lymphocytic leukemia (CLL), the hypoxia-inducible factor 1 (HIF-1) regulates the response of tumor cells to hypoxia and their protective interactions with the leukemic microenvironment. In this study, we demonstrate that CLL cells from TP53-disrupted (TP53 dis) patients have constitutively higher expression levels of the α-subunit of HIF-1 (HIF-1α) and increased HIF-1 transcriptional activity compared to the wild-type counterpart. In the TP53 dis subset, HIF-1α upregulation is due to reduced expression of the HIF-1α ubiquitin ligase von Hippel-Lindau protein (pVHL). Hypoxia and stromal cells further enhance HIF-1α accumulation, independently of TP53 status. Hypoxia acts through the downmodulation of pVHL and the activation of the PI3K/AKT and RAS/ERK1-2 pathways, whereas stromal cells induce an increased activity of the RAS/ERK1-2, RHOA/RHOA kinase and PI3K/AKT pathways, without affecting pVHL expression. Interestingly, we observed that higher levels of HIF-1A mRNA correlate with a lower susceptibility of leukemic cells to spontaneous apoptosis, and associate with the fludarabine resistance that mainly characterizes TP53 dis tumor cells. The HIF-1α inhibitor BAY87-2243 exerts cytotoxic effects toward leukemic cells, regardless of the TP53 status, and has anti-tumor activity in Em-TCL1 mice. BAY87-2243 also overcomes the constitutive fludarabine resistance of TP53 dis leukemic cells and elicits a strongly synergistic cytotoxic effect in combination with ibrutinib, thus providing preclinical evidence to stimulate further investigation into use as a potential new drug in CLL.

The tumor microenvironment is the cellular and molecular environment in which the tumor exists and with which it continuously interacts. In B-cell lymphomas, this microenvironment is intriguing in that it plays critical roles in the regulation of tumor cell survival and proliferation, fostering immune escape as well as the development of treatment resistance. The purpose of this review is to summarize the proceedings of the Second Annual Summit on the Immune Microenvironment in Hematologic Malignancies that took place on September 11–12, 2014 in Dublin, Ireland. We provide a timely overview of the composition and biological relevance of the cellular and molecular microenvironment interface and discuss the role of interactions between the microenvironment and neoplastic cells in a variety of B-cell lymphomas. In addition, we focus on various novel therapeutic strategies that target the tumor microenvironment, including agents that modulate B-cell receptor pathways and immune-checkpoints, chimeric antigen receptor T cells and immunomodulatory agents.

Hairy cell leukemia is an uncommon hematologic malignancy characterized by pancytopenia and marked susceptibility to infection. Tremendous progress in the management of patients with this disease has resulted in high response rates and improved survival, yet relapse and an appropriate approach to re-treatment present continuing areas for research. The disease and its effective treatment are associated with immunosuppression. Because more patients are being treated with alternative programs, comparison of results will require general agreement on definitions of response, relapse, and methods of determining minimal residual disease. The development of internationally accepted, reproducible criteria is of paramount importance in evaluating and comparing clinical trials to provide optimal care. Despite the success achieved in managing these patients, continued participation in available clinical trials in the first-line and particularly in the relapse setting is highly recommended. The Hairy Cell Leukemia Foundation convened an international conference to provide common definitions and structure to guide current management. There is substantial opportunity for continued research in this disease. In addition to the importance of optimizing the prevention and management of the serious risk of infection, organized evaluations of minimal residual disease and treatment at relapse offer ample opportunities for clinical research. Finally, a scholarly evaluation of quality of life in the increasing number of survivors of this now manageable chronic illness merits further study. The development of consensus guidelines for this disease offers a framework for continued enhancement of the outcome for patients.