Rearrangement of Cyclin D1 (CCND1-R) is the hallmark genetic lesion of mantle cell lymphoma (MCL). However, recently diffuse large B-cell lymphomas (DLBCL) have been described carrying a CCND1-R, often with additional rearrangements of BCL2, BCL6 and/or MYC raising the question if these are bona fide DLBCL or pleomorphic MCL. Protein expression and fluorescence in situ hybridisation (FISH) screening of 708 aggressive B-cell lymphomas failed to disclose CCND1-R, demonstrating the rarity of such cases. Fifteen large B-cell tumours, with CCND1-R were collected from different institutions and characterized by immunohistochemistry and for their molecular features. Three of 15 cases were CD5 positive, and all cases were negative for SOX11 but exhibited cyclin D1 staining and CCND1-R by FISH. In 10/15 cases IG could be determined as rearrangement partner by FISH or WGS with occurrence of both aberrant VDJ rearrangement and IGH class-switch recombination (CSR). Eight of 15 tumours had additional translocations involving MYC, BCL2, or BCL6. 8/12 evaluable cases showed significantly mutated IGHV genes and evidence of intraclonal variations in their rearranged IGHV genes. WES disclosed a mutational spectrum typical of DLBCL in 14/14 evaluable cases. We conclude that DLBCL CCND1-R do exist and that CCND1-R in DLBCL can occur without additional translocations.

DDX41-mutant myeloid neoplasia (MN) is characterized by unique clinical-molecular characteristics and prognosis. However, it is poorly understood how DDX41 mutational constellations drive MN outcomes. We leveraged collaborative resources to test the new 2022 MN diagnostic and prognostic schemes and account for the diverse mutational configurations of DDX41-mutant MN. Diagnostic re-classification from 2016 to 2022 schemes showed an overall shift of 14.9% and 29.7% for DDX41-mutant MDS and AML, respectively. Current prognostic systems (IPSS-R/M and ELN 2017/22) showed poor applicability to DDX41-mutant MN when compared to wild-type counterparts. Dissecting all possible DDX41 configurations, we assigned the greatest prognostic impact to R525H somatic and germline truncating hits. The former impacted most survival outcomes, while the latter were enriched in AML, independently predicting leukemic evolution. Such features had synergistic effects, albeit with different treatment interactions, and were included in DDX41-specific multivariable outcome models, which alleviated the shortcomings of the current prognostic MN algorithms. We here show that current prognostic tools are not able to adequately assess leukemic evolution and survival outcomes in DDX41-mutant MN. Additional risk factors inherent to this MN subentity hold a prognostic significance beyond the consideration of traditional disease-specific variables, substantiating the need for a dedicated risk scoring system.

Runt-related Transcription Factor 1 (RUNX1) is essential for definitive hematopoiesis and is among the most frequently mutated genes in leukemia. Previous work from our lab demonstrated that Histone Deacetylase 1 (HDAC1), a known RUNX1 partner, is unexpectedly required for active transcription suggesting a non-histone role for HDAC1 in regulating components of the RUNX1 complex. Here, we use proteomics, genomics, and long-read transcriptomics to identify novel RUNX1 interacting partners and decipher their role in gene regulation and RNA splicing in leukemia cells. We demonstrate that Polypyrimidine Tract Binding Protein 1 (PTBP1) interacts with RUNX1 in an HDAC1-dependent manner. Chromatin profiling revealed extensive genome-wide overlap in sites occupied by RUNX1 and PTBP1, with significant enrichment at promoters of actively transcribed genes. Loss of PTBP1 in AML cells led to widespread alterations in RNA splicing and decreased expression of genes whose promoters are bound by both factors, including metabolic genes. In agreement with these findings, we found that loss of PTBP1 reduced glycolysis and glucose uptake and ultimately caused cell death. Based on our data, we propose that the interaction between RUNX1 and PTBP1 facilitates expression of metabolic proteins essential for leukemia cell growth and survival.

Treatment-free remission is one of the most important goals of CML treatment but so far, the best treatment to reach this aim is still undefined, even though it is widely accepted that a sustained DMR is the prerequisite to discontinue TKI. Here we report on the depth of the molecular response, the first co-primary end point of the SUSTRENIM study, in a cohort of newly diagnosed CP-CML patients randomized 1:1 to be treated with nilotinib or with imatinib followed by switching to nilotinib in absence of optimal response. Of the 448 enrolled patients, 228 and 220 were randomized to the nilotinib (NIL) and imatinib (IM) arms, respectively, and followed for a median of 45.9 months. Eighty-two (37.2%) of the 220 patients on the IMarm did not fulfill the ELN criteria for optimal response of treatment and switched to nilotinib therapy. At the 24 months of follow-up, 107 of the 448 patients reached an MR4.5 response with a significantly higher frequency within the patients on the nilotinib arm (65 vs 42; p = 0.02). The analysis of the first primary endpoint indicates that, despite the early switch in the IM-randomized patients, NIL therapy is more effective to induce DMR.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by high incidence, poor prognosis, and marked genetic and clinical heterogeneity. This heterogeneity poses a significant challenge to effective treatment and underscores the urgent need for tailored therapeutic strategies. Here, we identified the EvC ciliary complex subunits EVC and EVC2 as aberrantly overexpressed in a subset of AMLs, particularly those harboring ASXL1 mutations or the t(8;21) translocation, with elevated expression correlating with poor patient prognosis. Functional studies demonstrated that EVC/EVC2 are essential for maintaining the leukemogenic properties of AML cells, while being dispensable for the function of normal hematopoietic stem/progenitor cells. Loss of EVC/EVC2 impairs leukemia cell proliferation, promotes differentiation, and effectively blocks AML progression in vivo. Mechanistically, we revealed that elevated EVC/EVC2 expression is associated with gained AML1-ETO occupancy or enhanced chromatin interactions at EVC/EVC2 promoter regions in AML cells carrying t(8;21) or ASXL1 mutations, respectively. Notably, we demonstrate that the leukemogenic role of EVC/EVC2 is mediated through MYC pathway activation, independent of their canonical role in Hedgehog signaling. Collectively, our findings demonstrate an oncogenic event of overexpressed EVC/EVC2, identifying novel therapeutic vulnerabilities in AML.

Diverse haematological neoplasms are driven by tyrosine kinase (TK) fusion genes formed by recurrent or non-recurrent genomic rearrangements. The resulting chimeric proteins often present excellent targets for treatment with kinase inhibitors, and the fusion transcripts or genomic junctions can be used as specific targets for molecular monitoring. Whilst the TK genes involved are generally well characterised (e.g. ABL1, PDGFRA, FGFR1), the fusion partners are very diverse, presenting a challenge for detection and characterisation of these structural variants (SV) using current diagnostic methods. We assessed the ability of targeted nanopore sequencing using adaptive sampling to detect fusion genes in myeloid neoplasms. We sequenced genomic DNA from patients (n = 20) with a known or suspected TK gene fusion and identified rearrangements in 18 cases, including all cases with a known TK fusion, typical and atypical BCR::ABL1 rearrangements, an 843Kb deletion causing a FIP1L1::PDGFRA fusion, novel AGAP2::PDGFRB and NFIA::PDGFRB fusions, and a complex CCDC88C::PDGFRB rearrangement with multiple translocation events. The approach was fast (<72 h/sample from DNA to result), flexible with minimal hands-on laboratory time, and provided accurate, patient-specific characterisation of genomic breakpoints.

Multiple myeloma (MM) is the second most common blood malignancy, with several lines of evidence supporting an inherited genetic component. Here, we sequenced 177 affected individuals from 128 families, and 170 early-onset MM cases diagnosed before 55 years of age. Samples were identified and collected through nationwide efforts in France, Sweden, and Greece. We focused on rare germline protein truncating and likely deleterious missense variants in genes harboring variants in at least two families showing variant-disease segregation, and in additional index (≥2) and/or early-onset (≥2) cases. We identified likely pathogenic variants in ATM (N = 12), ANGPTL6 (N = 5), and FBXW9 (N = 6). Additionally, we detected variants in previously reported MM predisposition genes, including DIS3, EP300, and KDM1A. Our results represent the largest sequencing study on familial and early-onset MM to date, and further illuminate the constitutional genetic basis of MM.

Anaplastic large cell lymphoma (ALCL), an aggressive T-cell malignancy, is marked by elevated expression of CD30 and the immune checkpoint molecule PD-L1. While CD30-directed chimeric antigen receptor (CAR) therapies have demonstrated clinical promise, therapeutic resistance remains a major hurdle. Here, we conducted integrated genome-wide CRISPR-Cas9 loss-of-function screens using CD30-specific CAR-engineered natural killer (CAR-NK) cells, alongside a complementary PD-L1 regulator screen, and uncovered a critical role for interleukin-1 receptor (IL-1R) signaling in modulating CAR therapy efficacy in both ALK⁺ and ALK⁻ ALCL. Mechanistically, IL-1R signaling drives an NFKBIZ - IL-17F - MAPK axis that sustains PD-L1 expression via an autocrine loop, while simultaneously inducing proinflammatory cytokines and chemokines that reinforce immune evasion and shape an immunosuppressive tumor microenvironment. Notably, NFKBIZ (IκBζ) emerges as a central transcriptional regulator orchestrating this immune suppression program upstream of IL-17F. Importantly, pharmacologic inhibition of IL-1R signaling significantly enhances the antitumor activity of CD30-specific CAR therapies both in vitro and in ALCL xenograft models. Collectively, our findings uncover a novel mechanism of immune resistance and nominate IL-1R blockade as a promising combinatorial strategy to improve CAR-based immunotherapy in ALCL.