An atlas of cells in the human tonsil.

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

Infrequent Presentations of Chronic NPM1-Mutated Myeloid Neoplasms: Clinicopathological Features of Eight Cases from a Single Institution and Review of the Literature.

Non-acute myeloid neoplasms (MNs) with NPM1 mutations (NPM1mut-MNs) pose a diagnostic and therapeutic dilemma, primarily manifesting as chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS). The classification and treatment approach for these conditions as acute myeloid leukemia (AML) are debated. We describe eight cases of atypical NPM1mut-MNs from our institution and review the literature. We include a rare case of concurrent prostate carcinoma and MN consistent with chronic eosinophilic leukemia, progressing to myeloid sarcoma of the skin. Of the remaining seven cases, five were CMML and two were MDS. NPM1 mutations occur in 3-5% of CMML and 1-6% of MDS, with an increased likelihood of rapid evolution to AML. Their influence on disease progression varies, and their prognostic significance in non-acute MNs is less established than in AML. Non-acute MNs with NPM1 mutations may display an aggressive clinical course, emphasizing the need for a comprehensive diagnosis integrating clinical and biological data. Tailoring patient management on an individualized basis, favoring intensive treatment aligned with AML protocols, is crucial, regardless of blast percentage. Research on the impact of NPM1 mutations in non-acute myeloid neoplasms is ongoing, requiring challenging prospective studies with substantial patient cohorts and extended follow-up periods for validation.

Genomic classification and outcomes of young patients with polycythemia vera and essential thrombocythemia according to the presence of splanchnic vein thrombosis and its chronology.

To elucidate the role of splanchnic vein thrombosis (SVT) and genomic characteristics in prognosis and survival, we compared patients with polycythemia vera (PV) or essential thrombocythemia (ET) presenting SVT at diagnosis (n = 69, median age 43 years) or during follow-up (n = 21, median age 46 years) to a sex- and age-matched control group of PV/ET without SVT (n = 165, median age 48 years). The majority of patients presenting with SVT at diagnosis were classified as myeloproliferative neoplasm with heterozygous JAK2 mutation (87% of cases vs. 69% in PV/ET control group, p < 0.05), characterized by low JAK2 allele burden and no high-risk mutations. Despite this lower molecular complexity, patients presenting with SVT showed a higher risk of death (HR 3.0, 95% CI 1.5-6.0, p = 0.003) and lower event-free survival (HR 3.0, 95% CI 1.9-4.8, p < 0.001) than age- and sex-matched PV/ET controls. In patients presenting with SVT, molecular high-risk was associated with increased risk of venous re-thrombosis (HR 5.8, 95% CI 1.4-24.0, p = 0.01). Patients developing SVT during follow-up were more frequently allocated in molecular high-risk than those with SVT at diagnosis (52% versus 13%, p < 0.05). In the whole cohort of patients, molecular classification identified PV/ET patients at higher risk of disease progression whereas DNMT3A/TET2/ASXL1 mutations were associated with higher risk of arterial thrombosis. In conclusion, clinical and molecular characteristics are different in PV/ET patients with SVT, depending on whether it occurs at diagnosis or at follow-up. Molecular characterization by NGS is useful for assessing the risk of thrombosis and disease progression in young patients with PV/ET.

BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

Chronic lymphocytic leukemia patient-derived xenografts recapitulate clonal evolution to Richter transformation.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm with a heterogeneous clinical behavior. In 5-10% of patients the disease transforms into a diffuse large-B cell lymphoma known as Richter transformation (RT), which is associated with dismal prognosis. Here, we aimed to establish patient-derived xenograft (PDX) models to study the molecular features and evolution of CLL and RT. We generated two PDXs by injecting CLL (PDX12) and RT (PDX19) cells into immunocompromised NSG mice. Both PDXs were morphologically and phenotypically similar to RT. Whole-genome sequencing analysis at different time points of the PDX evolution revealed a genomic landscape similar to RT tumors from both patients and uncovered an unprecedented RT subclonal heterogeneity and clonal evolution during PDX generation. In PDX12, the transformed cells expanded from a very small subclone already present at the CLL stage. Transcriptomic analysis of PDXs showed a high oxidative phosphorylation (OXPHOS) and low B-cell receptor (BCR) signaling similar to the RT in the patients. IACS-010759, an OXPHOS inhibitor, reduced proliferation, and circumvented resistance to venetoclax. In summary, we have generated new RT-PDX models, one of them from CLL cells that mimicked the evolution of CLL to RT uncovering intrinsic features of RT cells of therapeutical value.

Characteristics and long-term outcome in a large series of chronic myelomonocytic leukaemia patients including 104 formerly referred to as oligomonocytic.

Recently modified diagnostic criteria for chronic myelomonocytic leukaemia (CMML) have lowered the cut-off for absolute monocytosis. In the largest series to date, we have analysed 313 CMML patients, including 104 with oligomonocytic (OM)-CMML. Five-year survival was longer for OM-CMML than for other patients (p < 0.001). Multivariate analysis identified OM-CMML as a favourable prognostic factor (HR 0.58; p = 0.002). The 5-year cumulative incidence of progression to classical CMML was 47%. Older age and transfusion dependence were adverse prognostic factors for OM-CMML. Our results support the inclusion of OM-CMML in the CMML category as a subtype with superior outcomes.

Clinical Characteristics and Outcomes of Patients with Primary and Secondary Myelofibrosis According to the Genomic Classification Using Targeted Next-Generation Sequencing.

Myelofibrosis (MF) is a heterogeneous disease regarding its mutational landscape, clinical presentation, and outcomes. The aim of our work is to evaluate the genomic classification of MF considering whether it is primary or secondary. One-hundred seventy-five patients, 81 with primary MF (PMF) and 94 with secondary MF (SMF) were hierarchically allocated into eight molecular groups. We found that TP53 disruption/aneuploidy (n = 16, 9%) was more frequent (12% versus 6%) and showed higher allele burden (57% versus 15%, p = 0.01) in SMF than in PMF, and was associated with shorter survival (median 3.5 years). Mutations in chromatin/spliceosome genes (n = 72, 41%) represented the most frequent genomic group in PMF. Homozygous JAK2 mutation (n = 40, 23%) was enriched with old patients with SMF after long-standing polycythemia vera, whereas MF with heterozygous JAK2 mutation (n = 22, 13%) was similarly distributed among PMF and SMF. MF with CALR mutation (n = 19, 11%) predominated in post-essential thrombocythemia MF. The remaining genomic groups were infrequent. TP53 disruption, chromatin/spliceosome mutation, and homozygous JAK2 mutation were associated with significantly shorter survival and higher risk of progression. In conclusion, genomic classification reveals different pathogenic pathways between PMF and SMF and provides relevant information regarding disease phenotype and outcomes.

SF3B1 mutation-mediated sensitization to H3B-8800 splicing inhibitor in chronic lymphocytic leukemia.

Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in SF3B1 are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic SF3B1 WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3' splice sites upon SF3B1 mutation, including an event in the MAP3K7 gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in SF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards SF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.

Germ line variants in patients with acute myeloid leukemia without a suspicion of hereditary hematologic malignancy syndrome.

Germ line predisposition in acute myeloid leukemia (AML) has gained attention in recent years because of a nonnegligible frequency and an impact on management of patients and their relatives. Risk alleles for AML development may be present in patients without a clinical suspicion of hereditary hematologic malignancy syndrome. In this study we investigated the presence of germ line variants (GVs) in 288 genes related to cancer predisposition in 47 patients with available paired, tumor-normal material, namely bone marrow stroma cells (n = 29), postremission bone marrow (n = 17), and saliva (n = 1). These patients correspond to 2 broad AML categories with heterogeneous genetic background (AML myelodysplasia related and AML defined by differentiation) and none of them had phenotypic abnormalities, previous history of cytopenia, or strong cancer aggregation. We found 11 pathogenic or likely pathogenic variants, 6 affecting genes related to autosomal dominant cancer predisposition syndromes (ATM, DDX41, and CHEK2) and 5 related to autosomal recessive bone marrow failure syndromes (FANCA, FANCM, SBDS, DNAJC21, and CSF3R). We did not find differences in clinical characteristics nor outcome between carriers of GVs vs noncarriers. Further studies in unselected AML cohorts are needed to determine GV incidence and penetrance and, in particular, to clarify the role of ATM nonsense mutations in AML predisposition.

Novel Tools for Diagnosis and Monitoring of AML.

In recent years, major advances in the understanding of acute myeloid leukemia (AML) pathogenesis, together with technological progress, have led us into a new era in the diagnosis and follow-up of patients with AML. A combination of immunophenotyping, cytogenetic and molecular studies are required for AML diagnosis, including the use of next-generation sequencing (NGS) gene panels to screen all genetic alterations with diagnostic, prognostic and/or therapeutic value. Regarding AML monitoring, multiparametric flow cytometry and quantitative PCR/RT-PCR are currently the most implemented methodologies for measurable residual disease (MRD) evaluation. Given the limitations of these techniques, there is an urgent need to incorporate new tools for MRD monitoring, such as NGS and digital PCR. This review aims to provide an overview of the different technologies used for AML diagnosis and MRD monitoring and to highlight the limitations and challenges of current versus emerging tools.

A novel patient-derived 3D model recapitulates mantle cell lymphoma lymph node signaling, immune profile and in vivo ibrutinib responses.

Mantle cell lymphoma (MCL), a rare and aggressive B-cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T-cells self-organize in disc-shaped structures, where B and T-cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproduces in vivo responses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features and in vivo responses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.

Decoding the molecular heterogeneity of pediatric monomorphic post-solid organ transplant lymphoproliferative disorders.

Posttransplant lymphoproliferative disorders (PTLDs) represent a broad spectrum of lymphoid proliferations, frequently associated with Epstein-Barr virus (EBV) infection. The molecular profile of pediatric monomorphic PTLDs (mPTLDs) has not been elucidated, and it is unknown whether they display similar genetic features as their counterpart in adult and immunocompetent (IMC) pediatric patients. In this study, we investigated 31 cases of pediatric mPTLD after solid organ transplantation, including 24 diffuse large B-cell lymphomas (DLBCLs), mostly classified as activated B cell, and 7 cases of Burkitt lymphoma (BL), 93% of which were EBV positive. We performed an integrated molecular approach, including fluorescence in situ hybridization, targeted gene sequencing, and copy number (CN) arrays. Overall, PTLD-BL carried mutations in MYC, ID3, DDX3X, ARID1A, or CCND3 resembling IMC-BL, higher mutational burden than PTLD-DLBCL, and lesser CN alterations than IMC-BL. PTLD-DLBCL showed a very heterogeneous genomic profile with fewer mutations and CN alterations than IMC-DLBCL. Epigenetic modifiers and genes of the Notch pathway were the most recurrently mutated in PTLD-DLBCL (both 28%). Mutations in cell cycle and Notch pathways correlated with a worse outcome. All 7 patients with PTLD-BL were alive after treatment with pediatric B-cell non-Hodgkin lymphoma protocols, whereas 54% of patients with DLBCL were cured with immunosuppression reduction, rituximab, and/or low-dose chemotherapy. These findings highlight the low complexity of pediatric PTLD-DLBCL, their good response to low-intensity treatment, and the shared pathogenesis between PTLD-BL and EBV-positive IMC-BL. We also suggest new potential parameters that could help in the diagnosis and the design of better therapeutic strategies for these patients.

Unraveling the genetics of transformed splenic marginal zone lymphoma.

The genetic mechanisms associated with splenic marginal zone lymphoma (SMZL) transformation are not well defined. We studied 41 patients with SMZL that eventually underwent large B-cell lymphoma transformation. Tumor material was obtained either only at diagnosis (9 patients), at diagnosis and transformation (18 patients), and only at transformation (14 patients). Samples were categorized in 2 groups: (1) at diagnosis (SMZL, n = 27 samples), and (2) at transformation (SMZL-T, n = 32 samples). Using copy number arrays and a next-generation sequencing custom panel, we identified that the main genomic alterations in SMZL-T involved TNFAIP3, KMT2D, TP53, ARID1A, KLF2, 1q gains, and losses of 9p21.3 (CDKN2A/B) and 7q31-q32. Compared with SMZL, SMZL-T had higher genomic complexity, and higher incidence of TNFAIP3 and TP53 alterations, 9p21.3 (CDKN2A/B) losses, and 6p gains. SMZL and SMZL-T clones arose by divergent evolution from a common altered precursor cell that acquired different genetic alterations in virtually all evaluable cases (92%, 12 of 13 cases). Using whole-genome sequencing of diagnostic and transformation samples in 1 patient, we observed that the SMZL-T sample carried more genomic aberrations than the diagnostic sample, identified a translocation t(14;19)(q32;q13) present in both samples, and detected a focal B2M deletion due to chromothripsis acquired at transformation. Survival analysis showed that KLF2 mutations, complex karyotype, and International Prognostic Index score at transformation were predictive of a shorter survival from transformation (P = .001; P = .042; and P = .007; respectively). In summary, SMZL-T are characterized by higher genomic complexity than SMZL, and characteristic genomic alterations that could represent key players in the transformation event.

Prognostic impact of MYD88 and CXCR4 mutations assessed by droplet digital polymerase chain reaction in IgM monoclonal gammopathy of undetermined significance and smouldering Waldenström macroglobulinaemia.

Waldenström macroglobulinaemia (WM) is characterized by recurrent somatic mutations in MYD88 and CXCR4 genes. However, limitations arise when analysing these mutations in IgM monoclonal gammopathy of undetermined significance (MGUS) or smouldering WM (SWM) given the lower tumour load. Here, we used droplet digital polymerase chain reaction (ddPCR) to analyse MYD88 L265P and CXCR4 S338* mutations (C1013G and C1013A) in unsorted bone marrow (BM) or cell-free DNA (cfDNA) samples from 101 IgM MGUS and 69 SWM patients. ddPCR was more sensitive to assess MYD88 L265P compared to allele-specific PCR, especially in IgM MGUS (64% vs 39%). MYD88 mutation burden correlated with other laboratory biomarkers, particularly BM infiltration (r = 0.8; p < 0.001). CXCR4 C1013G was analysed in MYD88-mutated samples with available genomic DNA and was detected in 19/54 (35%) and 18/42 (43%) IgM MGUS and SWM cases respectively, also showing correlation with BM involvement (r = 0.9; p < 0.001). ddPCR also detected 8 (38%) and 10 (63%) MYD88-mutated cfDNA samples in IgM MGUS and SWM respectively. Moreover, high BM mutation burden (≥8% MYD88 and ≥2% CXCR4) was associated with an increased risk of progression to symptomatic WM. We show the clinical applicability of ddPCR to assess MYD88 and CXCR4 in IgM MGUS and SWM and provide a molecular-based risk classification.

Real-World Data on Chronic Myelomonocytic Leukemia: Clinical and Molecular Characteristics, Treatment, Emerging Drugs, and Patient Outcomes.

Despite emerging molecular information on chronic myelomonocytic leukemia (CMML), patient outcome remains unsatisfactory and little is known about the transformation to acute myeloid leukemia (AML). In a single-center cohort of 219 CMML patients, we explored the potential correlation between clinical features, gene mutations, and treatment regimens with overall survival (OS) and clonal evolution into AML. The most commonly detected mutations were TET2, SRSF2, ASXL1, and RUNX1. Median OS was 34 months and varied according to age, cytogenetic risk, FAB, CPSS and CPSS-Mol categories, and number of gene mutations. Hypomethylating agents were administered to 37 patients, 18 of whom responded. Allogeneic stem cell transplantation (alloSCT) was performed in 22 patients. Two-year OS after alloSCT was 60.6%. Six patients received targeted therapy with IDH or FLT3 inhibitors, three of whom attained a long-lasting response. AML transformation occurred in 53 patients and the analysis of paired samples showed changes in gene mutation status. Our real-world data emphasize that the outcome of CMML patients is still unsatisfactory and alloSCT remains the only potentially curative treatment. However, targeted therapies show promise in patients with specific gene mutations. Complete molecular characterization can help to improve risk stratification, understand transformation, and personalize therapy.