Leukemia

High genomic complexity is linked to poor prognosis in chronic lymphocytic leukaemia (CLL), but its independent prognostic value remains uncertain amid emerging biomarkers. We analysed copy number alterations (CNA) in 495 treatment-naive patients from three randomized trials (CLL4, ADMIRE, ARCTIC), incorporating IGHV status, telomere length (TL), targeted sequencing, and DNA-methylation subtypes. Patients harboured low (LGC, 0-2 CNAs; n=334), intermediate (IGC, 3-4 CNAs; n=97), or high (HGC, [&ge;]5 CNAs; n=64) genomic complexity. U-CLL (81%, p<0.001) and short TL (61%, p<0.05) were enriched in HGC, and TL inversely correlated with CNA burden ({tau} = -0.147, p<0.001). 62% of HGC patients were n-CLL. TP53 dysfunction was associated with HGC (36%, p<0.001). Trisomy 12 and NOTCH1 mutations, were enriched in LGC (p<0.001). HGC predicted shorter progression-free and overall survival in all univariate models but only remained independently prognostic for OS only in CLL4 (HR=1.61, p=0.02). Of 64 HGC patients, 23 had TP53 dysfunction; 92% of TP53 wild-type cases had other high-risk features (TL-S, U-CLL, or n- CLL). HGC is associated with adverse outcomes but may reflect underlying biological risk rather than serve as an independent biomarker. Its interplay with telomere attrition, immunogenetics, and epigenetic subtype warrants further validation in targeted therapy-treated cohorts.

BackgroundPatients with active acute myelogenous leukemia (AML) are at risk for leukemic infiltration (LI) into the lung and acute tumor lysis pneumopathy (ATLP) following chemotherapy. Fulminant presentations of these leukemic lung diseases are well-described, but indolent forms have not yet been studied. Therefore, we sought to elucidate the clinical features of mild-to-moderate LI and ATLP. MethodsA retrospective cohort analysis was performed on 51 hospitalized patients with AML, circulating blast count [&ge;]3%, non-critical illness, and receipt of bronchoscopy between 2015-2019. Diagnoses of LI and ATLP were made via retrospective chart review by a multidisciplinary team of physicians. Results19 cases of leukemic lung disease were identified: 14 with LI and 5 with ATLP. The clinical presentations closely resembled pneumonia, with the majority demonstrating respiratory symptoms (63%), hypoxemia (63%), fever (84%), and pulmonary opacities (100%). All patients were presumptively diagnosed with infection, leading to an average of 18 days of broad-spectrum antibiotic therapy and multiple instances of delayed chemotherapy in treatment candidates. Although most patients were near the end-of-life (90% died within 1 year), transitions to comfort care were infrequent (25%) and hospitalizations were protracted (median 25 days). ConclusionsLI and ATLP are common yet under-recognized pulmonary complications in patients with active AML. When presenting indolently, these conditions are difficult to distinguish from lung infection, leading to missed diagnosis, inappropriate antibiosis, chemotherapy deferrals, and prolonged hospitalizations. Greater awareness and consensus definitions of LI and ATLP are therefore needed to improve care of this population.

HighlightsO_LISingle-cell RNA sequencing of bone marrow from TP53-mutated AML patients showed a decrease in cells expressing anti-apoptotic genes like BCL2 and MCL1 after venetoclax and azacitidine treatment. C_LIO_LIImmune cells increased both in number and in gene expression levels associated with cytotoxicity post venetoclax combination therapy, verifying immune recovery. C_LIO_LIResidual AML cells expressed CD47 and CLL1, suggesting a role for ancillary treatment targeting antigens expressed on residual TP53-mutated AML cells. C_LI The BCL-2 inhibitor venetoclax combined with the hypomethylating agent azacitidine or with low-dose cytarabine significantly improves response rates and overall survival (OS) for newly diagnosed unfit and relapsed / refractory (R/R) acute myeloid leukemia (AML) patients. We retrospectively analyzed our experience with venetoclax combination therapy in 41 unfit AML patients (23 untreated, 18 R/R). Overall response rates were 78.3% for untreated patients and 61.1% for R/R patients. TP53 alterations were observed in 13 patients (31.7%) and were identified as an independent predictor of poor outcome (p=0.0008). We further conducted single-cell RNA sequencing in bone marrow, sampled before and after venetoclax and azacitidine treatment, of three TP53-mutated AML patients who achieved complete remission (CR) or CR with incomplete hematologic recovery. After treatment, numbers of cells expressing anti-apoptotic genes such as BCL2 and MCL1 decreased. CD4 T cells, cytotoxic CD8 T cells, and NK cells significantly increased both in number and in levels of gene expression associated with cytotoxicity post-treatment, confirming immune recovery in the tumor microenvironment. Residual AML cells expressed CD47 and CLEC12A (CLL1). These results indicate that venetoclax combination therapy of AML is promising in real-world clinical practice and suggest a role for ancillary treatment targeting antigens expressed on residual AML cells as a therapeutic strategy in TP53-mutated AML.

WHO5-2022 classification of B-lymphoblastic leukemia (B-ALL) incorporates several novel entities requiring high-throughput sequencing for their accurate characterization. The clinical relevance of this classification in the context of contemporary MRD-directed therapy is unclear. We analyzed 533 pediatric B-ALL uniformly treated with ICiCLe-ALL-14 protocol as defined by WHO2016 and reclassified them as per WHO5-2022 using targeted sequencing, FISH, and cytogenetics. Subtype-defining genetic abnormalities were identified in 81.2% of the cohort as per the WHO5 classification. Among the new subtypes, PAX5alt, MEF2D-r, and BCR::ABL1-like(ABL-class) were associated with an inferior 2-year event-free survival (EFS) of 39.1% (p<0.0001), 53.8% (p=0.024) and 60.6% (p=0.043), respectively. We developed a 3-tier genetic risk stratification model incorporating 15 genetic subtypes and the IKZF1 deletion. Children with standard, intermediate, and high genetic risk demonstrated 2-year EFS of 92.6%, 71.0%, and 50.7% (p<0.0001), and 2-year overall survival of 94.3%, 81.9%, and 71.6% (p<0.0001), respectively. Genetic risk further identified heterogeneous outcomes among ICiCLe risk groups (p<0.0001). Standard genetic risk was associated with superior OS and EFS irrespective of MRD status. We demonstrate the applicability of the WHO5 classification in routine practice and create a general framework for incorporating the WHO5 classification in risk-adapted therapy for childhood B-ALL.

BackgroundRare oncogenic driver events, particularly affecting the expression or splicing of driver genes, are suspected to substantially contribute to the large heterogeneity of hematologic malignancies. However, their identification remains challenging. MethodsTo address this issue, we generated the largest dataset to date of matched whole genome sequencing and total RNA sequencing of hematologic malignancies from 3,760 patients spanning 24 disease entities. Taking advantage of our dataset size, we focused on discovering rare regulatory aberrations. Therefore, we called expression and splicing outliers using an extension of the workflow DROP (Detection of RNA Outliers Pipeline) and AbSplice, a variant effect predictor that identifies genetic variants causing aberrant splicing. We next trained a machine learning model integrating these results to prioritize new candidate disease-specific driver genes. ResultsWe found a median of seven expression outlier genes, two splicing outlier genes, and two rare splice-affecting variants per sample. Each category showed significant enrichment for already well-characterized driver genes, with odds ratios exceeding three among genes called in more than five samples. On held-out data, our integrative modeling significantly outperformed modeling based solely on genomic data and revealed promising novel candidate driver genes. Remarkably, we found a truncated form of the low density lipoprotein receptor LRP1B transcript to be aberrantly overexpressed in about half of hairy cell leukemia variant (HCL-V) samples and, to a lesser extent, in closely related B-cell neoplasms. This observation, which was confirmed in an independent cohort, suggests LRP1B as a novel marker for a HCL-V subclass and a yet unreported functional role of LRP1B within these rare entities. ConclusionsAltogether, our census of expression and splicing outliers for 24 hematologic malignancy entities and the companion computational workflow constitute unique resources to deepen our understanding of rare oncogenic events in hematologic cancers.

Precision medicine holds great promise to improve outcomes in cancer, including haematological malignancies. However, there are few biomarkers that influence choice of chemotherapy in clinical practice. In particular, multiple myeloma requires an individualized approach as there exist several active therapies, but little agreement on how and when they should be used and combined. We have previously shown that a transcriptomic signature can identify specific bortezomib- and lenalidomide-sensitivity. However, gene expression signatures are challenging to implement clinically. We reasoned that signatures based on the presence or absence of gene mutations would be more tractable in the clinical setting, though examples of such signatures are rare. We performed whole exome sequencing as part of the CARDAMON trial, which employed carfilzomib-based therapy. We applied advanced machine learning approaches to discover mutational patterns predictive of treatment outcome. The resulting model accurately predicted progression-free survival (PFS) both in CARDAMON patients and in an external validation set of patients from the CoMMpass study who had received carfilzomib. The signature was specific for carfilzomib therapy and was strongly driven by genes on chromosome 1p36. Importantly, patients predicted to be carfilzomib-sensitive had a longer PFS when treated with carfilzomib/lenalidomide/dexamethasone than with bortezomib/carfilzomib/dexamethasone. However, in those predicted to be carfilzomib-insensitive, the latter therapy may have been capable of eradicating carfilzomib-resistant clones. We propose that the signature can be used to make rational therapeutic decisions and could be incorporated into future clinical trials.

Occasional complete responses to immune checkpoint inhibitor therapies suggest that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are immune-sensitive when appropriately targeted. Here, we analyzed AML/MDS patients treated with anti-TIM3 sabatolimab and decitabine in a phase Ib clinical trial (NCT03066648) using single-cell RNA and T cell receptor (TCR) sequencing and functional co-culture assays. Unlike T cell restricted CTLA4 and PD1, TIM3 was broadly expressed across natural killer (NK), myeloid, and T cell populations. Therapy induced expansion of cytotoxic NK cell subsets and enhanced type I interferon signaling. Less than 1% of bone marrow CD8+ T cells showed canonical exhaustion phenotypes, and the treatment preferably expanded small CD8+ T cell clones in responders. Responders had more cytotoxic CD4+ T and B cells which was exemplified by a patient with an outstanding complete response of 23-months. Over >20% of this patients lymphocytes were CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) cells that expressed a TCR capable of recognizing autologous blasts. SignificanceAnti-TIM3+decitabine offers a distinct mechanism compared to anti-PD1 and anti-CTLA4: it preferentially enhances innate and unconventional cytotoxic lymphocyte populations. Furthermore, our findings suggest that clonal expansion of self-antigen-specific T cells, such as T-LGLL, may augment anti-leukemic immunity in the context of immunotherapy.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) constitutes a rare and aggressive malignancy originating from plasmacytoid/common dendritic cells (pDCs/cDCs) with a primarily cutaneous tropism followed by dissemination to the bone marrow and other organs. We conducted a genome-wide analysis of the tumor methylome in an extended cohort of 45 BPDCN patients supplemented by WES (n=54) and RNA-seq (n=54) as well as ATAC-seq on selected cases (n=4). We determine the BPDCN DNA methylation profile and thereby identify a reliable means to discriminate BPDCN from AML, CMML and T-ALL. DNA methylation profiling characterizes disruption of oncogenic pathways whilst unraveling the proliferative history as well as the prognostically relevant composition of the tumor microenvironment. Beyond the two recently established BPDCN subtypes (C1/C2), we identified a transcriptional reliance on JAK/STAT and NF{kappa}B-signaling in atypical C2 versus C1-BPDCN cases through RNA-sequencing. Our integrative characterization of BPDCN offers novel molecular insights and potential diagnostic applications.

DIRECT was a prospective, multisite study assessing the feasibility and utility of circulating tumor DNA (ctDNA) in 188 patients with aggressive B-cell non-Hodgkin lymphoma using a lymphoma-customized assay and open-source pipeline. CtDNA fraction assessed by copy number alterations in pre-treatment plasma identified high-risk patients more effectively than existing clinical risk scores. In 74.5% of cases ctDNA was equivalent or superior to biopsy for genetic profiling. Patients not suitable for ctDNA genotyping had low tumor volumes and could be predicted from simple clinical factors. Finally, a phased variant-supported minimal residual disease (MRD) assay was predictive of outcomes in all groups analyzed. Patients achieving ctDNA clearance at end of treatment had an extremely low 2-year progression rate (<5%). However, false positive MRD results were common in patients with transformed indolent lymphoma. This study highlights the considerable potential, but also the caveats and limitations, of ctDNA technology when applied to aggressive B-cell lymphoma.

BackgroundProcarbazine-containing chemotherapy regimens associate with cytopenias and infertility, suggesting stem cell toxicity. Procarbazine in eBEACOPP (escalated dose bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisolone) is increasingly replaced with dacarbazine (eBEACOPDac) to reduce toxicity, although limited genomic and clinical data support this substitution. MethodsTo assess mutagenic and clinical consequences of dacarbazine-procarbazine substitutions, we compared mutational landscapes in haematopoietic stem and progenitor cells (HSPCs) from patients treated with different Hodgkin regimens and children, sperm and bowel tissue from procarbazine-treated patients. We compared efficacy and toxicity data of a multicentre eBEACOPDac-treated patient cohort, with eBEACOPP clinical trial and real-world datasets. ResultseBEACOPP-treated patients exhibit a higher burden of point mutations, small insertions and deletions in HSPCs compared to eBEACOPDac and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine)-treated patients. Two novel mutational signatures, SBSA (SBS25-like) and SBSB were identified in HSPCs, neoplastic and normal colon from only procarbazine-treated patients. SBSB was also identified in germline DNA of three children conceived post-eBEACOPP and sperm of an eBEACOPP-treated male. The dacarbazine substitution did not appear to compromise efficacy; 3-year progression-free survival of 312 eBEACOPDac patients (93.3%; CI95=90.3-96.4%) mirrored that of 1945 HD18-trial eBEACOPP patients (93.3%; CI95=92.1-94.4%). eBEACOPDac-treated patients required fewer blood transfusions, demonstrated higher post-chemotherapy sperm concentrations, and experienced earlier resumption of menstrual periods. ConclusionsProcarbazine induces a higher mutational burden and novel mutational signatures in eBEACOPP-treated patients and their germline DNA raising concerns for hereditary consequences. However, replacing procarbazine with dacarbazine appears to mitigate gonadal and stem cell toxicity while maintaining comparable clinical efficacy.

Nucleophosmin-1 (NPM1) mutations define a major molecular subtype of acute myeloid leukemia (AML) and is generally associated with favorable prognosis. However, the impact of myelodysplasia-associated mutations (MDSm+) on patient outcomes within this subgroup remains uncertain. We retrospectively analyzed 271 NPM1-mutated AML patients from three independent cohorts (SWOG, Fred Hutch, and Beat AML) to assess the prognostic significance of MDSm+ and its interaction with age. MDSm+ occurred in 17% of cases, most commonly involving SRSF2 and SF3B1. Although MDSm+ was associated with inferior overall survival compared to MDSm-in ELN2022 favorable-risk patients (HR 2.0, p=0.008), this effect was largely driven by worse outcomes in older patients ([&ge;]65 years) as older ELN22 favorable-risk patients had poor OS regardless of presence of MDSm+ compared to younger patients. After stratification of patients by age, there was not a significant difference between MDSm+ and MDSm-in either younger patients (HR 0.99, p=0.98) or older patients (HR 1.42, p=0.33). These findings indicate that MDSm+ in NPM1+ AML is not independently associated with adverse risk after adjusting for age and highlight the need for age-adjusted AML risk models.

Precision medicine can significantly improve outcomes for cancer patients, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here we describe somatic biallelic TET2 mutation (focal deletion and nonsense mutation) in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine, but acutely sensitive to 5-azacitidine (5-Aza) hypomethylating monotherapy, resulting in long-term morphological remission (overall survival (OS) 850 days). Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5-Aza compared to cells with monoallelic mutation. We subsequently identified 29 additional patients from the Study Alliance Leukemia biobank with chromosome 4 abnormalities and identified two further patients with complex biallelic TET2 mutations, including one with trisomy 4, homozygosity across the long arm and an inactivating point mutation. We also screened patients recruited to the PETHEMA FLUGAZA phase 3 clinical trial and identified three patients with biallelic TET2 mutations, two of whom had responded very well to single agent 5-Aza (OS 767 and 579 days) despite having adverse risk AML and poor performance status. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for cancer patients. Key PointsO_LIMutant TET2 allele dosage affects response to 5-azacitidine in acute myeloid leukemia in vitro and in a xenograft model. C_LIO_LIOur data highlight the importance for screening of biallelic mutations to predict response to therapy in acute myeloid leukemia. C_LI

Accurate and comprehensive genetic characterization of acute myeloid leukemia (AML) is essential for diagnosis, prognostication, and treatment selection. We report here, in 255 adults with AML enrolled in a prospective clinical protocol at 18 major cancer centers across the USA, the results of whole genome DNA-sequencing (WGS) at diagnosis and post-treatment remission. WGS effectively recapitulated, and frequently identified genetic alterations missed by, conventional standard of care clinical testing. These new findings included important prognostic and predictive biomarkers, copy number alterations, regulatory element, splicing, and structural variants including partial tandem duplications within KMT2A. All patients had a pathogenic variant detected at diagnosis, and approximately ten percent also had evidence of a potential inherited myeloid malignancy predisposition. This comprehensive atlas of adult AML genomics provides novel insights into disease biology, creates an evidentiary basis to support clinical testing improvements, and is a resource for both diagnostics and drug development. <This work is embargoed, with agreement of medRxiv, until 7th December 2025>. Statement of SignificanceAcute myeloid leukemia is a diagnostic category encompassing multiple rare hematological malignances. We show, in this nationwide multicenter study, that standardized unbiased whole genome DNA-sequencing and disease-optimized bioinformatics can replicate conventional "standard of care" AML clinical testing results, while also revealing currently underdiagnosed AML disease biology and potential genetic predisposition.

Richters transformation (RT) is a progression of chronic lymphocytic leukemia (CLL) to aggressive lymphoma. MGA (Max gene associated), a functional MYC suppressor, is mutated at 3% in CLL and 36% in RT. However, genetic models and molecular mechanisms of MGA deletion driving CLL to RT remain elusive. We established a novel RT mouse model by knockout of Mga in the Sf3b1/Mdr CLL model via CRISPR-Cas9 to determine the role of Mga in RT. Murine RT cells exhibit mitochondrial aberrations with elevated oxidative phosphorylation (OXPHOS). We identified Nme1 (Nucleoside diphosphate kinase) as a Mga target through RNA sequencing and functional characterization, which drives RT by modulating OXPHOS. As NME1 is also a known MYC target without targetable compounds, we found that concurrent inhibition of MYC and ETC complex II significantly prolongs the survival of RT mice in vivo. Our results suggest that Mga-Nme1 axis drives murine CLL-to-RT transition via modulating OXPHOS, highlighting a novel therapeutic avenue for RT. Statement of SignificanceWe established a murine RT model through knockout of Mga in an existing CLL model based on co-expression of Sf3b1-K700E and del(13q). We determined that the MGA/NME1 regulatory axis is essential to the CLL-to-RT transition via modulation of mitochondrial OXPHOS, highlighting this pathway as a novel target for RT treatment.

Patients with hematologic malignancies are a high priority for SARS-CoV-2 vaccination, yet the benefit they will derive is uncertain. We investigated the humoral response to vaccination in 53 non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), or CLL patients. Peripheral blood was obtained 2 weeks after first vaccination and 6 weeks after second vaccination for antibody profiling using the multiplex bead-binding assay. Serum IgG, IgA, and IgM antibody levels to the spike specific receptor binding domain (RBD) were evaluated as a measure of response. Subsequently, antibody-positive serum were assayed for neutralization capacity against authentic SARS-CoV-2. Histology was 68% lymphoma and 32% CLL; groups were: patients receiving anti-CD20-based therapy (45%), monitored with disease (28%), receiving BTK inhibitors (19%), or chemotherapy (all HL) (8%). SARS-CoV-2 specific RBD IgG antibody response was decreased across all NHL and CLL groups: 25%, 73%, and 40%, respectively. Antibody IgG titers were significantly reduced (p < 0.001) for CD20 treated and targeted therapy patients, and (p = 0.003) for monitored patients. In 94% of patients evaluated after first and second vaccination, antibody titers did not significantly boost after second vaccination. Only 13% of CD20 treated and 13% of monitored patients generated neutralizing antibodies to SARS-CoV-2 with ICD50s 135 to 1767, and 445 and > 10240. This data has profound implications given the current guidance relaxing masking restrictions and for timing of vaccinations. Unless immunity is confirmed with laboratory testing, these patients should continue to mask, socially distance, and to avoid close contact with non-vaccinated individuals. Statement of Translational RelevanceNon Hodgkin lymphoma (NHL) and Chronic Lymphocytic leukemia (CLL) patients who are treated with anti-CD20 antibody therapy, BTK inhibitor therapy, or who are monitored with active disease, have decreased antibody response to SARS-CoV-2 vaccination and decreased antibody titers compared to healthy controls. Antibody titers do not boost following second vaccination, and very few patients generate neutralizing antibodies against SARS-CoV-2. This data is of particular importance, given the recent guidance from the CDC that vaccinated patients no longer need to be masked indoors as well as outdoors. Patients with NHL or CLL who fall into these categories should not consider their immunity from vaccination to be assured. If infected with SARS-CoV-2, they should be a high priority for monoclonal antibody directed therapy. Unless immune response to vaccination is confirmed with laboratory testing, they should continue to mask, socially distance, and to avoid close contact with non-vaccinated individuals.

Acute myeloid leukemia (AML) patients rarely have long first remissions (> 5 years) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, short remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing and functional immunologic studies, we characterized 28 Normal Karyotype (NK)-AML patients with >5 year first remissions after chemotherapy (Long First Remissions, LFR) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). Our combined analyses indicated that genetic risk profiling at presentation (as defined by ELN 2017 Criteria) was not sufficient to explain the outcomes of many SFR cases. Single cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T-cells expressed an exhaustion signature and were resistant to activation by T-cell receptor stimulation in the presence of autologous AML cells. T-cell activation could be restored by removing the AML cells, or blocking the inhibitory MHC Class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ T-cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

CD8+ T cells are critical players in anti-tumor immunity. In higher-risk myelodysplastic neoplasms (HR-MDS) and acute myeloid leukemia (AML), CD8+ T cells exhibit altered functionality, however whether this affects disease course is poorly understood. Herein, we aimed to identify immune cell signatures in the bone marrow (BM) associated with disease progression and treatment outcomes. In-depth immunophenotypic analysis utilizing mass and flow cytometry on 104 pre-treatment BM samples from patients with myeloid neoplasms, revealed an increased frequency of a CD57+CXCR3+ subset of CD8+ T cells in patients with HR-MDS and AML who failed AZA therapy. Furthermore, increased baseline frequency (>29%) of the CD57+CXCR3+CD8+ T cell subset correlated with poor overall survival. We further engaged scRNA-seq to assess the transcriptional profile of BM CD8+ T cells from treatment-naive patients. We observed an increased abundance of cells within cytotoxic CD8+ T lymphocytes (CTL) cluster in secondary AML compared to HR-MDS. Additionally, response to AZA was positively associated with enrichment of IFN-mediated pathways, whereas enhanced TGF-{beta} signaling signature in CTL clusters was observed in non-responders. Our results support that targeting of CD8+ T cells with inhibitors of TGF-{beta} signaling in combination with AZA is a potential future therapeutic strategy in HR-MDS and AML.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal mature B lymphocytes in peripheral blood, bone marrow, lymphoid tissues, and extranodal sites. Genes involved in RNA splicing such as SF3B1 and U1 are frequently mutated in CLL, leading to altered splicing and generation of tumor neoepitopes. To study the impact of these mutations on the tumor microenvironment (TME), we have developed a comprehensive single-cell atlas of unmutated CLL encompassing 26 bone marrow and lymph node tumor samples from 23 U-CLL patients with mutations in U1 (n=7), SF3B1 (n=8), or without mutations in splicing genes (n=10). We observed high intra-tumor heterogeneity, discerning 12 transcriptional programs, one linked to the U1 g.A3>C mutation and characterized by NFKB hyperactivation. T cell and NK compartments exhibited site- and mutation-specific enrichment, with increased CD4+ regulatory cells (Treg) and CD8+ exhausted cells in lymph nodes, while U1-mutant tumors showed increased CD8+ cytotoxic activity, with a predominance of effector-like CD8+ cells. Single-cell T cell receptor sequencing revealed clonotype expansion in U1-mutated tumors, particularly in CD8+ effector and exhausted cells, suggesting a neoantigen-driven immune response. Cell-to-cell interaction analysis identified CD44 as a key mediator in U1-mutated tumors, showing pro-B survival interactions as those involving MIF-CD44-CD74. Furthermore, interactions between CD80 on CLL cells and CTLA4 on Tregs and CD8+ exhausted were upregulated, reflecting an immunosuppressive phenotype associated with U1 mutated CLL. These findings highlight the complex interplay between mutations in CLL and the TME, offering novel avenues for alternative therapeutic strategies for U-CLL with mutations in U1.

TP53 mutations represent one of the strongest adverse prognostic factors in myelodysplastic neoplasms (MDS). While multi-hit TP53 (TP53multiHit) alterations uniformly lead to very poor outcomes, the prognostic relevance of monoallelic TP53 (TP53mono) mutations remains controversial. TP53 variants can cause loss-of-function, dominant-negative, or gain-of-function effects. We hypothesized that functional heterogeneity among TP53 variants contributes to the variable clinical behavior observed in monoallelic TP53-mutated MDS. Therefore, we analyzed pretreatment samples from 4,505 patients with MDS from two independent cohorts (IWG, n=3,173; J-MDS, n=1,332), including 271 patients with TP53mono and 499 with TP53multiHit. Functional annotation of TP53 variants was performed using a previously published phenotype score (PS) derived from saturation mutagenesis screens, capturing dominant-negative and loss-of-function effects. Median overall survival (OS) differed significantly by TP53 allelic state (TP53 wild-type (TP53wt) 42.4 months; TP53mono 22.9 months; TP53multiHit 9.2 months; p < 0.001). Within the TP53mono subgroup, functional annotation identified marked heterogeneity. Patients with high PS ([&ge;]7) showed significantly inferior OS compared with those with low PS (median OS: 13.8 vs. 39.2 months; HR 1.68, 95% CI 1.16-2.42; p = 0.006), particularly for IPSS-R and IPSS-M low-risk cases. Combining PS and variant allele frequency (VAF) further improved risk stratification. TP53mono patients with PS [&ge;]7 and VAF [&ge;]22% had outcomes comparable to TP53multiHit (median OS: 8.8, p = 0.2), whereas those with PS <7 and VAF <22% exhibited survival similar to TP53wt (median OS: 49.7, p = 0.9). Overall, functional annotation of TP53 variants refines prognostication in TP53mono-mutated MDS and may enhance individualized risk assessment.

We previously used a disease-specific B cell receptor (BCR) point mutation (IGLV3-21R110) for selective targeting of a poor-risk subset of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR) T cells. Since CLL is a disease of the elderly and a significant fraction of patients is not able to physically tolerate CAR T cell treatment, we explored bispecific antibodies as an alternative for precision targeting of this tumor mutation. Heterodimeric IgG1-based antibodies consisting of a fragment crystallizable region (Fc) attached to either an anti-IGLV3-21R110 Fab or an anti-CD3 (UCHT1) single chain variable fragment (R110-bsAb) selectively killed cell lines engineered to express high levels of the neoepitope as well as primary CLL cells using healthy donor and CLL patient-derived T cells as effectors. R110-bsAb spared polyclonal human B cells (as opposed to CD19-targeting Blinatumomab) as well as CD34+ human stem cells. Yet, R110-bsAb induced lower T cell activation than Blinatumomab with primary CLL cells likely due to lower expression of target antigen. In vivo, R110-bsAb specifically killed IGLV3-21R110-expressing cell lines and CLL cells while sparing peripheral blood mononuclear cells. These findings highlight bispecific antibodies as a promising, off-the-shelf immunotherapy for high-risk CLL patients, offering selective targeting while preserving healthy B cells.