Abstract Richter transformation (RT) is a rare but clinically challenging event in the evolution of chronic lymphocytic leukemia (CLL), characterized by transformation of CLL into an aggressive lymphoma, commonly diffuse large B-cell lymphoma. Although the molecular profiles of CLL and RT are well characterized, their evolutionary trajectories remain uncertain. A better understanding of CLL and RT dynamics could enable earlier identification and intervention in high-risk patients. Thus, our aim was to trace CLL/RT clonal histories over the lifespan of the patients through tumor phylogenies. To that aim, we applied primary template-directed amplification-based single-cell whole-genome sequencing to 5 patients, analyzing 349 single cells from up to 6 sequential time points spanning up to 19 years from CLL diagnosis to RT. A bioinformatic workflow that integrates existing algorithms with an in-house PTA-artifact filtering strategy was developed to optimize the sensitivity and specificity of the variant calling. A median sensitivity of 87.7% was obtained for point mutations at a sequencing depth of ∼20×, with only ∼5% of cell-specific artifacts remaining. Our analyses revealed an increased mutational burden from normal B-cells to CLL and from CLL to RT explained by the combination of therapy-related and cell-intrinsic mutational processes. In addition, a high intratumor heterogeneity was detected both in terms of somatic mutations and copy number alterations, which was not previously captured by bulk sequencing. The phylogenetic reconstruction revealed remarkable intrapatient parallel genomic evolution. This was exemplified by the independent acquisition of multiple del(13q) in 3 cases, del(9p21) in 2 cases, or 7 LOH(17p) in 4 different phylogenetic clades with distinct TP53 mutations in 1 patient. Next, we used clock-like mutations to time the acquisition of genetic drivers, the diversification steps between CLL and RT, and their clonal bursts, which are indicative of periods of rapid proliferation. These analyses revealed that initial CLL genetic drivers were acquired up to 30 years before diagnosis. However, abrupt clonal bursts were not triggered by these genetic drivers, suggesting that cell-extrinsic factors such as B-cell receptor stimulation or microenvironmental changes could initiate the expansion of CLL. On the other hand, RT diversifies from the CLL early, including before CLL diagnosis. However, these clones follow a years-long process of clonal evolution in which the acquisition of high genomic complexity at advance stages facilitates their final outgrowth. Overall, this study provides a roadmap of the origin, early diversification steps, and evolution of CLL and RT with potential clinical implications for early detection and intervention, while providing a framework for the study of the evolution of other hematological and solid tumors. Citation Format: Ian Márquez-López, Aleix Real, Núria Russiñol, Nicholas Williams, Romina Royo, Markus van Roosmalen, Heribert Playa-Albinyana, Juan Antonio Piñeyroa, Melika Bashiri, Pablo Mozas, Armando López-Guillermo, Julio Delgado, Dolors Colomer, Ruben van Boxtel, Jyoti Nangalia, Elias Campo, Ferran Nadeu. Clonal trajectories of chronic lymphocytic leukemia and Richter transformation over the patients' lifespan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4116.

BACKGROUND & AIMS:Patients with non-cirrhotic non-tumoral portal venous system thrombosis (NCNT-PVT) require long-term anticoagulation to prevent rethrombosis (either splanchnic or at any site, referred to as overall rethrombosis - oRT) when there is an underlying high-risk thrombophilic disorder or history of previous thrombotic episodes. However, up to 25% of patients without such indications still develop oRT. Elevated factor VIII (≥150%), D-dimer (≥500 ng/ml) or the presence of high molecular risk (HMR) variants have been proposed as risk predictors for oRT in these patients. This study aimed to compare the prognostic value of these three biomarkers in a cohort of patients with NCNT-PVT. METHODS:We performed a multicenter, retrospective, observational study including 123 patients with NCNT-PVT without an indication for long-term anticoagulation. RESULTS:During a median follow-up of 89.3 months (IQR 36-129), 33 patients (27%) developed venous oRT, with a cumulative incidence of 7%, 17% and 26% at 1, 5 and 10 years. Factor VIII ≥150% and HMR were associated with oRT risk (hazard ratio 5.92, 95% CI 2.67-13.15, p <0.01; and hazard ratio 2.07, 95% CI 1.01-4.24, p = 0.04, respectively) while D-dimer ≥500 ng/ml showed a non-significant trend. Multivariate analysis confirmed factor VIII and HMR as independent predictors. CONCLUSIONS:Patients with unprovoked or local factor-associated PVT have a clinically relevant long-term risk of recurrence, with a 25% cumulative incidence of oRT at 10 years. Elevated factor VIII (≥150%) identifies a subgroup at particularly increased risk, and its combination with HMR variants may further refine risk stratification. IMPACT AND IMPLICATIONS:This study addresses an important clinical gap by evaluating whether prothrombotic and genetic biomarkers can help identify patients with non-cirrhotic, non-tumoral portal vein thrombosis who remain at risk of recurrent thrombosis despite the absence of classical thrombophilia. Our results show that persistently elevated factor VIII levels, alone or combined with high molecular risk variants, are strong predictors of rethrombosis in this population. These findings may help refine recurrence-risk stratification in patients traditionally considered low risk and could support more individualized decisions regarding long-term anticoagulation and follow-up intensity.

Chronic lymphocytic leukaemia (CLL) presents a complex biological and clinical landscape, strongly influenced by the tumour microenvironment (TME). To better capture this heterogeneity, we have developed patient-derived lymphoma spheroids from CLL samples (CLL-PDLS), which recapitulate the cross-talk between malignant cells and the autologous immune microenvironment enabling efficient drug screening for further studies. CLL spheroids incorporate tumour B cells together with autologous T cells, which self-organize into a three-dimensional (3D) disc-shaped structure. Cultures can be maintained for up to 6 days under optimal conditions, preserving cell viability and high proliferative activity. T cells exhibit a range of activation and exhaustion phenotypes across both CD4+ and CD8+ subsets, including expression of Programmed Cell Death Protein 1 (PD-1), CD25, T cell immunoreceptor with Ig and ITIM domains (TIGIT) and CD69. The cellular composition remains stable, with tumour B cells representing approximately 90% of the population and T cells comprising 10%. The model was further validated by assessing responses to conventional therapies, including ibrutinib and venetoclax. In conclusion, this 3D spheroid model captures key functional aspects of B-T interactions in CLL, maintaining the dynamic interactions between malignant B cells and their autologous immune components, partially reproducing microenvironmental features, and offering a promising platform for preclinical therapeutic evaluation.

Disease persistence and resistance remain major challenges in chronic myeloid leukemia (CML). We evaluated pharmacological SOS1 inhibition with BI-3406, alone or in combination with BCR::ABL1 tyrosine-kinase inhibitors (TKI), across complementary preclinical models. In p210BCR/ABL transgenic mice, BI-3406 plus imatinib was well-tolerated, markedly prolonged survival, improved hematologic indices, reduced splenomegaly, and decreased bone-marrow LSK/hematopoietic stem and progenitor cells compared to either agent alone. In human CML cell lines and primary patient-derived bone-marrow cells, combined therapeutic treatments with BI-3406 and imatinib (standard of care BCR-ABL TKI) or next-generation TKIs showed strong synergistic suppression of cellular proliferation and increased apoptosis relative to single-agent treatment. Remarkably, drug combinations containing BI-3406 also restored TKI sensitivity to primary bone-marrow cell samples derived from a variety of CML patients that had been initially identified as clinically resistant to imatinib. Mechanistically, BI-3406 attenuated RAS/RAC-ERK/AKT signaling, while SOS1 knockdown phenocopied BI-3406-mediated imatinib sensitization, supporting an on-target SOS1-dependent therapeutic effect. Upregulation of the membrane transport protein SLC22A4 following combination treatment may contribute to the rescued TKI sensitivity but is probably not the sole underlying mechanism as BI-3406+imatinib retained strong synergistic activity despite SLC22A4 knockdown. Comparison of the transcriptomic profiles revealed that the combined BI-3406+imatinib drug regimen triggered the strongest repression of oncogenic, metabolic and stemness-associated transcriptional programs in treated CML cells. Consistently, serial replating MethoCult assays functionally confirmed the impairment of clonogenic self-renewal capacity and the depletion of murine LSK and human Lin⁻CD34⁺CD38⁻ stem/progenitor-enriched compartments after dual SOS1 and BCR::ABL1 inhibition. Our data support SOS1 targeting as a rational therapeutic strategy to potentiate TKI efficacy, impair CML stem/progenitor self-renewal, and restore drug responsiveness in clinically relevant TKI-resistant settings.

Chronic lymphocytic leukemia (CLL) is a common lymphoid neoplasm with heterogeneous biological features and clinical behavior, influenced by acquired genetic alterations and microenvironment interactions. We explored non-coding RNAs expression to understand their role in CLL pathogenesis and identify new clinically relevant biological biomarkers that could improve CLL prognosis and management. We initially profiled coding and non-coding RNA families in different CLL cohorts (N = 21) to identify those modulated by a combination of agonist microenvironment stimuli. Then we focused on lncRNAs that were overexpressed in response to the agonist stimuli, even in the presence of Ibrutinib, and whose baseline expression was associated with a shorter time-to-treatment in several independent CLL series (N = 266/72). Three lncRNAs (LINC00152, LINC00158 and RP11-161H23.5) were selected for functional validations in MEC-1 CLL cell line, where they demonstrated to regulate genes and pathways activated by microenvironmental stimuli, including induction of miR-155, a key oncomiR also enhanced by agonist treatment. LINC00152 expression was increased in more proliferative B-cell populations, including pre-treated as BTK-mutated Ibrutinib-relapsed primary CLL samples. Its silencing ex vivo reduced cell growth under combined microenvironmental stimulation and Ibrutinib in a subset of Ibrutinib-relapsed primary CLL samples, underscoring its potential relevance for further studies to improve CLL therapy.

Although first-line immunotherapy achieves remission in most patients with follicular lymphoma (FL), improved biomarkers and therapeutic strategies are required to identify and manage those who relapse. We investigated the immune microenvironment at diagnosis in uniformly treated FL patients with extended follow-up (>11 years), comparing relapsed and relapse-free cases. Transcriptomic profiling revealed enrichment of inflammatory response pathways in relapsed patients, including cytokine overexpression and upregulation of CD70, a molecule implicated in immune activation and inflammation. Multiplex immunofluorescence confirmed CD70 overexpression at diagnosis in tumor cells as well as in CD8+ and CD4+ T follicular helper (TFH) cells, correlating with inferior progression-free survival. Functional studies demonstrated that CD70+ tumor cells were more proliferative and induced CD70 expression in T cells, suggesting a feed-forward loop sustaining immune activation. To target these cells and enhance current CAR-T approaches, we engineered dual CD19–CD70 chimeric antigen receptor (CAR) T cells by co-transducing the CD19-CAR-T ARI-0001 with a CD27-based anti-CD70 CAR. Dual CAR-T cells exhibited enhanced in vitro cytotoxicity against patient-derived spheroids of FL, diffuse large B-cell lymphoma, and mantle cell lymphoma. In FL xenograft models, dual CAR-T treatment achieved superior disease control compared to monotargeted CAR-T cells, inducing complete tumor clearance in spleen and bone marrow. Collectively, these findings provide strong preclinical rationale for the clinical development of CD19–CD70 dual CAR-T therapy to improve outcomes in high-risk FL and potentially other B-cell non-Hodgkin lymphomas.

Criteria for the diagnosis of polycythemia vera (PV) require evidence of erythrocytosis, defined by increased hemoglobin and/or hematocrit. Although the International Consensus Classification also recognizes increased red cell mass (RCM), it is not included in the World Health Organization classification and is not widely available. We characterized a group of patients harboring JAK2 mutation with increased RCM, but hemoglobin/hematocrit values below diagnostic thresholds – referred to as low-hemoglobin PV (lhPV) – and evaluated red blood cell (RBC) count as a surrogate marker of RCM. We analyzed 288 patients: 79 essential thrombocythemia (ET), 31 lhPV and 178 PV. lhPV displayed features between ET and PV, but clustered closely with PV. Splenomegaly and thrombosis at diagnosis were more frequent in lhPV. JAK2 variant allele frequency (VAF) and high molecular risk abnormalities increased progressively from ET to lhPV and PV. In women, RBC count identified increased RCM with greater accuracy (AUC, 0.93) than hemoglobin, hematocrit or JAK2 VAF. RBC thresholds of >5.6×10^12/L (women) and >5.9×10^12/L (men) showed high diagnostic performance. lhPV represents a form of PV, with features closer to PV than ET. RBC count is an accurate surrogate marker of RCM and can improve recognition of PV patients who do not meet hemoglobin/hematocrit criteria.

MALAT1 is a long non-coding RNA (lncRNA) with altered expression in many cancers but poorly studied in B-cell neoplasms like mantle cell lymphoma (MCL), a very aggressive B-cell lymphoma. To elucidate the role of MALAT1 in MCL we have investigated its expression in 219 primary tumors using microarray data. We also analyzed the expression of its natural antisense transcript (TALAM1), which is key for the processing of MALAT1 RNA to its active isoform. High expression of MALAT1/TALAM1 lncRNAs was consistently linked to favorable clinical behavior in MCL, independently of other alterations previously described to influence MCL prognosis. In concordance with the observed clinical association, we found that the increased expression of these lncRNAs was inversely associated with gene signatures related to a proliferative and activation phenotype (MCL35-proliferation/BCR signaling signatures) in MCL samples from lymph nodes as peripheral blood. Finally, functional studies of MALAT1 in MCL primary samples showed that its levels were downregulated by microenvironmental stimuli and EZH2 activity, a known epigenetic factor induced by pro-proliferative stimuli and previously related with poor MCL prognosis. Altogether, these data support the favorable prognostic value of MALAT1 expression in MCL, and the MALAT1 dual role as oncogene or tumor suppressor in different neoplasms.

Background:MYD88 L265P is an early mutation in IgM monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic Waldenström macroglobulinemia (WM). Given the high prevalence of the MYD88 mutation observed in epidemiological studies, its presence is not sufficient to drive disease progression. In fact, a recent risk model of progression reported that the impact of other laboratory biomarkers was superior to the MYD88 mutation's presence. Due to the low incidence of these clinicopathological entities, there is a need for a better characterization of tumor and immune cells that can help to identify novel biomarkers. We hypothesize that the characterization of the risk groups in asymptomatic patients could improve the discovery of drivers of disease progression. Methods:We characterized the genomic and immune landscape of the most recent prognostic risk categories in 19 IgM MGUS and 17 asymptomatic WM patients. We performed targeted next generation sequencing (NGS) on CD19+ cells from bone marrow samples at diagnosis using a panel of 54 lymphoma-driver genes. Whole bone marrow samples were also used to measure mRNA gene expression in tumor and immune cells using the PanCancer ImmuneProfiling panel on the nCounter platform (NanoString). Results:We observed that low-risk patients were only characterized by the presence of MYD88 L265P, while intermediate- and high-risk groups harbored additional mutations on CXCR4, KMT2D, ARID1A and EP300. Regarding the mRNA expression analyses, we found an increased proportion of myeloid cells in the low-risk group, with monocytes having a significant decrease in low versus high-risk patients. The high-risk group also upregulated genes involved in the activation of NF-κB and B-cell receptor (BCR) signaling, while low-risk patients upregulated genes associated with an alternative activation of B cells or a decrease of the BCR signaling, such as TOLLIP, CEACAM1 and CR1. Conclusions:Beyond the MYD88 mutation, we described novel molecular mechanisms associated with high-risk patients, as an effort moving towards easy-to use new biomarkers in IgM gammopathy.

Ibrutinib and acalabrutinib are first- and next-generation Bruton Tyrosine Kinase inhibitors (BTKi), respectively, approved for chronic lymphocytic leukemia (CLL). Ibrutinib has been associated with cardiovascular events, including atrial fibrillation (AF) and hypertension. Acalabrutinib has demonstrated non-inferior progression-free survival than ibrutinib in relapsed/refractory CLL patients, with a lower cardiovascular event incidence. These adverse events seem to be derived from off-targets rather than BTK inhibition. Machine learning algorithms were applied to identify targets likely to trigger AF and hypertension in simulated CLL patients receiving acalabrutinib or ibrutinib. Common ibrutinib and acalabrutinib off-targets showed association with AF through structural remodeling and electrophysiology/ectopic activity mechanisms (TEC and ERBB4). There was association with hypertension through inflammation (ERBB4) and oxidative stress and endothelial dysfunction (ERBB4 and RIPK2). Ibrutinib-specific off-targets showed association with AF through structural remodeling (HCK, FGR, LYN, FYN, YES1, and FLT3) and electrophysiology activity (LYN and SRC), and with hypertension through inflammation (LCK, JAK3, and FLT3) and oxidative stress and endothelial dysfunction (ERBB2, BLK, SRC, and CSK). No acalabrutinib-specific off-targets were identified for AF or hypertension. This study supports that BTKi off-target selectivity may justify the different AF and hypertension incidences, suggesting their association with several ibrutinib-specific off-targets and identifying no acalabrutinib-specific ones.

In the last few years, whole-transcriptome sequencing has shown a high number of low-frequency fusion transcripts (FTs) involved in acute myeloid leukemia (AML) pathogenesis. Most of them are not identifiable through conventional diagnostic techniques. In this research, using RNA sequencing, we have investigated FTs in 109 cases of AML without recurrent genetic abnormalities (as defined by the fourth edition of the World Health Organization Classification of Hematolymphoid Tumours). We identified and validated 6 well-known AML-causing FTs (Tier-1), 9 FTs in which recurrently affected genes in AML were involved (Tier-2), and 4 Tier-3 FTs, along with other FTs found in healthy tissue databases (Tier-4). We highlighted 2 previously unknown FTs (ARHGAP11A::NUTM1 and RAP1B::GPC3) that constitute putative driver fusion genes in AML after performing a thorough analysis of their intrinsic properties, expression pattern, and clinical data correlation. Altogether, 15 patients from our cohort (14%) presented at least 1 validated FT, half of which had diagnostic and/or therapeutic implications. Furthermore, we were able to monitor 8 FTs during disease evolution, finding a good correlation with tumor burden. Nevertheless, the significance of many FTs remains unknown, which makes it necessary to enlarge curated FT databases to implement whole-transcriptome sequencing in clinical practice.

Introduction Molecular testing is increasingly essential in lymphoid malignancies to support future efforts in molecular classification and precision oncology. A custom 60-gene, DNA-only amplicon NGS panel using Ion AmpliSeq™ technology was developed for genomic profiling of lymphomas and lymphoid leukemias. The panel targets key exons from 36 genes and full coding sequences from 24 genes, including some relevant non-coding regions (e.g., NOTCH1 3' UTR). Gene selection was informed by the 4th and 5th WHO classifications, 2022 ICC classification, NHS Genomic Test Directory, disease incidence, and key literature. The panel is compatible with fully automated workflows on the Ion Genexus™ (GX5 chip) and Ion GeneStudio™ (Ion 530 chip) platforms. A network of 9 laboratories across Europe and Africa were involved in the panel evaluation. Methods A 2-phase, multicentre study was conducted to assess the LLN-panel performance, across a variety of sample types (including formalin fixed paraffin embedded tissue, bone marrow aspirates, and peripheral blood) using 5 Genexus and 4 GeneStudio platforms. Phase 1 focused on amplicon performance and variant detection, using commercial controls and a heterogenous set of available pre-characterized clinical research haematological samples, and samples from unrelated solid tumours known to carry similar variants. Orthogonal data sources included amplicon and hybrid capture based NGS assays and qPCR. Of 148 samples, 137 met the QC criteria (>1500x mean coverage per sample; >98.65% raw read accuracy). Phase 2 assessed inter-laboratory concordance through a sample sharing strategy using clinical research samples. Three sets containing extracted DNA from 16 lymphoma clinical research samples were cross shared between 8 labs. All phase 2 samples were analyzed blind, using IonReporterTM with the Lymphoma_Network_530_ver_1.0 (5.18) pipeline. Results Phase 1, sample level sequencing metrics for the Ion Genexus/GeneStudio platforms across all sample types showed average of 3,037,788/3,697,164 reads, mean read length of 124 bp/129 bp, 93.08%/95.96% uniformity, mean read coverage of 2118x/3117x, 97.71%/98.60% amplicons at 100x coverage, 88.88%/94.75% reads at 500x coverage, respectively. Regarding variant calling performance, SeraSeq® Lymphoma DNA Mutation Mix control data reported 100% sensitivity and specificity. Across 68 samples with orthogonal data available, 163 of 174 expected variants with limit cut-off set at 5% variant allele frequency (VAF) were detected, yielding a sensitivity of 93.68%. Among the pre-characterized lymphoid samples, 136 variants had comparative information, showing 93.4% of detection rate. KMT2D, ATM, and NOTCH1 emerged as the most frequently altered genes within the benchmarked lymphoid subset, with most expected variants detected. Significant findings included a p.Cys481 BTK alteration associated with resistance to covalent BTK-inhibitors in a CLL sample; BRAF V600E altered in 2 HCL research samples. Furthermore, most TP53 variants detected by the LLN panel were concordant with orthogonal data; the few discrepancies observed corresponded to anon-lymphoid tumor sample. A small number of expected alterations across several genes were not identified, potentially due to suboptimal performance of specific amplicon regions. Phase 2 results showed an inter-lab concordance of 92,8% for variants with VAF>5% (96.4%; 97.8%; and 84.2%, for each set). Lower concordance in the third set suggests sample quality issues. Conclusions The Ion AmpliSeq™ Liverpool Lymphoid Network (LLN) NGS panel is a research tool that demonstrates strong sequencing performance, coverage, variant detection, and inter-lab concordance. Ongoing refinements to the panel's gene content is informed by emerging research to support improved molecular characterization of lymphoid malignancies. While the inclusion of additional gene targets may expand biological insights, it will also increase the number of variants of uncertain significance (VUS). Feedback from the LLN research network is used to inform updates to the bioinformatics workflow, with the aim of enhancing analytical sensitivity, specificity, and variant annotation.

ABSTRACT:To identify genetic variants that influence myeloproliferative neoplasm (MPN) phenotypes, we undertook a 2-stage patient-only genome-wide association study. MPN subtypes (essential thrombocythemia [ET]; polycythemia vera [PV]) were compared with each other to healthy controls and stratified analyses was performed for chromosome 9p aberrations, JAK2 V617F mutation burden, and sex. The ET vs PV analysis identified known associations: (1) at HBS1L-MYB that increased ET risk (Pmeta = 7.93 × 10-6, odds ratio [OR] = 1.28) and reduced PV risk (Pmeta = 9.43 × 10-5, OR = 0.81) and (2) at GFI1B-GTF3C5 that predisposed to PV only (Pmeta = 1.43 × 10-9, OR = 1.38). Two further linked intronic variants, rs2425786 and rs2425788, at CDH22/CD40 were significant in females only (Pmeta = 2.67 × 10-8), with predisposition to PV (Pmeta = .0006, OR = 1.3) and reduction of ET risk (Pmeta = 7.82 × 10-5, OR = 0.75). A polygenic risk score consisting of 48 variants from 31 loci demonstrated moderate discriminative performance for ET and PV (area under the curve [AUC] = 0.718) and was improved by optimization for disease subtype (AUCET = 0.724 and AUCPV = 0.755). Overall, our results reveal that multiple germline variants influence MPN phenotype, with HBS1L-MYB and a novel sex-specific association with CDH22/CD40 being the strongest determinants.

PURPOSE:The Precision Oncology Program (POP) in Catalonia aims to provide equitable access to molecular testing for individuals with cancer, integrating Next-Generation Sequencing (NGS) into clinical practice to inform diagnosis, prognosis, and treatment decisions for both adult and pediatric patients with solid and hematologic malignancies, including somatic and germline alterations. This study evaluates the program's outcomes and impact. METHODS:This evaluation covers the period from the program's implementation in July 2021 through December 2023, with a more detailed analysis focusing on 2022-2023. The program involved 12 reference centers utilizing NGS technology for cancer genetic analysis, coordinated by CatSalut, the regional public health service payer. Data collected from each reference laboratory included the number of tests performed, types of tumor panels used, clinical indications, and associated outcomes. RESULTS:Between July 2021 and December 2023, a total of 23,135 molecular tests were performed on 22,501 patients. The most frequently analyzed panels were for solid tumors (38.1%), hematologic cancers (17.3%), and germline mutations (42.2%). Pediatric patients accounted for 2.4% of the total. Notably, 24.7% of patients underwent a change in clinical management, contributing to more targeted treatment strategies, particularly in solid tumors (58.7%). Reports were delivered within an average of four weeks, meeting program benchmarks and facilitating timely decision-making. Sample submission compliance was high, reaching 98.5%. CONCLUSIONS:This POP successfully addressed operational, financial, and logistic challenges, ensuring equitable access to molecular testing. This program led to more efficient and personalized clinical management, with growing impact on cancer care and patient outcomes.

CD6 is a lymphocytic receptor expressed by all T cells and a subset of B and natural killer (NK) cells. It physically associates with the antigen-specific clonotypic receptor of T (TCR) cells, where it modulates the activation and differentiation signals delivered along lymphocyte development and upon peripheral antigen recognition. CD6 is also expressed in some B-cell malignancies (e.g., chronic lymphocytic leukemia [CLL]), though its biological role and clinical performance is largely unknown. To this end, we have evaluated the potential impact of CD6 differential expression in a CLL patient cohort. 270 CLL patient case histories from the CLL-ES project with available RNA-Seq data have been analyzed. High CD6 expression was found to be associated with mutated IGHV status and predictive of longer time to first treatment in a uni- and multi-variable model. Ten-year probability of receiving treatment was 33% vs. 55% in the CD6hi and CD6lo groups, respectively (P=0.0003), along with the lymphocyte count and the CLL International Prognostic Index. Further Gene Set Enrichment Analyses showed association of high CD6 expression with downregulation of MYC-regulated, mitotic spindle-related, and RNA splicing-associated genes, all positively related to cancer progression. Interestingly, CD38, a widely studied adverse prognostic marker in CLL, was significantly down-regulated in the CD6hi group, in agreement with flow cytometry data. These results reinforce the notion that CD6 may play a pivotal role in neoplastic B-cell biology and lay the ground to further explore CD6 expression in the context of CLL prognoses.

ABSTRACT:The transcription factor lymphoid enhancer-binding factor 1 (LEF1) is aberrantly expressed across all subtypes and stages of chronic lymphocytic leukemia (CLL), yet the molecular mechanisms underlying its contribution to CLL pathogenesis remain poorly defined. Here, we conducted a comprehensive mechanistic dissection of LEF1 function in CLL using extensive functional analyses of patient-derived samples. We identified that, although LEF1 messenger RNA levels remain stable, patients with clinically aggressive disease show elevated LEF1 protein levels due to enhanced protein stability. LEF1 protein abundance is selectively modulated by lymph node-derived stimuli, including T-cell interactions and B-cell receptor signaling. Importantly, we uncovered a dual, context-dependent role for LEF1 that is determined by its protein levels. Low LEF1 protein, characteristic of indolent disease, supports B-cell activation, whereas increased protein abundance in aggressive disease promotes proliferation through the binding and induction of cell cycle and metabolic gene networks. We further showed that LEF1 exon 6 skipping is enriched in proliferative and aggressive CLL. Both in vitro and in vivo experiments revealed that LEF1-driven proliferation is mediated by these short, alternative spliced isoforms. Although all LEF1 isoforms bind to a core set of proliferation- and activation-related genes, they induce distinct transcriptional programs; full-length LEF1 promotes a quiescence gene signature and limits leukemic growth, whereas exon 6-skipping isoforms drive proliferation. Our findings establish LEF1 as an oncogenic transcription factor in CLL whose biological and clinical effects are modulated posttranscriptionally by both protein abundance and isoform composition.

Despite the remarkable clinical successes of chimeric antigen receptor (CAR) T-cell therapies in treating B-cell malignancies and multiple myeloma, similar outcomes have not been achieved in other indications. For patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) or T-cell acute lymphoblastic leukemia (T-ALL), treatment options are limited, yet CART-cell therapies offer significant potential to address this unmet need. Here, we introduce a first-in-class CART-cell therapy targeting CD84, a novel antigen, for the treatment of R/R AML and T-ALL. CD84 is highly expressed on leukemic blasts, with limited expression on hematopoietic stem progenitor cells (HSPC), and is largely absent in healthy human tissues. Our second-generation CARTs targeting CD84 (CART84) demonstrate potent cytotoxicity against AML and T-ALL cells both in vitro and in vivo in patient-derived xenograft (PDX) models. Furthermore, CART84 eliminated primary leukemic blasts while exhibiting low cytotoxicity against CD34+ HSPC in vitro and in humanized mouse models in vivo, suggesting a low risk of myelotoxicity. These results support CD84 as a promising target for AML and T-ALL and provide the foundation for our upcoming first-in-human phase I/II clinical trial using CD84-directed CAR T cell therapy for patients with R/R AML and T-ALL (EudraCT 2024-519966-31-00).

Mantle cell lymphoma (MCL) is considered one of the most aggressive B-cell lymphoid neoplasms. The transcription factor SOX11 is aberrantly expressed in conventional aggressive MCL, while it is not or weakly expressed in the leukemic non-nodal MCL subtype with a predominantly indolent clinical evolution. SOX11 is a key driver of MCL through the regulation of several oncogenic mechanisms, suggesting that it may be interacting with different protein complexes to exert its multiple actions. Using proteomic strategies, we characterized the SOX11-interactome and validated its physical interaction with SMARCA4, the catalytic subunit of the SWI/SNF chromatin-remodeling complex. SMARCA4 expression is directly regulated by SOX11, and its upregulation significantly associates with worse outcomes of patients. Integration of global DNA-binding and transcriptomic profiles revealed that SOX11 and SMARCA4 share binding sites enriched in open chromatin and active promoters and regulate common key oncogenic pathways crucial for MCL progression and aggressiveness. The SMARCA4-specific PROTAC-degrader AU-15330 significantly reduced SOX11 binding to specific regulatory regions and diminished the activation of BCR-, NIK-, and BCL2-signaling pathways. Moreover, SMARCA4 degradation significantly reduced proliferation and induced apoptosis of SOX11-positive MCL cells, highlighting AU-15330 as a promising therapeutic approach for patients who may relapse from current target therapies in MCL.

The 2022 WHO revision and the ICC classification have recently modified the diagnostic criteria for chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia. However, there is no consensus on whether CMML with NPM1 mutation (NPM1mut) should be diagnosed as AML. Nowadays, it is a subject of discussion because of its diagnostic and therapeutic implications. Therefore, we describe a case of a patient diagnosed with CMML NPM1mut and briefly review the literature to highlight the uncertainty about how to classify a CMML with NPM1 mutation. We emphasize the importance of a comprehensive molecular study, which is crucial to optimize the individualized treatment of patients, enabling them to access targeted therapies.