PURPOSE:This treat-and-resect clinical study evaluated a specialized form of pulsed electric field (PEF) ablation's potential to modulate antitumor immunity in early-stage non-small cell lung cancer (NSCLC). Tertiary lymphoid structures (TLS) are lymphoid aggregates that recruit immune cells into the tumor microenvironment (TME) and serve as immunity-generating neighborhoods. TLS with germinal centers (GC) have strong prognostic value in most cancers and promote tumor control and responsiveness to immune-based therapies, but no commercially available therapeutics induce their formation. The Aliya System is a proprietary electrosurgical technology that delivers microsecond electrical pulses to tissue, achieving tumor clearance while maintaining stromal architecture. PATIENTS AND METHODS:Treatment group patients received ablation immediately after diagnostic biopsy versus a biopsy-only control group. Herein, we report on exploratory endpoints examining immune modulation in blood and tumor samples collected from patients in both groups. RESULTS:Histopathologic assessments revealed the presence of TLS with GC in resected tumors. Tissue cytokine assessments and single-cell RNA sequencing demonstrated upregulation of cytokines capable of recruiting and organizing TLS-associated lymphocytes, including CXCL13, and significant increases in GC-related immune populations, including class-switched memory B and plasma cells in ablated tumors versus preablation biopsies. Ablation group patients had more tumors containing TLS with GC (47%) versus control (24%). Ablation group patients exhibited enhanced systemic immunity, with increased serum high mobility group box 1 and circulating cytotoxic T cells. CONCLUSIONS:This specialized PEF may orchestrate multiple simultaneous changes that promote a more immunologically active TME and represent a new approach for improving immunotherapeutic regimen responses in patients with NSCLC.

Systemic mastocytosis (SM) is a protean hematologic disorder characterized by uncontrolled expansion and accumulation of tissue mast cells (MC) in various organs, including skin, bone marrow, spleen, and gastrointestinal tract. In most cases, neoplastic cells exhibit the transforming KIT mutation D816V. Clinical symptoms arise from organ infiltration by neoplastic MC and/or pro-inflammatory mediators and cytokines released by these cells. Indeed, in a majority of the patients, acute or chronic mediator-induced symptoms are recorded, and in some instances, symptoms are severe and drug-resistant or even manifest as life-threatening anaphylaxis. In this article, we review the role of MC and basophils in anaphylactic reactions in patients with SM and the impact of genetic variables, co-morbidities, specific IgE, and factors counteracting MC activation. Whereas avoidance of all known and potential triggers of MC activation is crucial in the management of anaphylaxis in SM patients, specific therapy is also standard, including histamine receptor blockers, other anti-mediator-type drugs and drugs suppressing MC activation and/or expansion. Novel KIT D816V-targeting drugs can potentially decrease the anaphylaxis risk by reducing the MC burden and by targeting KIT-dependent and IgE-receptor-dependent signaling processes in neoplastic MC. Application of such drugs often leads to sustained responses and an increase in the quality of life in these patients.

The contribution of 9p deletion to B cell acute lymphoblastic leukemia (B-ALL) has remained elusive since its discovery more than 40 years ago. Here we show that loss of CD72 is recurrent in B-ALL cases containing PAX5 deletions, and that Cd72 haploinsufficiency drives B-ALL development in Pax5+/- mice. Mechanistically, Cd72+/-;Pax5+/- precursor B cells exhibited an inflammatory transcriptional profile characterized by a decrease in Myd88 expression, a finding that aligns with our previous studies of B-ALL development in Pax5+/- mice following exposure to immune stressors. These combined genomic analyses and functional models provide compelling evidence that co-deletion of 2 contiguous genes, Pax5 and Cd72, drives B cell leukemogenesis.

BACKGROUND:Primary immunodeficiencies (PIDs) are rare disorders caused by immune system defects that are commonly screened using multi-parameter flow cytometry (FCM). To counter the subjective and time-consuming manual data analysis of FCM data, we present PIDgeon, a fully automated computational pipeline based on artificial intelligence (AI) techniques. PIDgeon is designed to characterize PID immune profiles, suggest PID subtypes based on altered immune profiles, age, and immunoglobulin levels, and generate interpretable reports. METHODS:The PIDgeon pipeline, including FlowSOM and extreme gradient boosting models, was trained and tested on standardized FCM data generated according to EuroFlow procedures on 74 healthy controls and 399 patients (281 lymphoid-PID patients and 118 non-PID diseased controls) collected by the Ghent University Hospital. Subsequently, multi-centric validation was performed on internal (n = 211) and external (n = 338) independent data sets collected across 4 EuroFlow centers. RESULTS:Validation demonstrated high accuracy in cell count enumeration, achieving correlation scores above 0.90 for the major lymphocyte subsets. Interestingly, PIDgeon showed high sensitivity (93% to 100%) in predicting PID with severe T-cell defects, such as severe combined immunodeficiency and late-onset combined immunodeficiency, and low false-negative rates (1.5% to 5.4%) for distinguishing other lymphoid-PID vs non-PID diseased controls across data sets. Additionally, PIDgeon gives a first hint toward prediction of subtypes of primary antibody deficiencies, such as common variable immunodeficiency. CONCLUSIONS:In summary, PIDgeon is an accessible and explainable AI-pipeline aligned with current clinical needs, aiding laboratory immunologists in early PID diagnostics and increasing data analysis efficiency.

BACKGROUND:Varnimcabtagene autoleucel (var-cel) is an autologous CD19-directed chimeric antigen receptor (CAR) T-cell therapy with adaptive intra-patient dose escalation and was approved in Spain in 2021 for patients older than 25 years with relapsed or refractory B-cell precursor acute lymphoblastic leukaemia. We report activity and safety from var-cel's pivotal trial in this patient population. METHODS:This multicentre, single-arm, phase 2 trial was conducted at nine academic centres in Spain. Eligible patients had CD19+ B-cell precursor acute lymphoblastic leukaemia in the first relapse or refractory disease, either ineligible for allogeneic haematopoietic cell transplantation (HCT) or with relapsed disease after allogeneic HCT; centralised measurable disease in peripheral blood or bone marrow, or both, using flow cytometry; were aged 18-70 years; and had an Eastern Cooperative Oncology Group performance status of 0-2. Patients received a single course of intravenous lymphodepleting chemotherapy with fludarabine (30 mg/m2 per day for 3 days) plus cyclophosphamide (300 mg/m2 per day for 3 days), followed by intravenous fractionated var-cel escalation (0·1, 0·3, 0·6, and 2·0 × 106 CAR T cells per kg) separated by at least 24 h, with each fraction contingent on safety criteria. The primary endpoint was complete response with undetected measurable residual disease (MRD; sensitivity of ≤10-5) determined by flow cytometry by day 28 from var-cel administration, assessed in the efficacy population (defined as all patients who received at least one var-cel fraction). Safety was analysed in patients that initiated lymphodepletion. This study is registered with ClinicalTrials.gov, NCT04778579 (completed). FINDINGS:Between May 19, 2021, and July 6, 2023, 50 patients were assessed for eligibility, 12 were ineligible, and 38 were enrolled. 37 (97%) of 38 underwent leukapheresis (ITT population), 33 (87%) initiated lymphodepleting chemotherapy (safety population), and 32 (84%) received at least one var-cel fraction (efficacy population). Five patients who underwent leukapheresis did not receive var-cel due to rapid disease progression (n=3), death due to veno-oclusive disease (n=1), and persistent COVID-19 (n=1). Patients receiving var-cel had a median age of 40 years (33-48); of whom, 16 (50%) were female and 16 (50%) were male. By data cutoff (Jan 18, 2024), the median follow-up from infusion was 8·6 months (IQR 5·1-14·4) and 27 (84·4% [95% CI 67·2-94·7]) of 32 patients who received at least one dose of var-cel had a complete response with undetected MRD by day 28. Treatment-emergent adverse events occurred in 31 (94%) of 33 patients. The most common grade 3 or higher adverse events were neutropenia in 15 (45%) patients, thrombocytopenia in seven (21%), anaemia in five (15%), and cytokine release syndrome in four (12%). Immune effector cell-associated neurotoxicity syndrome and cerebral oedema was observed in one patient (3%; grade ≥3), and immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome occurred in two (6%; one resulted in death after protocol violation due to infusion during uncontrolled sepsis). INTERPRETATION:Var-cel induced deep remissions with low incidence of severe cytokine release syndrome and any-grade immune effector cell-associated neurotoxicity syndrome, supporting fractionated dose escalation as a strategy that preserves activity, limits acute toxic effects, and supports a hospital-based approach that could expand access to CAR T-cell therapy. FUNDING:Instituto de Salud Carlos III Subdirección General de Evaluación y Fomento de la Investigación Sanitaria and Fondo Europeo de Desarrollo Regional, CaixaResearch, Servei Català de la Salut, Servicio Madrileño de Salud, Servicio Murciano de Salud, Salud de Castilla y León, Servicio Andaluz de Salud, and Servicio Navarro de Salud-Osasunbidea. TRANSLATION:For the Spanish translation of the abstract see Supplementary Materials section.

ABSTRACT:In 5% to 15% of patients with systemic mastocytosis (SM), SM coexists with an associated hematological neoplasm (AHN). Most patients with SM-AHN harbor a KIT mutation, mainly KIT p.D816V, together with other AHN-related genetic alterations; however, limited data exist about the frequency and clinical impact of the coexistence of both types of genetic alterations in the same vs distinct bone marrow (BM) cell compartments. Here, we analyzed 79 patients with SM-AHN classified into patients who (1) displayed distinct unrelated genetic alterations in BM mast cells (MC) and AHN cells (21/79 [27%]); (2) shared the KIT mutation as a first genetic event (24/79 [30%]) involving both BM MC and AHN cells, together with or without AHN-associated alterations restricted to the latter cells; (3) had AHN-associated alterations as a first event (25/79 [32%]) present in both BM MC and AHN cells; and (4) had both alterations involving virtually all SM and AHN cells (9/79 [11%]). Overall, patients with genetically unrelated SM and AHN more frequently showed clinical manifestations associated with SM (ie, anaphylaxis, osteoporosis, and skin lesions) together with prolonged progression-free survival (PFS), whereas those with shared genetic alterations displayed signs of more advanced disease (ie, cytopenias or organomegalies) and shorter PFS and overall survival. Our findings confirm the clinical, genetic, and prognostic heterogeneity of SM-AHN and point to its association with the underlying oncogenic profile of neoplastic MC and AHN cells.

Infection remains a leading cause of morbidity in multiple myeloma. Preventing infections is paramount and immune profiling could reflect the cumulative effect of host, tumor and treatment-related immunosuppression. However, current understanding of immune dysfunction and its association with infection is limited. To address this gap in knowledge and identify immune biomarkers of increased infection risk, we performed immune profiling using next-generation flow cytometry in bone marrow and peripheral blood samples from 1,786 patients at various disease stages and treatment scenarios. Patients developing infection had significantly lower percentages of CD27+ B cells and CD27- NK cells, as well as increased CD27-/CD27+ T-cell ratio in bone marrow. These immune risk factors were validated in three independent datasets. An immune score was developed to stratify patients with ≤1 vs ≥2 of the aforementioned risk factors, which was associated with higher infection incidence (35% vs 60%, P <.001). The immune score (odds ratio: 2.31, P <.001), disease stage and CD38, BCMA or GPRC5D targeted therapy were independently associated with infection incidence. All cell types detectable in bone marrow and peripheral blood were significantly correlated, suggesting that immune biomarkers of increased infection risk could be monitored using minimally-invasive methods that are available in routine laboratories.

Measurable residual disease (MRD) assessment is a key determinant of risk stratification and treatment modification in B-cell acute lymphoblastic leukemia (B-ALL). Multicolour flow cytometry (MFC) is a globally adopted rapid and sensitive MRD technique. However, technical practices remain significantly variable across laboratories, compromising assay sensitivity, reproducibility, and comparability. Currently, systemic recommendations providing guidelines on the technical aspects of MFC-MRD in B-ALL are unavailable. The Cytometry Society (TCS)-India undertook a structured consensus process to standardize technical aspects of MFC-MRD testing. A two-round Delphi survey was conducted among laboratory professionals and clinicians across India to capture the prevailing practices and priorities. Consensus recommendations were prepared through survey findings and further refined during an in-person consensus congress with national and international experts from Australia, Egypt, Spain, and Russia. This document provides comprehensive recommendations across pre-analytic, analytic, and post-analytic phases of MFC-MRD including minimal antibody panel, best practices for sample collection, transport, and processing, Instrument setup, quality control, acquisition targets, gating strategies, approaches for LOD/LLOQ definition, hemodilution assessment, etc. A structured MRD report format with quality caveats is also provided. The recommendations are categorized into "Must," "Preferred," and "Acceptable" levels to suit diverse resource settings while promoting harmonization. Implementation of these standards will enhance assay sensitivity, reproducibility, and comparability, thereby strengthening MRD-guided clinical decision-making in B-ALL.

Adult B-cell precursor acute lymphoblastic leukemia (B-ALL) is genetically heterogeneous, comprising numerous subtypes. Standard methods leave many patients unclassified, and routine implementation of whole-transcriptome sequencing (WTS) or whole-genome sequencing (WGS) is limited. We evaluated a combined workflow of Optical Genome Mapping (OGM) and targeted next-generation sequencing (t-NGS) for genetic characterization of 100 adult BCR::ABL1-negative B-ALL patients enrolled in PETHEMA-LAL-19. This approach was compared with protocol genetic testing. The integration of OGM and t-NGS enabled the classification of 84% of BCR::ABL1-negative patients into 15 distinct subtypes, reducing the proportion of B-other/unclassified case from 50% to 16%. OGM was useful in identifying BCR::ABL1-like (26% of cases), associated with post-induction minimal residual disease (MRD) positivity (72% vs. 34%, p=0.0011), while t-NGS contributed to the detection of mutation-defined subtypes (PAX5 P80R and PAX5alt), correlated with MRD negativity (91% vs. 51%, p=0.012). OGM and t-NGS identified genetic patterns of certain subtypes, including a higher prevalence of IKZF1plus in BCR::ABL1-like (62% vs. 12%, p<0.001), JAK-STAT mutations only in patients with CRLF2-rearrangements and a high prevalence of PAX5-biallelic events (92% vs. 2%, p<0.001) in PAX5-subtypes. Therefore, the integration of OGM and t-NGS represents a feasible approach that enables comprehensive genetic characterization of adult BCR::ABL1-negative B-ALL, supporting its implementation in routine diagnostic workflows.

Abstract Myelodysplastic syndromes (MDS) are clinically and biologically diverse disorders, emphasizing the need for personalized treatment approaches. The International Working Group for Prognostication of MDS (IWG_PM) recently introduced a molecular classification, referred to as the MDS taxonomy, that categorizes patients into 16 subgroups based on 21 gene mutations, 6 cytogenetic abnormalities, and loss of heterozygosity (LOH) at TP53 and TET2 loci. This study sought to validate and enhance the clinical relevance of the MDS taxonomy by analyzing a large retrospective cohort (n = 5136) and transcriptomic data from a prospective cohort (n = 477). The taxonomy successfully identified subgroups with distinct clinical characteristics and disease progression patterns. However, incorporating gene interactions from taxonomy subgroups did not improve the prognostic performance of the Molecular International Prognostic Scoring System (IPSS‐M). We further assessed whether the taxonomy could guide management in patients receiving disease‐modifying therapies. Except for the “TP53‐complex” subgroup, taxonomy classifications were not predictive of hypomethylating agent response or transplant outcomes. Nonetheless, they correlated with overall survival, suggesting that while both IPSS‐M and the taxonomy capture disease biology, other non‐genetic factors may influence treatment response. RNA sequencing confirmed the biological distinctiveness of the taxonomy groups. Transcriptomic profiling of CD34+ bone marrow cells revealed unique, homogeneous gene expression patterns, particularly within the AML‐like, biTET2, SF3B1, and TP53‐complex subgroups. Further integration of multi‐omics data may refine MDS classification, improving clinical decision‐making and guiding the development of targeted therapies.

Breast cancer that arises in the postpartum period often carries a poor prognosis. The increasing trend of later-life pregnancies further exacerbates this risk. We urgently need chemoprevention strategies that reduce risk for postpartum period tumors, especially for more-aggressive ER-negative tumors and for those that arise in women with a genetic susceptibility. Here, we report that a single post-lactation dose of cabergoline, a dopaminergic agonist that blocks prolactin secretion, delays the onset and reduces the incidence of mammary cancer that arises postpartum in K14-Cre; Brca1 F/F , Trp53 F/F mice. In these mice, cabergoline treatment remodels the postpartum mammary gland by potentiating involution, reducing ductal area and proliferation, and reversing T cell exhaustion, potentially through modulation of ion channel expression. Notably, independent retrospective studies in two large cohorts of European women demonstrated a markedly lower incidence of postpartum breast cancer in those treated with cabergoline compared to a control group, with a meta-analysis indicating an approximate 69% reduction in risk. Our work underscores the importance of targeting post-lactational involution as a strategy for the prevention of postpartum breast cancer and identifies cabergoline as a novel, low-risk chemoprevention strategy during the vulnerable postpartum window by promoting physiological remodeling and mitigating immune dysfunction.

ABSTRACT:In adults with Philadelphia chromosome-negative (Ph-) acute lymphoblastic leukemia (ALL) genetic risk is usually combined with measurable residual disease (MRD) assignment to consolidation therapy. However, this combination is not uniform across trials and centralized assessment is not always performed. This study analyzed patient outcomes using centrally assessed MRD and genetics. Patients with high genetic risk (HGR), those who required 2 induction cycles for complete remission (CR), and patients with CR with end-of-induction (EOI) MRD of ≥0.01% were assigned to allogeneic hematopoietic stem cell transplant (allo-HSCT), whereas the remaining patients were assigned to delayed consolidation and maintenance. HGR for B-cell ALL included KMT2A rearrangements, low hypodiploidy and age of >35 years, homozygous TP53 mutations/deletions, or concomitant IKZF1 and CDKN2A/B deletions. HGR in T-cell ALL (T-ALL) included absence of NOTCH1/FBXW7 mutations and/or K/NRAS or PTEN alterations. Patients with early T-cell precursor (ETP) ALL received a different induction regimen, and all were assigned to allo-HSCT. Median age of 436 patients was 39 years (range, 18-60), 332 with B-lineage ALL and 104 with T-ALL. By intention to treat, 243 patients without ETP ALL (61%) were assigned to allo-HSCT and 157 (39%) to chemotherapy. The 3-year overall survival (OS) probability was 64% (95% confidence interval [CI], 58-69). For patients with CR and EOI MRD of <0.01% without HGR (n = 109), the OS probability was 81% (95% CI, 70-89), compared with 50% (95% CI, 34-63) for MRD-negative patients with HGR (n = 64). In patients with ETP ALL, the probability of 3-year OS was 61% (95% CI, 37-79). The combination of genetics and MRD allows accurate identification of adult patients with Ph- ALL who are candidates for allo-HSCT or chemotherapy. This trial was registered at www.clinicaltrials.gov as NCT04179929.

Monoclonal gammopathy (MG) of undetermined significance (MGUS) is the earliest and most prevalent stage of MG that may progress to symptomatic multiple myeloma (MM) or Waldenström's macroglobulinemia (WM) through intermediate smoldering forms of both diseases. We used next-generation flow cytometry (NGF) to identify and purify bone marrow (BM) clonal plasma cells (cPC) and B-cells/lymphoplasmacytic cells (cB-cells) from 25 asymptomatic individuals with MG diagnosed within the population-based iStopMM screening program for further molecular analysis by next-generation sequencing (NGS). IgM-MGUS cases consistently exhibited WM-like phenotypes by NGF, and MYD88 p.L265P and/or CXCR4 mutations in the coexisting cPC and cB-cells. Conversely, cPC from non-IgM MGUS and smoldering MM (SMM) displayed a MM-like phenotype associated with genetic drivers -i.e., t(11;14)-, frequently coexisting with genetically unrelated cB-cells. Interestingly, recurrent RUNX1 single nucleotide variants were detected in cPC, but not coexisting cB-cells, from WM-like cases, suggesting a role for RUNX1 in PC differentiation. In summary, NGF enabled detailed immunophenotypic characterization of BM cPC and cB-cell populations in all cases analyzed, subdivided in IgM vs non-IgM cases with WM-like or MM-like phenotypic and genetic profiles, respectively. These findings underscore the potential contribution of NGF and NGS for accurate classification or early-stage disease, beyond conventional criteria.

Background The Spanish Network on Mastocytosis (REMA) score was developed to predict clonal mast cell (MC) activation disorders (MCAD) in patients presenting with anaphylaxis without mastocytosis in the skin (MIS) and guide selection of patients for bone marrow (BM) examination. This study reassessed the diagnostic accuracy of the REMA score after 15 years of use and proposes refinements to optimize its predictive value.Methods Overall, 1204 patients with severe MC mediator-related symptoms without MIS referred to the REMA were retrospectively analyzed. All underwent clinical and allergologic evaluation, serum tryptase, BM histology, immunophenotyping, and KIT mutational analysis. REMA scores were calculated and diagnostic accuracy assessed across the score ranges. Multivariable analysis was applied to identify independent predictors and refine prediction.Results Clonal MCAD was diagnosed in 64% of patients who underwent a BM study. Overall, the REMA score showed excellent sensitivity (87%-90%) but limited specificity (34%-35%), with the poorest accuracy in the score 0-2 range. In this subgroup, independent predictors were elevated tryptase without hereditary alpha tryptasemia (OR 5.42, 95% CI 2.86-10.28) and insect venom as trigger (OR 2.18, 95% CI 1.28-3.72). KIT p.D816V in blood, although detected in only 29% of all clonal MCAD cases, allowed identification of additional low-score patients requiring BM evaluation. A refined algorithm combining these predictors improved specificity to 57% while maintaining sensitivity at 86%.Conclusions The REMA score remains highly sensitive for early detection of clonal MCAD presenting without MIS. Incorporation of additional clinical and molecular predictors into an updated diagnostic algorithm enhances specificity and ensures more accurate selection of patients with a REMA score of 0-2 for subsequent BM evaluation.

In March 2023 and 2024, a panel of international experts convened at the first and second Intercepting Blood Cancers (IBC) Workshops, with the aim of better appreciating the diagnostic challenges, pathophysiology, and potential therapeutic interventions for precursor malignant hematology conditions. Here, we report a summary of the proceedings from the sessions focused on monoclonal B-cell lymphocytosis (MBL)/chronic lymphocytic leukemia (CLL). We highlight four main content areas: biology of MBL, clinical implications of MBL, progression of MBL and transformation from indolent CLL to aggressive disease, and opportunities for therapeutic intervention in early CLL. We additionally outline key consensus management recommendations and research goals.

IntroductionThe interactions between T-cell chronic lymphoproliferative disorder (T-CLPD) tumor cells and the bystander immune cells may play a critical role in the failure of immune surveillance and disease progression, but the altered blood immune profiles of T-CLPD remain unknown.MethodsHere we analyzed the distribution of residual non-tumoral immune cells in blood of 47 T-CLPD patients -14 T-prolymphocytic leukemia (T-PLL), 7 Sézary syndrome/mycosis fungoides (SS/MF) and 26 T-large granular lymphocytic leukemia (T-LGLL)-, as tumor models of neoplastic T-cells that resemble naive/central memory (N/CM), memory and terminal effector T-cells, respectively, compared to 110 age- and sex-matched healthy donors, using spectral flow cytometry.ResultsOverall, our results showed deeply altered immune cell profiles in T-PLL, characterized by significantly increased counts of monocytes, dendritic cells, B-cells, NK-cells and innate lymphoid cells (ILC) -particularly ILC3-, together with reduced normal T-cells. In contrast, SS/MF showed neutrophilia, associated with decreased numbers of dendritic cells and NK-cells, potentially reflecting their increased migration from blood to the skin. In turn, T-LGLL displayed the mildest immune impairment, dependent on the TCD4+ vs. TCD8+ nature of the clonal T-cells and presence of STAT3 mutations among TαβCD8+ T-LGLL cases. Further dissection of the normal T-cell compartment showed a significant reduction of the earliest T-cell maturation compartments (N/CM) in T-PLL and SS/MF, whereas T-cells remained within normal ranges in T-LGLL, with only a minor reduction of N/CM T-cells.ConclusionThese findings point out the existence of differentially altered innate and adaptive immune cell profiles in the distinct diagnostic subtypes of T-CLPD, with progressively less pronounced alterations from T-PLL and SS/MF to T-LGLL.

Background Recent advances in next-generation sequencing (NGS) and transcriptomic profiling have refined the molecular classification of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), leading to the identification of novel subtypes with distinct biological features and clinical implications. Among these, the BCR::ABL1-like subtype is characterized by a stem/progenitor cell gene expression profile, poor response to conventional therapy, higher levels of measurable residual disease (MRD), and an increased risk of relapse. Accurate and timely classification of this subtype is crucial for therapeutic decision-making, including the use of tyrosine kinase inhibitors and intensified treatment protocols.AimsOur primary objective was to develop artificial intelligence (AI) models that allow for rapid and automated classification of BCR::ABL1-like BCP-ALL patients using gene expression data obtained from our custom panel. In addition, we aimed to assess whether the combination of gene expression profiles and diagnostic clinical variables could be used to predict adverse outcomes, such as relapse or death, at diagnosis.MethodsRNA-seq data from 179 BCP-ALL patients were quantified using Salmon and normalized across samples. Normalized expression values were used to train predictive models. The dataset was randomly divided into training (80%) and test (20%) subsets. The BCR::ABL1-like subtype was encoded as 1; all other subtypes as 0. Clinical outcome (relapse and death) was available for a subset of 82 patients. Two machine learning (ML) strategies were applied: LightGBM (LGBM, Microsoft®) and a neural network (NN) built with Keras. Hyper-parameter tuning was performed for both models. Prediction accuracy was used to assess classification performance, and ROC_AUC variable (receiver operating characteristic - area under the curve) was used to evaluate outcome prediction, both measured on the test set. Feature importance and SHAP values were computed to interpret the LGBM models Results Both AI models demonstrated strong classification performance, with test set accuracies of 0.96 (LGBM) and 0.98 (NN). No overfitting was observed (training vs. test accuracy difference < 0.02). For outcome prediction, the LGBM model yielded ROC-AUC values of 0.68 (death) and 0.83 (relapse). The top genes associated with relapse (based on LGBM feature importance) were JCHAIN (8), CD99 (11), and SHOC2 (19). For death, the top features were SHOC2 (17), RBM47 (17), and LDB3 (14). SHAP analysis suggested a protective role for SHOC2 in both relapse (SHAP: –0.70) and death (–0.40), whereas RBM47 (+0.60) and LDB3 (+0.70) were associated with increased mortality risk. Summary/Conclusions AI models trained on targeted RNA-seq gene expression data can reliably classify BCR::ABL1-like BCP-ALL, supporting their potential role in rapid, automated molecular diagnosis. Additionally, baseline gene expression at diagnosis shows promise for predicting clinical outcomes, although larger cohorts are needed to improve prognostic model performance. Preliminary findings highlight SHOC2 as a potential protective biomarker for relapse and mortality in BCP-ALL, while RBM47, LDB3, and CD99 warrant further investigation as markers of poor prognosis.

ABSTRACT:Detection of KIT p.D816V is a cornerstone in the diagnosis and classification of mast cell activation syndromes (MCAS) and mastocytosis. However, KIT p.D816V may be undetected due to a low mutated-cell burden in the blood and bone marrow (BM) of many patients, particularly those without skin lesions. These findings underscore the need for ultrasensitive molecular techniques for the detection of KIT p.D816V in these clinical settings. Here, we evaluated, to our knowledge, for the first time, the sensitivity and specificity of a novel Flow-SuperRCA KIT p.D816V assay, compared with conventional allele-specific oligonucleotide quantitative polymerase chain reaction (ASOqPCR), through the analysis of 548 blood and BM samples from 337 adult patients with MCAS and mastocytosis. Our results demonstrated greater sensitivity of the new technique vs ASOqPCR (limit of detection: 0.001% vs 0.01% variant allele frequency), with higher rates of positivity for KIT p.D816V in whole blood and/or BM of 64% of patients with monoclonal MCAS and 55% of patients with BM mastocytosis (P < .0001). Notably, the sensitivity of the new assay went beyond that of ASOqPCR in purified BM mast cells (P < .0001). Additionally, clonality was newly identified in 18% of patients previously diagnosed with nonclonal MCAS, who presented with a unique cutaneous MCA-related symptomatic profile. These results confirm the high specificity and ultrasensitivity of the Flow-SuperRCA assay for the detection of KIT p.D816V, emerging as a well-suited whole-blood and whole-BM test for diagnostic screening and classification of patients with MCAS and mastocytosis. These findings further highlight the clonal nature of an unprecedently high fraction of patients who presented with anaphylaxis and MCAS, with important pathogenic, diagnostic, and clinical implications.