CD200 is a glycoprotein that binds with its receptor CD200R, providing immunosuppressive signals to T and NK cells. CD200 is expressed by normal stem cells and progenitors committed to B-lymphopoiesis and myeloid development. CD200 biological relevance in acute leukemias is only partially understood.The study included a consecutive series of four hundred thirty-one patients with acute myeloid leukemia (AML). Immunophenotype was established by multiparametric flow cytometry, and the genetic diagnosis was performed by PCR-based methods and a targeted resequencing method covering 42 genes.66% of AML patients expressed CD200 being significantly associated with CD34 reactivity. The frequency of CD200 positivity was higher in cases with core-binding factor genetic lesions such as RUNX1-RUNX1T1 (81.3%) fusions and CBFB-MHY11 (63.2%) rearrangements and also with biallelic CEBPA mutations (100%). The molecular AML group with the lowest CD200 reactivity (19.1%) corresponded to AML with NPM1 mutations. RNA seq showed no uniform pattern of infiltrating cells in CEBPA mutated AML. Deconvolution analysis may be used to assess the immunoregulatory mechanisms of AML.CD200 expression could help identify the more immature compartment and, combined with other markers, single out CEPA-mutated AML.

Background/Objectives: CD38-targeting monoclonal antibodies isatuximab and daratumumab have revolutionized multiple myeloma (MM) treatment, but a deeper understanding of their distinct mechanisms is crucial for therapeutic optimization. Methods: We used flow cytometry to assess isatuximab and daratumumab binding competition in MM cell lines and patient-derived bone marrow cells. The dynamics of CD38 expression were evaluated at different time points before and after antibody-mediated removal. The effects of IMiDs (pomalidomide, lenalidomide) on CD38 expression and isatuximab-induced apoptosis, either alone or in combination with IMiDs, were also examined. Moreover, MM cell migration was assessed through CXCR4-mediated assays, and cell adhesion was evaluated via CD49d-dependent assays. Results: Isatuximab and daratumumab did not compete for CD38 binding, confirming distinct epitope recognition. Following depletion with either antibody, CD38 expression on the MM cell surface began to recover within 2 h, suggesting a dynamic regulation of CD38 availability. While daratumumab lacked direct apoptosis, isatuximab induced significant direct cell death. Pomalidomide enhanced isatuximab-induced apoptosis by increasing CD38 expression, whereas lenalidomide had no significant effect. Additionally, both antibodies effectively inhibited MM cell migration and significantly reduced cell adhesion. Conclusions: Their non-competitive binding and shared impact on cell dynamics suggest opportunities for optimizing treatment strategies through combinatorial or sequential approaches in MM therapy.

Given the heterogeneity of acute myeloid leukemia patients, it is necessary to identify patients considered fit for intensive therapy but who will perform poorly, and in whom alternative approaches deserve investigation. We analyzed 1034 fit adults ≤70 years intensively treated between 2012 and 2022 in the CETLAM group. Young adults ( ≤ 60 years) presented higher remission rates and improved survival than older adults above that age (CR 79% vs. 73%; p = 0.03 and 4-yr OS 53% vs. 33%; p < 0.001). Remission and survival outcomes varied among different genetic subsets. An especially adverse genetic group included complex, monosomal karyotype, TP53 alterations (deleted/mutated), and MECOMr. Transplant feasibility in this very adverse risk group was low, and OS and EFS at 4 years were 14% and 12%, in contrast to 70% and 57% in the favorable group and 38% and 32% in all other patients. We integrated clinical and genetic data into the Intensive Chemotherapy Score for AML (ICSA) with 6-risk categories with significantly different remission rates and OS, validated in another cohort of 581 AML patients from a previous CETLAM protocol. In summary, we identified groups of fit patients that benefit differently from an intensive approach which may be helpful in future treatment decisions.

Background Measurable residual disease (MRD) detection in acute myeloid leukemia (AML) patients in complete morphological remission (CR) is associated with an increased risk of relapse and mortality. While molecular methods such as qPCR offer high sensitivity, most patients lack a molecular marker and are monitored using flow cytometry (FC). Next-generation sequencing (NGS) and digital PCR (ddPCR) represent a potential alternative for MRD assessment in AML, given that they offer high sensitivity and broad applicability. In this prospective study, we aimed to compare the clinical performance of NGS and ddPCR for MRD assessment in a cohort of intensively treated AML patients. Methods Adult patients with newly diagnosed AML treated with intensive chemotherapy (IC) according to CETLAM 2022 protocol were included prospectively in this study. Bone marrow (BM) samples of patients in CR were collected for MRD analysis at different time points: after two courses of IC (all), at the end of IC (favorable patients), at the time of hematopoietic stem cell transplant (HSCT) and 1 month after (intermediate/adverse patients). A custom NGS panel for MRD detection (NGS-MRD) was designed including 30 genes frequently mutated in AML and a specific assay to detect FLT3-ITD. NGS-MRD was performed on DNA extracted from BM. ddPCR assays were selected personally based on the mutational profile of each patient and performed on the same DNA samples. Dilution studies were conducted to assess the level of detection of both techniques, establishing a positivity threshold of 0.2% VAF for NGS-MRD and 0.1% VAF for ddPCR. MRD status was defined according to the European LeukemiaNet (ELN) guidelines (Heuser M et al, Blood, 2021). Results Forty-nine BM samples in CR from 23 AML patients were included in this study. The median age at diagnosis was 61 (range 21-73), and 52.2% were male. Fifteen (65.2%) patients had HSCT indication based on ELN 2022 intermediate or adverse-risk, while 8 (34%) were classified as favorable-risk. Standard of care (SoC) methods for MRD evaluation were flow cytometry in 16 (69.5%) of them, NPM1-qPCR in 6 (21.8%), and CBF-qPCR in 1 (4.3%). With a median follow-up of 17.9 (range 3.7-24.9) months, there were 4 (17.4%) relapses and 2 (8.7%) deaths. All 49 samples were analyzed with NGS-MRD. A median of 3 mutations (range 1-8) could be tracked for each patient with a median read depth of 6336x (range 576-26402x). Twenty-five (51%) samples had a targetable mutation for ddPCR analysis including IDH1, IDH2, NRAS, and FLT3-TKD. In the 25 samples studied by NGS and ddPCR, we found a strong correlation (r=0.925) between the VAF values obtained by both methods. The median VAF for positive MRD samples was 4.2% (range 0.1-42.1%). All NGS-MRD-positive samples were also considered positive by ddPCR, while 5 NGS-MRD-negative had low-level detectable ddPCR-MRD (range 0.1-0.5% VAF). Next, we compared our results with standard methods. After two cycles of IC, 33.7% of patients had detectable MRD by SoC techniques, while 52.1% by NGS and 63.6% by ddPCR. All SoC-MRD+ were also positive by NGS and ddPCR, but we could identify MRD persistence in 50% of patients with undetectable MRD by FC or qPCR. Moreover, in our cohort, NGS-MRD status after the second induction significantly predicted RFS (22.2 vs not reached; p=0.025). ddPCR-MRD status showed a positive trend (15.2 vs NR; p=0.191) but did not reach statistical significance. At the end of chemotherapy, all favorable-risk patients had undetectable MRD by SoC tests, but we could detect 3 (37.5%) MRD+ cases by both NGS and ddPCR. Two of these cases relapsed within 6 months. Regarding HSCT recipients, pre-transplant MRD was detectable in 35.7% of patients by FC, 50% by NGS, and 75% by ddPCR. However, pre-HSCT MRD by any method did not show an impact on RFS. This could be due to the high MRD clearance rate after HSCT: all FC-MRD+ patients converted to MRD- and 92.9% of MRD+ patients with NGS or ddPCR. Conclusions NGS and ddPCR showed a high correlation as MRD assessment methods. While ddPCR offered higher sensitivity, NGS-MRD status was more predictive of relapse-free survival. Also, NGS allowed us to track multiple mutations and even new clones, while ddPCR was restricted to 1-2 targets per patient. Applying these techniques in a prospective AML cohort, we could detect MRD persistence in a substantial number of MRD-negative patients by standard methods.

Increased WT1 mRNA levels are pervasive in acute myeloid leukemia (AML), and this marker has been used to assess the leukemic compartment size after chemotherapy or hematopoietic cell transplants. Little is known about the effects of WT1 on the leukemic cells and their targets. This work used data obtained from gene expression arrays performed on AML samples with high and low WT1 mRNA levels to pinpoint genes that WT1 can regulate. We singled out SGK1, which showed an inverse correlation between its mRNA levels and WT1 in leukemic cell lines and AML samples. In cellular models, forced expression of WT1 reduced mRNA and protein levels of SGK1. Furthermore, WT1 repressed the SGK1 promoter activity, and accordingly, WT1 knockdown showed an increased expression of SGK1. We also detected an inverse correlation between WT1 and SGK1 during leukemic cell-line differentiation. WT1 genetic knockdown displayed decreased cell viability under nutrient deprivation. By contrast, SGK1 knockdown or pharmacologic inhibition increased resistance to apoptosis in response to serum starvation, acting on cell cycle progression. The effects of SGK1 targeting in hematologic conditions merit further investigation.

Acute myeloid leukemia (AML) presents major clinical challenges due to chemoresistance and high relapse rates, primarily driven by therapy-resistant leukemic stem cells (LSCs) within the bone marrow. To address this issue, we developed T22-HSNBT-H6-MMAE, a novel humanized nanoconjugate designed to target CXCR4⁺ cells while delivering the cytotoxic payload monomethyl auristatin E (MMAE). CXCR4 signaling pathway plays a critical role in LSC survival by preserving stem-like properties and activating protective mechanisms that promote treatment resistance. Moreover, CXCR4 is overexpressed in approximately 50 % of AML patients and is associated with poor prognosis and high relapse rates. We evaluated the therapeutic potential of T22-HSNBT-H6-MMAE using in vitro assays with AML cell lines and primary patient samples, as well as in vivo studies in a disseminated AML mouse model. Our results demonstrated that T22-HSNBT-H6-MMAE exerts a CXCR4-dependent cytotoxic effect through mitotic catastrophe and apoptosis induction in CXCR4⁺ AML cell lines. In vivo evaluation in a disseminated CXCR4⁺ AML mouse model showed potent antineoplastic activity, with complete suppression of leukemic dissemination and significantly prolonged survival, all without systemic toxicity. Notably, the nanoconjugate remained effective even in models with extensive bone marrow involvement and exhibited activity against diverse patient-derived CXCR4⁺ AML blasts, while sparing healthy donor bone marrow and peripheral blood mononuclear cells (PBMCs) from significant toxicity. Collectively, these findings highlight T22-HSNBT-H6-MMAE as a promising therapeutic candidate for a broad range of AML patients, offering selective antineoplastic activity against CXCR4⁺ leukemic cells.

The mechanisms through which mutations in splicing factor genes drive clonal hematopoiesis (CH) and myeloid malignancies, and their close association with advanced age, remain poorly understood. Here we show that telomere maintenance plays an important role in this phenomenon. First, by studying 454,098 UK Biobank participants, we find that, unlike most CH subtypes, splicing-factor-mutant CH is more common in those with shorter genetically predicted telomeres, as is CH with mutations in PPM1D and the TERT gene promoter. We go on to show that telomere attrition becomes an instrument for clonal selection in advanced age, with splicing factor mutations 'rescuing' HSCs from critical telomere shortening. Our findings expose the lifelong influence of telomere maintenance on hematopoiesis and identify a potential shared mechanism through which different splicing factor mutations drive leukemogenesis. Understanding the mechanistic basis of these observations can open new therapeutic avenues against splicing-factor-mutant CH and hematological or other cancers.

Background Hairy cell leukemia (HCL) is a rare disorder characterized by splenomegaly, pancytopenia, and proliferation with "hairy" lymphocytes. Treatment is based on purine analogs and anti-CD20 antibodies, often resulting in significant adverse effects. Aims The objective of this study is to describe the frequency, clinical, and biological characteristics of a historic cohort of HCL patients in our center and the most common side effects related to treatment with purine analogs. Methods and Results This study analyzed 21 patients treated between 2009 and 2023, focusing on clinical characteristics, treatment response, complications, and survival outcomes. Cladribine treatment achieved complete response in 77.8% of patients. The 5-year OS and PFS were 100% and 91.7%, respectively. Infections, pathogens such as herpes viruses and mycobacteria, were major complications, impacting 38% of patients. Severe skin reactions were noted in patients treated with cladribine. Conclusion The study highlights cladribine's effectiveness in inducing remission in HCL patients, pointing out the significant risks of infections and other adverse effects. Introducing targeted treatments like BRAF inhibitors provides promising alternatives, especially for resistant patients or those intolerant to purine analogs. Future strategies should focus on integrating targeted therapies to reduce treatment-related morbidity.

Chronic myelomonocytic leukaemia (CMML) is a rare haematological disorder characterized by monocytosis and dysplastic changes in myeloid cell lineages. Accurate risk stratification is essential for guiding treatment decisions and assessing prognosis. This study aimed to validate the Artificial Intelligence Prognostic Scoring System for Myelodysplastic Syndromes (AIPSS-MDS) in CMML and to assess its performance compared with traditional scores using data from a Spanish registry (n = 1343) and a Taiwanese hospital (n = 75). In the Spanish cohort, the AIPSS-MDS accurately predicted overall survival (OS) and leukaemia-free survival (LFS), outperforming the Revised-IPSS score. Similarly, in the Taiwanese cohort, the AIPSS-MDS demonstrated accurate predictions for OS and LFS, showing superiority over the IPSS score and performing better than the CPSS and molecular CPSS scores in differentiating patient outcomes. The consistent performance of the AIPSS-MDS across both cohorts highlights its generalizability. Its adoption as a valuable tool for personalized treatment decision-making in CMML enables clinicians to identify high-risk patients who may benefit from different therapeutic interventions. Future studies should explore the integration of genetic information into the AIPSS-MDS to further refine risk stratification in CMML and improve patient outcomes.

Introduction: Recent advancements in the prognostication of Myelodysplastic Syndromes (MDS) have highlighted the importance of integrating genetic data with traditional clinical and laboratory parameters. MDS has traditionally been stratified using models such as the Revised International Prognostic Scoring System (IPSS-R) and, more recently, the molecular IPSS (IPSS-M). These models guide therapeutic decisions and clinical trial designs. Our previous work introduced the Artificial Intelligence Prognostic Scoring System for Myelodysplastic Syndromes (AIPSS-MDS) score, which demonstrated robust predictive accuracy using conventional data. This study aims to enhance AIPSS-MDS by incorporating molecular annotations. Objectives: The primary objective was to recalibrate the AIPSS-MDS model by integrating comprehensive molecular data to evaluate their prognostic significance alongside traditional clinical indices. We aimed to assess the model's ability to predict overall survival (OS) and leukemia-free survival (LFS) and identify specific genetic variables that independently affect prognosis. Methodology: We calculated the AIPSS-MDS scores for OS and LFS within a subset of patients in the IPSS-M cohort who had non-missing annotation (1,548 patients for OS and 1,427 for LFS). Then, we used the entire IPSS-M cohort (2,956 patients) to generate a random survival forest (RSF) model using all the 130 genetic variables. Afterwards, we conducted cox regression analyses to identify significant genetic predictors of OS and LFS independently of the AIPSS-MDS score, and integrated these into our models to create the molecular AIPSS score for MDS (AIPSSmol-MDS). Models were evaluated based on time-dependent Areas Under the Curve (AUCs) and out-of-bag concordance indexes (c-indexes). Results: The AIPSS-MDS yielded a c-index of 0.735 for OS and 0.801 for LFS, underscoring the robustness of traditional data inputs. The model based on the 130 genetic parameters (genetic-only) model showed a slightly lower c-index (0.685 for OS and 0.772 for LFS). The integrative model combining both data types achieved a c-index of 0.746 for OS and 0.805 for LFS, indicating that molecular data marginally enriches the prognostic landscape, and does not replace conventional data. Next, we used cox regression to identify gene mutations associated with prognosis independently of the AIPSS-MDS score. Mutations in ASXL1, CBL, EZH2, FLT3-ITD, KRAS, NRAS, RUNX1, SETBP1, and STAG2, as well as TP53 multihit mutations, emerged as significant predictors of OS; whereas mutations in ASXL1, EZH2, FLT3-ITD, MLL-PTD, RUNX1, STAG2, U2AF1, and WT1, as well as TP53 multi-hit mutations, were independent predictors of LFS. Incorporating these markers into AIPSSmol-MDS achieved c-indexes of 0.743 for OS and 0.812 for LFS, and improved moderately the time-dependent AUCs for LFS prediction. Independent validation efforts are ongoing and will be presented in ASH. Conclusion: Our study underscores the importance of conventional data in MDS prognostication and shows that adding layers of molecular annotations offers modest improvements in predictive accuracy. AIPSSmol-MDS, though slightly improved, remains a robust tool, especially where molecular data is unavailable. Future research should incorporate broader genomic data to further refine these models and enhance precision medicine in MDS.

BACKGROUND:Overall, the prognosis of patients with chronic lymphocytic leukemia (CLL) in the early phase of the disease (Rai 0, Binet A) is favorable; some patients never require therapy. However, some patients require intervention shortly after diagnosis. In the past decade, several risk scores (RS) have been developed to predict disease progression, yet some patients are misclassified. On the other hand, IGHV subset 2 (IGHV2) predicts poor outcomes. METHODS:A retrospective and multicentric study was conducted to compare the accuracy of five different RS (IPS-E, CR0, AIPS-E, CLL-IPI, and Barcelona-Brno) to predict disease progression in 781 stage A previously untreated patients with CLL. As an exploratory analysis, it was further investigated whether the inclusion of the IGHV2 as a poor prognostic parameter improved the accuracy of RS. RESULTS:All the scores identified a similar group of patients with CLL in early stage with low-, intermediate-, and high-risk progression. Discrimination was high and similar in all RS (c-index = 0.74-0.79, area under the curve = 0.7-0.75), as well as calibration (p = .98) and parsimony, although CLL-IPI showed the best results (Akaike information criterion = 441). A total of 34.4% of patients were categorized within the same RS and concordance was at least moderate between RS. CONCLUSION:Moreover, the results suggest that IGHV2 may improve the accuracy of RS.

Introduction Measurable residual disease (MRD) assessment has become a crucial prognostic tool in patients diagnosed with acute myeloid leukemia (AML) undergoing intensive chemotherapy. While its prognostic impact is well-documented in these patients, its value among those treated with venetoclax-based low-intensity combinations remains less certain. Some studies have individually validated the efficacy of flow cytometry (MFC) (Pratz K et al, JCO, 2022) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) in patients with AML with mutated NPM1 (Othman J et al, Blood, 2024) in this context. Methods This multicenter, retrospective study aims to validate the prognostic value of MRD in patients treated with low-intensity venetoclax combinations, unfit for consolidation with further strategies, following the ELN-MRD 2021 recommendations. Patients were treated in 5 centers belonging to the Spanish CETLAM Group from March 2019 to January 2024. MRD was measured following the updated ELN 2021 recommendations (Heuser M, et al. Blood 2021), using RT-qPCR for patients with NPM1 mutated AML, and an eight-color MFC panel for the rest of the patients. Impact of MRD results on leukemia-free survival (LFS) and overall survival (OS) was estimated considering MRD response as a time-dependent variable using the Mantel-Byar test and with a corresponding landmark analysis. Multivariate analysis (MVA) was performed using Cox's proportional hazards model, including relevant variables related to treatment success. Results Initially, 251 patients were included. The overall response rate (CR/CRi/MLFS/PR), including partial response (PR), was 66% (166 patients). Patients who achieved a PR (21 patients, 8.3%) or underwent allogeneic hematopoietic stem cell transplantation (28 patients, 11.5%) were excluded of the study. Additionally, MRD assessment during follow-up was not performed in 29 patients who achieved a response, reducing the final cohort size to 89 patients. The median age of the cohort was 73 years (range 48-85), with 52.8% being male. Patients received venetoclax combined with hypomethylating agents (VenHMA) as frontline therapy (ND) (74.1%) or in a relapsed/refractory (R/R) (25.9%) status. AML with myelodysplasia-related features was the most frequent diagnosis (42.7%, including specific gene mutations (n = 32) and cytogenetic abnormalities (n = 6)), followed by AML with mutated NPM1 (37.1%) and AML with mutated TP53 (11.2%). According to the ELN 2022 risk classification, 24.7% of the patients were classified in the favorable risk group, 19.1% in the intermediate, and 56.1% had an adverse risk. Additionally, 18 patients had adverse cytogenetic risks, including 6 patients presenting complex karyotypes. Regarding morphological complete response (CR/CRi/MLFS), median cycles to any response were 1 (range 1-8). Median follow-up was 11.5 months (range 2-52), with a OS and LFS of 18 (range 13.3-24.3) and 10.2 (range 8.2-14.8) months respectively. MRD response rate was 43.6%. Median number of cycles to MRD response were 3 (range 1-11). MRD response rates at 2, 4, and 6 cycles were 20.2%, 33.7% and 42.7% respectively. Responders who achieved a negative MRD were more frequently younger than 70 years old (p = 0.02) and NPM1 mutated (p = 0.003), with no other clinical nor biological differences found between the two cohorts. Mantel-Byar test (OS: 27 vs. 11 months, LFS: 21 vs. 9, both p <0.001) and Kaplan-Meier landmark analysis at 3 cycles (OS: 27 vs. 10, LFS: 18 vs.7, both p <0.001) showed that MRD negativity was prognostic for OS and LFS. The MVA analysis considering the most relevant characteristics related to VenHMA treatment [age <70 years (vs. younger), NPM1, FLT3-ITD, N/KRAS/PTPN11/CSF3R, adverse cytogenetic risk (vs. other), ND (vs. R/R) and CR (vs. CRi)] further confirmed MRD response as an independent prognostic value for OS (HR: 0.27 (95%CI: 0.12-0.59), p <0.001) and LFS (HR: 0.3 (95%CI: 0.15-0.58), p <0.001) in patients responding to VenHMA. Conclusion MRD response, evaluated accordingly to the ELN-MRD 2021 recommendations, holds significant prognostic value for both OS and LFS in patients treated with VenHMA in real-life clinical practice. These findings underscore the importance of MRD monitoring in guiding treatment follow-up and improving subsequent treatment decisions in low-intensity venetoclax-based therapies.

Topic: 6. Chronic lymphocytic leukemia and related disorders - Clinical Background: Chronic lymphocytic leukemia (CLL) is marked by severe immune dysfunction, which results in an increased risk of autoimmune disorders (AID). The association of autoimmune cytopenia (AIC) with the disease has been extensively studied. However, there is little information about its relationship with other non-hematologic autoimmune complications. Aims: To describe the prevalence and characteristics of patients with CLL spectrum (CLL, small lymphocytic leukemia, and monoclonal B-cell lymphocytosis) and non-hematologic AID. The secondary objective was to ascertain differences in clinical and prognostic variables between AIC and non-hematologic AID groups. Methods: We included 712 patients with CLL from the historical database of our center. The medical records were retrospectively reviewed to identify any AID diagnosis. AID were categorized based on the American Autoimmune Related Diseases Association classification. Those patients with both AIC and non-hematologic AID were classified into the AIC group. Results: The main characteristics of patients with CLL and AID are shown in Table 1. After a median follow-up of 6.4 years (0.1-36.4), 87/712 (12.2%) presented non-hematologic AID, and 45/712 (6.3%) showed AIC; 11/712 (1.5%) had both AIC and non-hematologic AID. In the non-hematologic AID group, endocrine disorders were the most frequent (48.3%; 42/87), followed by dermatological AID (33.3%; 29/87; including 5 patients with paraneoplastic pemphigus), and other AID such as rheumatoid arthritis (6.9%; 6/87), vasculitis (5.7%; 5/87), and neuromuscular syndrome (6.9%; 6/87). Most patients (51/87; 58.6%) presented the non-hematologic AID, with a median time of 1.3 years (-45.8 – 15.9) before CLL diagnosis. We found a significant association between the presence of poor biological variables (i.e., unmutated IGHV), the need of antileukemic therapy, and the development of AIC. However, female sex was the only variable significantly associated with presence of non-hematologic AID and CLL. In terms of prognosis, patients who developed non-hematologic AID either before, at the time of, or after the diagnosis of CLL had significantly longer OS compared to those who developed AIC (13.9 vs 10.9; p=0.002). Summary/Conclusion: In our series, 12.2% of patients with CLL presented non-hematologic AID, being the most frequent endocrine disorder. The high prevalence of AID observed in this series was not associated with CLL adverse features and these patients had the same prognosis as those with CLL without autoimmunity. In contrast, the appearance of AIC was linked to the presence of poor biological features and had a negative impact on patients’ outcomes. The different characteristics observed between patients with CLL and non-hematologic AID and AIC may indicate different pathogenic pathways. Further studies are needed to clarify the underlying mechanisms of these phenomena.Keywords: Autoimmunity, Chronic lymphocytic leukemia, Prevalence, Survival

Supplementary Data from Stereotyped B-Cell Receptor Is an Independent Risk Factor of Chronic Lymphocytic Leukemia Transformation to Richter Syndrome

Topic: 10. Myelodysplastic syndromes - Clinical Background: GATA2 deficiency is a complex multi-system disorder with high risk of developing myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with a nearly complete lifetime penetrance. GATA2 carriers within families show variable expressivity and heterogeneity in clinical features with manifestation of myeloid neoplasm at any age. This suggests that both cooperating genetic and environmental drivers shape the course of the disease. Aims: We applied NGS and bead-array techniques to identify acquired somatic mutations and aberrant DNA methylation patterns in 20 Spanish GATA2 carriers, including 16 patients and 4 asymptomatic carriers. Methods: To identify acquired somatic mutations, we performed NGS of total PB and BM samples in 17 of the 20 GATA2 carriers. Then, using the Infinium Human Methylation EPIC 850 K platform (Illumina), we profiled DNA of 8 BM and 8 PB samples and compared with a cohort of 12 age-matched healthy donor (HD) controls. Results: Median age at the diagnosis was 36 (6–75) years. The primary initial manifestation was MDS (n=12, 55%), followed by immunodeficiency (n=3, 15%), and AML (n=2, 10%). On cytogenetics, trisomy 8 was detected in 3 patients, complex karyotype in 2 patients, while 11 patients had normal karyotype. Somatic mutations in myeloid malignancy genes were identified in 71% (12/17) tested patients. This analysis confirmed the heterogeneity of acquired somatic mutations in GATA2 deficiency, with STAG2, ASXL1 and SETBP1 as recurrently affected genes. Furthermore, an unbiased genome-wide screen for aberrant CpG island methylation revealed a specific hypermethylated pattern in GATA2 patients when compared with HD. In detail, 2834 differentially methylated positions (DMPs) were identified in GATA2-mutant BM samples and 1406 DMPs in PB samples. revealed that the majority of DMPs are promoter-distant in both, BM (Open Sea: 60% hypomethylated DMPs and 72.5% hypermethylated DMPs) and PB samples (Open Sea: 52% hypomethylated and 82.3% hypermethylated). Overall, we observed that DNA methylation changes are enriched in gene-distant and intronic regions in GATA2 patients. Interestingly, we identified the presence of a hypermethylated DMP subcluster across all the GATA2 patients, including the asymptomatic youngest asymptomatic one (6-years old). Importantly, the 2-year longitudinal follow-up of this patients showed the evolution to MDS with multilineage dysplasia (MDS-MLD) and monosomy 7 without secondary mutations. In contrast, the other asymptomatic GATA2 carriers with not apparent symptoms had DNA methylation profile comparable with the HD group. This observation suggests the presence of a likely early aberrant DNA methylation at specific loci in GATA2 carriers that might have a potential prognostic utility in early identifying patients at risk for impending myeloid transformation. Furthermore, Hypergeometric Optimization of Motif EnRichment (HOMER) analysis revealed in the hypermethylated DMPs a significant enrichment in TFs motives of the ETS family, which are known to play a role in MDS. Finally, integration of hypermethylated genes with a GATA2 ChIPseq dataset revealed that GATA2 deficiency is associated with aberrant DNA methylation in GATA2 target genes. Summary/Conclusion: We identified an aberrant DNA hypermethylated signature in GATA2 deficiency. Specifically, we described the presence of a subset of aberrant hypermethylated set of genes present in GATA2 carriers at early disease stage, which could be potentially used as predictors of clonal evolution. Keywords: GATA-2, Genetic, MDS/AML, Epigenetic

Myelodysplastic neoplasms (MDS) are a heterogeneous group of hematological stem cell disorders characterized by dysplasia, cytopenias, and increased risk of acute leukemia. As prognosis differs widely between patients, and treatment options vary from observation to allogeneic stem cell transplantation, accurate and precise disease risk prognostication is critical for decision making. With this aim, we retrieved registry data from MDS patients from 90 Spanish institutions. A total of 7202 patients were included, which were divided into a training (80%) and a test (20%) set. A machine learning technique (random survival forests) was used to model overall survival (OS) and leukemia-free survival (LFS). The optimal model was based on 8 variables (age, gender, hemoglobin, leukocyte count, platelet count, neutrophil percentage, bone marrow blast, and cytogenetic risk group). This model achieved high accuracy in predicting OS (c-indexes; 0.759 and 0.776) and LFS (c-indexes; 0.812 and 0.845). Importantly, the model was superior to the revised International Prognostic Scoring System (IPSS-R) and the age-adjusted IPSS-R. This difference persisted in different age ranges and in all evaluated disease subgroups. Finally, we validated our results in an external cohort, confirming the superiority of the Artificial Intelligence Prognostic Scoring System for MDS (AIPSS-MDS) over the IPSS-R, and achieving a similar performance as the molecular IPSS. In conclusion, the AIPSS-MDS score is a new prognostic model based exclusively on traditional clinical, hematological, and cytogenetic variables. AIPSS-MDS has a high prognostic accuracy in predicting survival in MDS patients, outperforming other well-established risk-scoring systems.

Treatment of chronic lymphocytic leukemia (CLL) has dramatically evolved over the last decades thanks to the introduction of targeted therapies. We aimed to describe retrospectively the evolution in the frontline prescription in the CLL patients from our institution. As a secondary objective, the impact of frontline therapy on the time-to-next-treatment (TTNT) and overall survival (OS). After a median of 6.4 years (0.1-36.4) of follow-up from diagnosis, 323 of 780 CLL patients (41.4%) required therapy. Alkylating agents in monotherapy (chlorambucil) were the most used until 2012, and from then, chemoimmunotherapy. Since 2018, targeted therapies were the most common therapeutic strategy (74.1%). Patients who received targeted therapies had significantly longer TTNT compared to other regimens. In the multivariable analyses, mutated IGHV genes targeted therapies and chemoimmunotherapy regimens were related to longer TTNT, and sex female, age younger than 65, and mutated IGHV genes were associated with better OS.