BACKGROUND:A Phase 3 randomized trial compared immunosuppressive therapy with or without eltrombopag in untreated patients with aplastic anemia (AA) and showed that addition of eltrombopag increased the rate, rapidity, and durability of hematological response, without increasing transformation to myeloid malignancies. We sought to systematically investigate clonal hematopoiesis (CH) dynamics from patient samples from this trial. METHODS:Peripheral blood and bone marrow aspirates were collected at diagnosis (i.e., baseline) and at 6 and 24 months from patients with severe or very severe AA. Genetic testing for somatic mutations was performed using 31-gene "core" and 291-gene "extended" custom targeted panels and analyzed longitudinally. RESULTS:Samples were collected from patients at baseline (n=170) and 6 (n=150) and 24 months (n=103); 85 patients' samples were tested at all three timepoints. Somatic mutations were present in 30% of patients at baseline, 55.3% at 6 months and 79.6% at 24 months, with a mean number of mutations per patient of 0.4, 1.2, and 2.5, at baseline and 6 and 24 months, respectively. CONCLUSIONS:CH was frequent in patients with AA and its prevalence appeared to be higher at posttherapy timepoints than at diagnosis. CH in AA may reflect the survival and expansion of selected residual hematopoietic stem/progenitor cells associated with immune-mediated damage. (Funded by Cancer Research UK, Bloodwise UK, and Novartis AG; ClinicalTrials.gov number, NCT02099747; EudraCT number, 2014-000363-40.).

Introduction Somatic mutations in Hematopoietic Stem Cells and Progenitors (HSC/HCP) are very frequent in patients with acquired Immune-mediated Aplastic Anemia (IAA) being found in about a third of patients at diagnosis and up to 65-75% after Immuno-Suppressive Treatment (IST). In our systematic study investigating 170 patients enrolled in the EBMT RACE randomized, prospective clinical trial (NCT02099747) the CUB and Sushi Multiple Domains 1 (CSMD1) gene for the first time emerged as one of the most frequently mutated genes. Here we present a deep analysis of CSMD1 mutations in IAA patients enrolled in the RACE trial, looking for the frequency of mutations, its association with clinical variables and other somatic mutations, the dynamics of the mutated clones across the 2-year study period and a in silico structural analysis predicting functional consequences. Methods The RACE trial enrolled 197 IAA patients randomized to receive standard IST (anti-thymocyte globulin [ATG] and cyclosporine A) with or without eltrombopag. Bone marrow aspirate samples were collected at baseline, 6 months, and 24 months and analyzed at King's College London using custom targeted gene panels incorporating unique molecular identifiers. The HaloHS 31 gene panel was performed on 170, 150 and 103 patient samples collected at baseline, 6 and 24 months post randomization (TP6 and TP24), respectively. Appropriate variant caller protocols were utilized to identify candidate variants. An arbitrary Variant Allele Frequency (VAF) threshold of 0.2% was exploited to define mutations. Results At baseline (n=170), no CSMD1 mutation was found in any IAA patient. At later time points, mutations in the CSMD1 gene were found in 4/150 (2.7%) patients at TP6 and in 16/103 (15.5%) patients at TP24. Many patients harbored multiple mutations, with a total of 11 mutations detected in 4 patients at TP6, and 48 mutations in 16 patients at TP24. This resulted in a global mean number of CSMD1 mutations per patients increasing from 0 at baseline, to 0.07 and 0.47 at TP6 and TP24, respectively. The proportion of patients carrying CSMD1 mutations and the mean number of CSMD1 mutations per patients were not associated with age (≤40 vs >40 years), disease severity at baseline (severe vs very severe), presence of somatic mutations in myeloid-cancer genes at baseline, treatment arm (eltrombopag yes vs no) and hematological response. The VAF of CSMD1-mutated clones was usually very low, with 9/11 (81.8%) and 42/48 (87.5%) clones remaining below a 5% VAF at TP6 and TP24 (VAF range was 0.9-34.4% at TP6 and 0.8-48.5% at TP24), with unpredictable behavior. However, the general rule is that CSMD1-mutated clones become detectable only after IST, with the highest frequency at TP24, with very low VAF and often being multiple in the same patient. Patients with CSMD1 mutated clone often showed mutations in other myeloid cancer genes, especially ETV6, PHF6, PPM1D, PTPN11, RAD21 and STAG2. The CSMD1 protein consists of 14 CUB domains alternated with 14 Sushi domains, followed by further 15 Sushi domains, a transmembrane domain and an intracytoplasmic tail. The Sushi domains are known as Regulator of Complement Activation (RCA) which preferentially disable the classical complement pathway (CCP). Looking structurally at the specific mutations, no hotspot mutation was found; at TP6 frameshift and missense mutations were 8/11 (72.7%) and 3/11 (27.3), whereas at TP24 they were 42/48 (87.5%) and 6/48 (12.5%), respectively. These mutations result in major structural changes of CSMD1, which in most cases are truncated proteins lacking the transmembrane domain. ConclusionsCSMD1 mutations are commonly found after IST in IAA patients. We hypothesize that the secretion of truncated CSMD1 proteins retaining RCA domains may result in a paracrine effect which may spare mutated HSC/HCP from some CCP-mediated damage. The observation that all these clones emerge after IST makes ATG as the most likely trigger for such CCP activation via immune complexes. Although CSMD1 is a known tumor suppressor gene, in the context of IAA clones carrying specific CSMD1 mutation might have a transient survival advantage in the context of hematopoietic toxicity during ATG treatment. Thus, CSMD1-mutated HSC/HCP seem to be an example of pruning selection, where some intrinsic advantage transiently emerging in specific micro-environmental setting may eventually become evident at the time of hematological recovery.

Standardized reporting of clinical trials results for MDS is essential to improve clinical interpretation and cross-study comparison. However, reporting of patient characteristics, response criteria, and endpoints in publications is often inconsistent; there is no systemic analysis of MDS trial reporting available. We conducted a systematic review of published MDS trial manuscripts on behalf of the international consortium for MDS (icMDS) to assess variability in reporting practices. We searched ClinicalTrials.gov to identify all clinical trials registered for adults (≥18 years) with MDS that reported results between 2015-2024. Clinical trials that included acute myeloid leukemia (AML) or MDS/myeloproliferative neoplasms (MPN) were included if they enrolled ≥5 MDS patients. We excluded trials without an MDS cohort; non-therapeutic trials; trials focused on transplant interventions; or those which enrolled solid malignancies or other hematologic malignancies (chronic myeloid leukemia or lymphoid diseases). Finally, trials were required to have a manuscript available on Larvol, PubMed, or Google Scholar. Trials were categorized by disease risk (lower-risk [LR] vs. higher-risk [HR]) and trial phase (early phase [EP] vs. late phase [LP]). We identified 502 MDS trials on ClinicalTrials.gov, of which 351 did not meet inclusion criteria and 79 lacked a manuscript. A total of 72 trials (32 EP, 40 LP) with 80 publications (34 EP, 46 LP) were analyzed. Frequently reported baseline characteristics included age (100% EP, 100% LP), disease risk (74% EP, 74% LP), Eastern Cooperative Oncology Group performance status (85% EP, 72% LP), prior treatments (71% EP, 67% LP), and RBC transfusion dependency (32% EP, 70% LP). Less frequently reported characteristics included blood counts (hemoglobin [Hb; 38% EP, 54% LP], platelet count [38% EP, 54% LP], neutrophil count [24% EP and LP]) and bone marrow blasts (29% EP, 43% LP). Although disease risk was widely reported, definitions varied: IPSS and IPSS-R were each reported in 54% of manuscripts, while IPSS-M appeared in only 3%. Cytogenetic risk was separately reported in 39% of manuscripts (38% EP, 39% LP), and mutational data in 45% (47% EP, 43% LP). Responses per IWG 2006 criteria were reported in 84% of manuscripts. The primary endpoint involved safety/tolerability in 85% of EP manuscripts, though definitions were variable; recommended phase 2 dose was the most common (26% of EP manuscripts). In LP trials, primary endpoints were risk-specific: transfusion independence (TI) in 77% of LR, overall response rate (ORR) in 28% of HR, and complete remission (CR) or overall survival (OS) in 22% of HR. In HR manuscripts, CR was reported in 100%, ORR in 73%, hematologic improvement (HI) in 48%, RBC-TI in 23%, and OS in 80%. However, seven different definitions were used for ORR: CR + marrow CR + partial remission + HI per IWG 2006 was most common (35%). Event-free survival was reported in 25%; progression-free survival and relapse-free survival in 13%; and leukemia-free survival in 5%. Early mortality rate was reported in 35% (18% EP, 56% LP) and transplant rate in 58% (50% EP, 67% LP). Only 47% of HR and 90% of LR manuscripts identified HI-eligible patients. In LR-MDS, RBC-TI was reported in 72% of manuscripts but used six definitions. The most common were RBC-TI ≥8 weeks at any time during treatment (24%), ≥8 weeks within 28 weeks of treatment (13%), ≥8 weeks with Hb increase ≥1.0 (4%), ≥12 weeks with Hb increase ≥1.5 (4%), and ≥16 weeks within 24 weeks of treatment (12%). The remainder did not report RBC-TI, did not define it, or used a custom definition not used in another trial. HI was reported in 80% of LR papers with most of those reporting HI using IWG 2006 criteria (90%). Reporting of baseline characteristics, response criteria, and outcomes is inconsistent in MDS clinical trials, with wide variability in response definitions, including ORR and TI. This heterogeneity limits interpretability, hampers historical comparisons and may obscure efficacy signals. Our findings highlight the need for standardized reporting guidelines to ensure clarity and consistency in MDS trial publications. As a next phase of this effort, we will conduct a formal Delphi process among icMDS experts to establish consensus recommendations for minimal and optimal MDS clinical trial reporting in manuscripts by disease risk (LR/HR) and trial phase (EP/LP).

ABSTRACT:Pathogenic germ line variants causing excessive telomere shortening may result in bone marrow failure, hematopoietic malignancy, and extramedullary complications, such as pulmonary fibrosis, liver cirrhosis, and solid tumors. Patients with short telomeres also develop immunodeficiency with low CD4+ T cells and impaired general immunosurveillance, particularly against solid neoplasms. We investigated a broad spectrum of lymphocyte subsets and myeloid immune cells from human patients with telomere biology disorders (TBDs) and matched healthy volunteers to understand further how the immune system is affected by telomere dysfunction. We used mass cytometry for deep-immunophenotyping peripheral blood mononuclear cells, followed by high-dimensional data analysis. Cytokines, chemokines, and growth factors were assessed in serum. Our results showed profound immune alterations in TBDs beyond those observed in aging, with low naïve lymphocytes and thymic hypofunction. We further observed that T helper (Th) subsets were markedly skewed, with an inverted Th2/Th1 ratio, and low Th17 and Th17.1 levels. T-cell activation and exhaustion markers were upregulated, whereas circulating mucosal-associated invariant T cells were significantly decreased and overactivated. Several serum cytokine levels were positively correlated with telomere length and blood counts, suggesting an association with marrow function. In aggregate, these findings suggest a proinflammatory profile in TBDs. Our data provide new details on how TBD affects immune cells, particularly lymphocytes, which may contribute to the clinical phenotypes.

Myelodysplastic syndrome (MDS) comprises a group of clonal stem cell disorders characterized by ineffective haematopoiesis. Using humanized bone marrow (BM) niches within immunodeficient mice, we have previously demonstrated a critical dependence of MDS hematopoietic stem and progenitor cells (HSPCs) on the human BM niche (Leukemia 2017, BCD 2021). To further investigate this niche-specific dependency, we coupled high resolution omics to a dual approach combining an in-vivo humanized xenograft model and an ex-vivo co-culture system. HSPCs from MDS patients (n=11) and age-matched healthy donors (HD, n=8) were cultured with mesenchymal stromal cells (MSCs) derived from either MDS or HD donors, under both direct-contact and trans-well conditions. Bulk RNA sequencing and single-cell RNA sequencing allowed us to map the HSPC–MSC interactome and to target candidate genes via a large-scale siRNA knockdown approach in primary human MSCs. Notably, MDS HSPCs proliferated ~4 times more in direct contact with MSCs than in trans-well conditions (P<0.01). A similar proliferation advantage was observed when MDS HSPCs were co-cultured with HD MSCs. Interestingly, CD34⁺CD38⁻ HSCs from HDs exhibited significantly higher proliferation when cultured with HD MSCs compared to MDS MSCs (11.5% vs. 5.6%; P<0.03). Unsupervised clustering of bulk RNA sequencing data from MSCs revealed that MDS HSPCs could reprogram HD MSCs toward a ‘disease-like transcriptomic‘ profile, but only under direct-contact conditions. In contrast, MDS MSC co-cultured with HD HSPCs closely resembled the HD MSCs co-cultured with HD HSPCs. Further analysis of the bulk MSC RNA transcriptome revealed a group of differentially expressed genes (n≈1000, adjPvalue<0.05). Meta-enrichment analysis on the differentially expressed genes showed enrichment of gene sets related to cell-cell signalling, stress response, such as regulation of cellular activation/response and myeloid leukocyte activation. Using an unbiased systems biology approach Niche-net algorithm (Browaeys, 2020), we then identified a novel complex network of ligand-receptor interactions (n=84, P<0.05) between the HSPCs and MSCs, that could be responsible for rewiring of the intrinsic core pathways of the MSCs.Using an siRNA screening approach on primary MSC, we targeted these receptor genes (n=46) along with additional targets that were identified from our analysis. Notably, siRNA-mediated knockdown of a significant number of these genes led to growth arrest and apoptosis in MSCs, highlighting their critical role in maintaining MSC biology. Among the most promising candidates, Ephrin type-A receptor 3 (EPHA3) emerged as a key gene with minimal impact on proliferation of HD MSCs upon siRNA knockdown. Our interaction analysis from bulk-RNA sequencing revealed that EPHA3 receptor on MSCs is highly regulated by IL10 expressed by HSPCs. Single-cell RNA sequencing of humanized scaffolds seeded with patients MDS HSPC and MSC confirmed EPHA3 was specifically expressed in MSCs, while as IL10 was restricted to myeloid cells. These findings were further corroborated by publicly available human BM single-cell RNA sequencing dataset. Stimulation of HD MSCs (donors, n=7) with recombinant human IL-10 induced a dose-dependent increase in EPHA3 expression at both mRNA and protein levels. To functionally validate this further, we used OCI-AML3 cells (known to express and secrete IL10). Knockdown of IL10 in the OCI-AML3 cells, followed by co-culture with HD MSCs, abolished EPHA3 upregulation in MSCs. To understand the biological implication of lack of EPHA3-IL10 interactions on MDS HSPCs, we generated a knockdown of EPHA3 in BM MSCs. Long-term co-culture of BM CD34+ HSPCs from healthy donors (n=2) and MDS patients (n=2) resulted in up to 14-fold reduction in MDS HSPCs, with little impact on healthy donor HSPCs. Experiments are currently underway to better understand the biological impact of EPHA3 inhibitor on MSCs and HSPCs in MDS. In conclusion, our integrative and unbiased analysis of the MDS bone marrow niche reveals novel insights into the intrinsic dysregulation of MDS MSCs. We identify a complex network of ligand–receptor interactions driven primarily by MDS-HSPCs that modulate MSC behaviour, contributing to disease maintenance. Notably, we describe a novel IL10–EPHA3 HSPC and niche interaction that selectively supports MDS clones, offering a promising therapeutic target for disrupting the pathological HSPC–MSC crosstalk in MDS.

Matching HLA between donor and recipient pairs significantly improves engraftment and decreases GvHD. However, even in HLA matched siblings, the risk of graft failure and GvHD still exist and is partially attributed to the bidirectional alloreactivity generated from minor histocompatibility antigens (miHA) mismatching between donors and recipients. Consanguineous marriage increases the chance of miHA matching. This fact may be an advantage or disadvantage in consanguineous MSD transplants. We retrospectively reviewed our institutional transplant registry and selected AML and ALL patients who received a MSD allo-HCT between January 2006 and December 2020. The objective was to compare the outcomes of transplant among consanguineous vs non-consanguineous pairs. Analysis was conducted using RStudio. Version 1.4.1106 © 2009-2021 RStudio, PBC. 271 patients met the eligibility criteria were included in this study. Sixty-three were consanguineous and 208 were not. The median follow-up time was 72.6 months. The five years OS and DFS were not statistically different between consanguineous and non-consanguineous group. A trend toward a higher relapse rate in the consanguineous group was observed. The five years NRM for the whole group was 10.5% with no statistically significant difference between groups. The grade II-IV aGvHD incidence was 25.8% for the whole group with no statistically significant difference between groups. The all grade cGvHD incidence was 52.8% with no statistically significant difference between groups. Populations with high levels of consanguinity, complex consanguinity loops often arise from cousin marriages across multiple generations. This means that even individuals who consider themselves non-consanguineous may still share genetic traits associated with consanguinity. Therefore, high level of homozygosity probably exist in the non-consanguineous cohort and explain these results.

Introduction: Immune-mediated aplastic anaemia (AA) is bone marrow failure syndrome, where T-cell mediated destruction of hematopoietic stem and progenitor cells (HSPCs) results in pancytopenia. Overall, two third of patient respond to immune suppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine A (CsA), adding eltrombopag (EPAG) further improves response, but the immune signatures that predict outcome remain unclear. Methods: We studied at diagnosis 130 previously untreated severe/very-severe AA patients enrolled in a randomised phase-3 trial of hATG + CsA with (Arm B, n = 65) or without EPAG (Arm A, n = 65). Number of patients analysed at both baseline and 6 months was 95. The frequency of 11 CD8+ and 22 CD4+ T-cell subsets, as well as B cell, Myeloid and NK cells were defined by mass cytometry (CyTOF) using panel of 38 antibodies. The immune architecture was associated with patients' age, disease severity, treatment arms, treatment outcomes at 6 months and other patient specific features using non-parametric tests. Predictive features were identified using multinomial and multivariable logistic regression. Results: At baseline, across all the cohort exhibited broad immune dysregulation. Most patients presented reduced myeloid and NK cell frequencies, while activated effector T-cells, and particularly non-naïve CD8⁺ Tc1 and effector memory CD4⁺ T cells, were expanded. Patients with very severe AA (vsAA) had significantly lower myeloid cells and higher levels of effector Th1 CCR4⁺ and Th1 CCR4⁺ CD4⁺ subsets compared to those with severe AA (sAA). Immune environment skewed towards pro-inflammatory cells in vsAA patients, exhibiting elevated levels of activated cytotoxic and effector T-cell subsets, including CD38+PD1+ Tc1, Tc2, and CD103+ CD8+ cells. This shift was more pronounced in vsAA patients younger than 40 years compared to vsAA patients older than 60 years, while vsAA patients between 40 and 60 years showed intermediate findings. When stratified by 6-month treatment response, patients achieving complete response (CR) had a distinct baseline immune profile. Naïve Tregs CD45RA⁺CCR4⁻ were significantly enriched in CR patients, while memory Tregs CD45RA⁻CCR4⁺ were elevated in non-responders (NR). Effector Th1 CCR4⁺ cells and central memory CD45RA-CD27+ cells were also decreased in CR, and partial responders (PR) displayed intermediate frequencies across these subsets. Multinomial logistic regressions identified two regulatory-T-cell (Treg) signatures. Naïve Treg CD45RA+ CCR4- independently predicted better hematological response, while memory Treg CD45RA- CCR4+ was inversely associated. HLA-DRB1*15:01 (present in 47 %) co-segregated with older age and higher memory-Treg levels but did not affect response. At 6 months, both treatment groups shared immunological shifts; Myeloid cell, NK cell and Effector memory CD45RA-CD27- frequencies increased, indicating improved hematopoiesis, restored innate immunity and accumulation of differentiated T cells. Activated Tc CD8+ cells and effector Th1 CCR4+ were decreased however terminally differentiated effector memory Tcells, which can be identified by markers CD45RA+ CD27-, were increased in both treatment arms but significantly in arm B. Effector Th1 CCR4+ cells declined in CR at 6 month, this declined more pronounced in arm B; however, this may be influenced by the number of CR difference between arms. Responder profiles were marked by maintenance or enrichment of naïve Tregs and reduced memory Tregs post-treatment, reinforcing the prognostic value of baseline Treg subset balance. Conclusions: In summary, AA is characterized by a broadly shared immune activation state marked by reduced innate immunity and expanded effector T-cell subsets. Within this framework, two regulatory T-cell subsets, naïve and memory Tregs, emerge as independent predictors of treatment response, regardless of age. After treatment they remained stable in CR and PR; however, memory Treg slightly decreased, and Naïve Treg slightly increased in NR. These findings highlight immune profiling as a powerful tool for predicting outcome and tailoring therapy in AA.

IntroductionXeroderma pigmentosum (XP) is a rare autosomal recessive disorder caused by defects in the nucleotide excision repair pathway, resulting in genomic instability and increased cancer susceptibility. In Saudi Arabia, the prevalence of XP is significantly higher than the global average due to the high rate of consanguineous marriages. While XP is associated with skin malignancies, it might also be a cancer predisposition, including haematological malignancies (HMs) driven by inherited germline variants. In this study, we describe HMs cases among genetically confirmed XP patients in Saudi Arabia and present the germline variants that may contribute to this cancer predisposition.MethodsWe retrospectively identified 47 patients diagnosed with XP at King Faisal Specialist Hospital and Research Centre in Riyadh, Saudi Arabia, between 2003 and 2025. Of these, 29 patients belonged to 11 families. The cohort included 25 females and 22 males, with ages ranging from birth to 44 years (median age: 15 years). Parental consanguinity was reported in 35 patients (74.5%). Clinical and genetic data were reviewed, including the diagnosis of cancers, age at onset, cancer type, and germline variants identified through targeted gene panels, exome or genome sequencing.ResultsAmong the 47 patients, 23 (48.9%) developed basal or squamous cell carcinoma. The lower observed frequency of basal cell carcinoma in our cohort may be partly due to underreporting, as routine dermatological follow-up was not consistently performed. HMs including myelodysplastic syndrome or acute myeloid leukaemia were identified in 7 patients (14.9%). Additionally, one patient each developed ovarian cancer or small cell neuroendocrine carcinoma with liver metastases.The median age of patients with HMs was 22 years. All patients with HMs had a positive family history of XP and cancer, and six of the seven were born to consanguineous parents. Notably, each of these patients developed basal cell carcinoma prior to their HMs diagnosis.Notably, five of the seven HMs cases belonged to a single extended family and were homozygous for a novel germline pathogenic variant in the XPC gene (c.856A>T; p.R286*). Somatic TP53 mutations were detected in three of these patients, and complex karyotypes were observed in three cases. Interestingly, the same XPC variant was also found in a patient who did not develop HMs but was diagnosed with ovarian cancer.A deletion in the XPC protein (p.E34del) was also identified in another XP/HM patient who harbored also a JAK2 mutation. Another XP patient who developed basal cell carcinoma, ovarian high-grade serous carcinoma, and breast cancer harbored pathogenic variants in TP53, STAG2, BCOR, TET2, and ABL1, along with a complex karyotype; however, XPC gene status was not assessed in this individual due to the limitations of the testing panel.ConclusionOur findings highlight XP particularly in individuals harboring XPC variants, as a germline predisposition syndrome not only for skin cancers but also for HMs especially in populations with high rates of consanguinity. These results support the need for enhanced genetic surveillance and HMs screening in XP patients. Functional study is needed to confirm the deleterious effect of XPC c.856A>T (p.R286*) in the XP patients. Furthermore, our data provide a compelling rationale for considering XP, specifically XPC-related subtypes, among the WHO-classified germline predisposition syndromes to HMs.

Chimeric antigen receptor T cell (CAR-T) therapy is a genetically engineered cellular therapy that is currently integrated into the management of hematologic malignancies. Institutions treating patients with CAR-T therapy need to establish a framework of delivery that covers all the main components of the patient journey including intake of patients into the program from referring centers, patient selection according to established eligibility criteria, apheresis, logistics, bridging therapy, infusion, and postinfusion care. A CAR-T therapy program, with its unique requirements, needs to be delivered by a multidisciplinary team. Prior to the establishment of the program, a well-structured business plan should be developed with a clear financial and/or reimbursement model. Consideration should be given to overall capacity and staffing requirements. Standard operating procedures and guidelines are vital for ensuring that quality standards are clearly defined and adhered to. Institutions should develop a research plan for CAR-T therapy that may incorporate not only industry-sponsored trials, but also in-house manufacturing of investigational CAR-T products. This report presents recommendations from a group of international experts, highlighting the priorities and considerations when developing a new CAR-T program.

Loss-of-function (LoF) mutations frequently found in human cancers are generally intractable by classical small molecule inhibitor approaches. Among them are mutations affecting Polycomb-group (PcG) epigenetic regulators, enhancer of zeste homolog 2 (EZH2) and Additional sex combs like 1 (ASXL1), frequently found in hematological malignancies of myeloid or lymphoid lineage, and their concurrent mutations associates with particularly poor prognosis. Although there is a clear need to develop novel and effective treatments for these patients, the lack of appropriate disease models and mechanistic insights have significantly hindered the progress. Here, we show that genetic inactivation of Asxl1 and Ezh2 in murine hematopoietic stem/progenitor cells results in highly penetrant hematological malignancies as observed in corresponding human diseases. These PcG proteins regulate both coding and noncoding genomes, leading to marked reactivation of transposable elements (TEs) and DNA damage responses in PcG LoF-mutated cells, which create a novel vulnerability for poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi)-induced synthetic lethality. Using both mouse models and primary patient samples, we demonstrate that Asxl1/Ezh2-mutated cells are highly sensitive to PARPis that induce excessive DNA damage and significantly extend disease latency. Intriguingly, the observed PARPi sensitivity can be specifically overridden by reverse transcriptase inhibitors that interrupt target site-primed reverse transcription and life cycle of TEs. This mechanism is contrastingly different from the current concept of BRCAness associated PARPi-induced synthetic lethality, which largely rely on deficient homologous recombination, and is independent on reverse transcriptase inhibitors. Together, this study reveals a novel application and mechanism of PARPi-induced synthetic lethal targeting of blood cancers with reactivated TEs such as those carrying PcG epigenetic mutations.

Clonal Haematopoiesis (CH) with DNA damage repair pathway (DDRp, e.g. ATM, ATRX, PPM1D, TP53) gene mutations carries increased risk of myeloid malignancy. In our cohort of older patients having orthopaedic surgery (n=721, mean age 69 years) we found mutations (VAF≥0.2%) in DDRp in 9% of patients (n=64, ATM/ ATRX n=26, PPM1D n=21, TP53n=17). TP53 mutations were missense, with two cancer hotspots (R248Q and R282W). TP53-CH had larger clones (median VAF 2.9%) compared with other DDRp-CH (median VAF 1.4%, p<0.01). Compared with non-CH (n=366) patients with TP53-CH, but not other DDRp mutations, had significantly elevated MCV (94.7 vs. 92.1, p<0.05) without changes in haemoglobin or RDW. We studied erythropoiesis using scRNAseq in bone marrow (BM) from non-CH (n=27), ATM / ATRX (n=8), PPM1D(n=11) and TP53-CH (n=11). With the exception of one patient, >80% of cells analysed in CH samples did not carry a mutation. Erythroid populations in most TP53-CH patients, but not ATM / ATRX nor PPM1D-CH, upregulated HbF (HBG1, HBG2, log2FC 1.6x) compared with non-CH. We confirmed this finding with HPLC quantitation and surmised that it may be associated with erythroid cell stress. Along the erythroid differentiation trajectory, there is a switch from glycolytic to OXPHOS gene expression from megakaryocyte-erythroid progenitor (MEP) onwards, peaking in erythroid precursor (EPre) cells. Expression of OXPHOS and G1S / G2M cell cycle (CC) genes are also strongly positively correlated in non-CH samples. OXPHOS generates reactive oxygen species (ROS), and expression of antioxidant genes (e.g. GPX4, PRDX2, GSTP1) rise to compensate. Compared with non-CH, MEP and EPre in TP53-CH upregulate OXPHOS but had reduced expression of CC genes and reduced expression of GSTP1, suggesting inadequate response to oxidative stress. To assess function of haematopoietic stem-progenitor cells (HSPCs) in TP53-CH, we performed colony assays with flow-sorted CD34+ HSPCs from 4 TP53-CH samples with different mutations (C238Y, S241Y, R248Q, I255F). The GM-E ratios were no different from controls. However, consistent with reduced expression of CC genes, cloning efficiency was significantly lower in CH than in non-CH controls (mean 21% vs 40%, p<0.05). To investigate mutational effects, we genotyped colonies and found reduced frequency of TP53-mutant (MUTTP53) erythroid colony forming units (CFU-E, reflecting EPre activity) relative to burst-forming units (BFU-E, reflecting MEP activity), indicative of clonal impaired differentiation, in patient #126 (hotspot R248Q, VAF 36%) only. This patient had normal blood counts at index sampling but developed AML 11 months later. To examine if OXPHOS upregulation is mutant clone-autonomous, and if mutations conferred different biological effects, we compared scRNAseq data in 4 MUTTP53 and their ‘isogenic’ wild-type (WT) patient-derived BFU-E (with cells from erythroid precursors to polychromatic erythroblasts). Only patient #126 showed significantly increased OXPHOS and CC expression in the MUTTP53-R248Q versus its WT control in erythroblasts. MUTTP53-R248Q cells also upregulated thioredoxin (TXN), which scavenges ROS, and mitochondrial uncoupling protein 2 (UCP2) which uncouples OXPHOS from ATP synthesis to reduce ROS. Upregulation of these antioxidant genes may confer metabolic and survival advantage in MUTTP53-R248Q cells over WT counterparts. We predict that this advantage would be greatest in OXPHOS-dependent differentiating progenitors rather than HSC (which are preferentially glycolytic). Indeed, genotyping of HSPCs from #126 shows TP53-R248Q clonal enrichment in CMP, MEP and GMP (VAF 34-38%) versus HSC/MPP (VAF 27%). In summary, we report that erythroid cells from TP53-CH exhibit signs of decompensated oxidative stress that is not directly driven by mutant clones. We identify metabolic differences between MUT and WT erythropoiesis which may underlie competitiveness in TP53-CH patients. Intriguingly, compared with non-CH, TP53-CH patients are more likely to have metabolic syndrome-related disorders that are associated with oxidative stress which may exert clonal selective pressure.

Background The EBMT RACE trial (NCT02099747) has recently shown the superiority of Eltrombopag given with horse-ATG and cyclosporine (standard IST) (Arm B) in comparison to standard IST (Arm A) alone. Clonal haemopoiesis (CH) has been documented at diagnosis and after immunosuppressive therapy (IST) in idiopathic aplastic anaemia (AA); CH and its dynamics was systematically investigated in the RACE trial to ascertain its clinical significance in the biology of AA. Methods The somatic mutational profiles were assessed at diagnosis (Timepoint 0 - TP0) and sequentially, 6 and 24 months (TP6 and TP24) following treatment using a 31-gene ‘core’ (Haloplex HS, TP0, TP6, and TP24 with 170, 151, and 107 patients, respectively) and 291-gene ‘extended’ (Sureselect XT, TP0 with 140 and TP6 with 136 patients) custom targeted panel, with unique molecular identifiers (UMI) allowing identification of low-level clones ( VAF > 0.2 %). 141 patients were analysed at both TP0 and TP6, whilst 89 analysed at all three time points. The repertoire of somatic variants was correlated with treatment arms, age, severity of disease, response to treatment, and other patient-specific variables. Results At baseline, 6 and 24 months using the ‘core panel’, 83 mutations were detected in 59 (59/170, 34.7%), 193 mutations in 86 (86/151, 56.9%) and 265 mutations in 84 (84/107, 78.5 %) patients respectively, with significant increase over time (TP0 vs TP6 p= <0.00001, TP0 vs TP24 p= <0.00001, TP6 vs TP24 p= <0.00001). In patients with mutations, the mean mutation rates were 1.4 (TP0), 2.24 (TP6) and 3.15 (TP24). There is also a trend of median VAF increase from TP0 (8.44%) to TP6 (13.16%), with no significant difference in treatment arms. Mutational frequency at TP0 did not differ between treatment arms, and baseline mutations did not correlate with treatment response. PIGA (TP0 n=23, TP6 n=44, TP24 n=30), DNMT3A (TP0 n=18, TP6 n=33, TP24 n=25), BCOR/BCORL1 (TP0 n=12, TP6 n=35, TP24 n=24), TET2 (TP0 n=10, TP6 n=10, TP24 n=9), ASXL1 (TP0 n=2, TP6 n=22, TP24 n=23), CSMD1(TP0 n=4, TP6 n=15, TP24 n=54) were the most frequently mutated genes. The median age of patients with PIGA, BCOR, and BCORL1 mutations was lower than that of patients with other mutations ((TP0 44 vs 60 years, TP6 49 vs 58 years and TP24 49 vs 56 years). DNMT3A and TET2 mutations occurred more frequently in older patients (median 58 and 61 years at 6 months, 60.5 and 62.5 years at 24 months). Gene interactions, either co-occurrence or exclusivity patterns, were variable across the different time points, with no specific patterns observed. New ASXL1, BCOR and PIGA mutations tend to emerge in between TP0 and TP6, whilst new RAD21, ETV6, CSMD1 and STAG2 mutations mostly appeared between TP6 and TP24. The number of PIGA mutations increased over time with a significant change in Arm A at TP6 (p=0.008) and TP24 (p=0.001) compared to TP0, which also correlated with higher incidence of haemolytic PNH in standard IST arm. The ‘extended’ gene panel of 291 genes, detected pathogeneic somatic variants in 132 out of 140 patients (94%) at baseline and in all patients at TP6 (100%, 136/136); only 4.3% and 3.8% of the detectable mutations had VAF>2%. The low-level clones (<1% VAF), were also validated by ddPCR (n=3). Mutations (n=36) undetectable by ‘core’ panel at baseline were present at low level (<1%) using the more sensitive ‘extended’ XT panel. The larger panel also uncovered mutations in ATM, NCOR2 and epigenetic pathways regulators. Conclusions We observed a high prevalence of CH (94%) in AA patients at diagnosis, which tends to increase after therapy, irrespective of treatment arm, in the absence of progression to myeloid malignancies. These findings suggest CH in AA arises in a stochastic manner before and after immunosuppressive treatment and increase in mutations may represent a bottle neck effect in terms of residual hematopoietic stem cells, but longer-term clinical data is mandatory to ascertain the impact of mutations on malignant transformation

Pancytopenia with hypocellular bone marrow is the hallmark of aplastic anaemia (AA) and the diagnosis is confirmed after careful evaluation, following exclusion of alternate diagnosis including hypoplastic myelodysplastic syndromes. Emerging use of molecular cyto-genomics is helpful in delineating immune mediated AA from inherited bone marrow failures (IBMF). Camitta criteria is used to assess disease severity, which along with age and availability of human leucocyte antigen compatible donor are determinants for therapeutic decisions. Supportive care with blood and platelet transfusion support, along with anti-microbial prophylaxis and prompt management of opportunistic infections remain key throughout the disease course. The standard first-line treatment for newly diagnosed acquired severe/very severe AA patients is horse anti-thymocyte globulin and ciclosporin-based immunosuppressive therapy (IST) with eltrombopag or allogeneic haemopoietic stem cell transplant (HSCT) from a matched sibling donor. Unrelated donor HSCT in adults should be considered after lack of response to IST, and up front for young adults with severe infections and a readily available matched unrelated donor. Management of IBMF, AA in pregnancy and in elderly require special attention. In view of the rarity of AA and complexity of management, appropriate discussion in multidisciplinary meetings and involvement of expert centres is strongly recommended to improve patient outcomes.

Introduction The RACE study (NCT02009747) compared standard Immuno-Suppressive Treatment (IST) (as horse antithymocyte globulin plus ciclosporin A) ± eltrombopag [EPAG] as front-line treatment for Severe Aplastic Anemia (SAA). Primary analysis has shown that triple therapy improved rate and quality of hematological response, with higher rates of complete response (CR) at 3 months (21.9% vs. 9.9%, primary endpoint), and of overall response (OR) at 3 months and CR and OR at 6 months. Here we report the final analysis of this 2-year follow-up, prospective, randomized, phase III study, focusing on the long-term outcomes. Methods The trial population consisted of 197 treatment-naive patients randomized to receive either standard IST (arm A; n=101) or standard IST + EPAG (arm B; n=96) at the dose of 150 mg/d from day +14 until 6 months (m) (or 3m, in case of early complete response). Here we report final data on overall survival (OS), disease-free survival (DFS; events: death, no response at 6m, myeloid malignancy, relapse, transplant), event-free survival (EFS; events: as for DFS, plus new AA treatment), relapse (competing events: death, transplant, myeloid malignancy), and evolution to either myeloid malignancy or clinical paroxysmal nocturnal hemoglobinuria (PNH) (competing events for both: death and transplant). We report multi-variable analysis (MVA) hazard ratios (HR) for arm B compared to arm A, adjusted for age and disease severity. Results All the 197 patients were tracked and analyzed; 5 patients discontinued the study prematurely (withdrawn consent to data collection). A total of 22 deaths were observed, 14 in arm A and 8 in arm B; all deaths were due to SAA and its expected complications (mostly infectious or bleeding). For the remaining 170 patients reaching the 2-year end of study visit, the median follow up was 24 months (range 21.4-28.1). The 2-year OS was 91.4% (95% CI, 85.8-97.1%) in arm B vs 86.0% (95% CI, 79.2-92.8%) in arm A. In MVA, the HR for arm B was 0.54 (95% CI, 0.28-1.04, p=0.064); only disease severity was found to affect OS. The 2-year DFS was 54.7% (95% CI, 44.7-64.7%) in arm B vs 36.6% (95% CI, 27.2-46.0%) in arm A. In MVA, the effect of treatment arm varied in time: HR=0.50 (95%CI, 0.38-0.66, p<0.001) on average across the 2-year period, being significant only in the periods 0-6mo (HR=0.41) and 6-12mo (HR=0.61); both age and disease severity affected DFS. The 2-year EFS was 48.4% (95% CI, 38.4-58.5%) in arm B vs 32.7% (95% CI, 23.5-41.8%) in arm A (p<0.001). As for DFS in MVA the effect of treatment arm (on average across the 2-year period, HR=0.54 [95% CI, 0.43-0.69], p<0.001) was not significant after 12 months; only age reached significance for EFS. The cumulative incidence of relapse was comparable in the two arms, with a 2-year estimate of 22.7% (95% CI, 12.9-32.6%) in arm B vs 18.0% (95% CI, 6.7-29.3%) in arm A (adjusted HR=1.05 [95% CI, 0.87-1.27], p=0.60). The risk of clonal evolution remained negligible, with 1 patient in arm A and 2 in arm B developed karyotypic abnormalities. The 2-year cumulative incidence of clinical PNH was 1.1% (95% CI, 0-3.2%) in arm B vs 8.1% (95% CI, 2.7-13.5%) in arm A (adjusted HR=0.12 [95% CI, 0.04-0.33], p<0.001). Conclusion We demonstrate that improved rate and quality of hematological response resulting from the addition of EPAG to standard IST leads to improved 2-year OS, DFS and EFS, in absence of increased risk of secondary myeloid malignancies. While even longer follow up is appropriate to confirm the role of triple therapy in the scenario of disease-eradicating treatments for SAA, these data prove that EPAG on top of standard IST should be the preferred initial therapy for all SAA adult patients not eligible for first-line hematopoietic stem cell transplantation.

Retrotransposons (RTEs) have been postulated to reactivate with age and contribute to aging through activated innate immune response and inflammation. Here, we analyzed the relationship between RTE expression and aging using published transcriptomic and methylomic datasets of human blood. Despite no observed correlation between RTE activity and chronological age, the expression of most RTE classes and families except short interspersed nuclear elements (SINEs) correlated with biological age-associated gene signature scores. Strikingly, we found that the expression of SINEs was linked to upregulated DNA repair pathways in multiple cohorts. We also observed DNA hypomethylation with aging and the significant increase in RTE expression level in hypomethylated RTEs except for SINEs. Additionally, our single-cell transcriptomic analysis suggested a role for plasma cells in aging mediated by RTEs. Altogether, our multi-omics analysis of large human cohorts highlights the role of RTEs in biological aging and suggests possible mechanisms and cell populations for future investigations.

Mutational profiles of myelodysplastic syndromes (MDS) have established that a relatively small number of genetic aberrations, including SF3B1 and SRSF2 spliceosome mutations, lead to specific phenotypes and prognostic subgrouping. We performed a multi-omics factor analysis (MOFA) on two published MDS cohorts of bone marrow mononuclear cells (BMMNCs) and CD34 + cells with three data modalities (clinical, genotype, and transcriptomics). Seven different views, including immune profile, inflammation/aging, retrotransposon (RTE) expression, and cell-type composition, were derived from these modalities to identify the latent factors with significant impact on MDS prognosis. SF3B1 was the only mutation among 13 mutations in the BMMNC cohort, indicating a significant association with high inflammation. This trend was also observed to a lesser extent in the CD34 + cohort. Interestingly, the MOFA factor representing the inflammation shows a good prognosis for MDS patients with high inflammation. In contrast, SRSF2 mutant cases show a granulocyte-monocyte progenitor (GMP) pattern and high levels of senescence, immunosenescence, and malignant myeloid cells, consistent with their poor prognosis. Furthermore, MOFA identified RTE expression as a risk factor for MDS. This work elucidates the efficacy of our integrative approach to assess the MDS risk that goes beyond all the scoring systems described thus far for MDS.

Introduction AML genome in the western populations has provided pathogenetic insights, prognosticators and selection of appropriate therapies. AML genome in the Middle East may differ from that in the west, because of younger population, racial, environmental and cultural factors including high consanguinity. We report for the first time, Whole Genome Sequencing (WGS) results in AML patients treated at the King Faisal Specialist Hospital and Research Centre in Riyadh, Saudi Arabia. The study was approved by KFSHRC ethics committee. Method The clinical data of 786 adult AML patients (18-89 years; median age 38) seen at KFSHRC from 2005 to 2023 was reviewed. In 217 (median age 42 years), bone marrow (BM) samples were available for WGS. The median follow up was 16 months (range 2-202 months). 198 cases were analysed at diagnosis, 19 at first relapse and 10 at both. DNA was extracted from BM; libraries were prepared using TruSeq DNA PCR-Free and Illumina DNA Prep, followed by paired-end sequencing with NovaSeq 6000 at 30-50× depth at KFSHRC. Germline and somatic SNVs/Indels were identified, using Mutect2, VarDict, and Strelka2 pipelines. Pathogenic and likely pathogenic variants were identified from ClinVar, COSMIC, and ACMG databases. Variants with minor allele frequency>1% specific to the Saudi population were filtered out. VAF thresholds were ≥35% for germline and ≥5% for somatic variants. The ichorCNA algorithm detected CNVs >5 Mbp, and structural variants were identified using Manta4. Extrachromosomal circular DNA (eccDNA) was detected with Amplicon Architect, and Kaplan-Meier and log-rank tests were used to assess survival. Results 170 patients had denovo AML, 37 secondary AML, and 10 therapy-related AML. 112 patients had allogeneic and 16 autologous hematopoietic stem cell transplantation as consolidation. Of 198 patients at diagnosis, ELN risk of 46 were favourable (median survival, not reached), 80 intermediate (median survival 27 months), 63 adverse risk (median survival 15 months), and 5 APL, and not known in 4 patients. WGS revealed additional alterations in 44 patients, leading to the reclassification within ELN risk categories of 27 individuals. WGS detected genetic alterations led to the reassignment of 23 patients to the adverse risk group and 3 cases with normal karyotype (NK) to favourable risk. The adverse risk AML was frequent in the Saudi cohort (43.4%). A novel MECOM::BCL11A rearrangement was found in a patient with sickle cell disease. Parental consanguinity was observed in 77(48.4%);46 were first cousin marriages. Germline variants were identified in 17/217(median age 30). Pathogenetic variants were detected in RUNX1 (4), FANCA (3), XPC (3), ANKRD26 (1), DDX41 (1), GATA2 (1), TP53 (1) and PTPN11(1). One patient each had Downs syndrome and clinical features of Fanconi Anemia without pathogenetic mutations in the known FANC genes. Notably, 67 patients (30.8%) reported a past history of cancer; 14.9% had history of cancer, 44.7% in first-degree relatives, 31.3% in second-degree relatives and 7.5% in both. One patient had coexistent AML and colorectal cancer. Total of 360 somatic SNV/indels were detected in 152/182 (83.5%) patients at diagnosis. Mutations were seen in signalling pathway (26.4%), DNA methylation (26.4%), tumor suppressor (24.2%), FLT3 (21.4%), myeloid transcription factors(18.7%), chromatin modifiers(7.1%), NPM1(13.2%), (99% with insertion in exon12 ,75% with NK), spliceosome (6.6%), and cohesin-complex (2.7%) genes. Distinct differences emerged when comparing our cohort with the University of Washington and the BEAT datasets. NPM1 mutations were significantly less prevalent 13.2% vs 33.7% and 21.8% (p<0.05) and DTA, spliceosome, and cohesin-complex genes were also significantly less frequent (p<0.01). eccDNA was detected in 27 patients. Amplicons with AML-related genes (BCL2, FLT3, CALR, MYC, ASXL1) were present in four patients who failed to achieve CR, had a complex karyotype, and TP53 mutations. Conclusion Saudi AML patients are younger, with high consanguinity, family history of cancers and adverse risk features detected by WGS. Spectrum of germline mutations is different than in Western populations. NPM1, DTA, spliceosome and cohesin mutations are significantly less common than reported in Western literature, including those with NK. The study has important implications for pathogenesis and treatment of AML in Middle Eastern populations.