Clonal landscape and clinical outcomes of telomere biology disorders: somatic rescuing and cancer mutations.

Telomere biology disorders (TBD), caused by pathogenic germline variants in telomere-related genes, present with multi-organ disease and a predisposition to cancer. Clonal hematopoiesis (CH) as a marker of cancer development and survival in TBD is poorly understood. Here, we characterized the clonal landscape of a large cohort of 207 TBD patients with a broad range of age and phenotype. CH occurred predominantly in symptomatic patients and in signature genes typically associated with cancers: PPM1D, POT1, TERT promoter (TERTp), U2AF1S34, and/or TP53. Chromosome 1q gain (Chr1q+) was the commonest karyotypic abnormality. Clinically, multiorgan involvement and CH in TERTp, TP53, and splicing factor genes associated with poorer overall survival. Chr1q+, and splicing factor or TP53 mutations significantly increased the risk of hematologic malignancies, regardless of the clonal burden. Chr1q+ and U2AF1S34 mutated clones were pre-malignant events associated with the secondary acquisition of mutations in genes related to hematologic malignancies. Like known effects of Chr1q+ and TP53-CH, functional studies demonstrated that U2AF1S34 mutations primarily compensated for aberrant upregulation of TP53 and interferon pathways in telomere-dysfunctional hematopoietic stem cells, highlighting the TP53 pathway as a canonical route of malignancy in TBD. In contrast, somatic POT1/PPM1D/TERTp-CH had distinct trajectories unrelated to cancer development. With implications beyond TBD, our data show that telomere dysfunction is a strong selective pressure for CH. In TBD, CH is a poor prognostic marker associated with worse overall survival. The identification of key regulatory pathways that drive clonal transformation in TBD allows the identification of patients at a higher risk of cancer development.

Role of allogeneic hematopoietic cell transplantation in VEXAS syndrome.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a newly diagnosed syndrome comprising severe systemic inflammatory and hematological manifestations including myelodysplastic syndrome and plasma cell dyscrasia. Since its discovery four years ago, several groups have identified pleomorphic clinical phenotypes, but few effective medical therapies exist which include Janus Kinase (JAK) inhibitors, interleukin inhibitors (IL-1 and IL-6), and hypomethylating agents. Prospective trials are lacking at this time and most patients remain corticosteroid dependent. VEXAS has a high morbidity from frequent life threatening inflammatory symptoms and risk of progression to hematological malignancies and has an overall survival of 50% at 10 years. Allogeneic stem cell transplant (allo-HCT) is a curative option for this disease caused by somatic mutations in the UBA1 gene. Here we outline the role of allo-HCT in treating patients with VEXAS syndrome, highlighting the outcomes from several single-institution studies and case reports. Prospective trials will be required to precisely define the role of allo-HCT in the management of VEXAS syndrome.

VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic): clinical review in a rapidly emerging field.

VEXAS syndrome is a recently described entity characterized by systemic inflammatory and hematologic manifestations. The disease was first characterized by Beck et al. in 2020 in a study characterizing 25 patients with undiagnosed adult-onset inflammatory syndromes. While the literature regarding VEXAS syndrome has grown exponentially since 2020, there is still much to be understood. This lack of information leads to challenges in both the diagnosis and treatment of patients with VEXAS syndrome. Patients will often have a variety of clinical symptoms that can lead to missed or delayed diagnoses. Additionally, awareness of VEXAS syndrome is still developing among clinicians. In this comprehensive review, we summarize the current literature regarding VEXAS syndrome, and explore clinical updates of this emerging disease state. Our aim of this review is to increase awareness regarding this new disease state and identify research areas to better understand future treatment approaches for patients with VEXAS syndrome.

Immune-monitoring of myelodysplastic neoplasms: Recommendations from the i4MDS consortium.

Advancements in comprehending myelodysplastic neoplasms (MDS) have unfolded significantly in recent years, elucidating a myriad of cellular and molecular underpinnings integral to disease progression. While molecular inclusions into prognostic models have substantively advanced risk stratification, recent revelations have emphasized the pivotal role of immune dysregulation within the bone marrow milieu during MDS evolution. Nonetheless, immunotherapy for MDS has not experienced breakthroughs seen in other malignancies, partly attributable to the absence of an immune classification that could stratify patients toward optimally targeted immunotherapeutic approaches. A pivotal obstacle to establishing "immune classes" among MDS patients is the absence of validated accepted immune panels suitable for routine application in clinical laboratories. In response, we formed International Integrative Innovative Immunology for MDS (i4MDS), a consortium of multidisciplinary experts, and created the following recommendations for standardized methodologies to monitor immune responses in MDS. A central goal of i4MDS is the development of an immune score that could be incorporated into current clinical risk stratification models. This position paper first consolidates current knowledge on MDS immunology. Subsequently, in collaboration with clinical and laboratory specialists, we introduce flow cytometry panels and cytokine assays, meticulously devised for clinical laboratories, aiming to monitor the immune status of MDS patients, evaluating both immune fitness and identifying potential immune "risk factors." By amalgamating this immunological characterization data and molecular data, we aim to enhance patient stratification, identify predictive markers for treatment responsiveness, and accelerate the development of systems immunology tools and innovative immunotherapies.

When to consider inherited marrow failure syndromes in adults.

The inherited bone marrow failure syndromes (IBMFS) are a heterogenous group of disorders caused by germline mutations in related genes and characterized by bone marrow failure (BMF), disease specific organ involvement, and, in most cases, predisposition to malignancy. Their distinction from immune marrow failure can often be challenging, particularly when presentations occur in adulthood or are atypical. A combination of functional (disease specific assays) and genetic testing is optimal in assessing all new BMF patients for an inherited etiology. However, genetic testing is costly and may not be available worldwide due to resource constraints; in such cases, clinical history, standard laboratory testing, and the use of algorithms can guide diagnosis. Interpretation of genetic results can be challenging and must reflect assessment of pathogenicity, inheritance pattern, clinical phenotype, and specimen type used. Due to the progressive use of genomics, new IBMFS continue to be identified, widening the spectrum of these disorders.

Early activation of inflammatory pathways in UBA1-mutated hematopoietic stem and progenitor cells in VEXAS.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a pleiotropic, severe autoinflammatory disease caused by somatic mutations in the ubiquitin-like modifier activating enzyme 1 (UBA1) gene. To elucidate VEXAS pathophysiology, we performed transcriptome sequencing of single bone marrow mononuclear cells and hematopoietic stem and progenitor cells (HSPCs) from VEXAS patients. HSPCs are biased toward myeloid (granulocytic) differentiation, and against lymphoid differentiation in VEXAS. Activation of multiple inflammatory pathways (interferons and tumor necrosis factor alpha) occurs ontogenically early in primitive hematopoietic cells and particularly in the myeloid lineage in VEXAS, and inflammation is prominent in UBA1-mutated cells. Dysregulation in protein degradation likely leads to higher stress response in VEXAS HSPCs, which positively correlates with inflammation. TCR usage is restricted and there are increased cytotoxicity and IFN-γ signaling in T cells. In VEXAS syndrome, both aberrant inflammation and myeloid predominance appear intrinsic to hematopoietic stem cells mutated in UBA1.

Conditional survival and standardized mortality ratios of patients with severe aplastic anemia surviving at least one year after hematopoietic cell transplantation or immunosuppressive therapy.

Immunosuppressive treatment (IST) and hematopoietic cell transplant (HCT) are standard therapies for severe aplastic anemia (SAA). We report on conditional survival and standardized mortality ratios (SMR), which compare the mortality risk with the general population adjusted for age, gender, and race/ethnicity, in patients with SAA alive for at least 12 months after treatment with IST or HCT between 2000 and 2018. Given changes to treatment regimens and differences in length of follow-up, two treatment periods were defined a priori: 2000-2010 and 2011-2018. The SMR of patients treated during the period 2000-2010 and who survived one year were 3.50 (95% confidence interval [CI]: 2.62-4.58), 4.12 (95% CI: 3.20-5.21), and 8.62 (95% CI: 6.88-10.67) after IST, matched related donor HCT, and alternative donor HCT, respectively. For the period 2011-2018, the corresponding SMR were 2.89 (95% CI: 1.54-4.94), 3.12 (95% CI: 1.90-4.82), and 4.75 (95% CI: 3.45-6.38), respectively. For IST patients, their mortality risk decreased over time, and became comparable to the general population by five years. For patients who underwent HCT during 2000-2010 and 2011-2018, their mortality risk became comparable to the general population after ten years and after five years, respectively. Thus, 1-year survivors after IST or HCT can expect their longevity beyond five years to be comparable to that of the general US population.

Alemtuzumab in relapsed immune severe aplastic anemia: Long-term results of a phase II study.

Immune severe aplastic anemia (SAA) is characterized by pancytopenia and immune-mediated bone marrow destruction. SAA may be treated with hematopoietic stem cell transplantation (HSCT) or immunosuppressive therapy (IST). However, 30% of patients treated with IST relapse. We previously reported a clinical trial of alemtuzumab in which more than half of 25 relapsed SAA patients (56%) responded hematologically. Here, we present long-term results of a total of 42 patients. Participants with SAA who had previously completed antithymocyte globulin (ATG)-based IST, but had relapsed, were enrolled on this study. Alemtuzumab was administered intravenously (IV) (n = 28) or subcutaneously (SC) (n = 14). The primary endpoint was hematologic response at 6 months. Secondary endpoints included relapse, clonal evolution, and survival. This trial was registered at clinicaltrials.gov (NCT00195624). Patients were enrolled over 9 years, with median follow-up of 6 years. Median age was 32 years, with 57% being female. At 6 months, 18 patients (43%) achieved response; 15 (54%) of those who received IV compared with 3 (21%) who received SC therapy. Six patients (14%) had durable long-term response without need for subsequent AA-directed therapy or HSCT at last follow-up. Nine patients had clonal evolution, with high-risk evolution occurring in 6. Overall survival was 67% at median follow-up of 6 years. Prolonged iatrogenic immunosuppression was observed as long as 2 years after alemtuzumab administration. Alemtuzumab induces responses in relapsed SAA, some of which are durable long-term. However, immunosuppression can persist for years, requiring long-term monitoring.

Spectrum of clonal hematopoiesis in VEXAS syndrome.

Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is caused by somatic mutations in UBA1 (UBA1mut) and characterized by heterogenous systemic autoinflammation and progressive hematologic manifestations, meeting criteria for myelodysplastic syndrome (MDS) and plasma cell dyscrasias. The landscape of myeloid-related gene mutations leading to typical clonal hematopoiesis (CH) in these patients is unknown. Retrospectively, we screened 80 patients with VEXAS for CH in their peripheral blood (PB) and correlated the findings with clinical outcomes in 77 of them. UBA1mut were most common at hot spot p.M41 (median variant allele frequency [VAF] = 75%). Typical CH mutations cooccurred with UBA1mut in 60% of patients, mostly in DNMT3A and TET2, and were not associated with inflammatory or hematologic manifestations. In prospective single-cell proteogenomic sequencing (scDNA), UBA1mut was the dominant clone, present mostly in branched clonal trajectories. Based on integrated bulk and scDNA analyses, clonality in VEXAS followed 2 major patterns: with either typical CH preceding UBA1mut selection in a clone (pattern 1) or occurring as an UBA1mut subclone or in independent clones (pattern 2). VAF in the PB differed markedly between DNMT3A and TET2 clones (median VAF of 25% vs 1%). DNMT3A and TET2 clones associated with hierarchies representing patterns 1 and 2, respectively. Overall survival for all patients was 60% at 10 years. Transfusion-dependent anemia, moderate thrombocytopenia, and typical CH mutations, each correlated with poor outcome. In VEXAS, UBA1mut cells are the primary cause of systemic inflammation and marrow failure, being a new molecularly defined somatic entity associated with MDS. VEXAS-associated MDS is distinct from classical MDS in its presentation and clinical course.

Differential diagnosis of bone marrow failure syndromes guided by machine learning.

The choice to postpone treatment while awaiting genetic testing can result in significant delay in definitive therapies in patients with severe pancytopenia. Conversely, the misdiagnosis of inherited bone marrow failure (BMF) can expose patients to ineffectual and expensive therapies, toxic transplant conditioning regimens, and inappropriate use of an affected family member as a stem cell donor. To predict the likelihood of patients having acquired or inherited BMF, we developed a 2-step data-driven machine-learning model using 25 clinical and laboratory variables typically recorded at the initial clinical encounter. For model development, patients were labeled as having acquired or inherited BMF depending on their genomic data. Data sets were unbiasedly clustered, and an ensemble model was trained with cases from the largest cluster of a training cohort (n = 359) and validated with an independent cohort (n = 127). Cluster A, the largest group, was mostly immune or inherited aplastic anemia, whereas cluster B comprised underrepresented BMF phenotypes and was not included in the next step of data modeling because of a small sample size. The ensemble cluster A-specific model was accurate (89%) to predict BMF etiology, correctly predicting inherited and likely immune BMF in 79% and 92% of cases, respectively. Our model represents a practical guide for BMF diagnosis and highlights the importance of clinical and laboratory variables in the initial evaluation, particularly telomere length. Our tool can be potentially used by general hematologists and health care providers not specialized in BMF, and in under-resourced centers, to prioritize patients for genetic testing or for expeditious treatment.

Efficacy of JAK1/2 inhibition in murine immune bone marrow failure.

Immune aplastic anemia (AA) is a severe blood disease characterized by T-lymphocyte- mediated stem cell destruction. Hematopoietic stem cell transplantation and immunosuppression are effective, but they entail costs and risks, and are not always successful. The Janus kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T-cell activation and inhibits cytokine production in models of graft-versus-host disease. We tested RUX in murine immune AA for potential therapeutic benefit. After infusion of lymph node (LN) cells mismatched at the major histocompatibility complex [C67BL/6 (B6)⇒CByB6F1], RUX, administered as a food additive (Rux-chow), attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, preserved hematopoietic progenitors, and prevented mortality, when used either prophylactically or therapeutically. RUX suppressed the infiltration, proliferation, and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. Similar effects were obtained when Rux-chow was fed to C.B10 mice in a minor histocompatibility antigen mismatched (B6⇒C.B10) AA model. RUX only modestly suppressed lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice. Our data support clinical trials of JAK/STAT inhibitors in human AA and other immune bone marrow failure syndromes.

Clonality in immune aplastic anemia: Mechanisms of immune escape or malignant transformation.

Aplastic anemia (AA) is the prototypic bone marrow failure syndrome and can be classified as either acquired or inherited. Inherited forms are due to the effects of germline mutations, while acquired AA is suspected to result from cytotoxic T-cell mediated immune attack on hematopoietic stem and progenitor cells. Once thought to be a purely "benign" condition, clonality in the form of chromosomal abnormalities and single nucleotide variants is now well recognized in AA. Mechanisms underpinning this clonality likely relate to selection of clones that allow immune evasion or increased cell survival the marrow environment under immune attack. Widespread use and availability of next generation and other genetic sequencing techniques has enabled us to better understand the genomic landscape of aplastic anemia. This review focuses on the current concepts associated with clonality, in particular somatic mutations and their impact on diagnosis and clinical outcomes in immune aplastic anemia.

Predictors of clonal evolution and myeloid neoplasia following immunosuppressive therapy in severe aplastic anemia.

Predictors, genetic characteristics, and long-term outcomes of patients with SAA who clonally evolved after immunosuppressive therapy (IST) were assessed. SAA patients were treated with IST from 1989-2020. Clonal evolution was categorized as "high-risk" (overt myeloid neoplasm [meeting WHO criteria for dysplasia, MPN or acute leukemia] or isolated chromosome-7 abnormality/complex karyotype without dysplasia or overt myeloid neoplasia) or "low-risk" (non-7 or non-complex chromosome abnormalities without morphological evidence of dysplasia or myeloid neoplasia). Univariate and multivariate analysis using Fine-Gray competing risk regression model determined predictors. Long-term outcomes included relapse, overall survival (OS) and hematopoietic stem cell transplant (HSCT). Somatic mutations in myeloid cancer genes were assessed in evolvers and in 407 patients 6 months after IST. Of 663 SAA patients, 95 developed clonal evolution. Pre-treatment age >48 years and ANC > 0.87 × 109/L were strong predictors of high-risk evolution. OS was 37% in high-risk clonal evolution by 5 years compared to 94% in low-risk. High-risk patients who underwent HSCT had improved OS. Eltrombopag did not increase high-risk evolution. Splicing factors and RUNX1 somatic variants were detected exclusively at high-risk evolution; DNMT3A, BCOR/L1 and ASXL1 were present in both. RUNX1, splicing factors and ASXL1 somatic mutations detected at 6 months after IST predicted high-risk evolution.

Cell senescence and malignant transformation in the inherited bone marrow failure syndromes: Overlapping pathophysiology with therapeutic implications.

Fanconi anemia, telomeropathies and ribosomopathies are members of the inherited bone marrow failure syndromes, rare genetic disorders that lead to failure of hematopoiesis, developmental abnormalities, and cancer predisposition. While each disorder is caused by different genetic defects in seemingly disparate processes of DNA repair, telomere maintenance, or ribosome biogenesis, they appear to lead to a common pathway characterized by premature senescence of hematopoietic stem cells. Here we review the experimental data on senescence and inflammation underlying marrow failure and malignant transformation. We conclude with a critical assessment of current and future therapies targeting these pathways in inherited bone marrow failure syndromes patients.

Long-term eltrombopag for bone marrow failure depletes iron.

Eltrombopag (EPAG) has been approved for the treatment of aplastic anemia and for immune thrombocytopenia, and a subset of patients require long-term therapy. Due to polyvalent cation chelation, prolonged therapy leads to previously underappreciated iron depletion. We conducted a retrospective review of patients treated at the NIH for aplastic anemia, myelodysplastic syndrome, and unilineage cytopenias, comparing those treated with EPAG to a historical cohort treated with immunosuppression without EPAG. We examined iron parameters, duration of therapy, response assessment, relapse rates, and common demographic parameters. We included 521 subjects treated with (n = 315) or without EPAG (n = 206) across 11 studies with multiyear follow-up (3.6 vs. 8.5 years, respectively). Duration of EPAG exposure correlated with ferritin reduction (p = 4 × 10-14 ) regardless of response, maximum dose, or degree of initial iron overload. Clearance followed first-order kinetics with faster clearance (half-life 15.3 months) compared with historical responders (47.5 months, p = 8 × 10-10 ). Risk of iron depletion was dependent upon baseline ferritin and duration of therapy. Baseline ferritin did not correlate with response of marrow failure to EPAG or to relapse risk, and timing of iron clearance did not correlate with disease response. In conclusion, EPAG efficiently chelates total body iron comparable to clinically available chelators. Prolonged use can deplete iron and ultimately lead to iron-deficiency anemia mimicking relapse, responsive to iron supplementation.

Utility of plasma cell-free DNA for de novo detection and quantification of clonal hematopoiesis.

Although cell-free DNA (cfDNA) tests have emerged as a potential non-invasive alternative to bone marrow biopsies for monitoring clonal hematopoiesis in hematologic diseases, whether commercial cfDNA assays can be implemented for the detection and quantification of de novo clonal hematopoiesis in place of blood cells is uncertain. In this study, peripheral plasma cfDNA samples available from patients with aplastic anemia (n=25) or myelodysplastic syndromes (n=27) and a healthy cohort (n=107) were screened for somatic variants in genes related to hematologic malignancies using a Clinical Laboratory Improvement Amendments-certified panel. Results were further compared to DNA sequencing of matched blood cells. In reported results, 85% of healthy subjects, 36% of patients with aplastic anemia and 74% of patients with myelodysplastic syndromes were found to have somatic cfDNA variants, most frequently in DNMT3A, TET2, ASXL1 and SF3B1. However, concordance between cfDNA and blood cell findings was poor for the detection of clonal hematopoiesis when the allele frequency of the variants was <10%, which was mostly observed in the healthy and aplastic anemia cohorts but not in patients with myelodysplastic syndromes. After filtering data for potential artifacts due to low variant allele frequency and sequencing depth, the frequency of clonal hematopoiesis in cfDNA from healthy individuals and patients with aplastic anemia decreased to 52% and 20%, respectively. cfDNA and matched blood cells were not interchangeable for tracking changes in allele burdens as their agreement by Bland-Altman analysis was poor. A commercial cfDNA assay had good performance for de novo detection of clonal hematopoiesis in myelodysplastic syndromes, but showed no advantage over blood cells in diseases with low allele burdens or in healthy individuals.

Long-term outcomes in patients with severe aplastic anemia treated with immunosuppression and eltrombopag: a phase 2 study.

Patients with severe aplastic anemia (SAA) are either treated with bone marrow transplant (BMT) or immunosuppression (IST) depending on their age, comorbidities, and available donors. In 2017, our phase 2 trial reported improved hematologic responses with the addition of eltrombopag (EPAG) to standard IST for SAA when compared with a historical cohort treated with IST alone. However, the rates and characteristics of long-term complications, relapse, and clonal evolution, previously described in patients treated with IST alone, are not yet known with this new regimen, IST and EPAG. Patients were accrued from 2012 to 2020, with a total of 178 subjects included in this secondary endpoint analysis. With double the sample size and a much longer median follow-up (4 years) since the original publication in 2017, we report a cumulative relapse rate of 39% in responding patients who received cyclosporine (CSA) maintenance and clonal evolution of 15% in all treated patients at 4 years. Relapse occurred at distinct timepoints: after CSA dose reduction and EPAG discontinuation at 6 months, and after 2 years when CSA was discontinued. Most relapsed patients were retreated with therapeutic doses of CSA +/- EPAG, and two-thirds responded. Clonal evolution to a myeloid malignancy or chromosome 7 abnormality (high-risk) was noted in 5.7% of patients and conferred a poorer overall survival. Neither relapse nor high-risk evolution occurred at a higher rate than was observed in a historical comparator cohort, but the median time to both events was earlier in IST and EPAG treated patients. This trial was registered at www.clinicaltrials.gov as #NCT01623167.

Hematopoiesis under telomere attrition at the single-cell resolution.

The molecular mechanisms that drive hematopoietic stem cell functional decline under conditions of telomere shortening are not completely understood. In light of recent advances in single-cell technologies, we sought to redefine the transcriptional and epigenetic landscape of mouse and human hematopoietic stem cells under telomere attrition, as induced by pathogenic germline variants in telomerase complex genes. Here, we show that telomere attrition maintains hematopoietic stem cells under persistent metabolic activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of the innate immune signaling response, which directly compromises hematopoietic stem cells' self-renewal capabilities and eventually leads to their exhaustion. Mechanistically, we demonstrate that targeting members of the Ifi20x/IFI16 family of cytosolic DNA sensors using the oligodeoxynucleotide A151, which comprises four repeats of the TTAGGG motif of the telomeric DNA, overcomes interferon signaling activation in telomere-dysfunctional hematopoietic stem cells and these cells' skewed differentiation towards the megakaryocytic lineage. This study challenges the historical hypothesis that telomere attrition limits the proliferative potential of hematopoietic stem cells by inducing apoptosis, autophagy, or senescence, and suggests that targeting IFI16 signaling axis might prevent hematopoietic stem cell functional decline in conditions affecting telomere maintenance.