Venous and Arterial Thrombosis in Patients with VEXAS Syndrome.

VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, caused by somatic mutations in UBA1, is an autoinflammatory disorder with diverse systemic manifestations. Thrombosis is a prominent clinical feature of VEXAS. The risks factors and frequency of thrombosis in VEXAS are not well described, due to the disease's new discovery and paucity of large databases. We evaluated 119 VEXAS patients for venous and arterial thrombosis and correlated their presence with clinical outcomes and survival. Thrombosis occurred in 49% of patients, mostly venous thromboembolism (VTE; 41%). Almost two thirds of VTE were unprovoked, 41% were recurrent, and 20% occurred despite anticoagulation. The cumulative incidence (CI) of VTE was 17% at 1 year from symptom onset and 40% by 5 years. Cardiac and pulmonary inflammatory manifestations were associated with time to VTE. M41L was positively associated specifically with pulmonary embolism (PE) by univariate (OR: 4.58, CI 1.28-16.21; p=0.02) and multivariate (OR: 16.94, CI 1.99-144.3; p=0.01) logistic regression. The cumulative incidence of arterial thrombosis was 6% at 1 year and 11% at 5 years. The overall survival (OS) of the entire patient cohort at median follow up time of 4.8 years was 88% and there was no difference in survival between patients with or without thrombosis (p=0.8). Patients with VEXAS syndrome are at high risk of VTE; thromboprophylaxis should administered be in high-risk settings unless strongly contraindicated.

Clonal Hematopoiesis, Inflammation, and Hematologic Malignancy.

Somatic or acquired mutations are postzygotic genetic variations that can occur within any tissue. These mutations accumulate during aging and have classically been linked to malignant processes. Tremendous advancements over the past years have led to a deeper understanding of the role of somatic mutations in benign and malignant age-related diseases. Here, we review the somatic mutations that accumulate in the blood and their connection to disease states, with a particular focus on inflammatory diseases and myelodysplastic syndrome. We include a definition of clonal hematopoiesis (CH) and an overview of the origins and implications of these mutations. In addition, we emphasize somatic disorders with overlapping inflammation and hematologic disease beyond CH, including paroxysmal nocturnal hemoglobinuria and aplastic anemia, focusing on VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. Finally, we provide a practical view of the implications of somatic mutations in clinical hematology, pathology, and beyond.

Intrathecal hydrocortisone for treatment of children and young adults with CAR T-cell immune-effector cell-associated neurotoxicity syndrome.

Immune-effector cell-associated neurotoxicity syndrome (ICANS) is a significant toxicity occurring with chimeric antigen receptor (CAR) T-cell therapy, with first-line treatment options including supportive care and systemic corticosteroids. Sparse data exist on how to approach progressive/refractory cases of ICANS. We present five pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) who had progressively worsening ICANS despite systemic steroids, and received intrathecal hydrocortisone with rapid reversal of ICANS. Therapeutic lumbar punctures are routinely used in upfront ALL therapy in pediatrics, with a demonstrable safety profile, thus use of intrathecal hydrocortisone merits further prospective studies in patients with severe ICANS.

Diagnostic work-up of hematological malignancies with underlying germline predisposition disorders (GPD).

Hematological malignancies with underlying germline predisposition disorders have been recognized by the World Health Organization 5th edition and International Consensus Classification (ICC) classification systems. The list of genes and the associated phenotypes are expanding and involve both pediatric and adult populations. While the clinical presentation and underlying molecular pathogenesis are relatively well described, the knowledge regarding the bone marrow morphologic features, the landscape of somatic aberrations associated with progression to hematological malignancies is limited. These pose challenges in the diagnosis of low-grade myelodysplastic syndrome (MDS) to hematopathologists which carries direct implication for various aspects of clinical management of the patient, donor selection for transplantation, and family members. Here in, we provide a focused review on the diagnostic work-up of hematological malignancies with underlying germline predisposition disorders with emphasis on the spectrum of hematological malignancies associated with each entity, and characteristic bone marrow morphologic, somatic cytogenetic and molecular alterations at the time of diagnosis of hematological malignancies. We also review the key clinical, morphologic, and molecular features, that should initiate screening for these entities.

Germline Predisposition to Myeloid Neoplasms: Diagnostic Concepts and Classifications.

Molecular and sequencing advances have led to substantial breakthroughs in the discovery of new genes and inherited mutations associated with increased risk of developing myeloid malignancies. Many of the same germline mutated genes are also drivers of malignancy in sporadic cancer. Recognition of myeloid malignancy associated with germline mutations is essential for proper therapy, disease surveillance, informing related donor selection for hematopoietic stem cell transplantation, and genetic counseling of the patient and affected family members. Some germline mutations are associated with syndromic features that precede the development of malignancy; however, penetrance may be highly variable leading to masking of the syndromic phenotype and/or inherited etiology.

Natural history study of patients with familial platelet disorder with associated myeloid malignancy.

ABSTRACT: Deleterious germ line RUNX1 variants cause the autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM), characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematologic malignancies (HMs). We launched a FPDMM natural history study and, from January 2019 to December 2021, enrolled 214 participants, including 111 patients with 39 different RUNX1 variants from 45 unrelated families. Seventy of 77 patients had thrombocytopenia, 18 of 18 had abnormal platelet aggregometry, 16 of 35 had decreased platelet dense granules, and 28 of 55 had abnormal bleeding scores. Nonmalignant bone marrows showed increased numbers of megakaryocytes in 12 of 55 patients, dysmegakaryopoiesis in 42 of 55, and reduced cellularity for age in 30 of 55 adult and 17 of 21 pediatric cases. Of 111 patients, 19 were diagnosed with HMs, including myelodysplastic syndrome, acute myeloid leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, and smoldering myeloma. Of those 19, 18 were relapsed or refractory to upfront therapy and referred for stem cell transplantation. In addition, 28 of 45 families had at least 1 member with HM. Moreover, 42 of 45 patients had allergic symptoms, and 24 of 30 had gastrointestinal (GI) symptoms. Our results highlight the importance of a multidisciplinary approach, early malignancy detection, and wider awareness of inherited disorders. This actively accruing, longitudinal study will genotype and phenotype more patients with FPDMM, which may lead to a better understanding of the disease pathogenesis and clinical course, which may then inform preventive and therapeutic interventions. This trial was registered at www.clinicaltrials.gov as #NCT03854318.

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.

A novel GATA2 distal enhancer mutation results in MonoMAC syndrome in 2 second cousins.

Mutations in the transcription factor GATA2 can cause MonoMAC syndrome, a GATA2 deficiency disease characterized by several findings, including disseminated nontuberculous mycobacterial infections, severe deficiencies of monocytes, natural killer cells, and B lymphocytes, and myelodysplastic syndrome. GATA2 mutations are found in âˆ¼90% of patients with a GATA2 deficiency phenotype and are largely missense mutations in the conserved second zinc-finger domain. Mutations in an intron 5 regulatory enhancer element are also well described in GATA2 deficiency. Here, we present a multigeneration kindred with the clinical features of GATA2 deficiency but lacking an apparent GATA2 mutation. Whole genome sequencing revealed a unique adenine-to-thymine variant in the GATA2 -110 enhancer 116,855 bp upstream of the GATA2 ATG start site. The mutation creates a new E-box consensus in position with an existing GATA-box to generate a new hematopoietic regulatory composite element. The mutation segregates with the disease in several generations of the family. Cell type-specific allelic imbalance of GATA2 expression was observed in the bone marrow of a patient with higher expression from the mutant-linked allele. Allele-specific overexpression of GATA2 was observed in CRISPR/Cas9-modified HL-60 cells and in luciferase assays with the enhancer mutation. This study demonstrates overexpression of GATA2 resulting from a single nucleotide change in an upstream enhancer element in patients with MonoMAC syndrome. Patients in this study were enrolled in the National Institute of Allergy and Infectious Diseases clinical trial and the National Cancer Institute clinical trial (both trials were registered at www.clinicaltrials.gov as #NCT01905826 and #NCT01861106, respectively).

A phase III randomized crossover trial of plerixafor versus G-CSF for treatment of WHIM syndrome.

BACKGROUNDWarts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function CXCR4 mutations. Myelokathexis is a kind of neutropenia caused by neutrophil retention in bone marrow and in WHIM syndrome is associated with lymphopenia and monocytopenia. The CXCR4 antagonist plerixafor mobilizes leukocytes to the blood; however, its safety and efficacy in WHIM syndrome are undefined.METHODSIn this investigator-initiated, single-center, quadruple-masked phase III crossover trial, we compared the total infection severity score (TISS) as the primary endpoint in an intent-to-treat manner in 19 patients with WHIM who each received 12 months treatment with plerixafor and 12 months treatment with granulocyte CSF (G-CSF, the standard of care for severe congenital neutropenia). The treatment order was randomized for each patient.RESULTSPlerixafor was nonsuperior to G-CSF for TISS (P = 0.54). In exploratory endpoints, plerixafor was noninferior to G-CSF for maintaining neutrophil counts of more than 500 cells/µL (P = 0.023) and was superior to G-CSF for maintaining lymphocyte counts above 1,000 cells/µL (P < 0.0001). Complete regression of a subset of large wart areas occurred on plerixafor in 5 of 7 patients with major wart burdens at baseline. Transient rash occurred on plerixafor, and bone pain was more common on G-CSF. There were no significant differences in drug preference or quality of life or the incidence of drug failure or serious adverse events.CONCLUSIONPlerixafor was not superior to G-CSF in patients with WHIM for TISS, the primary endpoint. Together with wart regression and hematologic improvement, the infection severity results support continued study of plerixafor as a potential treatment for WHIM syndrome.TRIAL REGISTRATIONClinicaltrials.gov NCT02231879.FUNDINGThis study was funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

How I diagnose myeloid neoplasms with germline predisposition.

OBJECTIVES: Pathologists play a crucial role in the initial diagnosis of germline predisposition to myeloid neoplasia and subsequent surveillance for disease progression. The diagnostic workup can be challenging, particularly if clinical history, laboratory testing, or genetic studies are incomplete or unavailable. METHODS: Through case-based examples, we illustrate common diagnostic challenges and pitfalls encountered during bone marrow examination of patients being evaluated for myeloid malignancy with potential germline predisposition to myeloid neoplasia. RESULTS: Lack of familial disease, the absence of syndromic manifestations, and late-onset hematologic malignancy do not exclude an underlying germline predisposition syndrome. Targeted myeloid sequencing panels can help identify potential germline alterations but may not detect large deletions or insertions, noncoding, or novel variants. Confirmation of the germline nature of an alteration detected in the peripheral blood or bone marrow ideally requires genetic testing using nonhematopoietic germline DNA to definitively distinguish between germline and somatic alterations. The ideal tissue source for germline testing is cultured skin fibroblasts. Certain germline predisposition syndromes can contain characteristic baseline bone marrow dysplastic-appearing features associated with cytopenias without constituting myelodysplastic syndrome. CONCLUSION: Recognizing germline predisposition to myeloid neoplasia is critical for proper disease management. This recognition is particularly important for patients who will undergo hematopoietic stem cell transplantation to screen potential related donors. Integration of the clinical history, bone marrow findings, cytogenetic studies, and specialized laboratory and molecular genetic testing is often essential for accurate diagnosis and subsequent disease 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.

Constitutively active Lyn kinase causes a cutaneous small vessel vasculitis and liver fibrosis syndrome.

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of ß2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

Multiomics integration of 22 immune-mediated monogenic diseases reveals an emergent axis of human immune health.

Monogenic diseases are often studied in isolation due to their rarity. Here we utilize multiomics to assess 22 monogenic immune-mediated conditions with age- and sex-matched healthy controls. Despite clearly detectable disease-specific and "pan-disease" signatures, individuals possess stable personal immune states over time. Temporally stable differences among subjects tend to dominate over differences attributable to disease conditions or medication use. Unsupervised principal variation analysis of personal immune states and machine learning classification distinguishing between healthy controls and patients converge to a metric of immune health (IHM). The IHM discriminates healthy from multiple polygenic autoimmune and inflammatory disease states in independent cohorts, marks healthy aging, and is a pre-vaccination predictor of antibody responses to influenza vaccination in the elderly. We identified easy-to-measure circulating protein biomarker surrogates of the IHM that capture immune health variations beyond age. Our work provides a conceptual framework and biomarkers for defining and measuring human immune health.

The International Consensus Classification (ICC) of hematologic neoplasms with germline predisposition, pediatric myelodysplastic syndrome, and juvenile myelomonocytic leukemia.

Updating the classification of hematologic neoplasia with germline predisposition, pediatric myelodysplastic syndrome (MDS), and juvenile myelomonocytic leukemia (JMML) is critical for diagnosis, therapy, research, and clinical trials. Advances in next-generation sequencing technology have led to the identification of an expanding group of genes that predispose to the development of hematolymphoid neoplasia when mutated in germline configuration and inherited. This review encompasses recent advances in the classification of myeloid and lymphoblastic neoplasia with germline predisposition summarizing important genetic and phenotypic information, relevant laboratory testing, and pathologic bone marrow features. Genes are organized into three major categories including (1) those that are not associated with constitutional disorder and include CEBPA, DDX41, and TP53; (2) those associated with thrombocytopenia or platelet dysfunction including RUNX1, ANKRD26, and ETV6; and (3) those associated with constitutional disorders affecting multiple organ systems including GATA2, SAMD9, and SAMD9L, inherited genetic mutations associated with classic bone marrow failure syndromes and JMML, and Down syndrome. A provisional category of germline predisposition genes is created to recognize genes with growing evidence that may be formally included in future revised classifications as substantial supporting data emerges. We also detail advances in the classification of pediatric myelodysplastic syndrome (MDS), expanding the definition of refractory cytopenia of childhood (RCC) to include early manifestation of MDS in patients with germline predisposition. Finally, updates in the classification of juvenile myelomonocytic leukemia are presented which genetically define JMML as a myeloproliferative/myelodysplastic disease harboring canonical RAS pathway mutations. Diseases with features overlapping with JMML that do not carry RAS pathway mutations are classified as JMML-like. The review is based on the International Consensus Classification (ICC) of Myeloid and Lymphoid Neoplasms as reported by Arber et al. (Blood 140(11):1200-1228, 2022).

The spectrum of GATA2 deficiency syndrome.

Inherited or de novo germ line heterozygous mutations in the gene encoding the transcription factor GATA2 lead to its deficiency. This results in a constellation of clinical features including nontuberculous mycobacterial, bacterial, fungal, and human papillomavirus infections, lymphedema, pulmonary alveolar proteinosis, and myelodysplasia. The onset, or even the presence, of disease is highly variable, even in kindreds with the identical mutation in GATA2. The clinical manifestations result from the loss of a multilineage progenitor that gives rise to B lymphocytes, monocytes, natural killer cells, and dendritic cells, leading to cytopenias of these lineages and subsequent infections. The bone marrow failure is typically characterized by hypocellularity. Dysplasia may either be absent or subtle but typically evolves into multilineage dysplasia with prominent dysmegakaryopoiesis, followed in some instances by progression to myeloid malignancies, specifically myelodysplastic syndrome, acute myelogenous leukemia, and chronic myelomonocytic leukemia. The latter 3 malignancies often occur in the setting of monosomy 7, trisomy 8, and acquired mutations in ASXL1 or in STAG2. Importantly, myeloid malignancy may represent the primary presentation of disease without recognition of other syndromic features. Allogeneic hematopoietic stem cell transplantation (HSCT) results in reversal of the phenotype. There remain important unanswered questions in GATA2 deficiency, including the following: (1) Why do some family members remain asymptomatic despite harboring deleterious mutations in GATA2? (2) What are the genetic changes that lead to myeloid progression? (3) What causes the apparent genetic anticipation? (4) What is the role of preemptive HSCT?

A RUNX1-FPDMM rhesus macaque model reproduces the human phenotype and predicts challenges to curative gene therapies.

Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.

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.