p53 immunohistochemistry as an ancillary tool for rapid assessment of residual disease in TP53-mutated acute myeloid leukemia and myelodysplastic syndromes.

OBJECTIVES: Measurable residual disease flow cytometry (MRD-FC) and molecular studies are the most sensitive methods for detecting residual malignant populations after therapy for TP53-mutated acute myeloid leukemia and myelodysplastic neoplasms (TP53+ AML/MDS). However, their sensitivity is limited in suboptimal aspirates or when the immunophenotype of the neoplastic blasts overlaps with erythroids or normal maturing myeloid cells. In this study, we set out to determine if p53 immunohistochemistry (IHC) correlates with MRD-FC and next-generation sequencing (NGS) in the posttherapy setting and to determine the utility of p53 IHC to detect residual disease in the setting of negative or equivocal MRD-FC. METHODS: We retrospectively identified 28 pre- and posttherapy bone marrow biopsy specimens from 9 patients with TP53+ AML/MDS and a p53 overexpressor phenotype by IHC (strong 3+ staining at initial diagnosis). Next-generation sequencing and/or MRD-FC results were collected for each specimen. RESULTS: Using a threshold of more than ten 2-3+ cells in any one 400× field, p53 IHC detected residual disease with a sensitivity of 94% and a specificity of 89%. The threshold used in this study showed a high degree of concordance among 6 blinded pathologists (Fleiss κ = 0.97). CONCLUSIONS: Our study suggests that p53 IHC can be used as a rapid tool (within 24 hours) to aid in the detection of residual disease that may complement MRD-FC or NGS in cases in which the flow cytometry immunophenotype is equivocal and/or the bone marrow aspirate is suboptimal.

How I reduce and treat posttransplant relapse of MDS.

ABSTRACT: Allogeneic hematopoietic stem cell transplantation (HSCT) is the only potentially curative option for patients with high-risk myelodysplastic syndromes (MDS). Advances in conditioning regimens and supportive measures have reduced treatment-related mortality and increased the role of transplantation, leading to more patients undergoing HSCT. However, posttransplant relapse of MDS remains a leading cause of morbidity and mortality for this procedure, necessitating expert management and ongoing results analysis. In this article, we review treatment options and our institutional approaches to managing MDS relapse after HSCT, using illustrative clinical cases that exemplify different clinical manifestations and management of relapse. We address areas of controversy relating to conditioning regimen intensity, chemotherapeutic bridging, and donor selection. In addition, we discuss future directions for advancing the field, including (1) the need for prospective clinical trials separating MDS from acute myeloid leukemia and focusing on posttransplant relapse, as well as (2) the validation of measurable residual disease methodologies to guide timely interventions.

Shared and distinct mechanisms of UBA1 inactivation across different diseases.

Most cellular ubiquitin signaling is initiated by UBA1, which activates and transfers ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic) syndrome. Despite extensive clinical investigation into this lethal disease, little is known about the underlying molecular mechanisms. Here, by dissecting VEXAS-causing UBA1 mutations, we discovered that p.Met41 mutations alter cytoplasmic isoform expression, whereas other mutations reduce catalytic activity of nuclear and cytoplasmic isoforms by diverse mechanisms, including aberrant oxyester formation. Strikingly, non-p.Met41 mutations most prominently affect transthioesterification, revealing ubiquitin transfer to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. A similar E2 charging bottleneck exists in some lung cancer-associated UBA1 mutations, but not in spinal muscular atrophy-causing UBA1 mutations, which instead, render UBA1 thermolabile. Collectively, our results highlight the precision of conformational changes required for faithful ubiquitin transfer, define distinct and shared mechanisms of UBA1 inactivation in diverse diseases, and suggest that specific E1-E2 modules control different aspects of tissue differentiation and maintenance.

The clinical, molecular, and prognostic features of the 2022 WHO and ICC classification systems for myelodysplastic neoplasms.

Myelodysplastic neoplasms (MDS) are clonal disorders of bone marrow failure exhibiting a variable risk of progression to acute myeloid leukemia. MDS exhibit certain prognostic genetic or cytogenetic abnormalities, an observation that has led to both the pathologic reclassification of MDS in the 2022 World Health Organization (WHO) and International Consensus Classification (ICC) systems, as well as to an updated prognostic schema, the Molecular International Prognostic Scoring System (IPSS-M). This single-institution study characterized the molecular patterns and clinical outcomes associated with the 2022 WHO and ICC classification schemas to assess their clinical utility. Strikingly, with the exception of one individual, all 210 patients in our cohort were classified into analogous categories by the two pathologic/diagnostic schemas. Most patients (70%) were classified morphologically while the remaining 30% had genetically classified disease by both criteria. Prognostic risk, as assessed by the IPSS-M score was highest in patients with MDS with biallelic/multi-hit TP53 mutations and lowest in pts with MDS-SF3B1. Median leukemia-free survival (LFS) was shortest for those with MDS with biallelic/multi-hit TP53 (0.7 years) and longest for those with MDS with low blasts (LFS not reached). These data demonstrate the clear ability of the 2022 WHO and ICC classifications to organize MDS patients into distinct prognostic risk groups and further show that both classification systems share more similarities than differences. Incorporation of the IPSS-M and IPSS-R features provide additive prognostic and survival components to both the WHO and ICC classifications, which together enhance their utility for evaluating and treating MDS patients.

Shared and Distinct Mechanisms of UBA1 Inactivation Across Different Diseases.

Most cellular ubiquitin signaling is initiated by UBA1, which activates and transfers ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic) syndrome. Despite extensive clinical investigation into this lethal disease, little is known about the underlying molecular mechanisms. Here, by dissecting VEXAS-causing UBA1 mutations, we discovered that p.Met41 mutations alter cytoplasmic isoform expression, whereas other mutations reduce catalytic activity of nuclear and cytoplasmic isoforms by diverse mechanisms, including aberrant oxyester formation. Strikingly, non-p.Met41 mutations most prominently affect transthioesterification, revealing ubiquitin transfer to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. A similar E2 charging bottleneck exists in some lung cancer-associated UBA1 mutations, but not in spinal muscular atrophy-causing UBA1 mutations, which instead, render UBA1 thermolabile. Collectively, our results highlight the precision of conformational changes required for faithful ubiquitin transfer, define distinct and shared mechanisms of UBA1 inactivation in diverse diseases, and suggest that specific E1-E2 modules control different aspects of tissue differentiation and maintenance.

The trajectory of prognostication and risk stratification for patients with myelodysplastic syndromes.

ABSTRACT: Risk stratification and prognostication are crucial for the appropriate management of patients with myelodysplastic syndromes (MDSs) or myelodysplastic neoplasms, for whom the expected survival can vary from a few months to >10 years. For the past 5 decades, patients with MDS have been classified into higher-risk vs lower-risk disease phenotypes using sequentially developed clinical prognostic scoring systems. Factors such as morphologic dysplasia, clinical hematologic parameters, cytogenetics, and, more recently, mutational information have been captured in prognostic scoring systems that refine risk stratification and guide therapeutic management in patients with MDS. This review describes the progressive evolution and improvement of these systems which has led to the current Molecular International Prognostic Scoring System.

Myelodysplastic syndromes, thy name is heterogeneity.

Classification of the heterogeneous spectrum of myelodysplastic syndromes (MDS) requires both morphologic and molecular analysis to effectively subgroup patients. The paper by Zhang et al demonstrated the positive impact of combining the International Consensus Consortium (ICC) morphologic approach with prior clinical (IPSS-R) and mutational (IPSS-M) categorization to provide useful clinical evaluation of MDS patients. Commentary on: Zhang et al. Impact of the International Consensus Classification of Myelodysplastic Syndromes. Br J Haematol 2022 (Online ahead of print). doi: 10.1111/bjh.18628.

Recent Clinical and Molecular Advances for the Classification of Myelodysplastic Neoplasms.

Several major updates have recently occurred for the NCCN Guidelines for Myelodysplastic Syndromes (MDS) based on a number of prominent articles that have particular clinical and biologic impact for the field. These changes, which have been included in the current iteration of the NCCN Guidelines (Version 1.2023), include the WHO 2022 classification of MDS as well as the ICC suggestions for same. In addition, the molecular underpinning of MDS has been greatly updated with the generation of the Molecular International Prognostic Scoring System (IPSS-M) and an improved understanding to the prognostic implications of mutated TP53 subtypes, which are additive to the revised IPSS (IPSS-R) for stratification and management of patients with MDS. This report emphasizes the major components of the relevant changes to serve as a guide for therapeutic decision-making for patients with MDS.

Epidemiologic evaluation of clinical outcomes in ethnic minorities with myelodysplastic syndromes.

Since race/ethnicity and socioeconomic status (SES), including economic and educational disadvantage (EED), may impact overall survival (OS) in primary myelodysplastic syndromes (MDS), we analyzed the impact of these characteristics on OS in Non-Hispanic White (NHW), Non-Hispanic Black (NHB) and Hispanic patients. In this retrospective review using the SEER US population and American Community Survey data between 2001 and 2016, factor analysis was used for 23 county-level SES indicators to identify groups of interrelated measures of EED. For the 52,739 patients identified, increased age at diagnosis, male sex, higher prognostic risk category and higher EED were all associated with reduced OS. The most prominent SES factor extracted by factor analysis was EED. NHB patients had the highest OS compared to NHW and Hispanics. For all patients, EED analysis showed lower OS for most compared to least disadvantaged, most notably in lower-risk disease and Hispanics. More NHB and Hispanics (51% and 49%) were in the most disadvantaged category compared with NHW (30%). No significant differences were noted for causes of death by race/ethnicity. In conclusion, these data indicate that in addition to standard clinical measures for analyzing OS, race/ethnicity, SES and EED are critical determinants for assessing clinical outcomes in MDS patients.

NCCN Guidelines® Insights: Myelodysplastic Syndromes, Version 3.2022.

The NCCN Guidelines for Myelodysplastic Syndromes (MDS) provide recommendations for the evaluation, diagnosis, and management of patients with MDS based on a review of clinical evidence that has led to important advances in treatment or has yielded new information on biologic factors that may have prognostic significance in MDS. The multidisciplinary panel of MDS experts meets on an annual basis to update the recommendations. These NCCN Guidelines Insights focus on some of the updates for the 2022 version of the NCCN Guidelines, which include treatment recommendations both for lower-risk and higher-risk MDS, emerging therapies, supportive care recommendations, and genetic familial high-risk assessment for hereditary myeloid malignancy predisposition syndromes.

Molecular International Prognostic Scoring System for Myelodysplastic Syndromes.

BACKGROUND: Risk stratification and therapeutic decision-making for myelodysplastic syndromes (MDS) are based on the International Prognostic Scoring System­Revised (IPSS-R), which considers hematologic parameters and cytogenetic abnormalities. Somatic gene mutations are not yet used in the risk stratification of patients with MDS. METHODS: To develop a clinical-molecular prognostic model (IPSS-Molecular [IPSS-M]), pretreatment diagnostic or peridiagnostic samples from 2957 patients with MDS were profiled for mutations in 152 genes. Clinical and molecular variables were evaluated for associations with leukemia-free survival, leukemic transformation, and overall survival. Feature selection was applied to determine the set of independent IPSS-M prognostic variables. The relative weights of the selected variables were estimated using a robust Cox multivariable model adjusted for confounders. The IPSS-M was validated in an external cohort of 754 Japanese patients with MDS. RESULTS: We mapped at least one oncogenic genomic alteration in 94% of patients with MDS. Multivariable analysis identified TP53multihit, FLT3 mutations, and MLLPTD as top genetic predictors of adverse outcomes. Conversely, SF3B1 mutations were associated with favorable outcomes, but this was modulated by patterns of comutation. Using hematologic parameters, cytogenetic abnormalities, and somatic mutations of 31 genes, the IPSS-M resulted in a unique risk score for individual patients. We further derived six IPSS-M risk categories with prognostic differences. Compared with the IPSS-R, the IPSS-M improved prognostic discrimination across all clinical end points and restratified 46% of patients. The IPSS-M was applicable in primary and secondary/therapy-related MDS. To simplify clinical use of the IPSS-M, we developed an open-access Web calculator that accounts for missing values. CONCLUSIONS: Combining genomic profiling with hematologic and cytogenetic parameters, the IPSS-M improves the risk stratification of patients with MDS and represents a valuable tool for clinical decision-making. (Funded by Celgene Corporation through the MDS Foundation, the Josie Robertson Investigators Program, the Edward P. Evans Foundation, the Projects of National Relevance of the Italian Ministry of University and Research, Associazione Italiana per la Ricerca sul Cancro, the Japan Agency for Medical Research and Development, Cancer Research UK, the Austrian Science Fund, the MEXT [Japanese Ministry of Education, Culture, Sports, Science and Technology] Program for Promoting Research on the Supercomputer Fugaku, the Japan Society for the Promotion of Science, the Taiwan Department of Health, and Celgene Corporation through the MDS Foundation.)

Phase I First-in-Human Dose Escalation Study of the oral SF3B1 modulator H3B-8800 in myeloid neoplasms.

We conducted a phase I clinical trial of H3B-8800, an oral small molecule that binds Splicing Factor 3B1 (SF3B1), in patients with MDS, CMML, or AML. Among 84 enrolled patients (42 MDS, 4 CMML and 38 AML), 62 were red blood cell (RBC) transfusion dependent at study entry. Dose escalation cohorts examined two once-daily dosing regimens: schedule I (5 days on/9 days off, range of doses studied 1-40 mg, n = 65) and schedule II (21 days on/7 days off, 7-20 mg, n = 19); 27 patients received treatment for ≥180 days. The most common treatment-related, treatment-emergent adverse events included diarrhea, nausea, fatigue, and vomiting. No complete or partial responses meeting IWG criteria were observed; however, RBC transfusion free intervals >56 days were observed in nine patients who were transfusion dependent at study entry (15%). Of 15 MDS patients with missense SF3B1 mutations, five experienced RBC transfusion independence (TI). Elevated pre-treatment expression of aberrant transcripts of Transmembrane Protein 14C (TMEM14C), an SF3B1 splicing target encoding a mitochondrial porphyrin transporter, was observed in MDS patients experiencing RBC TI. In summary, H3B-8800 treatment was associated with mostly low-grade TAEs and induced RBC TI in a biomarker-defined subset of MDS.

Ex vivo drug screening defines novel drug sensitivity patterns for informing personalized therapy in myeloid neoplasms.

Precision medicine approaches such as ex vivo drug sensitivity screening (DSS) are appealing to inform rational drug selection in myelodysplastic syndromes (MDSs) and acute myeloid leukemia, given their marked biologic heterogeneity. We evaluated a novel, fully automated ex vivo DSS platform that uses high-throughput flow cytometry in 54 patients with newly diagnosed or treatment-refractory myeloid neoplasms to evaluate sensitivity (blast cytotoxicity and differentiation) to 74 US Food and Drug Administration-approved or investigational drugs and 36 drug combinations. After piloting the platform in 33 patients, we conducted a prospective feasibility study enrolling 21 patients refractory to hypomethylating agents (HMAs) to determine whether this assay could be performed within a clinically actionable time frame and could accurately predict clinical responses in vivo. When assayed for cytotoxicity, ex vivo drug sensitivity patterns were heterogeneous, but they defined distinct patient clusters with differential sensitivity to HMAs, anthracyclines, histone deacetylase inhibitors, and kinase inhibitors (P < .001 among clusters) and demonstrated synergy between HMAs and venetoclax (P < .01 for combinations vs single agents). In our feasibility study, ex vivo DSS results were available at a median of 15 days after bone marrow biopsy, and they informed personalized therapy, which frequently included venetoclax combinations, kinase inhibitors, differentiative agents, and androgens. In 21 patients with available ex vivo and in vivo clinical response data, the DSS platform had a positive predictive value of 0.92, negative predictive value of 0.82, and overall accuracy of 0.85. These data demonstrate the utility of this approach for identifying potentially useful and often novel therapeutic drugs for patients with myeloid neoplasms refractory to standard therapies.

SF3B1-mutant MDS as a distinct disease subtype: a proposal from the International Working Group for the Prognosis of MDS.

The 2016 revision of the World Health Organization classification of tumors of hematopoietic and lymphoid tissues is characterized by a closer integration of morphology and molecular genetics. Notwithstanding, the myelodysplastic syndrome (MDS) with isolated del(5q) remains so far the only MDS subtype defined by a genetic abnormality. Approximately half of MDS patients carry somatic mutations in spliceosome genes, with SF3B1 being the most commonly mutated one. SF3B1 mutation identifies a condition characterized by ring sideroblasts (RS), ineffective erythropoiesis, and indolent clinical course. A large body of evidence supports recognition of SF3B1-mutant MDS as a distinct nosologic entity. To further validate this notion, we interrogated the data set of the International Working Group for the Prognosis of MDS (IWG-PM). Based on the findings of our analyses, we propose the following diagnostic criteria for SF3B1-mutant MDS: (1) cytopenia as defined by standard hematologic values, (2) somatic SF3B1 mutation, (3) morphologic dysplasia (with or without RS), and (4) bone marrow blasts <5% and peripheral blood blasts <1%. Selected concomitant genetic lesions represent exclusion criteria for the proposed entity. In patients with clonal cytopenia of undetermined significance, SF3B1 mutation is almost invariably associated with subsequent development of overt MDS with RS, suggesting that this genetic lesion might provide presumptive evidence of MDS in the setting of persistent unexplained cytopenia. Diagnosis of SF3B1-mutant MDS has considerable clinical implications in terms of risk stratification and therapeutic decision making. In fact, this condition has a relatively good prognosis and may respond to luspatercept with abolishment of the transfusion requirement.

Wide variation in use and interpretation of gene mutation profiling panels among health care providers of patients with myelodysplastic syndromes: results of a large web-based survey.

Next-generation sequencing (NGS) is increasingly employed for diagnosis, risk stratification, and management of patients with myelodysplastic syndrome (MDS). We aimed to describe beliefs and practice patterns among providers who treat MDS patients with respect to the utility of NGS in diagnosis, risk stratification, prognosis, and treatment decisions at various points along the disease trajectory, response assessment, and development of institutional guidelines for MDS-specific molecular profiling. Using a 23-question web-based survey in May-June 2018, we identified a widespread use of molecular profiling with MDS-specific panels (N = 53; 39%) and general panels including MDS-related genes (N = 63; 47%), with the majority done at diagnosis (92%). We found substantial variations in genes tested in assays, providers beliefs, practices, testing logistics, and interpretation of results, and recognized multiple challenges limiting a wider utilization of molecular profiling. High-quality data are needed to develop evidence-based guidelines for the role of NGS in the care of MDS patients.

Profiling myelodysplastic syndromes by mass cytometry demonstrates abnormal progenitor cell phenotype and differentiation.

BACKGROUND: We sought to enhance the cytometric analysis of myelodysplastic syndromes (MDS) by performing a pilot study of a single cell mass cytometry (MCM) assay to more comprehensively analyze patterns of surface marker expression in patients with MDS. METHODS: Twenty-three MDS and five healthy donor bone marrow samples were studied using a 34-parameter mass cytometry panel utilizing barcoding and internal reference standards. The resulting data were analyzed by both traditional gating and high-dimensional clustering. RESULTS: This high-dimensional assay provided three major benefits relative to traditional cytometry approaches: First, MCM enabled detection of aberrant surface maker at high resolution, detecting aberrancies in 27/31 surface markers, encompassing almost every previously reported MDS surface marker aberrancy. Additionally, three previously unrecognized aberrancies in MDS were detected in multiple samples at least one developmental stage: increased CD321 and CD99; and decreased CD47. Second, analysis of the stem and progenitor cell compartment (HSPCs), demonstrated aberrant expression in 21 of the 23 MDS samples, which were not detected in three samples from patients with idiopathic cytopenia of undetermined significance. These immunophenotypically abnormal HSPCs were also the single most significant distinguishing feature between clinical risk groups. Third, unsupervised clustering of high-parameter MCM data allowed identification of abnormal differentiation patterns associated with immunophenotypically aberrant myeloid cells similar to myeloid derived suppressor cells. CONCLUSIONS: These results demonstrate that high-parameter cytometry methods that enable simultaneous analysis of all bone marrow cell types could enhance the diagnostic utility of immunophenotypic analysis in MDS.