Idiopathic Cytopenia Of Undetermined Significance Research Articles

Treatment Patterns and Outcomes of Patients with Lower-Risk Myelodysplastic Syndromes in the Connect ® Myeloid Disease Registry

Treatment Patterns and Outcomes of Patients with Lower-Risk Myelodysplastic Syndromes in the Connect ® Myeloid Disease Registry

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

Relationship between clone metrics and clinical outcome in clonal cytopenia

Clonal cytopenia of undetermined significance (CCUS) is associated with an increased risk of developing a myeloid neoplasm with myelodysplasia (MN). To identify the features of the mutant clone(s) that is associated with clinical phenotype and progression, we studied the following cohorts of individuals: 311 patients with idiopathic cytopenia of undetermined significance (ICUS), 532 community-dwelling individuals without hematologic phenotype (n = 355) or with unexplained anemia (n = 177), and 592 patients with overt MN. Ninety-two of 311 (30%) patients with ICUS carried a somatic genetic lesion that signaled CCUS. Clonal hematopoiesis (CH) was detected in 19.7% and 27.7% of nonanemic and anemic community-dwelling individuals, respectively. Different mutation patterns and variant allele frequencies (VAFs) (clone metrics parameters) were observed in the conditions studied. Recurrent mutation patterns exhibited different VAFs associated with marrow dysplasia (0.17-0.48), indicating variable clinical expressivity of mutant clones. Unsupervised clustering analysis based on mutation profiles identified 2 major clusters, characterized by isolated DNMT3A mutations (CH-like cluster) or combinatorial mutation patterns (MN-like cluster), and showing different overall survival (HR, 1.8). In patients with CCUS, the 2 clusters had different risk of progression to MN (HR, 2.7). Within the MN-like cluster, distinct subsets with different risk of progression to MN were identified based on clone metrics. These findings unveil marked variability in the clinical expressivity of myeloid driver genes and underline the limitations of morphologic dysplasia for clinical staging of mutant hematopoietic clones. Clone metrics appears to be critical for informing clinical decision-making in patients with clonal cytopenia.

Incidence and prognosis of clonal hematopoiesis in patients with chronic idiopathic neutropenia

AbstractThe incidence and prognosis of clonal hematopoiesis in patients with isolated neutropenia among patients with idiopathic cytopenia of undetermined significance (ICUS), known as ICUS-N or chronic idiopathic neutropenia (CIN) patients, is poorly defined. The current study sought to investigate the frequency and clinical significance of mutations of genes implicated in myeloid malignancies using next-generation sequencing in patients with CIN (n = 185) with a long follow-up. We found that 21 (11.35%) of 185 patients carried a total of 25 somatic mutations in 6 genes with a median variant allele frequency of 12.75%. The most frequently mutated genes were DNMT3A and TET2 involving >80% of patients, followed by IDH1/2, SRSF2, and ZRSR2. The frequency of transformation to a myeloid malignancy was low in the total group of patients (5 of 185 patients [2.70%]). However, from the transformed patients, 4 belonged to the clonal group (4 of 21 [19.05%]) and 1 to the nonclonal group (1 of 164 [0.61%]), indicating that the presence of mutation(s) confers a relative risk for transformation of 31.24 (P = .0017). The variant allele frequency of the mutant clones in the transformed patients was >10% in all cases, and the genes most frequently associated with malignant transformation were SRSF2 and IDH1. No significant differences were identified between the clonal and nonclonal groups in the severity of neutropenia. Patients with clonal disease were older compared with nonclonal patients. These data contribute to the better understanding of the heterogeneous entities underlying ICUS and highlight the importance of mutation analysis for the diagnosis and prognosis of patients with unexplained neutropenias.

Diagnostic Value of Flow Cytometry Technique By European Leukemianet for Idiopathic Cytopenia of Undetermined Significance

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous hematological disorders characterized by hematopoietic dysplasia and/or chromosomal aberrations. However, the diagnosis of dysplasia is subjective and chromosomal aberrations are not always present. Thus, the diagnosis of MDS is not always straightforward when lacking specific characteristics. To resolve this diagnostic problem, a novel flow cytometry technique standardized by the European LeukemiaNet (ELN) working group for MDS [Haematologica 94: 1124, 2009] was proposed. However, this ELN MDS index has not been validated for the diagnosis of idiopathic cytopenia of undetermined significance (ICUS).Purpose: To determine the diagnostic value of the ELN MDS index for ICUS, we analyzed the diagnostic accuracy of morphology, cytogenetics, and flow cytometry for cytopenia.Methods: We investigated 253 consecutive patients who underwent bone marrow aspiration to examine the cause of cytopenia at our institute between 2012 and 2015. We used the ELN MDS index verified by Ogata et al. for low-grade MDS [Haematologica 94: 1066, 2009]. The ELN MDS index consists of the following four parameters: (1) CD34+ myeloblasts on all nucleated cells, ≥2.4%; (2) CD34+ B cells on all CD34+ cells, ≤5.0%; (3) lymphocyte/myeloblast CD45 MFI ratio, ≤4.0 or ≥7.8; and (4) granulocyte/lymphocyte SCC peak ratio, ≤6.0. When two or more parameters met the criteria, the patient was diagnosed with MDS by cytometry. We evaluated the accuracy of diagnosis with cytometry by using the clinical diagnosis as a standard reference. The clinical diagnosis was determined by more than two hematologists/specialists. Patients with MDS and ICUS were included in this study. ICUS was defined as follows: bone marrow blast, <5.0%; single- or multi-lineage dysplasia, <10%; and no detection of clonal chromosomal abberation characteristic to MDS.Results: A total of 253 bone marrow samples were evaluated (117 females). The median patient age was 66 years (range, 1-92 years). The clinical diagnosis was MDS in 60 patients (24 females; median age, 69 years; range, 30-91 years) and ICUS in 17 patients (9 females; median age, 76 years; range, 54-88 years). Other patients were identified to have non-MDS/non-ICUS diseases and were used as a control group. MDS subcategories were as follows: RCUD (n = 14), RARS (n = 3), RCMD (n = 10), RAEB-1 (n = 9), RAEB-2 (n = 12), MDS-U (n = 8), and 5q− syndrome (n = 4). Two or more criteria of the ELN MDS index were met in 34 MDS (56.7%; 34/60) and 4 ICUS patients (23.5%; 4/17). Positivity with the ELN MDS index among the MDS subcategories varied as follows: 6 RCUD (42.9%; 6/14), 1 RARS (33.3%; 1/3), 5 RCMD (50.0%; 5/10), 7 RAEB-1 (77.8%; 7/9), 12 RAEB-2 (100%; 12/12), 2 MDS-U (25.0%; 2/8), and 1 5q− syndrome cases (25.0%; 1/4). When cutoff scores were set as the ELN MDS index of ≥2, sensitivities for diagnoses of MDS and ICUS were 56.7% (34/60) and 25.0% (4/16), specificities were 83.1% (147/177) and 83.1% (147/177), positive predictive values were 53.1% (34/64) and 11.8% (4/34), and negative predictive values were 85.0% (147/173) and 92.5% (147/159), respectively.Conclusion: Application of the ELN MDS index in MDS revealed sensitivity and specificity in line with those of previous studies, while 25.0% of ICUS patients were indicated to have MDS. This prevalence of MDS in ICUS may correspond to 27.5% (33/120) of patients with ICUS who had genetically clonal evidence identified by targeted sequencing of 22 MDS-associated genes [Kwok et al., Blood 2015; 126: 2355]. Our data suggest that the ELN MDS index is a practical tool to identify a subset of ICUS patients with MDS trait. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Correlation of Myelodysplasia-Related Flow Cytometric Findings with Mutations in Patients Undergoing Diagnostics for Suspected Myelodysplastic Syndrome

Introduction: Diagnostic procedures in patients with suspected myelodysplastic syndromes (MDS) have been broadened recently by adding flow cytometry and mutation analysis to cytomorphology and cytogenetics. In parallel the concept of pre-MDS conditions has been widely accepted in analogy to MGUS preceding multiple myeloma. Thus, idiopathic cytopenia of undetermined significance (ICUS) has been defined by cytopenia with minimal dysplasia in the absence of mutations and clonal cytopenia of undetermined significance (CCUS) is separated from ICUS by the presence of at least one MDS-related mutation. While this concept implies the outgrowth of a mutated cell clone and the acquisition of additional mutations as well as the gradual manifestation of the dysplastic phenotype as proven by cytomorphology and flow cytometry no comprehensive data is available yet on a multidisciplinary diagnostic approach taking into consideration ICUS and CCUS. Aim: To describe the correlation of the presence of mutations with the occurrence of flow cytometric aberrations in the context of cytomorphologic and cytogenetic findings in patients with reactive conditions, ICUS, CCUS, MDS and related malignancies. Patients and methods: In a retrospective analysis patients were identified in whom cytomorphology, cytogenetics, flow cytometry and mutation analysis had been performed in parallel for suspected MDS. Flow cytometric procedures followed ELN criteria. Mutation analysis was performed by next generation sequencing targeting the genes ASXL1, CALR, CBL, CSNK1A1, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2 . A total of 1607 patients were identified (median age 72 years, range 18-93; 1007 males, 600 females). Results: Cytomorphologic evaluation revealed reactive conditions in 522 (33%) patients, ICUS was diagnosed in 248 (15%), MDS in 631 (39%), MDS/MPN in 63 (4%), CMML in 108 (7%) and 35 (2%) were not evaluable. An aberrant karyotype was found in 350 (22%) patients. The numbers of flow cytometric aberrations were as follows: 0, 90 patients (6%); 1 to 2, 496 (31%); 3 to 4, 456 (28%); 5 to 7, 421 (26%); ≥8, 144 (9%). The myeloid progenitor cell compartment was not affected in 1292 (80%) and showed 1 aberration in 210 (13%), 2 in 86 (5%) and 3 to 4 in 19 (1%). The respective number for the granulocytic compartment were 0, 425 (26%); 1, 438 (27%); 2, 364 (23%); 3, 226 (14%) and 4 to 6, 154 (10%) and for the monocytic compartment 0, 451 (28%); 1, 513 (32%); 2, 307 (19%); 3, 161 (10%) and 4 to 7, 175 (11%). In 847 (53%) the erythroid compartment was not affected and in 760 (47%) 1 aberration was observed. The numbers of mutations were as follows: 0, 739 patients (46%); 1, 347 (22%); 2, 295 (18%); 3, 157 (10%); 4 to 6, 69 (4%). The genes most frequently mutated were TET2 (342, 21%), ASXL1 (226, 14%), SRSF2 (215, 13%), DNMT3A (129, 8%), SF3B1 (133, 8%), RUNX1 (108, 7%), TP53 (89, 6%), and U2AF1 (83, 5%). The number of flow cytometric aberrations was strongly correlated to the number of mutations (r=0.494, p Mean (±SD) numbers of flow cytometric aberrations and mutations, respectively, were highest in CMML (6.2±2.3, 2.6±1.2), MDS/MPN (5.0±2.7, 1.9±1.3) and MDS (4.5±2.4, 1.3±1.2). In contrast, patients with reactive conditions had the lowest numbers (2.4±2.0, 0.3±0.7). Interestingly, numbers for ICUS cases were in between (3.7±2.3, 1.0±1.1). Importantly, re-classifying ICUS cases as CCUS based on the presence of at least one mutation (n=141) not only by definition increased the number of mutations (1.8±1.0) but also increased the number of flow cytometric aberrations (4.4±2.4) to levels observed in MDS while the remaining ICUS cases had levels observed in those with reactive conditions (2.9±1.7). Mean (±SD) numbers of flow cytometric aberrations and mutations, respectively, in patients with and without cytogenetic aberrations were 4.7±2.5 vs. 3.7±2.4 and 1.4±1.2 vs. 1.0±1.2 (p Conclusions: This data confirms the concept of ICUS and CCUS preceding MDS and suggests to broaden this concept by integrating flow cytometric data showing strong correlation to observed mutations. Confirmatory analyses and follow up analyses correlating the findings of the different applied methods with the patients´ outcome should substantiate these results and build the basis for a clinically orientated classification of patients with cytopenia and MDS-related conditions. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Fasan: MLL Munich Leukemia Laboratory: Employment. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Chromosome Aberrations Detected By SNP Array Technique Indicating a High Risk of MDS Transformation in Patients with Icus and Poor Prognosis of Patients with MDS

AbstractPurpose: To evaluate the value of single nucleotide polymorphism array (SNP-A) analysis on the diagnosis and prognosis of Chinese patients with myelodysplastic syndromes (MDS).Methods: 416 cases were recruited from two medical centers in China; of which 200 cases were diagnosed with MDS, 25 with myeloproliferative neoplasm (MPN), 63 with primary acute myeloid leukemia(AML), 40 with aplastic anemia (AA), 62 with idiopathic cytopenia of undetermined significance (ICUS), and 26 were healthy individuals. We analyzed chromosome abnormalities in all the cases with both SNP-A and metaphase cytogenetics (MC) methods, and evaluated their clinical significance as well. Clinical data analyzed included age, sex, blood counts, marrow morphology, blast counts, progression free survival (PFS) and overall survival(OS).Results: 1. SNP-Aanalysis leaded to a significantly higher detection yield of chromosome abnormalities than MC examination. The abnormal detection rate for MDS, MPN and AML by MC was 42%, 48% and 36.5%, but by SNP-A significantly increased to 73.5%,72% and 69.8%, respectively. Moreover, SNP-A analysis revealed complex types and genomic distribution patterns of chromosome aberrations. 2. Notably, 20 of the 62 ICUS patients had abnormal detections by SNP-A, and interestingly 17 of these 20 ICUS patients with abnormal SNP-A detections transformed to typical MDS with a median follow-up time at 11months(Table 1). 3. Prognosis of the MDS patients with abnormal SNP-A detections was significantly worse than that of those patients without abnormal SNP-A observations in OS (24 months vs not reached (NR); P=0.004) and PFS (15 vs 40 months; P=0.002). Prognosis of the patients identified with abnormal SNP-A detections in addition to a normal or a good IPSS-R prognostic MC finding in a patient was significantly inferior than that of those patients with no such additional SNP-A aberrations in OS (25 months vs NR; P=0.004) and PFS (17 months vs NR; P=0.002). For the patients stratified as high-risk and very-high-risk MDS by the revised International Prognostic Scoring System(IPSS-R), there were no differences between the patients with and without SNP-A abnormalities in OS (13 vs 12 months; P =0.537) and PFS (14 vs 10 months; P =0.368); However, in the group of patients with very-low-risk, low-risk, and intermediate-risk MDS, the patients with SNP-A abnormalities showed significantly inferior survival than those without abnormal SNP-A detections in OS (NR vs NR; P=0.001) and PFS (25 months vs NR; P=0.008). 4. By multivariable analysis the presence of SNP-A aberrations additional to MC analyses in a patient and a higher number of SNP-A abnormalities (≥3 per patient) were independent predictors of poor prognosis for patients with MDS (HR=2.33, P=0.003; HR=2.40, P=0.002).Conclusion:SNP-A analysis can help significantly improve the diagnosis and better evaluate the prognosis of patients with MDS, and help better assess the risk of MDS transformation in patients with ICUS. SNP-A analysis is of great practical value in clinical management. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hypoplastic Myelodysplastic Syndrome: Clinical, Histopathological and Molecular Characterization

Introduction and AimsHypoplastic Myelodysplasic Syndrome (h-MDS) has distinct features compared to normo- or hypercellular MDS, with a higher response rate to immunosuppressive therapy and more favorable prognosis. However, to date, reproducible diagnostic criteria for h-MDS have not been clearly defined, making differential diagnosis from other bone marrow failure (BMF) syndromes challenging. Based on a large and well-annotated consecutive cohort of patients with BM hypocellularity from two tertiary centres, we aimed to delineate the clinical, histopathological and molecular features of h-MDS.MethodsOf 1262 consecutive adult patients analyzed at King's College Hospital, London, UK and IRCCS Policlinico San Matteo & University of Pavia, Italy: 533 patients had a hypocellular BM, including 205 MDS patients with BM cellularity ≤25%, 77 MDS with reduced age-adjusted BM cellularity, 139 with Aplastic Anemia (AA), 97 with a diagnosis of Idiopathic Cytopenia of Undetermined Significance (ICUS) and 15 with congenital BMF (c-BMF) and 729 with normo- or hypercellular MDS (n-MDS).ResultsComparison of clinical features and outcome of patients with h-MDS as defined by a BM cellularity ≤25% (n=205) and those with a reduced age-adjusted cellularity (n=77) did not detect any significant differences. Therefore, a reduced age-adjusted BM cellularity was adopted as a criterion to define h-MDS (n=282). Compared with patients with n-MDS, those with h-MDS were significantly younger, displayed higher levels of hemoglobin (p=.01), lower neutrophil and platelet counts and lower percentage of BM blasts, ring sideroblasts (RS) and CD34+cells as determined by immunohistochemistry (all p<.001). No significant difference was observed in the distribution of chromosomal abnormalities. A significantly higher prevalence of PNH clones was observed in h-MDS (24% vs 2%, p<.001). Those patients with h-MDS showed a significantly lower risk of leukemic evolution compared with n-MDS (4-year cumulative incidence 14% vs 26%, p=.003). Comparing h-MDS with AA, h-MDS showed a significantly higher percentage of RS (p=.004), BM blasts, dysmegakaryopoiesis and dysgranulopoiesis (p<.001), BM fibrosis (p=.002), clusters of CD34+ cells and chromosomal abnormalities (p<.001). H-MDS patients showed a significantly higher risk of leukemic evolution compared with AA (4-year cumulative incidence 14% vs 0%, p<.001).Targeted sequencing of 24 commonly mutated myeloid genes was performed on 358 patients (73 h-MDS, 45 AA and 240 n-MDS). Twenty-five patients with h-MDS (34%) showed one or more somatic mutation (median 1; range 1-4). Comparing h-MDS to n-MDS revealed a significantly lower number of mutations per subject and variant allele frequency (VAF) (p<.001). Moreover, a significantly lower number of mutations per patient and VAF were observed in AA compared to h-MDS (p=.031 and p=.003, respectively). Thirty-seven patients with a hypocellular BM had a peripheral blood telomere length (TL) ≤1st percentile and/or a germline mutation consistent with a diagnosis of c-BMF, leading to an estimated prevalence of unidentified c-BMF of 13% in cases without robust morphological evidence of MDS.Based on these findings, we defined a diagnostic score (h-score), including variables with the highest specificity for MDS: dysmegakaryopoiesis, dysgranulopoiesis, BM fibrosis, clusters of CD34+ cells, RS 2-14%, BM blasts 2-4% (score 1); RS≥15% and BM blasts ≥5% (score 2). By applying ROC analysis, a cut off value of 2 was associated with the highest specificity for MDS (0.98). Notably, 71% of patients with a hypoplastic BM and low h-score (0-1) had no evidence of clonal disease by cytogenetic or mutation analyses or had a mutation pattern consistent with age-related clonal hematopoiesis. These patients showed a significantly better OS and lower risk of leukemic evolution (P<.001).ConclusionsIntegration of cyto-histological and genetic features in patients with a hypocellular BM identifies two distinct groups, one with clinical and genetic features highly consistent with a clonal disease with high risk of leukemic evolution, and one with features more consistent with a non-malignant bone marrow failure. Furthermore, we identified relevant enrichment of patients with previously undiagnosed c-BMF. DisclosuresKulasekararaj:Akari Therapeutics Plc: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Honoraria; Ra Pharmaceuticals: Consultancy, Honoraria; Achillion pharmaceuticals: Consultancy, Honoraria.

Immunophenotypically Aberrant Stem Cells in Myelodysplastic Syndromes: A Biomarker for Leukemic Progression

Introduction: Myelodysplastic syndromes (MDS) are generally referred to as a spectrum of myeloid malignancies that are notorious for their heterogeneity. Subsequently, the question arises as to what is the best method to dissect the heterogeneity of these syndromes, which may range from a normal life expectancy and MDS-unrelated dead to rapid progression to acute myeloid leukemia (AML). Most effort is still being put into the development of morphological classification and prognostic models like the classification of the World Health Organization (WHO) and the revised International Prognostic Scoring System (IPSS-R). However, the need for a more refined classification based on new insights into MDS pathophysiology is emerging.Methods: Hitherto we propose a new approach to predict leukemic progression among MDS patients using hematopoietic stem cell immunoprofiles. In this study, leukemic stem cell associated antigens - CD7, CD11b, CD22, CD33, CD44, CD45RA, CD56, CD123, CD366, CD371 - were analyzed on CD34+CD38- hematopoietic stem cells (HSCs) derived of 130 patients and controls at time of diagnosis.Results: Aberrant expression of these antigens was found in 33% (24/72) and in 20% (1/5) of the MDS and chronic myelomonocytic leukemia (CMML) patients, respectively. Even in patients diagnosed with idiopathic cytopenia of undetermined significance (ICUS) based on inexplicable cytopenia but less than 10% dysplasia, expression of aberrant antigens was found in 50% (4/8). In contrast to MDS, CMML, and ICUS patients, aberrant antigen expression was absent on HSCs of healthy, age-matched controls (0/9) and pathological controls (0/36), i.e. patients with non-myeloid clonal disorders. In MDS patients, the presence of aberrant immunophenotypic HSCs (AIP-HSCs) at diagnosis was negatively associated with the leukemia free and overall survival time (Figure 1a-b, p<.001), and increased the likelihood of leukemic progression and death by 99.5% (HR=0.005, 95%CI: 0-10.0) and 87.2% (HR=0.128, 95%CI: 0.04-0.41). The robust performance of flow cytometric detection of AIP-HSCs as diagnostic test to identify MDS patients prone to AML was illustrated by a high sensitivity of 88% and specificity of 69% at a follow-up time of six months. Although a slide trend toward increased AIP-HSCs prevalence for higher risk categories, MDS patients with AIP-HSCs were found across almost all WHO 2016 and IPSS-R categories (Figure 2a-b). Interestingly, multivariate regression analyses, including AIP-HSC percentage as well as the IPSS-R variables cytogenetic abnormalities, bone marrow blasts, hemoglobin, absolute neutrophil count, and platelets, revealed AIP-HSC percentage to be the only determinant with a significant effect on leukemia free survival. In predicting overall survival, apart from AIP-HSCs, cytogenetic abnormalities rendered statistical significant value.Conclusion: Flow cytometric detection of AIP-HSCs can be used as a biomarker to predict which MDS patients will finally develop AML. As a result, the presence of AIP-HSCs may improve currently used classification models and substitute the 20% blast cut-off to discriminate between MDS and AML. Addition of molecular features will be needed to validate the malignant nature of these AIP-HSCs. In the near feature, we anticipate that flow cytometric detection of AIP-HSCs will not only contribute to the dissection of MDS heterogeneity and an increased understanding of MDS stem cell biology, but also to therapy decision making, disease monitoring, and the clinical outcome of MDS patients. [Display omitted] DisclosuresOssenkoppele:J&J: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Roche: Honoraria; Karyopharm: Consultancy, Research Funding; Novartis: Research Funding.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

British Society for Haematology guidelines for the diagnosis and evaluation of prognosis of Adult Myelodysplastic Syndromes.

Unique ethnic features of DDX41 mutations in patients with idiopathic cytopenia of undetermined significance, myelodysplastic syndrome, or acute myeloid leukemia.

DDX41 mutations are associated with hematologic malignancies including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but the incidence in idiopathic cytopenia of undetermined significance (ICUS) is unknown. We investigated the incidence, genetic characteristics, and clinical features of DDX41 mutations in Korean patients with ICUS, MDS, or AML. We performed targeted deep sequencing of 61 genes including DDX41 in 457 patients with ICUS (n=75), MDS (n=210), or AML (n=172). Germline DDX41 mutations with causality were identified in 28 (6.1%) patients, of whom 27 (96.4%) had somatic mutations in the other position of DDX41. Germline origins of the DDX41 mutations were confirmed in all of the 11 patients in whom germline-based testing was performed. Of the germline DDX41 mutations, p.V152G (n=10) was most common, followed by p.Y259C (n=8), p.A500fs (n=6), and p.E7* (n=3). Compared with non-mutated patients, patients with a DDX41 mutation were more frequently male, older, had a normal karyotype, low leukocyte count, and hypocellular marrow at diagnosis. Three of the four ICUS patients with germline DDX41 mutations progressed to MDS. The incidence of DDX41 mutations in Korean patients was high and there was a distinct mutation pattern, in that p.V152G was a unique germline variant. ICUS harboring germline DDX41 mutations may be regarded as a hereditary myeloid neoplasm. Germline DDX41 mutations are not uncommon and should be explored when treating patients with myeloid malignancies.

Significance of Targeted Sequencing Assay for Patients with Suspected Myeloid Malignancies

To investigate the clinical significance of the targeted next-generation sequencing assay for patients with suspected myeloid malignancies. A total of 39 hematopenia patients with suspected myeloid malignamies in Department of Hematology of The Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University from January 2018 to April 2019 were treated, 20 hot spot genes of myelodysplastic syndrome (MDS) were detected. Regarding the diagnostic type, there were 7 cases of idiopathic cytopenia of undetermined significance (ICUS), 8 cases of clonal cytopenias of undetermined significance (CCUS) and 24 cases of myeloid myeloid malignancies which included 18 cases of MDS, 4 cases of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and 2 cases of acute myeloid leukemia. Positive mutation was detected in 70.8% (17/24) of myeloid malignancy patients , and 72.7% (16/22) in MDS and MDS/MPN patients. The main mutation types were ASXL1, TET2 and RUNX1. Compared with gene negative group, there were no significant differences in sex, age (＜60 years old or ≥60 years old), proportion of bone marrow blast cells (＜5% or≥5%) and cytogenetics (good, medium and poor) (P＞0.05). Furthermore, all 8 CCUS patients showed positive mutation, and the incidence of double or multiple mutation in CCUS group was significantly lower than that of the MDS and MDS/MPN group (37.5% vs 54.5%) (P=0.002). The mutation types between the two groups were similar, and there was no significant difference in variant allele frequency (P＞0.05). Our results suggest that there are high rates of double or multiple mutations in myeloid malignancies, especially in patients with MDS and MDS/MPN. Targeted sequencing assay can improve the diagnosis of myeloid malignancies, and guide clinical treatment.

Predictors of Survival and Time to Progression to Myeloid Neoplasm in Patients with Clonal Cytopenias

Background: Clonal cytopenias of Undetermined Significance (CCUS) have a higher risk of progression to myeloid neoplasm (MN) compared to idiopathic cytopenias of undetermined significance (ICUS) (Malcovati et al., Blood, 2017). The implementation of flow cytometric (FC) immune-phenotyping and next generation sequencing (NGS) in clinical practice has improved the diagnosis of these entities but the clinical significance and interaction of these abnormal results are still unknown. In this study we investigated predictive factors that play role in survival and progression to MN in patients with ICUS/CCUS. Methods: Patients (Pts) who had undergone evaluation for unexplained cytopenia and had undergone FC and bone marrow morphological assessment between 3/2015-3/2020 at Mayo Clinic Rochester were included. Pts were excluded if a malignant hematological disorder was diagnosed prior to the time of FC. FC included evaluation of the following parameters: Abnormal expression of HLA-DR/CD13, CD2, CD7, CD45, and CD56 on blasts; and abnormal CD13/CD16 expression and side scatter on granulocytic cells. NGS panel up to 2018 included 34 genes, ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, and ZRSR2; in 2018, BRAF was omitted and 12 genes were added, ANRD26, DDX41, ELANE, ETNK1, KDM6A, MYD88, NOTCH1, RAD21, SH2B3, SRP72, SMC3, and STAG2. Results: Characteristics: Of 490 consecutive pts, 238 (median age 69 years (range 19-92), 66% males) met our inclusion criteria as ICUS. 86 (36%) pts had CCUS (abnormal cytogenetics and/or tested positive for pathogenic mutations on NGS). After a median follow-up of 28 months (95% CI: 20 to 31 months), 21 (25%) patients developed MN and 23 (27%) died during follow-up. Comparing ICUS vs CCUS: upon comparing CCUS pts to ICUS, several factors were found to be significantly different: CCUS pts were older (p= .02); majority male (p= .04), had more abnormal HLA-DR/CD13 (p &amp;lt;.0001), more side-scattered light by FC (p &amp;lt;.0001), more pancytopenia on follow up (p= .02) and more morphologic atypia (&amp;lt;.0001). Overall survival (OS) outcomes in CCUS: Several covariates were significant in univariate models, and model selection was used to generate a risk score based on abnormal CD7, abnormal CD13/CD16, abnormal HLA-DR/CD13, splenomegaly and history of prior chemotherapy or radiotherapy (range: 0 - 4; HR=2.58, [95 CI: 1.69 - 3.94], p&amp;lt;.0001). Risk scores were grouped as low risk (score=0), intermediate risk (score=1), and high risk (score=2+), with estimated 2-yr-OS of 95%, 84%, 40%, respectively (Figure 1). An extended risk score model including NGS factors added ASXL1 and IDH1 mutation status to the previous model (range: 0 - 6; HR=2.72, [95 CI 1.82 - 4.06], p&amp;lt;0.0001). MN-free survival (MNFS) in CCUS: 37 pts either progressed to MN and/or died with a median follow up time for MNFS of 22.4 months. Similar for OS, model selection approaches yielded a composite risk score: splenomegaly, BCOR mutation status, mutation in RAS signaling pathway, abnormal expression of flow markers CD13/CD16, HLA-DR/CD13 or CD7 (HR=3.23, [95 CI: 1.90 - 5.49], p=&amp;lt;0.0001). Risk scores were grouped as low risk (score=0), intermediate risk (score=1), and high risk (score ≥ 2), with estimated 1-yr MNFS of 84%, 74%, and 31%, respectively (Figure 2). Conclusion: CCUS has unique features compared to ICUS. Several factors, including clinical characteristics, immune-phenotyping by FC, and somatic mutations have important impact on risk of progression to MN and on overall survival. Our findings serve as proof-of-concept that such integrated models could help identify CCUS patients at higher risks for progression to MN or death. They can guide treatment accordingly and should be evaluated further. Disclosures Shah: Dren Bio: Consultancy.

The Potential of Molecular Genetic Analysis for Diagnostic and Prognostic Decision Making in Clonal Cytopenia of Undetermined Significance (CCUS) and MDS - a Study on 576 Patients

Background: Several specific therapies for MDS are now available. Molecular genetics plays a key role, as some therapies target distinct mutations and others are particularly effective for molecularly defined subgroups (e.g. SF3B1). In parallel, broad mutation studies suggest a continuous progression from clonal cytopenia of undetermined significance (CCUS) to MDS. Since panel sequencing is available for routine laboratory workflows, we evaluated its potential for diagnosis of CCUS and extended the question to possible (future) therapeutic decisions. Aim: (1) Identify the frequency of clonality (mutations) in a real life MDS vs. cytopenia cohort; (2) subdivide CCUS with a low and high risk of developing MDS; (3) identify the subset of patients qualifying for personalized treatments Patients and Methods: We performed morphology and cytogenetic analysis according to current WHO gold standard algorithms in 576 patients (F:221, M:355; median age: 72 [17-94]) with cytopenia and clinical conditions requiring bone marrow biopsy to exclude or diagnose MDS. Individuals with other possible explanations (e.g. lymphoma, PNH) had been excluded upfront by combining morphology and flow cytometry. DNA was isolated from bone marrow; sequencing was performed on NovaSeq after NextFlex library preparation (Illumina, ILMN, San Diego, CA) and hybrid capture of a 41 gene panel (IDT Inc. Coralville, IA). Data was analyzed with Pisces and Pindel (for FLT3-ITD) (BaseSpace, ILMN) using a minimum sensitivity of 3%. Genetic variants were classified by combining databases and in silico predictions. Results: We found 499 mutations in 181/213 (85%) patients with diagnosis of MDS, and 259 mutations in 132/363 (36%) other cytopenic patients (p &amp;lt;0.01). Cases with mutations are by definition CCUS and without mutation ICUS (idiopathic cytopenia of undetermined significance). CCUS and MDS patients were significantly older than ICUS (CCUS: 74, MDS: 75 vs. ICUS: 66 years, p &amp;lt;0.02, each). MDS patients had significantly more mutations per case than CCUS (average: 2.8 vs. 2.0, p&amp;lt;0.01). By contrast, the typical aging pattern (DNMT3A, TET2 and ASXL1 mutations [DTA] with VAF &amp;lt;10%) as the only molecular genetic aberration was significantly more frequent in CCUS than in MDS with mutations: 25% vs. 4% (33/132 vs. 7/181; p&amp;lt;0.01). The International Working Group for the Prognosis of MDS - IWG-PM - (Malcovati, et al. Blood 2020) suggested SF3B1 mutations to define the first subgroup of patients by molecular genetics. Using their criteria, 12% of our MDS (26/213) patients fall into this novel category of SF3B1-mutated MDS. Due to the high risk of developing MDS, the IWG-PM suggests to use the mutation as presumptive evidence of MDS even without definitive morphological features. In our CCUS cohort the same genetic SF3B1 constellation was significantly less frequent, however 5% of individuals (7/132, p=0.03) qualified for MDS diagnoses by molecular genetics. In addition, CCUS patients show other MDS typical mutations: e.g. 4% (5/132) had RUNX1 mutations and 30% (39/132) had splicing factor mutations other than SF3B1. Malcovati et. al. (Blood 2017) defined a "highly specific pattern" of mutations for patients with a 20% risk of developing a myeloid malignancy per year. This pattern was frequent in MDS (71%, 151/213), but was detected in 55% of CCUS (72/132), which corresponds to 20% of cytopenia patients without a definitive MDS diagnosis (72/363). Next, we subdivided the cohorts according to their potential treatment options (Figure 1). (A) SF3B1 mutations would suggest use of Luspatercept. (B) According to Bejar et al. (ASH 2015), 95/213 (45%) of our MDS and 37/132 (28%) of our CCUS have a high-risk genetic profile. Individuals with high-risk mutations could be considered for alloSCT depending on progression of disease or clinical settings. (C) A potential for targeted therapies (mutations in CSNK1A1, FLT3 [ITD, TKD], IDH1, IDH2, KRAS, NRAS) was found in 6% (12/213) of the remaining MDS and 3% (4/132) of the CCUS cohort. Conclusions: We suggest that molecular genetic markers should be recognized as presumptive evidence of MDS and allow the diagnoses to be based on three cornerstones: morphology, cytogenetics and molecular genetics. In two thirds of MDS and one third of CCUS patients mutations can be identified that allow personalized treatment based on respective molecular genetics even independent of other currently used diagnostic labels. Disclosures No relevant conflicts of interest to declare.

A Personalized Clinical-Decision Tool to Improve the Diagnostic Accuracy of Myelodysplastic Syndromes

Background While histo- and cytomorphological examinations are central to the diagnosis of myelodysplastic syndromes (MDS), significant inter-observer variability exists. The diagnosis can be challenging in pancytopenic patients (pts) without evidence of dysplasia and is contingent on observer expertise. We developed and externally validated a geno-clinical model that uses mutational data and peripheral blood counts/clinical variables to distinguish MDS from other myeloid malignancies. Methods Clinical and genomic data, including commercially available next-generation sequencing panels, were obtained for patients (pts) treated at the Cleveland Clinic (CC; 652 pts), Munich Leukemia Laboratory (MLL; 1509 pts), and the University of Pavia in Italy (UP, 536 pts). All patients had carried a diagnosis of MDS, chronic myelomonocytic leukemia (CMML), MDS/myeloproliferative neoplasm overlap (MDS/MPN), myeloproliferative neoplasm (MPN; either polycythemia vera, essential thrombocythemia, or myelofibrosis), clonal cytopenia of undetermined significance (CCUS), or idiopathic cytopenia of undetermined significance (ICUS). All diagnoses were established with bone marrow aspiration and according to World Health Organization 2017 criteria. The training cohort included data from CC and UP and randomly divided into learner (80%) and test (20%) cohorts. The final model was independently validated in the MLL cohort. A machine learning algorithm was used to build the model; multiple extraction algorithms were used to extract genomic/clinical variables on both the cohort and individual levels. Performance was evaluated according to the area under the curve of the receiver operating characteristic (ROC-AUC) and accuracy matrices. Results Among the 2697 pts included from all sites, the median age was 70 years [36 - 86]. Median hemoglobin (Hb) was 10.4g/dl [6.9 - 15.7], median platelet count (PLT) was 132 k/dL [14 - 722], median WBC count was 5.3 k/dL [1.4 - 49.9], median ANC was 2.8 k/dL [0.3 - 27.7], median monocyte count was 0.3 k/dL [0 - 9.9], and median lymphocyte count (ALC) was 1.1 k/dL [0.1 - 5.4], and median peripheral blast percentage 0% [0 - 8]. The most commonly mutated genes in all patients were (list top 5 genes) and among pts with MDS were SF3B1 (27%), TET2 (25%), ASXL1 (19%), SRSF2 (16%), and DNMT3A (11%); among patients with MDS-MPN/CMML, the most commonly mutated genes were MDS-MPN/CMML (TET2 46%, ASXL1 34%, SRSF2 29%, RUNX1 13%, CBL 12%) ; among patients with MPNs, the most commonly mutated genes were (JAK2 64%, ASXL1 27%, TET2 14%, DNMT3A 8%, U2AF1 7%); among patients with CCUS the most commonly mutated genes were (TET2 41%, DNMT3A 27%, ASXL1 19%, SRSF2 17%, ZRSR2 10%). The most important features for model predictions (ranked from the most to the least important) included: number of mutations detected/sample, peripheral blast percentage, AMC, JAK2 status, Hb, basophil count, age, eosinophil count, ALC, WBC, EZH2 mutation status, ANC, mutation status of KRAS and SF3B1, platelets, and gender. The final model achieved an average AUROC of 0.95 (95% CI 0.93-0.96) when applied to the test cohort and 0.93 (95% CI 0.91 - 0.94) when it was applied to the MLL cohort. The model also provides individual-level explanations for predictions, providing top differential diagnoses and individual-level explanations of how features influence a putative diagnosis (Figure 1b). Conclusions We developed and externally validated a highly accurate and interpretable model that can distinguish MDS from other myeloid malignancies using clinical and mutational data from a large international cohort. The model can provide personalized interpretations of its outcome and can aid physicians and hematopathologists in recognizing MDS with high accuracy when encountering pts with pancytopenia and with a suspected diagnosis of MDS. Disclosures Sekeres: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda/Millenium: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees. Mukherjee:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Honoraria; Bristol Myers Squib: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Aplastic Anemia and MDS International Foundation: Honoraria; Celgene/Acceleron: Membership on an entity's Board of Directors or advisory committees; EUSA Pharma: Consultancy. Gerds:Sierra Oncology: Research Funding; Imago Biosciences: Research Funding; Apexx Oncology: Consultancy; Celgene: Consultancy, Research Funding; Incyte Corporation: Consultancy, Research Funding; Roche/Genentech: Research Funding; CTI Biopharma: Consultancy, Research Funding; AstraZeneca/MedImmune: Consultancy; Gilead Sciences: Research Funding; Pfizer: Research Funding. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria. Nazha:Jazz: Research Funding; Incyte: Speakers Bureau; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee.

Genotype-Phenotype Correlations in Patients with Myeloid Malignancies Using Explainable Artificial Intelligence

Background: Myelodysplastic syndromes (MDS) and related myeloid malignancies are highly variable in both their clinical manifestations and underlying genetic abnormalities. While few mutations in myeloid malignancies are considered disease-defining, significant and complex associations between these genes exist and can influence the clinical characteristics and disease phenotype. Here, we took advantage of a large, international cohort of patients with myeloid malignancies to define genotype-phenotype relationships using state of the art machine learning models. Methods Data were collected for patients (pts) from the Cleveland Clinic (CC; 652 pts), Munich Leukemia Laboratory (MLL; 1509 pts), and the University of Pavia in Italy (UP; 536 patients). Clinical data including CBC at time of diagnosis and a genomic panel of 20 commonly mutated genes in myeloid malignancies were analyzed. Gene-gene correlations within disease subtypes, individual genes' co-occurrence or exclusivity within disease subtypes, and the co-occurrence or exclusivity of individual genes with clinically meaningful features including karyotypic abnormalities and severe cytopenias (defined as hemoglobin &amp;lt; 8 g/dL, platelets &amp;lt; 50k/dL, and ANC &amp;lt; 1k/dL) were evaluated using multiple machine learning/correlation/feature extraction algorithms. Results 2697 pts were included, 1630 (60%) with MDS, 399 (15%) with chronic myelomonocytic leukemia (CMML), 142 (5%) with idiopathic cytopenia of undetermined significance (ICUS), 129 (5%) with MDS-MPN overlap syndromes (MDS-MPN), 95 (4%) with primary myelofibrosis (PMF), 93 (3%) with clonal cytopenia of undetermined significance (CCUS), 52 (2%) with essential thrombocythemia (ET), 41 (2%) with polycythemia vera (PV), and 26 (1%) with other myeloproliferative neoplasms (MPNs). The median age at diagnosis for the entire cohort was 70 years [36 - 86]. Of patients with karyotype data available, 1091 pts (50%) had a normal karyotype, 17 (1%) had chromosome 17 abnormalities, 96 (4%) had chromosome 7 abnormalities, 145 (7%) had chromosome 5 abnormalities, and 123 (6%) had a complex karyotype. The most commonly mutated genes were: TET2 (28%), ASXL1 (22%), SF3B1 (22%), SRSF2 (19), JAK2 (11%), DNMT3A (9%), RUNX1 (9%), and U2AF1 (6%) SF3B1 mutations were associated with normal karyotype (NK), age &amp;lt;65 years, ANC &amp;gt;1 k/dL, platelets (plts) &amp;gt;50 k/dL, marrow blasts (MB) &amp;lt;10% and hemoglobin (hb) &amp;lt;8 g/dL. TP53 mutations were associated with complex karyotype, chro 5, 7, or 17 abnormalities. Clinical characteristics were also associated with specific genomic alterations (Figure 1). For example, NK correlated with the presence of SF3B1, ZRSR2, DNMT3A, a higher number of mutations, and absence of TP53, ASXL1, or KRAS; chromosome 5, 7, and 17 abnormalities were associated with a lower mutation number and the presence of TP53 mutations; complex karyotype correlated with the absence of TET2 and SF3B1 and the presence of TP53; age &amp;lt; 65 was associated with the presence of NRAS and JAK2 mutations and the absence of TET2, SF3B1, and SRSF2 mutations; hemoglobin &amp;lt; 8 g/dL positively correlated with mutation number and SF3B1 mutations and negatively correlated with TET2 mutations; ANC &amp;lt; 1 negatively correlated with JAK2, SF3B1, and DNMT3A mutations; platelets &amp;lt; 50k/dL negatively correlated with SF3B1 and JAK2 mutations, and positively correlated with the number of mutations; and MB &amp;lt;10% positively correlated with SF3B1 mutations and negatively correlated with number of mutations and ASXL1, RUNX1, TP53, and STAG2 mutations (Figure 1). Conclusions We applied machine learning techniques to reveal the complex relationships between mutational data and the clinical characteristics of several myeloid malignancies using a large, international patient cohort. In addition to correctly identifying previously described genotype-phenotype relationships, we identified several other intriguing relationships such as the relationship of particular mutations to the development of different cytopenias, demonstrating the potential utility for machine learning approaches in interrogating genomic data. Disclosures Sekeres: BMS: Consultancy; Pfizer: Consultancy; Takeda/Millenium: Consultancy. Gerds:Gilead Sciences: Research Funding; Imago Biosciences: Research Funding; CTI Biopharma: Consultancy, Research Funding; Pfizer: Research Funding; Sierra Oncology: Research Funding; AstraZeneca/MedImmune: Consultancy; Incyte Corporation: Consultancy, Research Funding; Apexx Oncology: Consultancy; Celgene: Consultancy, Research Funding; Roche/Genentech: Research Funding. Mukherjee:Aplastic Anemia and MDS International Foundation: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squib: Honoraria; Partnership for Health Analytic Research, LLC (PHAR, LLC): Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; EUSA Pharma: Consultancy; Celgene/Acceleron: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria. Nazha:Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Jazz: Research Funding; Incyte: Speakers Bureau.

Spectrum of myelodysplastic syndrome in patients evaluated for cytopenia(s). A Report from a Reference Centre in Saudi Arabia

Background/ObjectiveMyelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells, characterized by ineffective hematopoiesis, peripheral cytopenias along with hypercellularity of the bone marrow, and marked dysplastic features. Establishing MDS diagnosis is difficult due to nonspecific clinical presentation and imprecise morphological criteria. In anticipation to improve the diagnostic approach in this field, we aimed to characterize the clinical and morphological features of patients presented with cytopenias with a special focus on MDS. MethodsWe comprehensively reviewed all medical record of patients who were referred to the hematology laboratory at KFSH-RC, Riyadh, Saudi Arabia, between January 2009 and March 2016 for evaluation of bone marrow aspirates and trephine biopsies due to severe and persistent cytopenia(s) to rule out MDS. ResultsA total of 183 patients, 155 adult and 28 pediatric, were identified. In the adult group, MDS was diagnosed in 82 (52.9%) patients, with a male-to-female (M:F) ratio of 1.6:1 and mean age at diagnosis of 50 years. According to the World Health Organization (WHO) 2017 criteria, MDS subtypes were as follows: MDS with single lineage dysplasia (SLD, 5%), MDS with ring sideroblasts and SLD (MDS-RS-SLD 7%), MDS with multilineage dysplasia (MDS-MLD 21%), MDS with deletion of chromosome 5q (MDS del(5q), 2%), MDS unclassifiable (MDS-U7%), hypoplastic MDS (h-MDS 4%), MDS with excess blasts-1 (MDS-EB1, 20%), MDS with excess blasts-2 (MDS- EB2, 28%), and therapy-related MDS (6%). Laboratory and morphological features were described. In both groups, cytogenetic abnormalities were classified according to the Revised International Prognostic Scoring System cytogenetic risk groups. In adults, the dominating cytogenetic abnormalities were monosomy 5 and monosomy 7 seen in 20.7% and 24.4% of patients, respectively. Peripheral cytopenia not due to MDS was diagnosed in 54 (34.8%) patients, with a mean age of 43 years and M:F ratio of 1:1. The cause of these cytopenias were as follows: bone marrow failure (BMF, 22%), peripheral destruction (20%), drug induced (20%), anemia of chronic disease (16%), B12 deficiency (7%), infection (7%), paroxysmal nocturnal hemoglobinuria (4%), idiopathic cytopenia of undetermined significance (2%), and idiopathic dysplasia of undetermined significance (2%). A definite diagnosis of MDS was not possible in 19 patients due to insufficient clinical data. In the pediatric group, MDS was diagnosed in 14/28 (50%) patients, with M:F ratio of 1.8:1 and mean age at diagnosis of 4 years. MDS subtypes (WHO 2017) in 14 patients were as follows: refractory cytopenia of childhood (RCC, 42.8%), MDS-EB1 (42.8%), and MDS-EB2 (14.2%). Laboratory and morphological features were described. The prevalent cytogenetic abnormality was monosomy 7 in six/14 (42.8%) patients. Cytopenias due to other causes were diagnosed in eight/28 patients (28.5%), with a mean age of 6.5 years and M:F ratio of 1.6:1. The causes of non-MDS related cytopenia were: congenital BMF (4 patients), peripheral destruction (2 patients), immune deficiency (1 patient), and viral infection (1 patient). A definite diagnosis of MDS could not be made in six/28 (21.4%) patients. ConclusionMDS is the cause of cytopenia in a significant number of patients referred for evaluation of cytopenias, appears at younger age, and tends to be more aggressive than that reported in international studies. Anemia, dysplastic neutrophils in the peripheral blood, and dysplastic megakaryocytes in the bone marrow trephine biopsy are the most reliable features in distinguishing MDS from other alternative diagnoses.

Single-Nucleotide Polymorphism Array Technique Generating Valuable Risk-Stratification Information for Patients With Myelodysplastic Syndromes.

Background: Chromosomal abnormalities play an important role in the diagnosis and prognosis of patients with myelodysplastic syndromes (MDSs). The single-nucleotide polymorphism array (SNP-A) technique has gained popularity due to its improved resolution compared to that of metaphase cytogenetic (MC) analysis.Methods: A total of 376 individuals were recruited from two medical centers in China, including 350 patients and 26 healthy individuals. Among these patients, 200 were diagnosed with de novo MDS, 25 with myeloproliferative neoplasm (MPN), 63 with primary acute myeloid leukemia (AML), and 62 with idiopathic cytopenia of undetermined significance (ICUS). We evaluated the significance of abnormal chromosomes detected by SNP-A in the diagnosis and prognosis of MDS-related disorders.Results: (1) When certain chromosomal abnormalities could not be detected by conventional MC methods, these abnormalities could be detected more efficiently by the SNP-A method. With SNP-A, the detection rates of submicroscopic or cryptic aberrations in the MDS, MPN, and AML patients with normal MC findings were 32.8, 30.8, and 30%, respectively. (2) The chromosomal abnormalities detected by SNP-A had a very important value for the prognosis of patients with MDSs, especially in the low-risk group. The survival of patients with abnormal chromosomes detected by SNP-A was significantly lower than that of patients with no detected chromosomal abnormalities; this difference was observed in overall survival (OS) (P = 0.001) and progression-free survival (PFS) [24 months vs. not reach (NR); P = 0.008]. The patients with multiple chromosomal abnormalities detected by SNP-A had an inferior prognosis, and SNP-A abnormalities (≥3 per patient) were found to be an independent predictor of poor prognosis in patients with MDSs [hazard ratio (HR) = 2.40, P = 0.002]. (3) Patients with ICUS may progress to myeloid malignancies, but most patients often maintain a stable ICUS status for many years without progression. An ICUS patient found to have an MDS-related karyotype would be rediagnosed with MDS. SNP-A can efficiently detect chromosomal abnormalities, which would be important for assessing the evolution of ICUS. In our study, 17 ICUS patients with SNP-A-detected abnormalities developed typical MDSs.Conclusions: SNP-A can help evaluate the prognosis of patients with MDSs and better assess the risk of disease progression for patients with ICUS.

Roles of immune responses in the pathogenesis of immunorelated pancytopenia.

Some patients with pancytopenia do not conform to any diagnostic criteria of known haematological or non-haematological diseases; however, they respond well to corticosteroid, high-dose intravenous immunoglobulin and rituximab treatment. This abnormality is termed immunorelated pancytopenia (IRP). Later studies indicated that IRP might be a kind of autoimmune disease in which T helper (Th) type 2 cell function is enhanced, resulting in the hyperfunction of B lymphocytes, which then produce excess autoantibodies that attack the bone marrow (BM) and cause cytopenia. Hypofunction of regulatory T (Treg) cells and enhanced Th17 cell function, an elevated percentage of plasmacytoid dendritic cells (pDCs) and a decreased percentage of natural killer (NK) cells help to promote the process. Moreover, increased expression of a synergistic stimulator of B lymphocytes, CD70 and the reactive overexpression of the BCR inhibitory coreceptor CD22 also support this claim. Candidate autoantigens targeted by autoantibodies on haematopoietic cell membranes have also been reported in IRP. This review is focused on studies that demonstrate the role of immune responses in the pathogenesis of IRP. Current diagnostic criteria and treatments for IRP are also referenced to provide a thorough understanding. Distinguishing IRP from idiopathic cytopenias of undetermined significance (ICUS) and other haematological disorders, for example myelodysplastic syndrome (MDS), aplastic anaemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and Evans syndrome, may help patients with pancytopenia benefit from proper treatment. Further studies are required to achieve new insight into the pathophysiology of IRP with regard to the immune system, which will be instrumental for the development of novel therapies for inhibiting disease initiation and/or progression.

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

BackgroundWe 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.MethodsTwenty‐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.ResultsThis 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.ConclusionsThese 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.