Low-grade Myelodysplastic Syndromes Research Articles

Abstract 5853: (R)-2-hydroxyglutarate inhibits KDM5 histone lysine demethylases to drive tumorigenesis in IDH-mutant cancers

Abstract Gain-of-function mutations in isocitrate dehydrogenase enzymes IDH1 and IDH2 occur in ∼10% of acute myeloid leukemias (AML) and >80% of gliomas. The mutant enzymes convert 2-oxoglutarate (2OG) to the oncometabolite R-2-hydroxyglutarate (R-2HG). R-2HG promotes cellular transformation by modulating the activities of 2OG-dependent dioxygenases (2OGDDs). The only functionally validated direct target of R-2HG is TET2, a 2OGDD myeloid tumor suppressor that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Interestingly, in clonal myeloid disorders the patterns of IDH and TET2 mutations are vastly different. TET2 mutations occur at similar frequencies in both low- and high-grade disease. IDH mutations, on the other hand, are associated with higher-grade and blast-phase MPN and MDS and with de novo AML and are rare in CHIP and low-grade MDS. This suggests that mutant IDH promotes a more aggressive disease phenotype and that R-2HG has additional targets other than TET2 that contribute to its leukemogenic activity. To identify other 2OGDD myeloid tumor suppressors that could be contributing to R-2HG-mediated transformation, we performed a positive-selection CRISPR-Cas9 screen under cytokine-poor conditions in TF-1 cells. We identified three H3K4 histone lysine demethylases, KDM5A, KDM5C and KDM5D, as genes whose sgRNAs were enriched upon cytokine withdrawal. Triple knockout of KDM5A, KDM5C and KDM5D (TKO) in TF-1 cells induces robust cytokine independence as does treatment of TF-1 cells with KDM5c70, a KDM5 inhibitor. We further found that R-2HG is a more potent inhibitor of KDM5A, KDM5C and KDM5D than of TET2. We then assessed the effect of mutant IDH1 expression, TKO, R-2HG treatment and KDM5c70 treatment on H3K4 trimethylation by ChIP-seq and found that each of these perturbations results in a significant enrichment in H3K4me3 peaks relative to controls, with the same sites being enriched. We then asked if mutant IDH positivity is associated with increased levels of H3K4 methylation in glioma. We performed ChIP-seq on a panel of IDH wild-type and IDH mutant glioma MCTS lines and found H3K4me peaks to be highly enriched in the IDH mutant lines when compared to IDH wild-type lines. Trimethyl-H3K4 levels were likewise increased in isogenic normal human astrocyte (NHA) cells ectopically expressing mutant IDH1. Expression of mutant IDH or treatment KDM5 inhibitor was sufficient to cause increased colony formation in NHA cells relative to controls. Collectively, these data suggest that R-2HG inhibits KDM5 histone lysine demethylases to promote mutant IDH-mediated transformation in AML and glioma. These studies identify a novel direct target of R-2HG in IDH mutant tumors and provide a functional link between IDH mutations and dysregulated histone lysine methylation in cancer. Citation Format: Kathryn E. Gunn, Matti Myllykoski, John Cao, Bofu Huang, Betty Rouaisnel, Bill Diplas, Michael M. Levitt, Ryan Looper, John G. Doench, Keith L. Ligon, Harley I. Kornblum, Hai Yan, Samuel McBrayer, Lucy A. Godley, Peppi Koivunen, Julie-Aurore Losman. (R)-2-hydroxyglutarate inhibits KDM5 histone lysine demethylases to drive tumorigenesis in IDH-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5853.

Morphologic, immunophenotypic, and molecular genetic comparison study in patients with clonal cytopenia of undetermined significance, myelodysplastic syndrome, and acute myeloid leukemia with myelodysplasia-related changes: A single institution experience.

Next-generation sequencing (NGS) analysis showed clonal cytopenia of undetermined significance (CCUS) as an immediate precursor to myelodysplastic syndrome (MDS). We evaluated and compared morphologic, multiparametric flow cytometry (MFC), and molecular genetic findings in patients with CCUS (n=37), MDS (n=75), and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC, n=24). CCUS patients showed variable MFC abnormalities including >2% CD34+ myeloblasts (5.8%), altered antigen expression on myeloblasts, monocytes, and granulocytes (1.2, 1.5, and 0.2/case), abnormal maturation of myeloblasts (45.8%), decreased hematogones (17.6%), and decreased side scatter (SSC) of granulocytes (11.4%). CCUS patients with high-risk mutations showed significantly more MFC abnormalities. However, CCUS patients with >20% variant allelic fraction (VAF) did not show more MFC aberrations than the rest of the group. MDS patients showed significantly more MFC abnormalities compared with CCUS patients (p=7.8E-05-0.047). Low-grade MDS patients showed significantly fewer MFC abnormalities compared with high-grade MDS or AML-MRC patients (p=1.89E-05-0.04). AML-MRC patients showed significantly elevated blast counts, more antigen aberrations, decreased hematogones, and decreased SSC of granulocytes compared with CCUS patients (p=2.0E-05-0.01). CCUS patients carried predominantly TET2/DNMT3A/ASXL1 mutations. They harbored fewer mutations in gene coding splicing factors compared with MDS patients (p=.0001-.02) and fewer mutations in tumor suppressor and transcription factor genes compared with AML-MRC patients (p=.0006-.02). CCUS is an immediate precursor to low-grade MDS. The progression from CCUS to MDS to AML-MRC is a stepwise process that requires acquisition of mutations in splicing, transcription factor, and tumor suppressor genes with accumulations of additional MFC abnormalities.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Integration of the Ki-67 proliferation index into the Ogata score improves its diagnostic sensitivity for low-grade myelodysplastic syndromes

BackgroundAlthough flow cytometric detection of myelodysplastic syndrome (MDS) with the Ogata score has a high specificity, its sensitivity for low-grade MDS is low. Additional markers are needed to improve its diagnostic reliability. Therefore, we investigated the diagnostic performance of the Ki-67 proliferation index in bone marrow (BM) cell populations for detection of MDS. MethodsBM aspirates from 50 MDS patients and 20 non-clonal cytopenic controls were analyzed with flow cytometry to determine the Ogata score and the Ki-67 proliferation indices in different cell populations. ResultsKi-67 proliferation indices alone could be used to detect MDS with a sensitivity of up to 80 % and specificity of up to 70 %. Combining the Ogata score with the Ki-67 proliferation index of erythroid cells significantly improved its sensitivity for detection of MDS from 66 % to 90 %, while maintaining a specificity of 100 %. Particularly, the sensitivity for detection of low-grade MDS improved from 56 % to 91 %. ConclusionsThis is the first study using Ki-67 proliferation indices to detect MDS and shows their particularly high diagnostic sensitivity for detection of low-grade MDS. Integration of the Ki-67 proliferation index of erythroid cells into the Ogata score significantly improved its sensitivity without loss of the high specificity.

Topographic Analysis of Low-Grade Myeloid Neoplasms By Multiparametric in Situ Imaging of Human Bone Marrow Core Biopsy Tissues

Diagnosis of low-grade myelodysplastic syndromes (LG-MDS) is one of the most challenging in hematopathology as it relies predominantly on morphologic assessment of dysplasia. Prior studies have demonstrated poor interobserver agreement among pathologists. Histomorphological evaluation of bone marrow core biopsy samples remains the gold standard for diagnostic workup of LG-MDS, including myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, this approach may be subjective, and cannot quantitatively assess subtle differences in marrow topography and the cellular microenvironment. Multiparametric in situ imaging (MISI) through various techniques enables multiple biomarker detection in a single tissue. BostonGene has developed an AI-based image analysis platform to reveal spatial information and subtle histomorphologic features in an objective, quantitative fashion. Here, we demonstrate the potential for automated AI-based imaging analysis of MISI to assist in the differentiation of LG-MDS samples from normal marrow tissues (NBM).Decalcified human bone marrow core biopsy tissues from LG-MDS (n=6) and uninvolved staging marrows (NBM, n=4) were first prepared by immunofluorescence-based MISI via staining with DAPI and CD34, CD38, CD117, CD71, CD15, and CD61 antibodies. BostonGene analyzed the resulting images (fig.1) using a proprietary AI-based imaging platform to identify cells by segmentation performed with the pre-trained instance segmentation neural network. Cell types were identified with mean marker expression values using an accelerated version of BostonGene's phenograph clustering algorithm. Pathologists manually masked fat and bone trabeculae.Using a combination of cell size/shape parameters and antigen expression levels, the following unique cell types were identified: myeloblasts, proerythroblasts, erythroid normoblasts, maturing granulocytes, megakaryocytes, mast cells, plasma cells, and B-cell precursors (hematogones). Data revealed differences in the cellular content of NBM and LG-MDS samples, and separation of LG-MDS samples with the del(5q) subtype (n=2). While linear slender islet-like small clusters of erythroid normoblasts were detected in NBM, we observed a chaotic arrangement of them in LG-MDS samples. In LG-MDS samples, we found an increase in the total number of erythroid normoblasts from 17% to 31%, LG-MDS-del(5q) had 14%.The ratio of maturing granulocytes to erythroid normoblasts (M:E ratio) was significantly lower in LG-MDS (0.63) than in NBM (1.95). The M:E ratio generated by MISI strongly correlated to the M:E ratio produced by manual differential count of bone marrow aspirate samples (R=0.83, p < 0.003). Additionally, fewer hematogones were identified in LG-MDS marrows compared to NBM samples, as reported by others using orthogonal methods.Topographic analysis showed the fat to cellular tissue area ratio was higher in NBM (0.73) than LG-MDS (0.41), but the ratio of trabecular area to total tissue area was higher in LG-MDS (1.67) than NMB (0.74). Spatially, myeloblasts and megakaryocytes were found closer to trabeculae in NBM than LG-MDS;12 different cell communities were identified;2 of them (cluster 3 - erythroid normoblasts enriched, cluster 5 - erythroid normoblasts contacting proerythroblasts) were distributed statistically significantly differently in NBM and LG-MDS samples, indicating the use of MISI with AI-based imaging to distinguish LG-MDS from NBM. Patients of MDS-del(5q) subtype differ significantly from other MDS samples and are more similar to NBM.AI-based image analysis applied to MISI of bone marrow tissue revealed multiple cell types in single tissue sections, along with histologically subtle differences in topography between NBM and LG-MDS samples. These results highlight the importance of integrating in situ tissue analysis with techniques that examine single cell characteristics for a more comprehensive picture of the differences between normal tissue and tumor samples. Coupling sophisticated imaging analytics with this imaging method may provide a more powerful tool for novel biomarker discovery of prognostic and therapeutic significance in the management of MDS and other marrow-based disorders. [Display omitted] DisclosuresSvekolkin: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Varlamova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Galkin: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Akaeva: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Smirnova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Ovcharov: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Polyakova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Tabakov: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Postovalova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Koltakova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Gunn: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Bagaev: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene.

Compromised Host Stem Cell Competitiveness Affords Fanconi Stem Cell Engraftment in C-Kit Mutant Humanized Mice

Humanized mouse model is a powerful tool for the study of human hematopoiesis and hematologic diseases in vivo. However, existing models continue to be limited by absent engraftment of stem cells with inherent growth defects, especially in bone marrow failure (BMF) disorders such as low-grade myelodysplastic syndromes (MDS) and Fanconi Anemia (FA). Previously, we presented a highly efficient MDS/AML PDX model in cytokine-humanized MISTRG mice, that express human M-CSF, IL-3, GM-CSF and THPO in the background of the SIRPα - humanized rag -/- IL2Rg -/- mice. MISTRG mice support efficient, faithful and long-term MDS hematopoietic stem and progenitor cell (HSPC) engraftment with multi-lineage representation. Similar to MDS, FA stem cells are defective and sensitive to toxic stress conferred by inflammation or irradiation. Irradiation, required in conventional mice as pre-conditioning, increases the risk of secondary malignancies, induces activation of inflammatory pathways and may damage the bone marrow microenvironment.In this study, we present MISTRG6kit W41 mice that in addition to the above cytokines express human IL-6, which improves lymphoid development, and that were CRISPR modified to introduce the white spotting 41 mutation in c-Kit (c-kit W41), which results in partial loss of c-kit function in the murine host stem and progenitor cells. To characterize this mouse model, we compare the engraftment capacity between MISTRG6 and MISTRG6kit W41 mice. We find that the HSPCs from both healthy donor and low grade MDS patient can be more efficiently propagated in MISTRG6Kit W41 mice and that they can be serially transplanted. Human cells are detectable at higher levels in PB of MISTRG6Kit W41 mice as early as 8 weeks post transplantation. As previously described, c-kit mutation enhances human erythroid engraftment while multi-lineage engraftment is maintained in engrafted MISTRG6Kit W41 mice. Therefore, MISTRG6Kit W41 mice offer a better host environment that supports human hematopoietic expansion without irradiation.FA is an inherited genetic disease carrying FA gene mutations associated with DNA damage and cancer predisposition and marked by BMF, MDS and AML. FA HSPCs cannot be expanded or grown for sufficient periods in vitro, and existing mouse models have failed to demonstrate engraftment of primary cells from FA patient. We transplanted a MDS sample from a FANCC mutant patient into MISTRG6Kit W41 mice giving rise to a myeloid predominant graft that could be successfully serially transplanted. Moreover, non-transformed FA HSPCs from 2 FANCA and 2 FANCG mutant patients were successfully engrafted in MISTRG6Kit W41 mice.In summary, MISTRG mice with human IL-6 expression and c-Kit W41 mutation provide a more efficient and convenient way for pre-clinical studies of BMFs such as FA and low-grade MDS with maintenance of an intact humanized stem cell niche, and this is the first time we saw primary cells from FA patient propagate in mouse model. We believe that our model would be highly beneficial to the study of additional diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita or Schwachman-Diamond syndrome. DisclosuresNo relevant conflicts of interest to declare.

Clinical Utility of Hematopathologist-Triaged NGS Testing When Investigating Patients with Suspected MDS

The recognition of MDS is challenging in early stages, where diagnosis may rely solely upon morphological criteria for dysplasia, a non-specific finding prone to inter-observer variation. Patients with equivocal bone marrow (BM) findings may be discharged from Hematology clinics and lost to follow up, or subjected to serial, invasive BM investigations and diagnostic delays. We therefore aimed to demonstrate the importance of hematopathologist-triaged, targeted NGS in identifying clonal cytopenias of undetermined significance (CCUS) in cases where MDS diagnostic criteria are not met based on morphology or cytogenetic analysis. We explored this using three REB-approved cohorts.Our first cohort was retrospective with BM samples ranging from 2010-14, involving cases that were previously suspicious for but non-MDS diagnostic. This included 70 patients from Sunnybrook (SHSC) and Kingston Health Sciences Centres (KHSC): 16 age-matched controls (8 negative lymphoma staging, 8 non-MDS cytopenias); 18 suspicious for MDS; 20 MDS; and 16 MDS/MPN. DNA was extracted and NGS was performed using our custom 48-gene Ion Torrent AmpliSeq myeloid panel (ThermoFisher).We identified suspected mutations in 2/16 (13%) controls (i.e. CHIP), 12/18 (67%) suspicious cases, 17/20 (85%) MDS cases, and 16/16 (100%) MDS/MPN cases. The mean and median number of mutations per suspicious patient (respectively 0.89 and 1; most commonly in SF3B1, TET2, RUNX1, and ASXL1) were lower than MDS (1.85 and 2; p=0.011) and MDS/MPN (3.13 and 3; p<0.0001). There was a significant difference in the average variant allele frequency (VAF) per patient (those with ≥1 mutation) between control and suspicious groups (p=0.022), however, there were no significant differences in the average VAF between suspicious, MDS, and MDS/MPN cases. Furthermore, of the 16 patients with BM suspicious for MDS, 7 went on to get MDS. 4 of these patients had at least 1 clinically relevant somatic variant, while 3 had none. Of those with at least 1 variant, 3 had IPSS-level cytopenias at the time, indicating that had their mutational status been known at the time of their assessment, they would have been diagnosed with the provisional CCUS entity (while the rest would be classified as CHIP).To supplement these findings, we are amassing a prospective cohort involving cases at SHSC where patients have either idiopathic cytopenias (ICUS), or confirmed MDS diagnoses with one or more previously non-diagnostic BM. To date, we have performed sequencing for 36 of these patients, including 23 ICUS and 13 diagnosed MDS cases. Of the ICUS cases, 10 (44%) had at least 1 variant (mean # variant/patient = 1, mean variant allele frequency (VAF) = 34.0%) consistent with CCUS, while 12/13 (92%) of MDS patients had at least 1 variant (mean # variants/patient = 2, mean VAF = 42.3%). These findings are consistent with CCUS being common in suspicious MDS cases, with similar clonal size but lesser mutational burden than diagnosed MDS. In addition to these preliminary findings, 15/36 patients have serial samples that we are currently processing for NGS (among other cases we are accruing to present at the ASH meeting). By exploring serial cases with molecular results pre- and post- MDS diagnosis, we aim to further elucidate which features of CCUS may predict progression to MDS.Finally, we assessed clonality in cases suspicious for myeloid malignancy in our existing prospective myeloid NGS cohort at KHSC (Ferrone et al, JMD 2021). In this cohort of 168 patients, when focusing on cytopenias yet to be diagnosed, 71 patients had suspected MDS, MPN, or MDS/MPN prior to NGS (completed using the Oncomine Myeloid Assay; ThermoFisher). 36/71 (51%) were found to have variants that indicate clonality. This facilitated diagnoses of either myeloid malignancies or pre-malignant states, with nine cases in total of ICUS resulting in the identification of variants that were non-diagnostic of MDS (mainly in TET2), but indicative of CHIP (n=2) or CCUS (n=7). Furthermore, for the limited number with available follow up data, we found no significant difference in survival between individuals with low-grade MDS (n=10) and CCUS (n=6) (p=0.457).This evidence is in keeping with recent findings that the clinical features of CCUS may be consistent with low-risk MDS, emphasizing the importance of closely monitoring these patients, and even the possibility of assessing and treating them similarly to those with low-risk MDS. DisclosuresNo relevant conflicts of interest to declare.

Antibody Response to Vaccination with BNT162b2, mRNA-1273, and ChADOx1 in Patients with Myeloid and Lymphoid Neoplasms

Background:In general, patients with hematological diseases are predisposed to develop infections. Severe COVID-19 infection associated with high mortality is more likely in these patient cohorts compared to the general population. Due to immune defects related to the primary disease and/or to immunosuppressive treatment regimes, vaccination efficacy may be reduced in patients with hematological diseases. So far, data on this area are limited.Aim:To evaluate vaccination-related antibody response to BNT162b2, mRNA-1273, and ChADOx1 in patients with hematological disorders.Patients and methods:In this interim analysis of a prospective, observational single-center study, we report antibody levels at least 2 weeks after COVID-19 vaccination. A FDA/CE approved electrochemiluminescent assay (ECLIA) (Elecsys®, Roche, Mannheim, Germany) was used to quantify antibodies, pan Ig (including IgG) against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The assay has a measurement range of 0.4 to 250 U/mL, with a concentration ≥0.8 U/ml considered as positive. Data were analyzed for patients without detection of anti-N (nucleocapsid) SARS-CoV-2 antibody (i.e., without having passed SARS-CoV-2 infection). All tests were performed according to the manufacturer's instructions in an accredited laboratory at the University Hospital Mannheim.Results:Between February 2021 and July 2021, a total of 175 patients with hematological diseases were included in this study. The median age was 66 years (range 21-90 years), and 81 (46.3%) were female. The antibody levels were measured at least 14 days (median, 58 days) after the 2 nd vaccination. The patients were vaccinated with BNT162b2 (BioNTech, n=134), mRNA-1273 (Moderna, n=19), ChADOx1 (AstraZeneca, n=12), or got the first vaccination with BNT162b2 and the second with ChADOx1 (n=10). Overall, 145/175 (82.9%) were diagnosed with a malignant hematological disease (myeloid neoplasms, n=108; lymphoid neoplasms, n=37) and 30/175 with a non-malignant hematological disease (autoimmune disease, n=24; benign, n=6). 124 patients (70.1%) were on active therapy, and 51 patients (29.1%) were previously treated or treatment naïve. Correlation to specific therapies is ongoing and will be presented.In general, vaccination-related antibody response was positive (≥0.8 U/mL) in 148/175 (84.6%) patients with a median level of 208.6 U/mL (range 0.8-250.00) and negative (<0.8 U/mL) in 27/175 (15.4%) patients. The distribution of the negative cohort regarding the disease subgroups were as followed: myeloid neoplasms 7/27 (25.9%), lymphoid neoplasms 16/27 (59.3%), non-malignant hematological disease 4/27 (14.8%). Within the negative cohort, 21/27 (77.8%) were treated on active therapy, 6/27 (22.2%) were previously treated or treatment naïve.In myeloid neoplasms, patients with classical myeloproliferative neoplasm (MPN) had the highest negative result for antibodies with 4/7 (57.1%) followed by myelodysplastic syndrome (MDS) 2/7 (28.6%). Interestingly, all patients with chronic myeloid leukemia (CML) had a measurable immune response.In lymphoid neoplasms, patients with low-grade non-hodgkin lymphoma (NHL) (predominately chronic lymphocytic leukemia, CLL) had the highest negative antibody result 13/16 (81.3%) followed by high-grade NHL 4/8 (50%; predominately diffuse large b-cell lymphoma, DLBCL).In non-malignant hematological diseases, only patients with autoimmune diseases had a negative result.Conclusion:A remarkable group of patients with hematological disease were measured with no or low immune response after 2 nd COVID-vaccination, especially those with low-grade NHL, MDS and autoimmune disease. It seems that the percentage of patients with MPN and low response is less critical. No problems appeared in CML patients.Further explorations are needed with focus on potential risk of COVID infections despite full vaccination: The potential of 3 rd booster vaccination should be explored within clinical trials. DisclosuresReiter: AOP Orphan Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Abbvie: Membership on an entity's Board of Directors or advisory committees; Deciphera: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses. Kreil: Novartis: Research Funding. Hofmann: Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Jawhar: Takeda: Honoraria, Other: Travel support; Blueprint Medicines: Honoraria; Stemline: Consultancy, Honoraria; Celgene: Other: Travel support; Novartis: Consultancy, Honoraria, Other: Travel support, Speakers Bureau. Saussele: Roche: Honoraria; Pfizer: Honoraria; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

DIAGNOSTIC APPLICATION AND CLINICAL SIGNIFICANCE OF FCM WELLS SCORING SYSTEM IN MYELODYSPLASTIC SYNDROMES

Myelodysplastic syndromes (MDS) are group of clonal diseases of the hematopoietic system characterized by ineffective hematopoiesis, dysmyelopoiesis, a high frequency karyotype abnormalities and the risk of transformation into acute leukemias. Cytopenic and dysplastic changes are not pathognomonic for MDS, and there are many diseases that can imitate MDS. According to various sources, clonal karyotypic abnormalities are present only in 20-60% of MDS. The diagnosis of MDS is not difficult if blasts or sideroblasts are present in the bone marrow, or there are chromosomal aberrations as evidence of clonal hematopoiesis. The diagnostic problem arises in cases of MDS without sideroblasts, with normal karyotype and/or bone marrow hypoplasia. Since 2012, the ELNet Working Group has proposed and subsequently supplemented guidelines for Flow Cytometry as a complementary diagnostic tool. The aim of the study was to compare the results of the FCM Wells score MDS with the results of the İPSS-R score MDS The study included 30 patients initially diagnosed with MDS . The classification was carried out according to the WHO Classification of MDS 2016: MDS SLD-6 (20%), MDS-MLD-5 (16.7%), MDS RS-MLD-2 (6.7%), MDS-EB1-9 (30%), MDS EB2-8(27%). According to the IPPS-R, patients were scored based on blasts, cytogenetic examination, hemoglobin/platelet/absolute neutrophil count and scored as very-low, low, intermediate, high, very-high. Using the Wells evaluation criteria, which takes into account cytometric analysis of the cells of the main myelopoiesis lines, changes were found in the compartment of granulocytes in 93%, monocytes in 40% and erythrocytes in 73% of cases. High scores on the Wells scale (> 4) were obtained in 89% of (8/9) MDS-EB1, 100%(8/8) MDS-EB2, 80% (4/5) MDS MLD patients, 17% (1/6) MDS –SLD, 50%(1/2) MDS RS-MLD. According to IPPS-R, MDS patients received a score <1.5 very low risk group include 50%(3/6) MDS –SLD, 20%(1/5) MDS-MLD, score > 1.5-3 - Low risk group include MDS –SLD 50%(3/ 6), MDS-MLD-80% (4/5), MDS RS-MLD 50% (1/2), MDS-EB1-78%(7/ 9), score > 3- 4.5-intermediate risk qroup got MDS-EB1 22%(2/ 9), MDS EB2- 25% (2/8), MDS RS-MLD- 50%(1/2), Score > 4.5 respectively high risk group got patients MDS –SLD- 17%(1/6), MDS EB2- 50%(4/8), Score > 6 very high risk group got MDS EB2- 25%(2/8). The Pearson's correlation coefficient (PCC) showed high correlation between İPSS-R and FCM Wells score was 0.83, p<0.002. In our study, the FCM score had a positive correlation with the IPSS-R prediction. Expanded analysis of the main compartments of the bone marrow (early precursors of myelopoiesis, the population of granulocytes and monocytes, erythrocytes) using the Wells scale as an additional tool improves the diagnosis and distinguish low-grade MDS from non-clonal cytopenias.

Programmed necroptosis is upregulated in low-grade myelodysplastic syndromes and may play a role in the pathogenesis

Myelodysplastic syndrome (MDS) is characterized by persistent cytopenias and evidence of morphologic dysplasia in the bone marrow (BM). Excessive hematopoietic programmed cell death (PCD) and inflammation have been observed in the bone marrow of patients with MDS, and are thought to play a significant role in the pathogenesis of the disease. Necroptosis is a major pathway of PCD that incites inflammation; however, the role of necroptosis in human MDS has not been extensively investigated. To assess PCD status in newly diagnosed MDS, we performed immunofluorescence staining with computational image analysis of formalin-fixed, paraffin-embedded BM core biopsies using cleaved caspase-3 (apoptosis marker) and necroptosis markers (receptor-interacting serine/threonine-protein kinase 1 [RIPK1], phospho-mixed lineage kinase domain-like protein [pMLKL]). Patients with MDS, but not controls without MDS or patients with de novo acute myeloid leukemia, had significantly increased expression of RIPK1 and pMLKL but not cleaved caspase-3, which was most evident in morphologically low-grade MDS (<5% BM blasts) and in MDS with low International Prognostic Scoring System risk score. RIPK1 expression highly correlated with the distribution of CD71+ erythroid precursors but not with CD34+ blast cells. We found that necroptosis is upregulated in early/low-grade MDS relative to control participants, warranting further study to define the role of necroptosis in the pathogenesis of MDS and as a potential biomarker for the diagnosis of low-grade MDS.

Flow Cytometric Findings in Clonal Cytopenia of Undetermined Significance.

To examine flow cytometric (FCM) findings in clonal cytopenia of undetermined significance (CCUS) in relation to variant allele fraction (VAF) and mutation risk. Nine FCM parameters, including 5 FCM metrics (Meyerson-Alayed scoring scheme [MASS] parameters) we previously used to identify myelodysplastic syndromes (MDS), were compared among 96 CCUS samples, 100 low-grade MDS samples and 100 samples from patients without somatic alterations (controls). FCM findings did not differ between CCUS samples with less than 20% VAF and controls. CCUS samples with more than 20% VAF (CCUS >20% VAF) demonstrated more than 1 abnormal FCM parameter at a frequency between MDS and controls. Abnormalities in CCUS with high-risk alterations (CCUS(hi)) were similar to MDS, with no statistical difference in the percentage of cases with more than 1 FCM abnormality or a positive MASS score. The positive predictive value (PPV) for clinically significant myeloid processes; MDS, CCUS(hi), and CCUS >20% VAF compared with other CCUS samples and controls was 94.8%, with 96.5% specificity and 61% sensitivity using a modified MASS score. A subset of MDS (43%) was distinguished from CCUS(hi) and CCUS >20% VAF using 3 parameters, with a 93.5% PPV and 83.3% specificity. FCM abnormalities can distinguish high-risk CCUS based on VAF or alteration type from low-risk CCUS and MDS in many cases. The findings are of potential utility in the evaluation of patients with cytopenias.

The utility of a myeloid mutation panel for the diagnosis of myelodysplastic syndrome and myelodysplastic/myeloproliferative neoplasm.

The diagnosis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) is based on morphology and cytogenetics/FISH findings per 2017 WHO classification. With rare exceptions, somatic mutations have not been incorporated as the diagnostic criteria. We analyzed the utility of mutational analysis with a targeted 54-gene or 40-gene next-generation sequencing (NGS) panel in the diagnosis of MDS and MDS/MPN. We retrospectively collected92 patientswho presented withunexplained cytopenia with or withoutcytosis, including 32 low-grade MDS (MDS-L), 18 high-grade MDS (MDS-H), 5 therapy-related MDS (MDS-TR), 19 MDS/MPN, and 18 negative cases. Of 92 patients, 197 somatic mutations involving 38 genes were detectedand hadvariant allele frequency (VAF) ranging from 3%to99%.The most common mutated genes wereTET2, ASXL1, RUNX1, TP53, SRSF2, and SF3B1. MDS-L, MDS-H, MDS-TR, and MDS/MPN showed an average number of somatic mutations with a mean VAF of 1.9/33%, 2.6/30%, 2/36%, and 4/41%,respectively. SF3B1 mutations were exclusively observed in MDS-L and MDS/MPN. TP53 gene mutations were more frequently seen in MDS-H and MDS-TR. Among 34 patients with a diagnosis of MDS or MDS/MPN with normal cytogenetics, 31 patients (91%) had at least 1 mutation and 24 patients (71%) had ≥2 mutations with ≥10% VAF. A myeloid mutational panel provides additional evidence of clonality besides cytogenetics/FISH studies in the diagnosis of cytopenia with or without cytosis. Two or more mutations with ≥10% VAF highly predicts MDS and MDS/MPN with a positive predictive value of 100%.

Oxidative Stress in Stem / Progenitor Cells and Specific Antioxidant Response in Bone Marrow from Myelodysplastic Syndrome Patients

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous group of blood diseases with varying degrees of severity, which can be classified into one of several subgroups based on features of the abnormal cells. The progression of MDS to secondary AML (sAML) is a good example of the multi-step theory of leukemogenesis in which a series of mutations occur in an initially normal cell and transform it into a cancer cell. Reactive Oxygen Species (ROS) are highly involved in normal hematopoiesis, a low amount being associated with quiescence and self-renewal, while a higher level is requested for ROS signaling pathways. The level of intracellular ROS is finely tuned by the expression of ROS-related enzymes that are suggested as major players in human MDS / AML. Altogether, these elements highlight the potential implication of redox metabolism in the pathophysiology of MDS / sAML.Methods: This study focused on ROS level and antioxidant gene expression in bone marrow (BM) cells from 97 patients compared to 25 healthy controls. All subjects were informed and consenting following a procedure approved by the ethical committee and provided a written informed consent. sAML patients were characterized at diagnosis by proliferation of blasts (>20%) and dysplasia in BM samples. The sternal BM samples were obtained at the time of diagnosis (University hospital of Tours and Cochin hospital in Paris), and were compared with BM from aged-matched healthy donors. Flow cytometry analyses and RNA extraction were processed within the first hour following BM aspiration. We quantified: (i) the level of ROS by flow cytometry (DCFDA staining) in the subpopulations of BM cells identified with anti-CD45-APC-H7, anti-CD34-PE-Cy7 and anti-CD38-APC antibodies; (ii) the expression level of 28 transcripts encoding for major enzymes implicated in the antioxidant cellular response by qRT-PCR using Universal Probe Library technology. All statistical analyses were conducted using R v3.2.2 software. Differences in ROS levels between pathologies was tested using Kruskall-Wallis test followed by Dunn's post hoc test. Differences in expression data between patients and controls were tested using Mann-Whitney test on ΔCt and RQ. Principal component analyses were performed using FactoMineR package on ΔCt values. Significant differences were identified on a change of at least 2-fold and P value less than .05.Results: The results highlight increased ROS level in BM non-lymphoid cells, especially in CD34high CD38low stem and progenitor cells. This increase is also much important in case of sAML compared to low grade (MDS-SLD and MDS-MLD) or high grade MDS (MDS-EB1 and MDS-EB2). Interestingly and as expected, the most important oxidative stress was found in erythroblast subpopulation in MDS-SLD-RS and MDS-MLD-RS, enriched in ring sideroblasts. Moreover, we identified a specific antioxidant signature in the different MDS / sAML bone marrow samples. More precisely, the progression of the disease was characterized by 3-step antioxidant cellular response: (i) overexpression of enzymes reducing disulfides bonds in protein (low-grade MDS - GLRX family), then (ii) enzymes detoxifying H2O2 (high-grade MDS - PRDX and GPX families) and finally (iii) decreased expression of these enzymes in sAML.Conclusion: We report for the first-time progressive increase in ROS level in CD34posCD38low stem andprogenitor cells from MDS patients compared to healthy controls, and higher level in sAML compared to MDS patients. In the light of these findings, antioxidant treatments should be explored in MDS with low blast counts, to avoid the risk of promoting transformation. Concomitantly, MDS BM cells initiate antioxidant program by overexpressing enzymes reducing disulfide bonds in proteins (GLRX family) then reinforced by H2O2 detoxifying enzymes (PRDX and GPX families), these enzymes being dramatically decreased when transformation in sAML. This specific molecular antioxidant response could be considered as biomarkers useful for diagnosis and follow-up of the disease. DisclosuresFontenay:Celgene: Research Funding.

Immune Clearance of Senescent Cells Via Activation of Endogenous Retroviruses and the Viral Recognition Pathway

Expression of genomic repeat element (RE) is known to induce interferon signaling termed ‘viral mimicry’. RE activates double-stranded RNA (dsRNA) recognition via mitochondrial antiviral signaling protein (MAVS), leading to activation of interferon-stimulated genes (ISG) and subsequent cell death. We recently showed that the expression of RE is dysregulated during acute myeloid leukemia (AML) development and in myelodysplastic syndrome (MDS). RE expression is suppressed in leukemic stem cells (LSCs) compared to pre-leukemic stem cells and blasts. Similarly, high-grade MDS showed significant suppression of RE and immune pathways compared to low-grade MDS. These findings suggest that the viral recognition pathway plays a significant role in hematopoietic disorders.Senescent cells activate interferon response and secrete inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP). To investigate whether the inflammatory phenotype seen in senescent cells is mediated by RE expression,we analyzed the RNA sequencing data of senescent cell lines for expression of RE. We found significant upregulation of REs following multiple modalities of senescence induction. We induced senescence in Human HSPCs and 3 different cell lines (K562, HCT116 and IMR90) using an alkylating agent, mitomycin (MMC), and/or radiation, and found significant upregulation of the measured REs. dsRNA is known to trigger the type 1 interferon response through the dsRNA sensor RIG-1/MDA5. Upon recognition of dsRNA by RIG-1, MDA5 is activated, which in turn leads to aggregation of MAVS in the outer mitochondrial membrane. Following senescence, we found aggregation of MAVS in the mitochondrial fraction of murine HSPCs and HCT116 cell. Aggregated MAVS phosphorylates IRF3 and IRF7, leading to translocation of IRF3, IRF7, and NF-κB into the nucleus, where these proteins induce expression of ISGs. Western blots of MMC-treated murine HSPCs showed increase in nuclear NF-κB and p-IRF7. qRT-PCR analysis of murine HSPCs and HCT116, IMR90, and K562 cells following MMC treatment showed increased expression of ISGs and NK ligands, suggesting activation of the dsRNA recognition pathway along with the transcription of ISGs and NK ligands upon senescence induction.We examined whether senescent cells activate NK cells. Mononuclear cells were isolated from the blood of healthy donors when incubated with HCT116 and IMR90 cells following MMC treatment, showed activation of NK cells (6-10-fold). To study NK-mediated cell killing of senescent cells, we incubated senescent IMR90 cells (target cells) with NK92Mi (human NK) cells. MMC-treated IMR90 cells were reduced by 40%. No significant reduction in mock cells was observed. This suggests that activation of ds-RNA recognition pathway in senescence (like in viral infection) leads to up-regulation of NK ligands, NK cell activation, and clearance of senescent cells by NK cells. In order to investigate if MAVS is critical for senescence mediated immune clearance, we employed cell lines with knock out or knock down of MAVS. Abrogation of the dsRNA recognition pathway diminished the interferon response, NK ligand expression and NK mediated clearance of senescent cells.Finally, to evaluate if senescence in human HSPCs also increases RE expression in vivo, we isolated human senescent HSPCs from healthy donors' blood using senescence associated □-galactosidase (SA-β-gal) and did RNA sequencing. Human senescent HSPCs showed overexpression of RE along with pathways of inflammation. % of SA-β-gal+ cells in HSPCs from 26 donors between 20 and 71 years of age (median = 39 years) showed no significant correlation with age (P= 0.25, R2 = 0.06), suggesting that senescent HSPCs do not accumulate with age; implying they are cleared as they are formed. Taken together, these findings suggest that RE expression and the subsequent activation of the dsRNA recognition pathway plays an important role in maintaining homeostasis during aging and dysregulation of this pathway could play a role in hematopoietic disorders such as AML and MDS. DisclosuresNo relevant conflicts of interest to declare.

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.

Only SF3B1 mutation involving K700E independently predicts overall survival in myelodysplastic syndromes.

SF3B1 mutations (SF3B1mut ) in myelodysplastic syndromes (MDS) frequently involve codon K700E and have a favorable prognosis. The prognostic effect of non-K700E SF3B1mut is uncertain. The authors analyzed the clinicopathological features and outcomes of a single-institution series of 94 treatment-naive SF3B1mut MDS patients (18%) and 415 treatment-naive SF3B1wt MDS patients and explored the differences between K700E and non-K700E SF3B1mut MDS. Fifty-five patients (59%) carried K700E. Recurrent non-K700E mutations (39 [41%]) included R625, H662, and K666. Compared with SF3B1mut K700E patients, non-K700E patients had a lower median absolute neutrophil count (1.8 vs 2.4; P = .005) and were frequently "high" according to the Revised International Prognostic Scoring System (19% vs 4%; P = .031). Non-K700E MDS was associated frequently with RUNX1 (26% vs 7%; P = .012) and exclusively with BCOR, IDH2, and SRSF2 mutations. A splicing analysis showed the differential distribution of alternatively spliced events and gene expression profiles between K700 and non-K700E MDS patients. The majority (at least 80%) of SF3B1mut K700E, SF3B1mut non-K700E, and SF3B1wt patients were treated with hypomethylating agents. Over a median follow-up of 16 months, SF3B1mut had superior overall survival (OS) in comparison with SF3B1wt in all MDS patients (not reached vs 25.2 months; P = .0003), in patients with low-grade MDS, and in patients with myelodysplastic syndromes with ring sideroblasts (MDS-RS). Compared with SF3B1wt , SF3B1mut K700E had superior outcomes in all MDS (median OS, 25 months vs not reached; P = .0001), in low-grade MDS (median OS, 41.3 months vs not reached; P = .0015), and in MDS-RS (median OS, 22.3 months vs not reached; P = .0001), but no significant difference was seen between non-K700E and SF3B1wt MDS. By multivariable analysis, the absence of SF3B1mut K700E mutations was independently associated with the prognosis. This study highlights the importance of the SF3B1 mutation subtype in MDS risk assessment. Myelodysplastic syndromes (MDS) with SF3B1 mutations are regarded as having a favorable prognosis by both the World Health Organization and the International Working Group for the Prognosis of Myelodysplastic Syndromes. However, this article shows that only MDS patients with SF3B1 K700E mutations have a favorable prognosis (and not MDS patients with SF3B1 mutations involving other codons). This has important implications for refining future MDS subclassification and risk assessment criteria.

Flow cytometry reveals the nuances of clonal haematopoiesis

Flow cytometry reveals the nuances of clonal haematopoiesis

Only Hotspot SF3B1 K700E Mutation Is an Independent Predictor of Overall Survival in Myelodysplastic Syndromes

Only Hotspot SF3B1 K700E Mutation Is an Independent Predictor of Overall Survival in Myelodysplastic Syndromes

Prodromal / Pre-Leukemic Acute Leukemia of B-Lymphoblasts in 7 Adult Patients

Background: Prodromal/pre-leukemic acute leukemia is rare, mostly described in childhood B-lymphoblastic leukemia (B-ALL), with its initial presentation confused with aplastic anemia (AA). Findings reported in pediatric prodromal/pre-leukemic B-ALL include female preponderance, bone marrow (BM) fibrosis and uniform hypocellularity, and transient recovery. Reports in adults are rarer still. We conducted a retrospective review of adult patients we diagnosed with B-ALL and mixed phenotype acute leukemia with B-lymphoid and myeloid components (MPAL-B/myeloid) with prodromal/pre-leukemic manifestations. We sought to identify common clinical features, peripheral blood (PB) and BM morphology, diagnostic considerations, and clinical follow-up (whenever available) to aid in improved recognition in the adult setting. Methods: Patients with unusual presentations preceding overt acute leukemia were identified from pathology and clinical records from our institution from 2010-2020. These include (a) incidental PB blasts and partial BM-based disease, followed by spontaneous, transient regression (STR); (b) pancytopenia with rare to absent PB blasts and unexpected detection of BM-based blasts (10% or lower), compatible with aleukemic prodrome (AP) cases; (c) pancytopenia without PB blasts yet unexpected excess BM-based blasts (50% or greater) followed by marked reduction of BM-based blasts (compared to initial BM) despite absence of treatment (AP--&amp;gt;SR). BM pathology during prodromal/pre-leukemic phases were reviewed to identify key morphologic features. Results: Seven patients were identified (4 M, 3 F; age range 20-75 years), including one previously described by our group (Table 1). All were of Hispanic ethnicity. They displayed a broad range of symptoms including fatigue, fevers, throat pain, and weight loss. Initial presentations were STR in 3, AP in 2, and AP--&amp;gt;SR in 2. Although variably cellular, all BM trephine biopsies in prodromal/pre-leukemic phase had at least focal hypocellularity with stromal damage, characterized by granular to fibrillary stroma, striking cellular dropout, and multiloculated fat cells with fibrinoid appearance. Relative erythroid hyperplasia was seen in 4, while dyserythropoiesis and dysmegakaryocytopoiesis were both observed in 2 cases, raising concern for myelodysplastic syndrome (MDS). AA and MDS were, in some cases, seriously considered in the initial differential diagnosis. Because of low level BM blasts at initial presentation in most (5/7), definitive lineage designation was sometimes challenging. Furthermore, flow cytometric data was limited and IHC stained only a sparse number of immature B-cells; thus normal precursor B-cell (hematogone) hyperplasia was a strong possibility. Workup in 2 cases during low numbers of BM-based blasts or spontaneous blast reduction (cases #6 and #7, respectively) revealed T-cell clones on PCR/NGS testing. In STR and AP cases, the disease proceeded from initial presentation to overt leukemia within a fairly short interval (range 1.5 - 7.0 months; median 2.5). Final diagnosis in the majority of cases (6/7) was B-ALL, with one case of MPAL-B/myeloid. Conclusion: Herein we describe a series of 7 adult prodromal/pre-leukemic cases of B-lymphoblasts (6 B-ALL; 1 MPAL-B/myeloid) with a variety of clinical presentations. In contrast to the female-predominant pediatric prodromal B-ALLs, most of our adult cases occurred in males. Given pancytopenia, relatively low numbers, and difficulty in assigning blast lineage, diagnostic considerations initially included MDS, AA, and hematogone hyperplasia with marrow regeneration. Key morphologic features that favored a prodromal/pre-leukemic case of B-lymphoblasts over MDS or AA were profound stromal damage and expansion (albeit low numbers) of immature B-cells - findings that would be unusual in low-grade MDS and AA. Intriguingly, in 2 of our cases, T-cell clones detected on PCR testing during low blast levels may indicate an immunologic role of T-cells in transiently reducing blasts in prodromal acute leukemia, as previously suggested by Zimmermannova and colleagues (Haematol2017;102:e225-228). Regardless of whether patients experienced STR, AP, or AP--&amp;gt;SR, close clinical follow-up is advised, as progression to overt leukemia occurs within a relatively short time frame (median of 2.5 months in our series). Disclosures No relevant conflicts of interest to declare.

Diagnostic significance of flow cytometry scales in diagnostics of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) can present a challenge for clinicians. Multicolor flow cytometry (MFC) can aid in establishing a diagnosis. The aim of this study was to determine the optimal MFC approach for MDS. The study included 102 MDS (39 low-grade MDS), 83 cytopenic patients without myeloid neoplastic disorders (control group), and 35 healthy donors. Bone marrow was analyzed using a six-color MFC. Analysis was conducted according to the "Ogata score," "Wells score," and the integrated flow cytometry (iFC) score. The respective sensitivity and specificity values were 77.5% and 90.4% for the Ogata score, 79.4% and 81.9% for the Wells score, and 87.3% and 87.6% for the iFC score. Specificity was not 100% due to deviations of MFC parameters in the control group. Patients with paroxysmal nocturnal hemoglobinuria (PNH) had higher levels of CD34+ CD7+ myeloid cells than donors. Aplastic anemia and PNH were characterized by a high proportion of CD56+ cells among CD34+ precursors and neutrophils. The proportion of MDS-related features increased with the progression of MDS. The highest number of CD34+ blasts was found in MDS with excess blasts. MDS with isolated del(5q) was characterized by a high proportion of CD34+ CD7+ cells and low granularity of neutrophils. In 39 low-grade MDS, the sensitivities were 53.8%, 61.5%, and 71.8% for Ogata score, Wells score, and iFC, respectively. The results support iFC as a useful diagnostic tool in MDS.