Idiopathic Cytopenia Of Undetermined Significance Research Articles

Clonal Hematopoiesis in Patients With Neuroendocrine Tumor Treated With Lutetium-177 and the Risk of Thrombocytopenia: A Prospective Study.

Thrombocytopenia is a relatively common dose-limiting toxicity during peptide receptor radionuclide therapy (PRRT) in patients with NET. Although uncommon, some patients develop persistent cytopenia and eventually therapy-related myeloid neoplasm (t-MN), which has a dismal prognosis. As the indications for PRRT are expanding, it is important to investigate factors that may predict cytopenias during/after PRRT. We prospectively evaluated the prevalence of clonal hematopoiesis (CH) and cytopenia in patients with NET undergoing PRRT. Patients with metastatic NET with plan to receive four cycles of lutetium-177 were enrolled. CH was evaluated before PRRT using a panel of 220 genes with a targeted depth of ≥1,000×. Patients were followed during PRRT and every 3 months thereafter. Of 37 patients enrolled, the median age was 68 years and 51.4% were male. Previous treatment exposures included alkylating agents in 30%, platinum agents in 8%, and external radiation in 13%. CH was detected in 35.1% using a variant allele frequency (VAF) cutoff of ≥2% and 45.9% with a VAF of ≥1%. The most common mutations were in age-related genes (DNMT3A, TET2). CH was not associated with anemia or neutropenia; however, it was associated with lower platelet count at baseline and more time spent in a thrombocytopenic state during/after PRRT. Five patients had bone marrow biopsies (BMBs) because of sustained hematologic dysfunction post-PRRT, and of those, diagnoses included clonal cytopenia of undetermined significance (CCUS) in three and idiopathic cytopenia of undetermined significance (ICUS) in two. CH is present in 35.1% of patients with NET and is associated with thrombocytopenia risk during PRRT. Future studies with long-term follow-up will delineate whether CH might be a predictor for higher risk of t-MN after PRRT.

Idiopathic Pancytopenia Caused By Immune Checkpoint Inhibitor

Immune checkpoint inhibitors (ICI) have demonstrated clinical utility in various cancers, but they also induce immune-related adverse events (irAEs) such as thyroiditis, pneumonitis, or colitis. Immune-related(IR) hematologic toxicities, including anemia, neutropenia, thrombocytopenia, or pancytopenia are rarely reported but can be fatal. However, the underlying immunobiology of IR hematologic toxicities by ICIs has been very poorly investigated. We report a case of IR pancytopenia by ICIs including an in-depth analysis of single-cell RNA sequencing for the bone marrow (BM). A 57-year-old man with metastatic clear cell renal cell carcinoma was treated with nivolumab (anti-PD-1) and ipilimumab (anti-CTLA-4) and showed rapid tumor reduction. After 7 months of ICI treatment, he showed fever and pancytopenia with hemoglobin - 8.3 g/dl, platelet count - 64,000 /㎕, WBC count - 2500 cells/㎕, absolute neutrophil count - 745 cells/㎕, MCH - 25.9pg, MCV - 82.0fl, MCHC - 31.5g/dl, RDW - 18.2% and reticulocyte count - 1.65%. Direct and indirect antiglobulin test was negative. On BM aspiration, the number of erythroid and megakaryocytic series was slightly increased and that of myeloid series was in the normal range. In BM biopsy, cellularity was 70-80% and three hematopoietic series showed mild hyperplasia. There were no abnormal findings in morphology and chromosomal analysis. BM diagnosis was idiopathic cytopenia of undetermined significance. Although he was treated with methylprednisolone (1mg/kg), hematologic improvement was minimal. Cyclosporine demonstrated the hematologic response in the case. After 5 months of immune suppressive therapy (6-month-discontinuation of nivolumab), he maintained anti-tumor responses by ICIs. We cautiously re-challenge nivolumab as maintenance therapy, but the pancytopenia reappeared. ICI was permanently terminated, and therapy was changed to axitinib (VEGF tyrosine kinase inhibitor). To gain comprehensive insights, we performed single-cell analysis on BM aspirate. Our approach aimed to retain the integrity of both mononuclear cells and granulocytes without cryopreservation by analyzing the sample promptly after collection. Following this, we segregated the sample into distinct fractions, namely bone marrow mononuclear cells (BMMC) and whole blood (BM), and subjected both fractions to in-depth single-cell analysis. This method allowed us to explore the cellular composition and heterogeneity within the BM aspirate, contributing valuable insights to our research. The analysis detected 19,227 cells and identified unique cellular subsets, including terminally differentiated CD8+ T cells (CD8 T EF) and T Helper 17 cells (T H17), which are not commonly found in BM. CD8 T EF cells expressed activation and exhaustion markers, HLA-DRA and KLRG1. Additionally, T H17 cells demonstrated elevated expression of typical T H17 markers RORC and CCR6, as well as polyfunctional markers, including KLRB1, TNFRSF4, and TNFRSF18. Interestingly, previous studies on IR-myocarditis have shown an increased presence of CD8 T EF in the peripheral blood. Moreover, the studies have associated aplastic anemia with the T H17 cell population. These findings offer significant insights into the molecular and cellular mechanisms, as well as the BM microenvironment, contributing to the IR hematologic toxicities by ICIs.

Predicting Disease Progression from Idiopathic Cytopenia of Undetermined Significance to Myeloid Malignancies Using a Machine Learning-Based Approach

Background Idiopathic cytopenia of undetermined significance (ICUS) is characterized by an unexplained, persistent cytopenia without a definite diagnosis of hematologic or non-hematologic conditions, and that does not meet criteria for myeloid neoplasms. The progression of ICUS to myeloid malignancies has been associated with certain clinical features, such as genetic mutations or allele frequencies. However, the incidence of progression and the risk factors vary among previous studies. While prognostic scoring systems are well-established in myelodysplastic syndrome (MDS) to predict progression to acute myeloid leukemia and survival outcomes, similar predictive tools for ICUS are currently lacking. Considering that a significant proportion of patients with ICUS share similar genetic abnormalities and clinical features with those with MDS, there is a need for predictive tools for ICUS. In this study, we aimed to establish a predictive model for disease progression from ICUS to myeloid malignancies. Methods Patients with ICUS were identified using electronic medical records, and those meeting the diagnostic criteria of ICUS were included. ICUS was defined as persistent cytopenia lasting for at least 4 months, with no underlying disease or condition that could explain the cytopenia which included history of cytotoxic chemotherapy or radiation therapy, blood or bone marrow (BM) disorders, autoimmune diseases, solid organ transplantation, active infection by atypical pathogens, and the use of immunosuppressant and chemotherapeutic agents. Cytopenia was defined as follows: hemoglobin <13 g/dL in males or <12 g/dL in females, neutrophil count <1.9 × 10 9/L, and/or platelets <150 × 10 9/L. For the predictive model, various features were considered, including medication history, laboratory data, comorbidities, family history of cancer and major disorders, smoking history, alcohol consumption, and physical measurements. Numeric data from the BM report were standardized using regular expressions, while free text or comments from the BM report and chromosomal analysis were extracted and processed using a pre-trained language model called PubMedBERT to generate embeddings. The primary objective of this study was to develop a predictive model for identifying progression from ICUS to myeloid malignancies. The performance of the model was assessed using machine learning algorithms, specifically XGBoost, support vector machine, and logistic regression. Results Among the initial 6,962 patients with medical records of the BM examination between January 2000 and December 2021, 5,147 patients were excluded from the modeling process due to secondary causes of cytopenia. Ultimately, 1,815 patients were finally included in the study. Of 1,815 patients, 47 (2.6%) were diagnosed with myeloid malignancies from further BM examination. The predictive model, utilizing XGBoost and incorporating standardized text and embedded features, demonstrated favorable performance, with a mean area under the receiver operating characteristic curve of 0.800 (Figure). Additionally, our analysis confirmed that the XGBoost model effectively processed features from text data, as we assessed the feature importance using Shapley values. Conclusion In conclusion, our study successfully established a predictive model for disease progression from ICUS to myeloid malignancies, demonstrating the potential utility of clinical predictive tools. Further integration of genetic data holds promise for enhancing the model's performance and providing clinical insights for managing patients with ICUS.

Targeted NGS on sequential bone marrow biopsies aids in the evaluation of cytopenias and monocytosis and documents clonal evolution—a proof of principle study

Differential diagnosis of clonal versus reactive cytopenia and monocytosis, respectively, frequently presents a diagnostic challenge. With the two recent classifications of myeloid disorders, mutational analysis has gained importance as a diagnostic tool. However, reports on its utility on trephine bone marrow biopsies (BMB) are sparse. The aim of our proof of principle study was to determine the suitability of targeted sequencing for the longitudinal evaluation of cytopenia and monocytosis and demonstration of clonal evolution on sequential BMB. Seventy-seven EDTA-decalcified BMB of 33 patients with peripheral cytopenia and/or monocytosis, including at least one follow-up biopsy/patient, were included. Initial morphological diagnoses were idiopathic cytopenia of undetermined significance (ICUS, 8 cases), MDS (without blast increase, 7 cases), MDS with increased blasts/excess blasts (MDS-IB/EB) (11 cases), and CMML (7 cases). Thirty-one genes relevant for myeloid disorders were examined using two custom AmpliSeq NGS panels. Mutations were found in the initial BMB of 5/8 cases of ICUS, thus changing the diagnosis to clonal cytopenia of unknown significance (CCUS), 5/7 MDS, 10/11 MDS-IB/EB, and 7/7 CMML. Clonal evolution was observed in 14/33 (42%) cases, mostly associated with disease progression. None of the wild-type patients acquired mutations during follow-up. NGS-based mutation profiling is a robust diagnostic tool for BMB and provides valuable additional information, especially for cases with no/minimal dysplasia, and for better risk stratification of MDS. Tracking variant allele frequency and appearance of mutations over time allows for observing clonal evolution or relapse.

Discordant pathologic diagnoses of myelodysplastic neoplasms and their implications for registries and therapies

AbstractMyelodysplastic neoplasms (MDS) are a collection of hematopoietic disorders with widely variable prognoses and treatment options. Accurate pathologic diagnoses present challenges because of interobserver variability in interpreting morphology and quantifying dysplasia. We compared local clinical site diagnoses with central, adjudicated review from 918 participants enrolled in the ongoing National Heart, Lung, and Blood Institute National MDS Natural History Study, a prospective observational cohort study of participants with suspected MDS or MDS/myeloproliferative neoplasms (MPNs). Locally, 264 (29%) were diagnosed as having MDS, 15 (2%) MDS/MPN overlap, 62 (7%) idiopathic cytopenia of undetermined significance (ICUS), 0 (0%) acute myeloid leukemia (AML) with <30% blasts, and 577 (63%) as other. Approximately one-third of cases were reclassified after central review, with 266 (29%) diagnosed as MDS, 45 (5%) MDS/MPN overlap, 49 (5%) ICUS, 15 (2%) AML with <30%, and 543 (59%) as other. Site miscoding errors accounted for more than half (53%) of the local misdiagnoses, leaving a true misdiagnosis rate of 15% overall, 21% for MDS. Therapies were reported in 37% of patients, including 43% of patients with MDS, 49% of patients with MDS/MPN, and 86% of patients with AML with <30% blasts. Treatment rates were lower (25%) in cases with true discordance in diagnosis compared with those for whom local and central diagnoses agreed (40%), and receipt of inappropriate therapy occurred in 7% of misdiagnosed cases. Discordant diagnoses were frequent, which has implications for the accuracy of study-related and national registries and can lead to inappropriate therapy. This trial was registered at www.clinicaltrials.gov as #NCT05074550.

Genomic Data Heterogeneity across Molecular Diagnostic Laboratories: A Real-World Connect Myeloid Disease Registry Perspective on Variabilities in Genomic Assay Methodology and Reporting

Genomic data variability from laboratory reports can impact clinical decisions and population-level analyses; however, the extent of this variability and the impact on the data's value are not well characterized. This pilot study used anonymized genetic and genomic test reports from the Connect Myeloid Disease Registry (NCT01688011), a multicenter, prospective, observational cohort study of patients with newly diagnosed myelodysplastic syndromes, acute myeloid leukemia, or idiopathic cytopenia of undetermined significance, to analyze laboratory test variabilities and limitations. Results for 56 randomly selected patients enrolled in the Registry were independently extracted and evaluated (data cutoff, January 2020). Ninety-five reports describing 113 assay results from these 56 patients were analyzed for discrepancies. Almost all assay results [101 (89%)] identified the sequencing technology applied, and 94 (83%) described the test limitations; 95 (84%) described the limits of detection, but none described the limit of blank for detecting false positives. RNA transcript identifiers were not provided for 20 (43%) variants analyzed by next-generation sequencing and reported by the same laboratory. Of 42 variants with variant allele frequencies ≥30%, 16 (38%) of the variants did not have report text indicating that the variants might be germline. Variabilities and lack of standardization present challenges for incorporating this information into clinical care and render data collation ineffective and unreliable for large-scale use in centralized databases for therapeutic discovery.

Abstract 5560: Prognostic effect of co-mutations on patients with NPM1+ acute myeloid leukemia (AML) enrolled in the Connect® Myeloid registry

Abstract Introduction: The 2017 World Health Organization classification of myeloid neoplasms and acute myeloid leukemia (AML) recognizes AML with mutated NPM1 as a distinct entity with favorable prognosis. However, as mutations other than NPM1 may further influence disease prognosis, here we assess the prognostic effect of NPM1 co-mutations in patients (pts) with AML enrolled in the Connect® Myeloid Registry (NCT01688011). Methods: The Registry is a large, US, multicenter, prospective observational cohort study of pts with newly diagnosed AML, myelodysplastic syndromes, idiopathic cytopenia of undetermined significance, or myelofibrosis. In this analysis, pts with AML ≥55 years of age were grouped by known NPM1 status (NPM1+ vs NPM1‒). The frequency of gene mutations was analyzed among patients with NPM1+ AML and NPM1‒ AML. Overall survival (OS) was evaluated using the Cox model adjusted for age and mutations in TP53 and RUNX1. Results: Overall, 123 pts with NPM1+ AML and 360 pts with NPM1‒ AML were included. Most pts were male (NPM1+, 61.5%; NPM1−, 60.7%) and White (NPM1+, 78.0%; NPM1−, 85.6%); median (range) age was 67 (55-87) and 71 (55-97) years for the NPM1+ and NPM1− groups, respectively. After adjusting for age, pts with NPM1+ AML had significantly longer median OS than pts with NPM1‒ AML (26 vs 15 months; HR [95% CI]: 0.58 [0.44-0.76]; P &amp;lt; 0.01; Table). Pts with NPM1+ vs NPM1‒ AML had a lower frequency of certain key mutations such as TP53 (1.6% vs 18.6%) and RUNX1 (0.8% vs 14.8%). The significant survival benefit with NPM1+ vs NPM1‒ initially observed after adjusting for age was not observed after further adjusting for presence of TP53 and RUNX1 mutations (NPM1+ vs NPM1‒; 23 vs 19 months; HR [95% CI]: 0.75 [0.48-1.17]; P = 0.21). Conclusions: TP53 plus RUNX1 mutation was identified as a pattern of co-mutational burden that were observed less frequently and potentially contributed to the improved OS of pts with NPM1+ AML and poorer OS of pts with NPM1‒ AML. NPM1+n = 123 NPM1‒n = 210 Overall survival, months Adjusted for age 26.0 15.0 Hazard ratio (95% CI) 0.58 (0.44-0.76) Adjusted for age, TP53, RUNX1 23.0 19.0 Hazard ratio (95% CI) 0.75 (0.48-1.17) Frequency of gene mutations, n (%)TP53 2 (1.6) 39 (18.6) ASXL1 3 (2.4) 37 (17.6) FLT3-ITD 44 (35.8) 36 (17.1) TET2 20 (16.3) 34 (16.2) DNMT3A 29 (23.6) 33 (15.7) IDH2 11 (8.9) 33 (15.7) RUNX1 1 (0.8) 31 (14.8) NRAS/KRAS 11 (8.9) 30 (14.3) IDH1 18 (14.6) 18 (8.6) CEBPA 4 (3.3) 15 (7.1) WT1 5 (4.1) 14 (6.7) FLT3-TKD 18 (14.6) 13 (6.2) PHF6 0 12 (5.7) PTPN11 4 (3.3) 12 (5.7) SF3B1 2 (1.6) 8 (3.8) C-KIT 1 (0.8) 7 (3.3) EZH2 1 (0.8) 4 (1.9) ETV6 0 3 (1.4) MLL-PTD 1 (0.8) 1 (0.5) Other 30 (24.4) 114 (54.3) Citation Format: Jaroslaw P. Maciejewski, Daniel A. Pollyea, Gail J. Roboz, Karen Seiter, Bart L. Scott, Irene S. DeGutis, Pavel Kiselev, Edward Yu, Ali McBride, Willem Heydendael, Harry P. Erba. Prognostic effect of co-mutations on patients with NPM1+ acute myeloid leukemia (AML) enrolled in the Connect® Myeloid registry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5560.

A Lower Frequency of Spliceosome Mutations Distinguishes Clonal Cytopenias of Undetermined Significance From Low-Risk Myelodysplastic Syndromes, Despite Inherent Similarities in Genomic, Laboratory, and Clinical Features

Clonal cytopenias of undetermined significance (CCUS) are associated with an increased risk of developing a myelodysplastic syndrome (MDS); however, the mechanism and factors associated with evolution remain unclear. We propose that next-generation sequencing (NGS) of cytopenic cases with equivocal morphologic dysplasia will improve patient clinical care and that serial sequencing of such equivocal cases could identify the factors that predict evolution to MDS. We performed targeted NGS of samples from 193 individuals with confirmed or suspected MDS or MDS/myeloproliferative neoplasm, including sequential investigation for 28 individuals at the time of diagnosis and during follow-up. NGS facilitated the diagnosis of all suspicious cases as myeloid neoplasm (21%), CCUS (34%), or idiopathic cytopenias of undetermined significance (45%) when no variants were detected. We found that there was no significant difference in most measured clinical features or clonal phenotypes, such as cell counts, number of variants, variant allele frequencies, and overall survival, between CCUS and International Prognostic Scoring System-Revised–defined low-risk MDS. However, there was a significant difference in the types of variants between CCUS and low-risk MDS, with a significantly lower number of splicing factor mutations in CCUS cases (P < .001). Moreover, we observed an increased probability of evolution to MDS of individuals with CCUS compared with that in those with idiopathic cytopenias of undetermined significance over the first 5 years (P = .045). Our analyses revealed no conclusive pattern associating clonal expansion or the number of variants with the evolution of CCUS to MDS, perhaps further supporting the similarity of these diseases and the clinical importance of recognizing and formally defining CCUS as a category of precursor myeloid disease state in the next revision of the World Health Organization guidelines.

CCUS and Low-Risk MDS Are Inherently Similar, Sharing Clonal and Clinical Features

Background Idiopathic cytopenia of undetermined significance (ICUS) is a condition of unexplained blood cytopenia not meeting the diagnostic criteria of myelodysplastic syndromes (MDS). Recently, the newer designation of clonal cytopenia of undetermined significance (CCUS) has been proposed to characterize patients with ICUS in the presence of one or more somatic mutations or copy number abnormalities. Clinically, CCUS has been associated with an increased risk of developing MDS; however, the mechanism and factors associated with evolution remain unclear. We sought to determine the molecular similarity of cytopenic cases with equivocal morphological dysplasia to bona fide MDS, as well as whether serial sequencing of ICUS and CCUS cases could identify factors that predict evolution to MDS. Methods We compared the incidence and frequency of somatic variants in 48 genes recurrently mutated in myeloid neoplasms (MNs), using targeted Next-Generation Sequencing (NGS) of bone marrow genomic DNA from 193 individuals with confirmed or suspected MDS or MDS/myeloproliferative neoplasm (MDS/MPN), including sequential investigation for 28 individuals at the time of diagnosis and during follow-up. Cytopenias qualifying patients as ICUS were based on WHO laboratory diagnostic criteria: hemoglobin levels <130g/L for males or <120 g/L for females; platelet count <150x109/L; and neutrophils <1.8x109/L. MDS patients were further subclassified as low risk (MDS-LR), including "Very Low” and "Low” International Prognostic Scoring System-Revised (IPSS-R) categories; intermediate risk (MDS-IR), including "Intermediate” category; or high risk (MDS-HR), including "High” and "Very High” risk categories. Results NGS further facilitated the diagnosis of all suspicious cases, either as MN (21%), CCUS (34%), or ICUS (45%). We found that there was no significant difference in most measured clinical features between CCUS and MDS-LR. While we saw significantly increased hemoglobin and platelet counts in individuals with CCUS compared with MDS-HR (Mann-Whitney U test; p=0.003 and p=0.012, respectively), no significant differences in cell counts were seen between CCUS and the MDS-LR or MDS-IR subgroups (Table 1). There was also no difference in overall survival between CCUS and MDS-LR when adjusted for age and sex in multivariable models (HR=0.575; 95% CI=0.256-1.294; p=0.330), while MDS-HR was associated with worse overall survival when compared with CCUS (HR=2.759; 95% CI=1.274-5.975; p=0.010). We saw no significant difference in clonal phenotypes, including number of variants or variant allele frequencies (VAFs), when comparing CCUS and MDS-LR. We further classified genes mutated in at least one patient in our cohort into functional groups for further analysis. These groups included epigenetic regulators: ASXL1, BCOR, DNMT3A, EZH2, IDH1, IDH2, KDM6A, PHF6, RAD21, STAG2, TET2; splicing factors: DDX41, SF3B1, SRSF2, U2AF1, ZRSR2; genes involved in signal transduction: BRAF, CALR, CBL, CSF3R, FLT3, GNB1, JAK2, KIT, KRAS, MPL, NF1, NPM1, NRAS, PTPN11, SETBP1; transcription factors: CEBPA, ETV6, GATA2, RUNX1; and genes involved in DNA damage repair: PPM1D and TP53. A significantly higher number of transcription factor mutations was seen when comparing CCUS to the MDS-IR and MDS-HR groups (p=0.007 and p=0.008, respectively), but not the MDS-LR group (p=0.754). We did, however, see significant significantly lower number of splicing factor mutations in CCUS cases compared with MDS-LR (p<0.001; Figure 1). Serial sequencing revealed no significant associations between number, type, or VAF of variants initially present at ICUS or CCUS diagnosis compared to those later acquired at the time of MDS evolution. Of note, we saw an increased probability of evolution to MDS of individuals with CCUS compared to ICUS over the first 5 years (HR=3.569; 95% CI=1.029-12.380, p=0.045). Conclusions Our analyses revealed no conclusive pattern associating clonal expansion or number of variants with evolution of CCUS to MDS, perhaps further supporting the similarity of these diseases and the proposal that CCUS should be classified as low-risk MDS in the next revision of the WHO guidelines. If future studies can better associate variants in specific types of genes with progression (such as splicing factors), this may also assist in predicting the probability of evolution and thus inform management strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Role of Cleaved Caspase-3 Reflecting Apoptosis in MDS Patients

Background: Myelodysplastic syndrome (MDS) is a heterogenous, clonal disorder in hematopoiesis. Pathophysiology of MDS is not clear yet, but there is growing interest in association with cellular proliferation and apoptosis. We hypothesized that Ki-67 and cleaved caspase-3 could be prognostic markers representative of proliferation and apoptosis in MDS patients. Methods: We performed retrospective study using bone marrow samples from 76 MDS patients who was diagnosed between 2004 and 2014 at Seoul National University Hospital. We compared Ki-67 and cleaved caspase-3 to evaluate cellular proliferative and apoptotic activities using immunohistochemical testing (IHC). And we additionally analyzed Ki-67 and cleaved caspase-3 between patients with MDS, idiopathic cytopenia of undetermined significance (ICUS) and health controls (HCs) based on data previously studied. And we also compared difference according to MDS risk scoring system. Results: Cleaved caspase-3 was highest in MDS patients, followed by ICUS patients and HCs (2.70, 95% CI: 2.56-2.85 vs. 2.19, 95% CI: 1.85-2.54 vs. 1.45, 95% CI: 1.17-1.73, p < 0.001). Similarly, the mean Ki-67 grade was also highest in MDS patients, followed by ICUS patients and HCs (1.72, 95% CI: 1.55-1.90 vs. 1.58, 95% CI: 1.30-1.86 vs 1.05, 95% CI: 0.95-1.16, p = 0.001) (Figure). Higher cleaved caspase-3 grade was significantly associated with lower IPSS-R score (p = 0.020), whereas Ki-67 was not. Interestingly, TET2 mutation was associated with decreased cleaved caspase-3 levels (p = 0.030). In terms of survival, there is no significant difference according to cleaved caspase-3 or Ki-67. In addition, we divided patients with MDS into four groups: lower-risk MDS patients with lower caspase-3 levels (A group : n = 32), lower-risk MDS patients with higher caspase-3 levels (B group : n = 13), higher-risk MDS patients with lower caspase-3 levels (C group : n = 28), and higher-risk MDS patients with higher caspase-3 levels (D group : n= 2). We compared PFS and OS between the four groups and found that high-risk MDS patients with lower caspase-3 showed significantly worse PFS than the other groups (p < 0.001; A vs B, p = 0.655; B vs C, p = 0.018; A vs C, p < 0.001) Conclusion: Our results suggest that apoptotic activity gradually increases from HCs, patients with ICUS to patients with MDS. Therefore, we could predict apoptotic activity from cleaved caspase-3. In addition, higher apoptotic activity was associated with better MDS patient prognostic scores and survival outcomes. Further studies are needed to reveal the the differences in apoptotic activity between lower- and higher-risk MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Therapy Related Myeloid Neoplasms Following PARP Inhibitors : Real-Life Experience

Purpose: To assess the hematologic toxicity of PARP inhibitors (PARPi) treatment, and provide insights into the diagnosis and management of therapy-related myeloid neoplasms (t-MN) following PARPi. Experimental Design: In a leading cancer center, we compared the profile of 13 t-MN diagnosis among 37 ovarian cancer (OC) patients treated with PARPi referred for hematological consultation. Next, we compared these 13 t-MN post PARPi among 37 t-MN post OC. Finally, we described the specificities of 69 t-MN post PARPi treated for gynecologic cancer (breast and/or ovarian cancer) in a national observatory. Results: From 2016 to 2021, cumulative incidence of t-MNs was 3.5% (13/373) among OC patients treated with a PARPi. At the time of referral for hematological consultation, patients who developed t-MNs had a longer PARPi exposure (9 months vs. 3, p= 0.01), lower platelet count (74 vs. 173 G/L, p=0.0005), and more cytopenias (2 vs 1, p=0.0005). After NGS evaluation of patients without t-MN, 5 patients (13.5%) were finally classified as CHIP (clonal hematopoiesis of indeterminate potential), 7 (18.9%) as CCUS (clonal cytopenia of undetermined significance) and 2 (5%) as ICUS (idiopathic cytopenia of undetermined significance). Compared to t-MN not exposed to PARPi, t-MN-PARPi patients had more BRCA1/2 germline mutation (61.5% vs 0% p=0.03) but similar OS [8.2 (95% CI: 2.03-18.7) months in the t-MN-PARPi group compared to 6.1 (95% CI: 3-12.2) months in the t-MN-non-PARPi group; p=0.8]. In the national observatory, t-MN post PARPi had mostly a complex karyotype (61%) associated with a high rate of TP53 mutation (71%) with a median VAF of 41% (IQR, 17-67) and 75% of multi-hit mutations (Figure 1). IPSS score was "Low and Intermediate-1" and "Intermediate-2 and High" MDS groups for 50% of patients, respectively. AMLs were mainly considered as adverse (75%) according to the ELN 2017 classification. Median OS from t-MN diagnosis was 9.7 months (95% CI: 5.3-13.9). In multivariate analysis, a longer time between the end of PARPi and t-NM (HR 1.046, p=0.02), olaparib treatment compared to others PARPi (HR 5.82, p=0.003) and AML (HR 2.485, p=0.01) were associated with shorter OS independently of the BRCA1/2 status (Figure 2). Conclusions: In a large series, we described a high incidence of t-MN post PARPi associated with unfavorable cytogenetic and molecular abnormalities leading to poor OS. Time to diagnose t-MN after PARPi interruption seems to influence OS, suggesting the importance of early diagnosis. Particular consideration should be done on BRCA1/2 patients, and in case of thrombocytopenia or more than one cytopenia and relatively long PARPi exposure. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sternal bone marrow cell morphology evaluation utility for diagnostic categorization in patients with acquired hypocellular bone marrow failure syndromes

Objective: Diagnostic value assessment of sternal bone marrow cell morphology in patients with acquired hypocellular bone marrow failure syndromes (BMFS) characterized by normal cytogenetics. Methods: A total of 194 eligible patients with an acquired hypocellular BMFS pre-sternum diagnosis in Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College from June 2014 to January 2019 were reviewed. Sternal bone marrow evaluation was performed, and a post-sternum diagnosis was made. Clinical characteristics and overall survival (OS) were then compared among patients with different post-sternum diagnosis. Binary logistic regression was used to develop a predictive scoring system. Results: In 152 patients with pre-sternum AA diagnosis, 29 patients with a pre-sternum idiopathic cytopenia of undetermined significance (ICUS) diagnosis, and 13 patients with a pre-sternum clonal cytopenia of undetermined significance (CCUS) diagnosis, sternal bone marrow evaluation resulted in a change of diagnosis to hypocellular myelodysplastic syndrome (hypo-MDS) in 42.8% (65/152) , 24.1% (7/29) , and 30.8% (4/13) , respectively. Patients with a post-sternum hypo-MDS diagnosis showed a significant difference in OS compared with patients with a post-sternum AA diagnosis (P=0.005) . Patients with ICUS/CCUS showed no difference in OS compared with AA and hypo-MDS (P=0.095 and P=0.480, respectively) . A 4-item predictive scoring system to identify hypocellular BMFS patients that need sternal bone marrow evaluation was developed, including age > 60 years old (OR=6.647, 95% CI 1.954-22.611, P=0.002, 2 points) , neutrophil alkaline phosphatase score ≤ 160 (OR=2.654, 95% CI 1.214-5.804, P=0.014, 1 point) , abnormal erythroid markers evaluated by flow cytometry on iliac bone marrow (OR=6.200, 95% CI 1.165-32.988, P=0.032, 2 points) , and DAT (DNMT3A, ASXL1, TET2) genes mutation (OR=4.809, 95% CI 1.587-14.572, P=0.005, 1 point) . The Akaike information criterin (AIC) was 186.1. Conclusion: Patients with a pre-sternum acquired hypocellular BMFS diagnosis characterized by normal cytogenetics may not reach accurate diagnostic categorization without sternal bone marrow cell morphology evaluation, which could be considered a diagnostic tool for this patient population. A predictive scoring system was developed, and when the total score is ≥ 2 points, sternal bone marrow evaluation should be performed for accurate diagnostic categorization that is critical to optimal patient care.

Bone marrow free immune checkpoints as a potential biomarker for differential diagnosis of acquired bone marrow failures.

ObjectiveClinically, to make a definite diagnosis of aplastic anemia (AA), idiopathic cytopenia of undetermined significance (ICUS) or myelodysplastic syndrome (MDS), they should be distinguished from each other. AA and ICUS have some incidence to transform into MDS. Immunosuppressive therapy (IST) is effective in AA and partial ICUS patients, while other ICUSs are more likely to progress to MDS without response to IST. To date, we neither found a technical method that could easily identify AA from hypoproliferative MDS, nor a simple parameter that could indicate ICUS with a response to IST. Here, we detected the concentration of free immune checkpoints in bone marrow supernatant of AA, ICUS, and MDS patients, analyzed the differences of immune status among these three diseases, to try to find a way to predict the response to IST in ICUSs.MethodsSeventy‐four novel patients were enrolled with newly diagnosed acquired bone marrow failure (including 29 AA patients, 11 ICUS patients, and 34 MDS patients), bone marrow supernatants were collected. Luminex liquid suspension array technology was used to measure the concentrations of 17 immune checkpoints to analyze the differences of immune status among these three diseases.ResultsThe levels of 17 free immune checkpoints were elevated in MDS and showed a strong correlation with each other, followed by ICUS, and with the weakest in AA. By drawing the ROC curve, we found eight immune checkpoints, including sCD40, sCD86/B7‐2, sCTLA‐4, sGITR, sHVEM, sPD‐1, sTIM‐3, and sTLR‐2, could easily distinguish AA from hypoproliferative MDS. ICUSs with lower concentrations of these eight free immune checkpoints predicted a better IST response.ConclusionIn conclusion, we found that there were notable differences in the immune status of AA, ICUS, and MDS. The concentrations of sCD40, sCD86/B7‐2, sCTLA‐4, sGITR, sHVEM, sPD‐1, sTIM‐3, and sTLR‐2 could be used to identify AA and hypoproliferative MDS patients, as well as to distinguish ICUS patients who could benefit from IST.

Increased Apoptotic Activity in Low-Risk Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous hematopoietic disorder associated with cellular proliferative and apoptotic activity. We retrospectively investigated these activities in bone marrow samples from 76 MDS patients using immunohistochemical staining for Ki-67 and cleaved caspase-3. We divided cleaved caspase-3 into two groups based on median value and compared the differences according to MDS risk scoring systems. We compared MDS patient indices with idiopathic cytopenia of undetermined significance (ICUS) and healthy control (HC) indices using our previously published data. Cleaved caspase-3 immunohistochemistry was highest in MDS patients, followed by ICUS patients and HCs. Similarly, the mean Ki-67 grade was also highest in MDS patients, followed by ICUS patients and HCs. Higher cleaved caspase-3 grade was significantly associated with lower IPSS-R score (p = 0.020), whereas Ki-67 was not associated with MDS. Interestingly, TET2 mutation was associated with decreased cleaved caspase-3 levels (p = 0.03). However, there was no significant association between proliferative/apoptotic activity and survival. Our results suggest that apoptotic activity gradually increases from healthy controls and ICUS patients to MDS patients. Furthermore, higher apoptotic activity was associated with better MDS patient prognostic scores. Further studies are needed to reveal the differences in apoptotic activity between lower- and higher-risk MDS.

P790: THE EXPRESSION LEVEL OF WT1 MRNA IN PATIENTS WITH IDIOPATHIC CYTOPENIA OF UNDETERMINED SIGNIFICANCE (ICUS) PROGRESSING TO MYELODYSPLASTIC SYNDROME (MDS)

Background: Idiopathic cytopenia of undetermined significance (ICUS) is defined as patients with one or more unexplained cytopenia who do not meet diagnostic criteria for myelodysplastic syndrome (MDS) or another hematologic disorder. In the clinical course of ICUS, some patients eventually have typical MDS changes. Until now, there is no effective method to predict the development of the disease. Recent studies have shown that the expression level of WT1 mRNA in peripheral blood has important clinical significance in the diagnosis, occurrence, development and prognosis of MDS. At the same time, some studies have proved that there is a significant difference in the expression level of WT1 mRNA between MDS and hemocytopenia caused by other reasons. Aims: In order to clarify the clinical significance of WT1 mRNA expression in the progression of ICUS to MDS. Methods: Between April 2017 and January 2020, untreated adult patients with ICUS or lower-risk MDS were enrolled in the study. All patients detected the WT1 mRNA expression of bone marrow and peripheral blood at diagnosis. Then patients with ICUS were followed up. Every three months, the patients with ICUS detected the WT1 mRNA expression of peripheral blood, until the patient progressed to MDS. The results of WT1 transcript levels are depicted as WT1 (copies/mL)/c-ABL (copies/mL) as a percentage. Results: Total of 80 patients with ICUS and 40 patients with lower-risk MDS were enrolled. At diagnosis, the median expression level of WT1 mRNA in MDS group was 5.99 (0.08-79.27), and that in ICUS group was 0.50 (0.16-22.48). There was significant difference between them (P = 0.000). Then 80 patients with ICUS were followed up to January 2022. The median follow-up time was 30 months (11-54 months). 8 patients developed MDS, the progression rate was 10%, and the median progression time was 6.5 months (2-19 months). The median expression level of WT1 mRNA at the initial diagnosis in ICUS group without progressing to MDS was 0.44 (0.16-7.58), and that in progressing to MDS group was 9.44 (0.26-22.48). There was significant difference between the two groups (P = 0.031). The median expression level of WT1 mRNA was 19.185 (0.73-42.30) when MDS was diagnosed. The median maximum expression level of WT1 mRNA in peripheral blood of ICUS group without MDS was 0.98 (0.22-7.58). There was significant difference between the two groups (P = 0.007). Summary/Conclusion: The study clarified the WT1 mRNA expression status of ICUS patients. There were significant differences in WT1 mRNA expression level between patients with ICUS and lower-risk MDS, and the WT1 mRNA expression level of patients with lower-risk MDS was relatively high. The study confirmed that patients with high WT1 mRNA expression level in the patients with ICUS (WT1 mRNA expression level > 9.44) are very likely to progressing to MDS; Dynamic monitoring of WT1 mRNA expression in peripheral blood is an effective method for following-up after ICUS diagnosis. The progressive increase of WT1 mRNA expression is one of the indicators for researchers to judge the progress of ICUS to MDS.

Clinical implications and genetic features of clonal cytopenia of undetermined significance compared to lower-risk myelodysplastic syndrome.

Clonal cytopenia of undetermined significance (CCUS) is characterized by persistent cytopenias with genetic aberrations, which do not meet the diagnostic criteria for myelodysplastic syndrome (MDS). We aimed to compare the clinical and genetic characteristics of CCUS with lower-risk MDS and identify patients with CCUS with a high risk of progression. We performed targeted sequencing of bone marrow (BM) samples from patients with idiopathic cytopenia of undetermined significance (ICUS) (n=139) and MDS (n=226). Overall survival (OS) of patients with CCUS (n=78) was worse than non-clonal ICUS (n=61) and superior to lower-risk MDS (n=99). Patients with CCUS showed similar characteristics to those with lower-risk MDS, except for higher haemoglobin, lower BM cellularity, and less frequent SF3B1 mutations. Lower haemoglobin, DDX41 (biallelic germline and somatic), ETV6, and RUNX1 mutations were independent prognostic factors for worse OS. Lower haemoglobin and DDX41 mutations were also associated with lower progression-free survival. Patients with CCUS with high-risk features showed similar or worse OS than patients with lower-risk MDS. Our findings suggest that patients with CCUS having certain clinical or genetic features should be regarded and treated as lower-risk MDS despite lacking significant dysplasia or MDS-associated chromosomal abnormalities.

Inflammatory Cytokine Profiles Do Not Differ Between Patients With Idiopathic Cytopenias of Undetermined Significance and Myelodysplastic Syndromes.

Immune dysregulation has been highlighted as a key player in the pathogenesis of myelodysplastic syndromes (MDS), but little is known about cytokine profiles in patients with unexplained cytopenia with or without mutations in MDS-associated genes (clonal cytopenias of undetermined significance [CCUS] and idiopathic cytopenias of undetermined significance [ICUS], respectively), which often precede MDS. Here, we study the cytokine profiles in 111 patients with ICUS (N = 41), CCUS (N = 30), lower-risk MDS (LR-MDS; N = 22) and higher-risk MDS (HR-MDS; N = 18), and in healthy elderly controls (N = 21). Twenty cytokines were examined in blood plasma at time of diagnosis using Luminex assays and enzyme linked immunosorbent assays. The cytokine levels were compared between patient groups, and in patients versus controls. Associations between cytokines and MDS-associated mutations were evaluated. An aberrant cytokine profile was observed in all patient groups relative to healthy elderly controls. Patients had significantly higher levels of IL-6 (P< 0 .001), tumor necrosis factor α (P < 0.001), IL-10 (P < 0.001), and C-X-C motif chemokine 10 (P < 0.001) and lower levels of transforming growth factor beta 1 (P < 0.001), CCL5/regulated on activation normal T-cell expressed and secreted (P < 0.001), and S100A4 (P < 0.001) compared with healthy controls. Survival was significantly shorter in CCUS and MDS patients with a high systemic inflammatory cytokine load (median overall survival [OS] 21 months) compared with those with low-moderate systemic inflammatory cytokine load (median OS 64 months; P < 0.0001). These data suggest that patients with ICUS and CCUS have cytokine levels as abnormal as in LR-MDS. Indeed, high cytokine levels are present before MDS is diagnosed and cytokine levels are elevated irrespective of the presence or size of the myeloid clones. Cytokines may have a prognostic impact at a very early premalignant stage of myeloid disorders.

Exclusive diagnosis of aplastic anemia

As an independent disease, aplastic anemia (AA) has been recognized for more than a century. When AA is diagnosed, other non-AA bone marrow failures should be excluded. It is termed as exclusive diagnosis of AA. The exclusive diagnosis of AA is helplessly based on that there is no parameter by which AA can be sensitively and specifically diagnosed now. So further searching for the meaningful diagnostic parameters of AA should be carried on to establish a direct diagnostic protocol of this disease and make it possible to differentiate it clearly from other bone marrow failure disease such as congenenital bone marrow failure, hypoplastic myelodysplastic syndromes, AA-paroxysmal nocturnal hemoglobinuria syndromes, large granules lymphocyte leukemia, clonal cytopenia of undetermined significance, immunorelated pancytopenia, acute hemopoietic arresting and idiopathic cytopenia of undetermined significance. The new markers and technologies being helpful for distinguishing AA from other bone marrow failures should be used in diagnosing AA. Correct understanding and application of exclusive diagnosis is not only related to the correctness of diagnosis and treatment of excluded diseases, but also to the quality of AA diagnosis, treatment and research.

The value of next-generation sequencing in routine diagnostics and management of patients with cytopenia.

We performed a single-center study of real-world health data to investigate the direct clinical consequence of targeted next-generation sequencing (NGS) results integrated in the clinicopathological evaluation of patients with cytopenia suspected of myelodysplastic syndrome (MDS). The study included 87 newly referred patients, who had a bone marrow examination, which included targeted NGS analysis. NGS was requested at the discretion of either examining pathologist or hematologist. Data were collected retrospectively from patient files including pathology reports with integrated NGS results. The NGS results had a diagnostic impact in 67 cases (77%) when combining both histopathological and final clinical evaluation and provided prognostic value in 19 cases (22%). NGS supported a confident or tentative histopathological diagnosis in 52 cases (60%). Twenty cases (23%) had a final diagnosis of either Clonal Cytopenia of Undetermined Significance (CCUS) or Idiopathic Cytopenia of Undetermined Significance (ICUS). In 4 cases, NGS results affected the choice of principal treatment strategy, including considerations of allotransplantation. Twenty-one patients (24%) could be discharged to primary care physician. In a multidisciplinary clinicopathological real-world setting, NGS analysis of bone marrow samples from selected patients contributed substantially to the diagnostic evaluation and management of patients with cytopenia suspected of MDS. Consequently, we have now included NGS analysis in most routine bone marrow examinations from patients with MDS or unexplained cytopenia.

COVID-19 Outcomes Among Participants in the NHLBI Myelodysplastic Syndromes (MDS) Natural History Study

COVID-19 Outcomes Among Participants in the NHLBI Myelodysplastic Syndromes (MDS) Natural History Study