Idiopathic Cytopenia Of Undetermined Significance Patients Research Articles

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

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.

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

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

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

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

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.

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

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

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

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

DDX41 mutation in Patients with Idiopathic Cytopenia of Undetermined Significance, Myelodysplastic Syndrome, and Acute Myeloid Leukemia

Background Following the advances in genetic tests, including next-generation sequencing, there have been new insights into hereditary hematopoietic malignancies. The germline mutation in DDX41 was included in a new category, myeloid neoplasms with germline predisposition, of the updated 2016 WHO classification. Based on the reported data to date, there seem to be racial differences in the mutation variants of DDX41 gene, which were found in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Idiopathic cytopenia of undetermined significance (ICUS) is known to be a precursor lesion of MDS, but the DDX41 mutations have not been evaluated in patients with ICUS. In this study, we aimed to reveal the incidence, genetic characteristics, and clinical features of the DDX41 mutations in patients with ICUS, MDS, and AML. Methods We performed targeted deep sequencing of 141 genes with a MiSeqDx sequencer (Illumina) using bone marrow (BM) samples obtained from the patients with ICUS (n=77), MDS (n=175), and AML (n=148) between May 2009 and June 2019. ICUS was defined by the proposed criteria of 2007 Consensus Group. The cut-off level of variant allele frequency (VAF) was set to 2.0% of mutant allele reads. We divided ICUS into clonal cytopenia of undetermined significance (CCUS), which was defined as ICUS with ≥ 2% VAF of somatic mutations of myeloid malignancy-associated genes and non-CCUS. Results Overall, DDX41 mutations were detected in 6 (7.8%) of 77 ICUS, 19 (10.9%) of 175 MDS, and 8 (5.4%) of 148 AML patients. Thirty-eight (49.4%) of 77 ICUS patients had CCUS. Of 6 DDX41 mutated patients with CCUS, 5 showed biallelic mutations with the median VAF of 44.7% (range, 29.3−50.0) and 10.2% (range, 3.3−25.4), indicating that one germline and one somatic mutation exists. Of 175 MDS patients, 78 were categorized into lower-risk MDS (revised international prognostic scoring system [IPSS-R] &lt; 3.5) and 97 into higher-risk MDS (IPSS-R ≥ 3.5), and DDX41 mutations were identified in 6 (7.7%) of 78 lower-risk MDS and 13 (13.4%) of 97 higher-risk MDS patients. Interestingly, biallelic mutations were found in 16 of 18 DDX41-mutated MDS patients with the median VAF of 47.75% (range, 43.4−55.6) and 13.8% (range, 2.7−35.8). In contrast, only one of 8 DDX41-mutated AML patients had biallelic mutation. Patients with DDX41 mutations typically showed hypocellular marrow (median BM cellularity, 30%; range, 5−95) with significant neutropenia (median neutrophil counts, 607/μL; range, 142−1675), male predominance (29/33, 87.9%), and relatively older age (median age, 64 years; range, 41−79) at diagnosis. In addition, we found novel mutation locations, which were different between presumed germline and somatic variants: V152G in germline, and T227M in somatic (Table 2). During a median follow-up duration of 2.9 years, 1 of 6 ICUS patients progressed to MDS-EB-1 after 17.3 months and 1 to non-severe aplastic anemia after 51.3 months. Conclusion Our data show that a significant proportion of ICUS, MDS, and AML patients had DDX41 mutations, many of which are presumably germline. These findings suggest that careful consideration of the predisposing germline mutation is important when selecting a familial donor for allogeneic HCT. We also found novel mutation locations of DDX41 gene which were different between somatic and germline variants. Further studies are warranted to define the clinical and molecular characteristics of DDX41 mutations and therapeutic implications in myeloid neoplasms. Disclosures No relevant conflicts of interest to declare.

Mutational Characteristics and Changing Pattern from Idiopathic Cytopenia of Undetermined Significance to High-Risk Myelodysplastic Syndrome Stratified By IPSS-R

Background: Unexplained cytopenia comprises a spectrum of hematological diseases from idiopathic cytopenia of undetermined significance (ICUS) to myelodysplastic syndrome (MDS). Revised International Prognostic Scoring System (IPSS-R) is the standard tool to assess risk in MDS. Here, we investigated the occurrence, characteristics, and changing pattern of mutations in patients with ICUS and MDS stratified by IPSS-R score. Methods: A total of 211 patients were enrolled: 73 with ICUS and 138 with MDS. We analyzed the sequencing data of a targeted gene panel assay covering 141 genes using the MiSeqDx platform (Illumina). The lower limit of variant allele frequency (VAF) was set to 2.0% of mutant allele reads. Bone marrow components were assessed for the revised diagnosis according to the 2016 WHO classification. Lower-risk (LR) MDS was defined as those cases with very low- or low-risk MDS according to the IPSS-R. Higher-risk (HR) MDS was defined as those cases with high- or very high-risk MDS according to the IPSS-R. Results: Patients with ICUS were classified as very low-risk (39.7%), low-risk (54.8%), and intermediate-risk (5.5%) according to the IPSS-R. Patients with MDS were classified as LR (35.5%), intermediate-risk (30.4%), and HR (34.1%). In the ICUS, 28 (38.4%) patients carried at least one mutation in the recurrently mutated genes in MDS (MDS mutation). The most commonly mutated genes were DNMT3A (11.0%), followed by TET2 (9.6%), BCOR (4.1%), and U2AF1, SRSF2, IDH1 and ETV6 (2.7% for each). IPSS-R classification was not associated with mutational VAF and the number of mutations in ICUS. In the 49 LR MDS, 28 (57.1%) patients carried at least one MDS mutation. The most commonly mutated genes were SF3B1 (20.4%), followed by TET2 (12.2%), U2AF1 (10.2%), DNMT3A (10.2%), ASXL1 (10.2%), and BCOR (6.1%). Higher VAF and number of mutations were observed in LR MDS compared to ICUS patients. In the 42 intermediate-risk MDS, 27 (64.3%) patients carried at least one MDS mutation. The most commonly mutated genes were ASXL1 (23.8%), followed by TET2 (21.4%), RUNX1 (16.7%), U2AF1 (14.3%), DNMT3A (14.3%), SF3B1 (9.5%), and SRSF2, BCOR, STAG2 and CBL (7.1% for each). In the 47 HR MDS, 36 (76.6%) patients carried at least one MDS mutation. The most commonly mutated genes were TET2 (25.5%), followed by DNMT3A (14.9%), TP53 (14.9%), RUNX1 (12.8%), U2AF1 (10.6%), ASXL1 (10.6%), and SRSF2 and KRAS (6.4% for each). As the disease progressed, VAF and number of the MDS mutations gradually increased, and mutations involving RNA splicing, histone modification, transcription factor or p53 pathway had a trend for increasing frequency. Specifically, ASXL1, TP53, and RUNX1 mutations were the most striking features in patients with advanced stage of the disease. Cohesin mutations were not detected in ICUS, whereas these mutations were detected at a relatively high frequency in HR MDS. Our data were summarized in Table 1. Conclusions: We demonstrate that on disease progression, MDS mutations are increased in number as well as are expanded in size. Furthermore, a subset of mutations tends to be enriched for intermediate- to HR MDS. The results of this study can aid both diagnostic and prognostic stratification in patients with unexpected cytopenia. In particular, characterization of MDS mutations can be useful in refining bone marrow diagnosis in challenging situations such as distinguishing LR MDS from ICUS. Disclosures No relevant conflicts of interest to declare.

The diagnostic and prognostic role of flow cytometry in idiopathic and clonal cytopenia of undetermined significance (ICUS/CCUS): A single-center analysis of 79 patients.

ObjectiveThe aim of this study was to evaluate the diagnostic and prognostic role of multiparameter flow cytometry (FC) in patients with idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS).MethodsWe performed FC using a standardized panel and two different diagnostic algorithms (Ogata, Wells) in a well‐characterized cohort of 79 patients with ICUS/CCUS and compared it with a retrospective blinded morphological evaluation and data from targeted next‐generation DNA sequencing of 20 myelodysplastic syndrome (MDS)‐related genes.ResultsOur data show that FC has low sensitivity in distinguishing CCUS from ICUS patients (40.5% for Ogata score and 59.5% for Wells score). The Wells score was suggestive of dysplasia in ICUS/CCUS patients with concurrent morphological signs of dysplasia in the bone marrow (following re‐evaluation by two hematopathologists) and in CCUS patients with a higher mutational burden. Eight patients with ICUS/CCUS from our cohort progressed to another myeloid malignancy (MDS, acute myeloid leukemia, or chronic myelomonocytic leukemia), all showing flow cytometric signs of dysplasia.ConclusionFC performs poorly in diagnosing CCUS versus ICUS. However, it can potentially provide prognostic information in cytopenic patients by identifying a subgroup of patients with a higher grade of dysplasia, higher mutational burden, and higher risk of progression and, together with mutational screening, also identify a group of patients who might require morphological reassessment of dysplastic changes in their bone marrow.

PF540 TERT PROMOTER METHYLATION AS A POTENTIAL PROGNOSTIC BIOMARKER FOR PROGRESSION OF UNEXPLAINED CYTOPENIA TO MDS AND AML

Background:Patients with myelodysplastic syndrome (MDS) often have mutations in epigenetic regulators and altered DNA methylation in their blood cells. Patients with unexplained cytopenia that do not fulfill the criteria for MDS are referred to as having idiopathic cytopenia of undetermined significance (ICUS) or, if they have MDS‐related mutations, clonal cytopenia of undetermined significance (CCUS).A previous study (Zhao et al. BJH 2016) showed that acute myeloid leukemia (AML) patients with preceding MDS (MDS/AML) have hypermethylation of the telomerase reverse transcriptase (TERT) promoter compared to healthy controls. Increased TERT promoter methylation may cause impaired telomerase activity and thereby shortening of telomeres and increased risk of progression to AML.Aims:We aimed to investigate DNA methylation of the TERT promoter to evaluate its potential as a biomarker of disease severity in patients with unexplained cytopenia and MDS.Methods:TERT promoter methylation of two regions (region 1 covering 5 CpG sites 456 to 424 bp upstream of the TSS and region 2 covering 6 CpG sites 384 to 328 bp upstream of the TSS) was analyzed by pyrosequencing in a cohort of 185 patients with ICUS (n = 65), CCUS (n = 49) or MDS (n = 71). Results are presented as DNA methylation percentage of region 1 and 2 (mean ± SD).Results:The 185 included patients (111 men, 74 women) had a median age of 69 years (ICUS: 65 years, CCUS: 70 years, MDS: 72 years) and the 15 healthy controls (6 men, 7 women) had a median age of 57 years.One‐way ANOVA analysis revealed significantly differences in TERT promoter methylation between all groups for both regions (region 1: p = 0.0004, region 2: p = 7.4 × 10–5).TERT promoter methylation in region 1 was significantly higher in all disease groups compared to healthy controls, with increasing methylation and significance levels with increased disease severity (ICUS: 8.3 ± 4.8, p = 0.03; CCUS: 10.2 ± 7.2, p = 0.0007, MDS: 11.3 ± 7.7, p = 1.9 × 10–6; Figure 1A). Furthermore, TERT promoter methylation levels of both CCUS and MDS patients were significantly higher than ICUS patients (p = 0.02 and p = 1.8 × 10–6, respectively.In region 2, MDS patients had significantly higher methylation (MDS: 8.8 ± 8.1, p = 0.001) compared to healthy controls (5.4 ± 3.2). TERT promoter methylation of region 2 in ICUS and CCUS patients was not significantly different from healthy controls or MDS patients (Figure 1B).Figure 1: Box‐and‐whisper plots of TERT promoter methylation percentage in region 1 (A) and region 2 (B) in 185 ICUS, CCUS, MDS patients and 15 age‐matched healthy controls.Summary/Conclusion:Promoter methylation of TERT increases gradually from healthy controls to ICUS, CCUS and MDS patients. Our study suggests that DNA methylation of the TERT promoter reflects disease severity of unexplained cytopenia and MDS patients. We are currently investigating if TERT promoter methylation can be used as a biomarker for survival and progression to MDS and AML.image

PF534 COPY NUMBER VARIATIONS AND UNIPARENTAL DISOMY PREDICT POOR SURVIVAL IN PATIENTS WITH IDIOPATHIC CYTOPENIA OF UNDETERMINED SIGNIFICANCE AND ARE ASSOCIATED WITH MACROCYTOSIS

Background:Approximately half of patients with idiopathic cytopenia of undetermined significance (ICUS) have somatic mutations associated with increased risk of progression to MDS. The cause of cytopenia in the remaining ICUS patients is unknown.Aims:We hypothesize that uniparental disomy (UPD) and submicroscopic copy number variations (CNVs), i.e., gains and deletions (dels), are present in ICUS patients and may be associated with prognosis.Methods:Patients (n = 154) referred with ICUS in Denmark between 2008–2017 were included if cytopenia persisted for &gt; 6 months, cytogenetics was normal and bone marrow (BM) morphology was not diagnostic. SNP‐A was performed on DNA from mononuclear cells or granulocytes using Illumina Infinium CytoSNP‐850K and analyzed with GenomeStudio v1.1 (Illumina). Only dels &gt; 30 markers, gains &gt; 90 markers and UPDs &gt; 5 Mb were reported.Results:A total of 33 structural variations (17 dels, 9 UPDs, and 7 gains), excluding deletion of the Y chromosome, were detected in 27/154 patients (18%) in all chromosomes except chromosome 5, 10, 13, 14, 18, 21 and X. Median sizes were 187 Kb, 1.9 Mb and 82.4 Mb of dels, gains and UPDs, respectively. Mutations were present in 12/27 patients (44%) with CNV/UPD. Twenty‐six deletions or UPDs (del/UPD) were detected in 21/154 patients (14%) of whom 11/21 (52%) harbored mutations.A hemizygous subclonal deletion of DNMT3A was found in a 72 year old male with anemia and neutropenia with no somatic mutations. Subclonal UPD involving almost the entire q arm of chromosome 11 and, thus, the CBL gene was identified in a 84 year old male with mutations in CBL and TET2. Three patients had structural variations involving the TET2 gene (subclonal deletion or UPD), and all three patients also had TET2 mutations.In a 25 year old bipenic male (hemoglobin 6 mM, platelet count 110 x 10^9/L) a 187 Kb hemizygous deletion of the KANK1 gene at chromosome 9p24.3 was detected as the only genetic abnormality.After a median follow‐up of 25 months (range, 2–114), median overall survival (OS) was 67 months (95% CI: 19‐not reached) in patients with del/UPD while it was not reached in the patients without (p = 0.003), Figure 1. The association with poor OS was also significant when including gains (p = 0.02), however, the impact seemed to be driven by del/UPD, and here only results with del/UPD are presented. In multivariate analysis, del/UPD (HR = 2.7, 95% CI: 1.22–5.9, p = 0.014) and deep anemia (hgb &lt; 6.2) (HR = 2.2, 95% CI: 1.10–4.4, p = 0.025) were independent adverse prognostic factors for OS, whereas mutational status was not (HR = 1.1, 95% CI: 0.55–2.3, p = 0.74). Interestingly, an almost identical pattern was observed between macrocytosis (MCV &gt; 100 fL) and del/UPD in Cox proportional hazard regression models indicating a linked impact on survival. Consequently, del/UPD and macrocytosis were not included in the same model due to multicollinearity. Patients with del/UPD had significantly higher MCV (p = 0.005) and P‐ferritin (p = 0.03). Importantly, macrocytosis was not associated with deep anemia (p = 0.317).Summary/Conclusion:To our knowledge this is the first study investigating CNVs/UPDs in ICUS patients. In a subset of patients with otherwise no detectable genetic abnormalities, we identified structural variations involving genes recurrently affected in MDS. Importantly, our results suggest that SNP‐A can aid the prognostics of ICUS patients by identifying submicroscopic structural variations as risk markers for poor OS. A possible role of macrocytosis as a surrogate marker should be explored in a validation cohort and will be prepared for the EHA meeting.image

Replicative senescence of hematopoietic cells in patients with idiopathic cytopenia of undetermined significance

We hypothesized that a subset of idiopathic cytopenia of undetermined significance (ICUS) is associated with an increased autonomous proliferation with exhaustion of hematopoiesis. The aim of this study was to investigate the cell turnover rate and replicative history of the bone marrow cells of ICUS patients. To this end, we examined telomere length (TL), proliferation, and apoptosis of the bone marrow cells of ICUS patients and healthy controls (HCs) using telomere quantitative fluorescence in situ hybridization and immunohistochemical staining for Ki-67 and cleaved caspase-3. We also performed targeted sequencing of 88 myeloid-associated genes. A total of 37 patients with ICUS were enrolled in this study, with a median age of 66 years (range: 31–83). TLs were significantly shorter in patients with ICUS than in the HCs (8.8, interquartile range [IQR] 6.8–12.1 vs 18.4, IQR 14.4–22.0, p < 0.0001). Proliferation (Ki-67-positive) and apoptosis (cleaved caspase-3-positive) were significantly increased in patients with ICUS compared to HCs (median = 20.0% vs 5.0%, p = 0.0003; 45.0% vs 22.5%, p = 0.0005, respectively). The shortening of TL and the increased proliferation and apoptotic activity was also prominent in patients with ICUS without mutation and dysplasia than in HCs (p < 0.0001, p < 0.0001, and p = 0.0093, respectively). TL was not associated with mutational profile and clinical characteristics as well in patients with ICUS. To our knowledge, this is the first study to show that ICUS is associated with premature replicative senescence with increased proliferation and apoptosis of bone marrow cells. Further study is needed to address the cause of replicative exhaustion in ICUS patients.

ASXL1 is a molecular predictor in idiopathic cytopenia of undetermined significance

We analyzed the mutational profile of idiopathic cytopenia of undetermined significance (ICUS) compared with that of myelodysplastic syndrome (MDS). Targeted sequencing of 88 genes associated with myeloid malignancies was performed using samples of bone marrow mononuclear cells from ICUS and MDS patients. Forty patients with ICUS and 128 patients with MDS were included in this study. The median mutational burden was 0.7 mutation/person in the ICUS group and 2.2 mutation/person in the MDS group. ASXL1 (seven patients) was the most frequently mutated gene. ASXL1 was an independent significant prognostic factor for event-free survival (EFS) and overall survival (OS) (hazard ratio (HR) = 10.07 and 30.63, p = .004 and .003, respectively). The ASXL1 mutation which is frequently detected in elderly patients is a molecular predictor for pancytopenia and survival in patients with ICUS. A larger prospective study is needed to validate the role of this genetic mutation in an ICUS prognosis.

Experience of Conventional Cytogenetics in Elderly Cytopenic Indian Patients Suspected with Myelodysplastic Syndromes

Idiopathic cytopenia of undetermined significance (ICUS) includes a highly heterogeneous population with unknown natural history; however, some patients develop clonal mutations of 1/2/3 cell-lineages and subsequently bona fide MDS/AML. A situation of blood-cytopenia and marrow-dysplasia often indicates MDS, which represent heterogeneous pre-malignant hematopoiesis with risk of AML. Cytogenetics has detected chromosomal alterations in MDS, including del(5q), -7/del(7q), del(9q), -13/del(13q), del(11q)/t(11q), del(12p)/t(12p), -17/del(17p)/i(17q), +19/t(19) and idic(Xq13) even in absence of morphological dysplasia. MDS-specific somatic mutations/chromosomal aberrations have been reported in >30% cytopenic patients. Present knowledge on mutational landscapes in mRNA-splicing, DNA-methylation and many other pathways in elderly individuals with hematopoietic and/or non-hematopoietic disease has redefined the clinical impact of clonal hematopoiesis of indeterminate potential (CHIP) and pathobiology of MDS/AML. Considering the power of conventional cytogenetics entrusted by recent WHO-2016 classification for risk-scoring, and also the resource setting available in developing countries, we performed cytogenetics for prognostication of on 1693 Indian cytopenic cases suspected with MDS.Conventional cytogenetics has described the novelty of comprehensive chromosomal picture as single and double independent clones and monosmal (MK)/complex karyotypes (CK) in explicit combinations indicating co-occurrence elderly presented with uni-/bi-/pancytopenia in peripheral blood. Unstimulated culture of bone marrow cells was carried out following the standard protocol for G-banding cytogenetics. Complete 20-25 metaphases were analyzed with karyotypic classification of ~10 metaphases as per ISCN using IKAROS software (MetaSystems, Germany). The frequency of del(3q/5q/7q/11q/12p/20q), monosomy 5/7, trisomy 8/19/21, i(17q), -Y, and translocations have been categorized as single and double independent clone and in co-occurrence of MK and CK for IPSS-R risk-scoring. The classification of single (other than del(5q/7q), -(7/-Y) and +(8/21)) and double (two separate clones with different aberrations) independent clones, CK with ≥3 aberrations include any 3 of these or others have been followed. The drawback was lack of WHO/FAB classification of the patients and monitoring progression and treatment outcome.Among the specific rearrangements, CK with ≥3 aberrations (8%), other single (7%) and double (5.7%) independent clones, +8 (5.3%), -7 (4%) and - Y (4.1%) were highly prevalent (Table 1a). The combined frequencies of single and double independent clones (12.6%) were higher than CK with ≥3 aberrations (8%), in which CK with >3 aberrations were frequently noted. MK with chromosome 5 (1.8%) was lower than 7 (12.2%). Five-tier IPSS-R risk grouping revealed the patient-prevalence with del(11q) and -Y is in good correlation (Table 1b); however, the incidence was significantly higher for ’intermediate’ and ’poor’ risk-groups and lower for ’good’ category, which is mostly influenced by variable frequencies of del(5)/-7/+8 in Indian patients. Co-occurrence of del7q and del20q was highly infrequent (Fig.1). Prognostic-signatures and pair-wise comparison of single exclusive and in combination were in concordance with literature, and are significant for understanding patho-biology and disease-spectrum in this population of different geographical location. Non-MDS ’cytogenetic-CHIP’ was evident in 12% patients as single and double independent clones, for which MDS-diagnosis remains unfulfilled. One patient showed at least 9 aberrations in all of 25 cells screened (Fig.2).This study recommends cytogenetic screening for cytopenic cases with ambiguous presentation and morphology. There could be significant delay in consulting a hematologist, especially for the villagers living below the border of sustainable economic condition, and therefore, opportunistic interaction of cooperating mutations and clonal expansions could be present at higher scale at pre-therapeutic stage. Cytogenetic risk-scoring of ICUS patients may be of tremendous help in absence of advanced technological resource setting.References1 Belli et al Am J Hematol 2011 86:5402 Ganguly&Kadam Mutat Res 2016 769:473 Kwok et al Blood 2015 126:23554 Steensma et al Blood 2015 126:9 [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IgG autoantibody subclasses altered in immuno-related hemocytopenia

Recently, we have detected autoantibodies to bone marrow (BM) hemopoietic cells in some idiopathic cytopenia of undetermined significance (ICUS), terming as immunorelated hemocytopenia. Immunoglobulin G consists of 4 subclasses which have different biological functions in many diseases. In this study, we analyzed the alterations of IgG subclasses in 27 IRH patients compared with 20 ICUS patients and 20 normal controls. The results showed that IgG1 and IgG3 levels were increased in the bone marrow supernatant in IRH patients, and had inverse correlations with hematopoietic function. Meanwhile IgG1 level had a positive correlation with the proportion of CD5+ B lymphocytes. Using immunohistochemical staining, IgG1 were also detected on bone marrow nucleated cells in IRH patients. All these results indicated that IgG1 on bone marrow cells in some IRH patients might be involved in the destruction of hematopoietic cells leading to cytopenia and might be a novel therapeutic target in future.

IgG Subclasses Abnormal in Immune-Related Pancytopenia

In recent years, we found some patients with idiopathic cytopenia of undetermined significance (ICUS)responded well to corticosteroid and/or high-dose intravenous immunoglobulin treatment. We had detected both IgG and IgM antibodies by bone marrow mononuclear-cell-Coombs test, flow cytometric and immunofluorescence analysis on the various bone marrow cell membrane. We also detected IgG in the BM supernatant of these ICUS patients, which can target several antigens on hematopoietic cells membranes by western blot. Then We termed this group of ICUS cases with autoantibodies as immuno-related hemocytopenia(IRP). IgG include IgGl, IgG2, IgG3, IgG4. IgG subclass defects, increased or imbalance in autoimmune diseases, infectious diseases, cancer and other diseases is an indication of the immune response, and IgG subclass in the development of the disease process also plays a different effect. Analysis of IgG subclass is beneficial for the disease etiology, pathogenesis and prognosis. 46 IRP patients and 20 normal controls were enrolled in this study. 58.69% patients’ hematopoietic cell antibody IgG were positive tested by flow cytometric. Hematopoietic cell antibody IgG-positive patients’ bone marrow supernatant were detected and analyzed, IgG1 and IgG3 were increased in the bone marrow supernatant of the patients with IRP, positively related to CD5+ B lymphocytes, and negative correlated with hematopoietic function. Further studies are needed to confirm which IgG subclass plays an important role in the pathogenesis of IRP. DisclosuresNo relevant conflicts of interest to declare.

Distinguishing immunorelated haemocytopenia from idiopathic cytopenia of undetermined significance (ICUS): a bone marrow abnormality mediated by autoantibodies.

In recent years we have observed that some patients with idiopathic cytopenia of undetermined significance (ICUS) responded well to corticosteroid and high-dose intravenous immunoglobulin treatment, indicating that some cytopenia in ICUS might be mediated by autoantibodies. In this study, we analysed 166 ICUS cases retrospectively, some of which were autoantibodies detected on haemopoietic cells in bone marrow (BM) by BM mononuclear cell (BMMNC)-Coombs test, flow cytometry (FCM), Western blot and immunofluorescence (IF). We found that 25·9% (43 of 166) of the cases had autoantibodies positive verified with BMMNC-Coombs test or FCM analysis, 72·1% (31 of 43) of whom had immunoglobulin (Ig)G autoantibody positive by Western blot. IgG could be detected in the erythroblastic islands on the BM smear of nine (32·1%, nine of 28) ICUS patients with autoantibodies by IF. Of these 43 patients, the median percentage of reticulocytes was 1·79%. More than half the patients had hyper-BM cellularity with a higher percentage of nucleated erythroid cells in the sternum. Total response rates to immunosuppressive therapy at 6, 12, 24 and > 36 months were 46·5% (20 of 43), 75% (30 of 40), 77·4% (24 of 31) and 66·7% (16 of 24), respectively. We termed this group of ICUS cases with autoantibodies as immunorelated haemocytopenia (or BMMNC-Coombs test-positive haemocytopenia).