ALBERT

Description: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages. Some patients exhibit leukemic transformation (LT) by unknown mechanisms, and chemotherapy may increase the risk of LT. To clarify the molecular mechanisms of LT, gene alterations involved in LT from patients in the chronic phase (CP) of MPNs were identified. Among 18 patients who progressed to leukemia, AML1/RUNX1 mutations were detected in 5 patients at the LT but in none at the CP. To investigate the leukemogenic effect of AML1/RUNX1 mutants, the AML1D171N mutant was transduced into CD34+ cells from patients in the CP of MPNs. The D171N transduction resulted in proliferation of immature myeloid cells, enhanced self-renewal capacity, and proliferation of primitive progenitors. Taken together, these results indicate that AML1/RUNX1 point mutations may have a leukemogenic potential in MPN stem cells, and they may promote leukemic transformation in MPN.

Description: Myelodysplastic syndrome (MDS) is a hematopoietic stem-cell disorder characterized by trilineage dysplasia and susceptibility to acute myelogenous leukemia (AML). Analysis of molecular basis of MDS has been hampered by the heterogeneity of the disease. Recently, mutations of the transcription factor AML1/RUNX1 have been identified in 15% to 40% of MDS–refractory anemia with excess of blasts (RAEB) and MDS/AML. We performed mouse bone marrow transplantation (BMT) using bone marrow cells transduced with the AML1 mutants. Most mice developed MDS and MDS/AML-like symptoms within 4 to 13 months after BMT. Interestingly, among integration sites identified, Evi1 seemed to collaborate with an AML1 mutant harboring a point mutation in the Runt homology domain (D171N) to induce MDS/AML with an identical phenotype characterized by marked hepatosplenomegaly, myeloid dysplasia, leukocytosis, and biphenotypic surface markers. Collaboration between AML1-D171N and Evi1 was confirmed by a BMT model where coexpression of AML1-D171N and Evi1 induced acute leukemia of the same phenotype with much shorter latencies. On the other hand, a C-terminal truncated AML1 mutant (S291fsX300) induced pancytopenia with erythroid dysplasia in transplanted mice, followed by progression to MDS-RAEB or MDS/AML. Thus, we have developed a useful mouse model of MDS/AML that should help in the understanding of the molecular basis of MDS and the progression of MDS to overt leukemia.

Description: Background Myelodysplastic syndromes (MDS), commonly seen in elderly patients, represent a heterogeneous group of clonal hematopoietic stem cell disorders caused by the accumulation of gene mutations. By contrast, congenital bone marrow failure syndromes and genetic predispositions associated with MDS are known in pediatric patients. However, little is known about the pathogenesis of MDS in adolescent and young adult (AYA) patients. Previous reports showed the patients with MDS aged under 40 or 41.5 years at allo-HSCT were associated with good survival compared to those among the older population (N Engl J Med. 2017;376:536-547, Blood. 2017;129:2347-2358). However, AYA-MDS is rare, and its clinical features and genetic abnormalities have not been analyzed enough. It is suspected that the clinical and genetic features of AYA-MDS patients might be different from those of elderly patients or pediatric patients. Therefore, we investigated the gene abnormalities of AYA-MDS patients and aimed to elucidate the genetic characteristics associated with the good outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We analyzed the patients younger than 50 years of age in order to reduce the variation of patient-related factors. Methods We analyzed the outcomes of all consecutive patients aged under 50 years who were diagnosed with MDS or acute myeloid leukemia evolving from MDS in our hospital between January 2005 and July 2018. The study was approved by the institutional review board, and patients gave written informed consent for the study, according to the Declaration of Helsinki. Cytogenetic analysis and genomic DNA extraction were carried out using diagnostic bone marrow samples. We performed targeted next-generation sequencing to identify mutations in 68 driver genes using AmpliSeq for Illumina Myeloid Panel and On-Demand Panel on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. Overall survival (OS) was analyzed for all patients, and the Kaplan-Meier survival curve was used to assess OS using the log-rank test. Additionally, the cumulative incidence of relapse (CIR) was analyzed for patients who underwent allo-HSCT. Gray's test was used to evaluate the CIR. Results A total of 85 patients with MDS aged under 50 years (U40 between 15 and 39 years old: N=37, 40s between 40 and 49 years old: N=48) were analyzed. The median follow-up time of survivors was 2,041 days (range 176-5,085). There were no significant differences in patient characteristics between U40 and 40s. The 3-year OS of U40 were superior to 40s (79.9% vs. 58.1%, P=0.018), especially lower risk IPSS categories (3-year OS, 95.5% vs. 50.8%, P=0.002). In total, 69 of 85 patients (U40: N=31, 40s: N=38) had undergone allo-HSCT. U40 patients had lower percentage of bone marrow blasts at just before HSCT than 40s patients (over 10%, 12.9% vs. 36.8%, P=0.048), and better 3-year OS from HSCT in lower-IPSS (88.8% vs. 53.8%, P=0.024); but not in higher-IPSS (45.0% vs. 43.2%, P=0.834). In this cohort, at least one driver mutation was detected in 61% of allo-HSCT recipients. Frequently mutated genes (more than 10%) were ASXL1 and RUNX1; however, both of the genes did not have significant impact on the outcomes. While, only one patient in 40s had TP53 mutation. We detected 0.8 (range 0-3) and 1.8 (range 0-6) mutations at average in U40 and 40s, respectively (P=0.06). The proportions of the patients without any gene mutations were 52% in U40 and 30% in 40s. Transplanted patients with 0 or 1 mutation showed lower relapse rate than those with 2 or more mutations (3-year CIR, 23.3% vs. 45.2%, P=0.049). Conclusions The clinical outcomes of U40 patients with MDS were favorable than those in the 40s, especially in lower disease risk. The number of driver mutations in U40 tended to be lower than that in 40s. MDS in adult is regarded as a stem-cell aging disease with gene mutations; however, MDS-associated mutations were not detected in the half of U40. Moreover, TP53 mutation that is associated with extremely poor posttransplant survival was not detected in U40 patients. MDS patients with less than 2 mutations showed lower relapse rate, which maybe indicate genetic mutations have a great impact on transplant outcomes between 15 and 49 years old. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1337 Erythroid hypoplasia or aplasia is a hematological condition observed in including idiopathic pure red cell aplasia (PRCA), thymoma-associated PRCA and aplastic anemia. Myelodysplastic syndrome (MDS) with erythroid hypoplasia/aplasia in bone marrow is a rare type of MDS that was not included in existing classifications of MDS. Patients with erythroid hypoplasia/aplasia have common characteristics; transfusion dependencies, immunologic abnormalities and successful immunosuppressive therapies with cyclosporin A (CsA). Thus, we may regard erythroid hypoplasia/aplasia as one of hematological disease entities. However, pathogenic mechanisms of erythroid hypoplasia/aplasia have not been fully elucidated, although T-lymphocyte-mediated inhibition of erythropoiesis is suspected to be the most possible mechanism of the pathogenesis. Recently, we reported that oligoclonal expansion of CD8+/perforin+ T cells was observed in patients with thymoma-associated PRCA and the oligoclonality was exclusively detected in CD8+ T cells, but not CD4+ T cells. To clarify the pathogenetic role of the T-cells, we analyzed the T-cell subsets and therapeutic responses in patients with erythroid hypoplasia/aplasia in bone marrow. Among 253 patients with MDS diagnosed at Hiroshima University Hospital between 2000 and June 2011, 12 patients (4.7%) showed erythroid hypoplasia/aplasia. A total of 22 patients with erythroid hypoplasia/aplasia, including 8 MDS with erythroid hypoplasia/aplasia, 3 idiopathic PRCA, 3 thymoma-associated PRCA and 8 aplastic anemia, were enrolled in this study. All patients were treated with CsA and improvement in anemia in this study followed the International Working Group (IWG) 2006 criteria. For T-cell subset analysis, mononuclear cells (MNCs) were purified from bone marrow (BM) or peripheral blood (PB) of the patients. MNCs were stained with fluorescent (FITC, PE, PerCP or APC)-conjugated antibodies for CD8, perforin, CCR7, CD62L, CD27, CD28 and CD45RO, CD45RA and were subjected to flow cytometric analysis. As controls, 30 patients with MDS without erythroid hypoplasia/aplasia and 30 patients without BM abnormalities were also analyzed. Among 22 patients with erythroid hypoplasia/aplasia, 10 patients (4 MDS with erythroid hypoplasia/aplasia, 1 idiopathic PRCA, 3 thymoma-associated PRCA and 2 aplastic anemia) responded to CsA therapy within 2 to 8 weeks. The median blood hemoglobin concentration increased from 6.5 g/dL at the baseline to 9.3 g/dL with treatment, with a median increase of hemoglobin of 2.8 g/dL from the baseline. We attempted to compare the T-cell subsets between CsA-responders and non-responders. All of 3 thymoma-associated PRCA showed good response to CsA therapy, suggesting that the oligoclonal expansion of a CD8+/perforin+ T-cell subset may be associated with the responses to immunosuppressive therapy. Thus, we focused on a T-cell subpopulation expressing CD8+/perforin+. Intriguingly, the CD8+/perforin+ T cells were significantly increased in the CsA-responders (44.3 ± 9.6%, n=10) compared to the non-responders (19.0 ± 9.3%, n=12, P

Description: A histone H3 Lysine 27 (H3K27)-methyltransferase, enhancer of zeste homolog 2(EZH2) is known as a tumor-associated gene. Physiological role of EZH2 is an enzymatic component of polycomb repressive complex 2 (PRC2) to inhibit expression of target genes. While EZH2 plays oncogenic roles by repressing the expression of tumor suppressors in solid tumors and some lymphomas, it plays rather tumor-suppressive roles in myeloid malignancies. We have generated a short-form EZH2 that lacks the catalytic SET domain (EZH2-dSET). Using this EZH2 mutant we could produce serially transplantable MDS-like diseases. Microarray analysis using the MDS-like bone marrow cells enabled us to identify novel targets of EZH2 in MDS tumorigenesis, including ATP-binding cassette (ABC) transporters. Derepression of Abcg2 via decreased H3K27-trimethylation was confirmed. Retroviral transduction of EZH2-dSET to MDS-like cell lines increased surface ABCG2-high populations and those cells functioned to exclude anticancer drugs as expected. Intriguingly, with Abcg2 expression alone, primary bone marrow cells could produce an MDS-like cytopenic disease in our BMT model. In our clinical specimens, ABCG2 high expressions were observed in MDS samples but not in de novo AML and CML samples. In two MDS patients, ABCG2 expression decreased along with leukemic transformation. Interestingly, two out of 33 MDS patients with extremely high expression of ABCG2 harbored the same U2AF1 mutation (Q157P). In addition, somatic mutations of EZH2 and those of either U2AF1 or SRSF2 were mutually exclusive in all investigated cases. Interestingly, U2AF1 mutants (S34F and Q157P) reduced EZH2 expression, leading the derepression of ABCG2 via decreased H3K27-trimethylation. These results indicate a link between U2AF1 mutations and ABCG2 expression via disrupted EZH2. In conclusion, different mechanisms are supposed to converge at dysregulated EZH2 in MDS. And a short form of EZH2 upregulates ABCG2 expression resulting in MDS advancing to secondary leukemia. Thus, either mutations affecting the EZH2 function or mutations of EZH2 itself could play an important role in MDS, and one of the downstream targets of EZH2 suppression in MDS pathogenesis is aberrant expression of ABCG2. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 183 RUNX1 transcription factor regulates hematopoietic ontogeny and is a frequent target of gene rearrangements in hematological malignancies. In addition to gene rearrangements, loss-of-function mutations of RUNX1 have been found in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Mutations of RUNX1 have been detected in about 10–20% patients classified as MDS/AML (high-risk MDS and AML following MDS). Although loss-of-function mutations of RUNX1 cause leukemia together with additional cooperating events in mouse models, the mechanisms, by which impaired RUNX1 functions led to the subsequent genetic alterations, remain unclear. Because DNA damage-repair response has an important role for prevention of many types of tumors, including hematological malignancies, we analyzed the role for RUNX1 in DNA repair system. First, we stably expressed a dominant-negative mutant of RUNX1, RUNX1dC, in a murine myeloid cell line 32Dcl3. RUNX1dC lacks the C-terminal 225 amino acids, which was originally found in a patient with MDS and suppresses functions of wild-type (WT) RUNX1 by inhibiting its DNA binding activity. To analyze the roles for RUNX1 in the DNA repair system, we took advantage of in vitro DNA repair assays with DNA cross-linking agents in 32D-neo and 32D-RUNX1dC cells. Since the cells that recovered from DNA damage make colonies after cisplatin exposure, we can evaluate DNA repair ability of test cells with this assay. As a result, clonogenic ability of 32D-RUNX1dC was significantly decreased by the 2-hour exposure of cisplatin (10nM treatment: 93.4% reduction) compared to that of 32D-neo cell (10nM treatment: 58.6% reduction) (p=0.0006). In addition, 32D-RUNX1dC showed significantly lower clonogenic ability than 32D-neo after exposure to UV-B and gamma-ray, respectively. To evaluate DNA-damage accumulation in 32D-neo and 32D-RUNX1dC cells, we performed immunofluorescent microscopic analysis using monoclonal antibodies for (6–4) photoproducts (6–4 PPs) and cyclobutane pyrimidine dimers (CPDs), which are major products of DNA damage induced by UV-B. These types of DNA lesions are repaired by nucleotide excision repair (NER) system. After six hours from UV-B exposure, both 6–4 PPs and CPDs accumulated in 32D-RUNX1dC cells more abundantly than in 32D-neo cells. These results suggest that RUNX1dC attenuates NER in 32D cells, thereby leading to the sustained accumulations of DNA lesions after exposure to UV-B and cisplatin. To identify the molecule(s) involved in DNA-damage signaling, we profiled expression of 84 genes involved in DNA damage signaling by real-time RT-PCR array. The expression profiling revealed that RUNX1dC repressed Gadd45a, a regulator of NER system in 32D cells. Because genetic alteration of RUNX1 is supposed to occur at a HSC level in MDS and AML, we next evaluated whether RUNX1dC modifies Gadd45 expression in murine Lineage−Sca1+c-Kit+ (LSK) cells. As a result, RUNX1dC-transduced LSK cells showed significantly lower expression of Gadd45a and Gadd45b compared to Mock-transduced LSK cells. Luciferase reporter and chromatin immunoprecipitation assays showed that RUNX1 directly regulates Gadd45a expression via two RUNX1-binding sites neighboring to the p53-binding site in the intron 3 of the human Gadd45a gene. To confirm the roles for endogenous RUNX1 in NER system, we next performed RUNX1-knockdown experiments by short hairpin RNA (shRNA) -mediated gene silencing. RUNX1-shRNA-transduced 32D cells showed significantly lower expression of Gadd45a and Gadd45b than non-silensing-shRNA-transduced 32D cells. As expected, RUNX1-shRNA-transduced 32D cells showed significantly lower clonogenic ability after UV-B exposure than non-silensing-shRNA-transduced 32D cells (p=0.0008). These results suggest that endogenous RUNX1 regulates Gadd45 expression, thereby controlling NER system. Finally, we screened mRNA expression of Gadd45a in the samples from 23 MDS/AML patients, and found that its expression was significantly decreased in MDS/AML patients harboring RUNX1-C-terminal mutation compared to those with WT RUNX1 (p=0.0233). In summary, we here demonstrated that RUNX1 participates in the DNA damage-repair response through transcriptional regulation of Gadd45a. Our study suggests that the impaired RUNX1 function deteriorates NER system and may cause additional mutation(s), which are required for multi-step leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Description: RUNX1/AML1 mutations have been frequently detected in patients with myeloid neoplasms, especially myelodysplastic syndromes (MDS) and chronic monocytic leukemia (CMML). Although the mutations have been analyzed thoroughly, its expression level has not been investigated. Therefore, we attempt to clarify the expression of RUNX1 in the pathogenesis of myeloid neoplasms. The study was approved by the institutional review board and patients gave written informed consent for the study, according to the Declaration of Helsinki. Several isoforms of RUNX1 mRNA are known and we analyzed RUNX1a (including exon 7a which has stop codon) and RUNX1b (skipping exon 7a and including exon 7b and 8). Expression levels of full length isoform (RUNX1b) and short isoform (RUNX1a which has a dominant negative effect on RUNX1b) in CD34+ cells from patients with myeloid neoplasms were examined. A part of patients with MDS or myelodysplastic syndrome / myeloproliferative neoplasms (MDS/MPN) including CMML showed RUNX1a overexpression. Average of relative RUNX1a expression level in MDS patients (n=34) and MDS/MPN patients (n=20) was 7.4-fold and 8.6-fold of the level in normal bone marrow (BM), respectively, whereas most of these patients showed almost same or slight increase of expression level of RUNX1b compared with normal BM. Interestingly, some patients showed high expression of RUNX1a and repression of RUNX1b. In both disease categories, patients with excess blasts displayed a significantly higher expression level of RUNX1a compared with normal BM and patients without excess blasts. During the disease progression in a single patient with MDS or MDS/MPN, the expression of RUNX1a became higher, while azacitidine treatment reduced RUNX1a expression. Genomic mutations of RUNX1 were also examined. RUNX1 mutations were detected in 16% of MDS and 35% of MDS/MPN. Surprisingly, a part of patients had both RUNX1 gene mutation and RUNX1a overexpression, and they showed rapid progression of disease. To evaluate the effects of RUNX1a overexpression, RUNX1a was transduced into CD34+ cells from MDS patients with low expression level of RUNX1a. RUNX1a-transduction resulted in cell proliferation on MS5 stromal cells. These results indicate that overexpression of RUNX1a may add growth advantage to CD34+ cells in patients with MDS or MDS/MPN. We next analyzed the mechanism of RUNX1a overexpression. Gene mutations affecting exon recognition were examined in the patients. Splicing factor mutations, SRSF2 and U2AF1, were detected frequently in MDS (15%) and MDS/MPN (50%). Patients with splicing factor mutations showed higher RUNX1a expression than patients without the mutations. To confirm that the splicing factor mutations affect the expression of RUNX1a, we performed enforced expression of SRSF2 p.P95H mutant using pMYs.IRES.EGFP retrovirus vector in a MDS-derived cell line, TF-1. After a single cell sorting, independent 13 expanding clones were analyzed. Most of the clones demonstrated higher expression of RUNX1a than mock cells, whereas RUNX1b expression was reduced in all clones. Increase of RUNX1a expression in SRSF2 mutant-transduced TF-1 cells was also confirmed by Western blot. Moreover, the clones with higher GFP intensity showed higher expression level of RUNX1a, suggesting that SRSF2 p.P95H expression level may affect the expression level of RUNX1a. Furthermore, SRSF2 mutant-transduced TF-1 cells showed phenotypic changes of higher CD11b and CD14 than mock TF-1 cells, suggesting that SRSF2 mutant may induce monocytic differentiation via RUNX1a overexpression. Gene mutations of RUNX1 in intron 6 and exon 7a were also analyzed. A 5' splice site change just after exon 6 was detected in a CMML patient with RUNX1a overexpression, which may be another mechanism of RUNX1a overexpression. Mutations of exon 7a or changes in 3' splice site just before exon 7a have not been detected yet. In conclusion, our data suggest that overexpression of RUNX1a may play a critical role in the progression of MDS and MDS/MPN, in addition to RUNX1 mutations. Splicing factor mutations are suspected to contribute to the mechanism of the dysregulation of RUNX1. Disclosures No relevant conflicts of interest to declare.

Description: Background Although the outcome after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) for the patients with acute myeloid leukemia (AML) in complete remission has been improved, the prognosis of patients with active disease is still dismal, generally with 20 - 30% of overall survival (OS) at 2 years. Prognostic value of gene mutations detected by the next generation sequencing (NGS) for this extremely poor group remains to be evaluated. Methods A total of 120 patients with AML not in hematological remission who received the first allo-HSCT at our institute between April 2005 and December 2017 were enrolled. For each patient, genomic DNA was extracted from the frozen bone marrow sample harboring leukemic blasts which was preserved at the nearest available date before the initiation of conditioning regimen. Sequencing was performed using TruSight Myeloid Sequencing Panel® on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. Charts were retrospectively reviewed on survival, relapse, and non-relapse mortality (NRM). The Kaplan-Meier method was used to assess OS using the log-rank test. Univariate and multivariate analysis were performed to identify potential prognostic factors. The Cox proportional hazards method was used for the multivariate analysis to assess OS. Gray's test and the Fine-Gray test were used to assess the cumulative incidence of relapse (CIR). Competing risks were relapse and NRM. Results Median follow-up of survivors was 1345 days (235 - 4888 days). Median age at transplant was 51 (range 21 - 71). Grafts were from bone marrow (n = 67, 55.8%), peripheral blood (n=42, 35.0%) and cord blood (n=11, 9.2%). Refined disease risk index (Blood. 2014;123:3664-71) scored high (n=61, 51.3%) and very high (n=58, 48.7%). OS at 2 years of the whole cohort was 27.3% (95% confidence interval [CI], 19.4% - 35.7%). There was no significant difference in OS between patients in primary induction failure and in relapse (OS at 2 years: 26.5% [n=50] vs 28.7% [n=70], p= 0.293). NGS analysis revealed TP53 loss-of-function mutation in 23 (19.2%) patients. Among all detected gene mutations, TP53 mutation was the most powerful predictor of poor OS after allo-HSCT (OS at 2 years: 13.5% vs 30.5% for TP53+ [n=23] vs TP53- [n=97], p= 0.0184). Consistent with previous reports, monosomal karyotype (MK, J Clin Ocnol 2010:26;4791-7) was significantly associated with positive TP53 mutation (13.3% of non-MK vs 37.9% of MK, p=0.006). Of note, all the patients (n=11) positive for both prognostic factors died within 1 year after allo-HSCT, whereas OS of the patients without either factor (n=78) was 33.6% at 2 years. Multivariate analysis on OS revealed MK, TP53 mutation, de novo AML (no prior history of MDS), ECOG performance status score 2 or more, C reactive protein 1.0 mg/dL or more, peripheral blood blast frequency of 1% or more at the initiation of conditioning regimen were independent prognostic factors for poor OS. Among them, to determine the prognostic factors critical for deciding the indication of allo-HSCT, we chose pre-transplant factors which were available around one months before transplant. From this point of view, multivariate analysis revealed independent prognostic factors for poor OS after allo-HSCT including MK (Hazard ratio [HR] 2.05; 95% confidence interval [CI] 1.30 - 3.25, p=0.00217), TP53 mutation (HR 1.72; 95% CI 1.04 - 2.86, p=0.035), and de novo AML (HR 1.67; 95% CI 1.04 - 2.86, p=0.036). Because the HR of these three factors were comparable, a score of 1 was assigned to each factor. OS at 2 years was 49.7%, 26.5% and 13.1% for patients with low (score 0, n=23), intermediate (score 1, n=63) and high (score 2 or 3, n=34) risk, respectively (p

Description: Background Constitutional partial trisomy 8 mosaicism (CT8M) is a congenital chromosomal abnormality with an estimated occurrence rate as one out of 25,000-50,000 pregnancies. CT8M has a wide variability in physical manifestation ranging from apparently normal to severe disablement. However, diagnosis of CT8M in adult without physical abnormality is difficult . Acquired trisomy 8, which is restricted to the malignant cells, is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), and clinical implication of carrying isolated trisomy 8 is considered as intermediate cytogenetic risk in MDS. However, isolated trisomy 8 without morphological dysplastic features is not definitive evidence for MDS. 15 to 20% of trisomy 8 in MDS are supposed to be derived from CT8M. We therefore diagnosed CT8M patients among patients with cytopenia and analyzed clinical and genetic features to uncover the association with MDS. Methods . Clinical features including cytogenetic analysis were analyzed regularly. Genomic DNA was extracted from whole PB cells or BM mononuclear cells. We performed targeted next-generation sequencing to identify mutations in 68 driver genes of myeloid neoplasms using AmpliSeq for Illumina Myeloid Panel and On-Demand Panel on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. The study was approved by the institutional review board and patients gave written informed consent for the study. Results We identified nine CT8M patients with cytopenia.They comprised 3 males and 6 females at a median age of 56 years (range 24-84 years) (Table). All the patients carried no physical abnormality nor conspicuous phenotypic features. Four patients (Patient #3, #4, #6 and #7) did not show apparent morphological dysplasia at the initial BM examination, and they were not diagnosed as MDS. Their cytopenia has not been exacerbated until now without any treatment, and the duration of stable cytopenia was from 2 to 12 years in these patients. By contrast, five patients with CT8M were diagnosed as MDS . Two patients (#5 and #8) were diagnosed as MDS-single lineage dysplasia (SLD), and their cytopenia has not become worse without any treatment for about 4 years. Other three patients diagnosed as MDS-multilineage dysplasia (MLD) showed various clinical courses. Patient #1 was treated with azacitidine and maintains complete hematological improvement after 34 courses of the treatment. Patient #2 was treated with erythropoietin stimulating agent and azacytidine but developed to AML 3 years after initial diagnosis but leukemic blasts has del(20), not +8. Patient #9 developed advanced pancytopenia in 3 months from initial diagnosis and received red blood cell transfusion regularly. Gene mutations were detected in five out of nine patients with CT8M. In three patients, gene mutations were detected at high (20 to 50%) variant allele frequency (VAF). Patient #2 who was analyzed at the AML phase had gene mutations of SRSF2, SF3B1, STAG2 and NOTCH1. BM sample from patient #9 showed ASXL1 mutation and two TET2 mutations. Patient #4 who did not show apparent myelodysplasia had a high VAF ASXL1 mutation, indicating clonal idiopathic cytopenias of undetermined significance. Two patients had low (2 to 5%) VAF mutations; patient #1 was analyzed after 34 courses of azacitidine had a TET2 mutation; patient #5 with MDS-SLD had a WT1 mutation and two PHF6 mutations. Four patients (#3, #6, #7 and #8) did not have any mutations. The clinical and genetic features showed that CT8M with cytopenia without MDS-related mutations were under 56 years old and did not develop to MDS or stayed at MDS-SLD. Patients with low VAF mutations were also stable. By contrast, patients with advanced diseases gained multiple MDS-related gene mutations with high VAF. One patient without dysplasia had a high VAF ASXL1 mutation. All the patients with gene mutations were age of 56 to 84 years. Conclusion Our results indicated that isolated trisomy 8 may cause cytopenia, but the cytopenia is not exacerbated without MDS-related driver gene mutations. CT8M patients with cytopenia might get gene mutations gradually with age, which leads to MDS or AML. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of HES1. MMP-9 is upregulated by Hes1, and MMP-9 deficiency impaired the development of CML-BC–like disease induced by BCR-ABL and Hes1 in mouse BMT.