De Novo Myelodysplastic Syndromes Research Articles

Age, Gender and Temporal Trends in Solid Tumor As Second Primary Malignancy Risk after De Novo Myelodysplastic Syndrome: A SEER Database Analysis

Age, Gender and Temporal Trends in Solid Tumor As Second Primary Malignancy Risk after De Novo Myelodysplastic Syndrome: A SEER Database Analysis

Soluble PD-L1 Predicts Poor Outcome and Disease Progression in Patients with De Novo Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) consist of a spectrum of myeloid malignancies typified by regular genetic abnormalities, clonal hematopoiesis, dysfunctional myelopoiesis, inefficient blood cell production, peripheral-blood cytopenia, and an elevated risk for progressing to acute myeloid leukemia (AML). Mounting evidence suggests that the dysregulation of immune checkpoints (ICs) plays critical roles in immune evasion of MDS, which thus led to a series of trials using HMAs in combination with immune checkpoints inhibitors (ICIs). Of these, programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway has been studied most extensively. Recently, forms of cell-free soluble PD-1/PD-L1 (sPD-1/sPD-L1) detection in plasm of patients with solid tumors has open a new paradigm for PD-1/PD-L1 investigations. However, within the context of MDS, the potential dysregulation and prognostic impact of sPD-1/sPD-L1 remains uncertain. This study was aimed to assess roles of sPD-1/sPD-L1 in newly diagnosed MDS patients. Method: Between July 2020 and March 2023, 130 MDS/sAML patients matched the inclusion criteria and 55 healthy individuals with no familial or personal history of autoimmune or cancer disorders were included. The criteria for patient inclusion included: (1) a diagnosis of MDS confirmed pathologically according to 2016/2022 World Health Organization (WHO) classification, or sAML patients with a verified prior MDS history; (2) absence of other conditions like autoimmune diseases or different types of tumors; (3) availability of complete clinical information and survival data. MDS patients were categorized by IPSS-R as: lower-risk (very low risk/ low risk; higher-risk (intermediate risk/high risk/very high risk. Follow-up was conducted until June 16th, 2023. The median follow-up was 14.32 months (range 3.2-34.63 months). Profile of sICs (sPD-L1, sPD-1, sCTLA-4, sGITR, sTIM-3, sOX40, sLAG-3, s4-1BB, sICOS) were tested by ELISA, cytokines (IFN-γ, IFN-α2, IL-2, IL-2α, IL-6, IL-7, IL-10, IL-15, IL-17, TNF-α) were detected using Luminex analysis, and the expression levels of PD-1 on T cells (CD3 +, CD4 +, CD8 +, DPT, DNT）were measured using flow cytometry (FCM). Result: In the plasma of 98 newly diagnosed MDS patients, sPD-L1 levels (median 66.80 pg/ml, range 25.00-219.5 pg/ml) were significantly elevated compared to 55 healthy blood donors (median 49.22 pg/ml, range 12.5-218.1 pg/ml). sPD-L1 level was higher in higher-risk IPSS-R groups ( P=0.0380). In addition, higher sPD-L1 levels also showed a correlation with lower hemoglobin concentration, implying a potential link between higher sPD-L1 levels and a more advanced MDS disease state. The AUC for sPD-1 was found to be 0.6333 [95% CI 0.5331-0.7334, P=0.0100] and for sPD-L1, it was 0.6785 [95% CI 0.5836-0.7733, P=0.0002]. The optimal cut-off values were established at 187.5 pg/mL for sPD-1 and 55.08 pg/mL for sPD-L1. Based on these cut-off values, patients with increased sPD-L1 experienced shorter OS ( P=0.0305). By using multivariate Cox model, high expression of sPD-L1 ( P=0.019, HR = 4.172, 95% CI: 1.265-13.755) and high IPSS-R scores ( P=0.030, HR = 2.898, 95% CI: 1.111-7.565) were found to be independent risk factors for newly diagnosed MDS patients. In addition, compared to healthy controls, the PD-1 + T cell subpopulations in MDS patients were significantly reduced, sPD-1 levels were positively correlated with proportion of CD4 + PD-1 + T cells, absolute count of CD4 + PD-1 + T cells and PD-1 + DPT cells, suggesting that CD4 + T cells and DPT cells may be potential sources of sPD-1 in plasma. Subsequently, we analysed the potential roles of cytokines and HMAs in sPD-1/sPD-L1 regulation. It was found that sPD-1 levels had a positive correlation with sPD-L1, IL-2Rα, IP-10, MIG, TNFα, GROα, and TRAIL. sPD-L1 was positively correlated with IL-2Rα, IP-10, MCP-1, MIG, MIP1α, and SCF (Figure 1 A). Notably. We found a reduced sPD-L1 levels in MDS patients who achieved remission after HMA treatment compared to treatment-naïve patients. ( P=0.0302) (Figure 1 B). Conclusions: In conclusion, we found sPD-L1 is an independent risk factors for overall survival in newly diagnosed MDS patients. The elevation of plasma sPD-L1 levels is associated with disease progression in MDS, and may be a potential target for MDS immunotherapy.

High Serum ST2 Level Predicts Progression and Poor Prognosis of Denovomyelodysplastic Syndrome

Background: Myelodysplastic syndromes (MDS) are heterogeneous hematological stem cell malignancies with a complex pathophysiology involving immune, genetic, and epigenetic changes induced by aging, oxidative stress, and genotoxicity. Dysregulated immune responses and aberrant inflammatory signaling have been identified as critical drivers of MDS development. Recent research has highlighted the significant association of the IL-33/ST2 signaling axis with tumorigenicity and tumor immunity in various cancers. Soluble ST2 (sST2), a transmembrane-free receptor induced by IL-33, negatively regulates the IL-33/ST2 signaling pathway, and elevated levels of sST2 have been reported to correlate with poor prognosis in malignant tumors. However, the specific level of soluble ST2 and the precise role of the IL-33/ST2 signaling axis in the context of MDS remain uncertain and require further investigation. Method: This prospective study enrolled 89 newly diagnosed, untreated outpatient individuals with MDS to assess their clinical prognostic risk using the International Prognostic Scoring System-Revised (IPSS-R) and the International Prognostic Scoring System-Molecular (IPSS-M). A control group of 45 healthy donors (HD) was also included. The levels of various cytokines (sST2, IL-33, IL-2Rα, IL-6, IL-15) were measured using ELISA. Result: Our study revealed that sST2 levels in MDS patients were significantly higher compared to HD (22.18 vs. 12.43 ng/ml, p = 0.0313), with no significant difference between low and high-risk groups. The Receiver Operating Characteristic (ROC) curve analysis showed an Area Under the Curve (AUC) of 0.6140 for sST2 (p = 0.0315, 95%CI 0.5199-0.7081). Using the maximum Youden index, the optimal cut-off value for sST2 (>14.71 ng/ml) was determined with a sensitivity and specificity of 51.69% and 73.33%, respectively. Progressive events include conversion to leukemia(n=8), transfusion-dependent occurrence(n=1) and death (n=29). Patients with higher sST2 levels in both high and low-risk groups demonstrated poorer Event-Free Survival (EFS) (p = 0.0193) and Overall Survival (OS) (p = 0.0130) (Figure 1). Further, multivariate analysis identified cytokine sST2(HR = 0.337, 95%CI 0.163-0.696, P=0.003), blast percentage(HR = 0.472, 95%CI 0.237-0.940, P = 0.033), and transfusion dependence(HR = 0.367, 95%CI 0.179-0.753, P = 0.006) as independent risk factors, indicating sST2's potential in predicting prognosis and adverse event risk in MDS patients. Furthermore, sST2 exhibited a significant correlation with other inflammatory cytokines (IL-2Rα, r = 0.4152, p = 0.0005; IL-6, r = 0.4897, p = 0.0013; IL-15, r = 0.3535, p = 0.0253). This suggests that sST2 plays an active role in immune system activation and chronic inflammation. Multiple attempts to detect IL-33 expression in the serum of MDS patients were unsuccessful, indicating the need for a more sensitive and specific assay. In our study, no statistically significant difference was observed in the level of sST2 between bone marrow and peripheral blood (P=0.2197), as well as before treatment and after chemotherapy (P=0.3965). These findings indicate that sST2 is an easily detectable and relatively stable indicator. Conclusions: sST2 emerges as a significant, independent negative prognostic factor at the time of MDS diagnosis, offering valuable predictive insights into disease progression and a poor prognosis.

Single-Hit TP53mut Is Associated with Poor Outcomes in Therapy-Related but Not De Novo Myelodysplastic Syndromes: Importance of Clinical History

BACKGROUND: Recent World Health Organization (WHO) classifications (5 th edition), International Consensus Classification (ICC) recognized TP53-mutated ( TP53mut) myeloid neoplasms (MNs) as a distinct subcategory, acknowledging their uniformly poor outcomes. However, there are critical differences between the two guidelines in how TP53 allelic status is assigned. Both WHO-5 and ICC acknowledged the poor outcome of multi-hit TP53mut but excluded single-hit TP53mut. These changes are driven by a pivotal study demonstrating favourable outcome of single-hit TP53mut, however this study predominantly included de novo myelodysplastic syndromes (MDS) cases (Bernard et al. Nat Med 2020). In contrast, we previously reported poor outcome of TP53mut therapy-related myeloid neoplasm (t-MN) irrespective of the allelic status of TP53 (Hiwase et al Blood 2022andShah et al BCJ 2023). Finally, the ICC removed the subcategory of “therapy-related,” substituting it with diagnostic qualifiers instead. The WHO has grouped t-MN with secondary MN and renamed it as “myeloid neoplasm post cytotoxic therapy,” with the assertion that only multi-hit T P53mut MDS occurring post-cytotoxic therapy have poor outcome compared with single-hit. METHODS: We analyzed one of the largest international cohorts of TP53mut MN (n=544) with full characterization of TP53mut allele status utilizing uniform ICC criteria and similar large numbers of de novo and t-MN cases managed at Mayo Clinic Enterprise (USA) and South Australian MDS Registry, Adelaide (Australia) with clinical, integrated genomic profiling, and long-term follow-up. RESULTS: In our cohort of TP53mut MN (n=544), 475 (87.3%) MN had TP53mut variant allele frequency (VAF) of ≥10% and thus satisfied ICC criteria for TP53mut MN. Majority of TP53mut t-MN presented with MDS (bone marrow (BM) blasts 0-9%) compared de novo MN (62% vs. 48%; P=0.0012). While acute myeloid leukemia (AML) (BM blasts ≥20%) were more prevalent in TP53mutde novo MN compared to t-MN (38% vs. 24%; P= 0.001) and there was no difference in frequency of MDS/AML (BM blasts 10-19%) between the two cohorts (14% vs. 14%; P=0.8) ( Figure 1A). Majority of TP53mut were observed in the DNA-binding domain in t-MN and de novo MN (95% vs. 96.2%; P=0.48). However, mutations in Tetramer Domain were more frequent in t-MN (4.3% vs. 0.3%; P=0.002), whereas those in the TAD2 domain were more frequent in de novo TP53mut MN (2.4 vs. 0%; P =0.006). Integrating the mutation, conventional G-banding cytogenetic, copy number variation (CNV), FISH, and SNP array analysis, TP53mut MDS (BM blast 0-9%; n=260) were classified as multi-hit (n=179, 68.9%) or multi-equivalent (n=35, 13.5%) and single-hit (n=46, 17.7%) (Arber et al. Blood 2022). Striking enrichment of multi-hit/multi-hit equivalent was observed in TP53mut t-MDS compared to de novo MDS (91% vs. 72%; P<0.0001). While single-hit TP53mut were prevalent in de novo MDS compared to t-MDS (28% vs. 9%; P<0.0001). Importantly, median overall survival (OS) of single-hit was significantly longer than multi-hit de novo MDS (25.4 vs. 7.2 months; P=0.0002). In contrast to this we did not observe OS difference between single-hit and multi-hit TP53mut t-MDS (11.6 vs. 10.9 months; P =0.73) ( Figure 1B). CONCLUSION: Our study based on a large international cohort of T53mut MN provides compelling evidence that in contrast to de novo MDS, single hit TP53mut t-MDS is associated with poor outcome. Our data supports the consideration of single hit t-MDS as a multi-hit equivalent.

Soluble PD-L1 Predict Poor Outcome and Disease Progression in Patients with De Novo Myelodysplastic Syndrome

Background: Myelodysplastic syndromes (MDS) are a group of myeloid neoplasms characterized by clonal proliferation of hematopoietic stem cells, recurrent genetic abnormalities, myelodysplasia, ineffective hematopoiesis, peripheral-blood cytopenia, and a high risk of evolution to acute myeloid leukemia (AML). Mounting evidence indicates that dysregulating immune checkpoint pathways are implicated in the pathogenesis of MDS, which thus promoting a number of trials employing immune checkpoints inhibitors (ICIs) in different settings. Soluble ICs (sICs) are soluble forms of ICs, which can be measured from circulation. In solid tumors, sICs have been identified with roles in predicting prognosis and mediating resistance to ICIs. However, in MDS, it is still unclear whether sICs levels are dysregulating in this disease and already has a prognostic influence at the time of diagnosis. Method: Seventy-nine adult patients with untreated de novo MDS were enrolled in this prospective study and managed upon clinical prognostic risk assessment by Revised International Prognostic Scoring System (IPSS-R), 10 healthy donors (HD) were enrolled as control. Profile of sICs (sPD-1, sPD-L1, sCTLA-4, sLAG-3, sTIM-3, sGITR, sOX40, s4-1BB, sST-2) and cytokines (IFN-γ, IFN-α2, IL-2, IL-2α, IL-6, IL-7, IL-10, IL-15, IL-17, TNF-α) were tested by ELISA, and the expression levels of PD-1 on T cells (CD3+, CD4+, CD8+, DPT, DNT）were measured using flow cytometry (FCM). Follow-up time ended on June 21, 2022. Result: MDS patients showed significant higher levels of sPD-1 and sPD-L1 (P＜0.01). Patients with high pretreatment sPD-L1 (>73.79 pg/mL) and high percent of PD-1+CD8+T cells (>7.3%) had worse OS (P < 0.05). Using a multivariate cox regression model adjusting established prognostic factors, including IPSS-R and transfusion dependency, high pretreatment sPD-L1 level was retained as a negative independent prognostic factor for OS (HR 3.151, 95%CI 1.061-9.364, P = 0.039). Moreover, to explore the roles of the PD-1/PD-L1 pathway in the pathogenesis of MDS, we examined the correlations between sPD-1, sPD-L1 and percent of PD-1+CD8+T cells and parameters associating with disease progression (Table. 1). Among clinico-pathological features, male patients showed higher levels of sPD-L1 and percent of PD-1+CD8+T cells, lower hemoglobin (＜80 g/L) significantly correlated to high sPD-L1 levels. In addition, in low- and very low- risk group according to IPSS-R, levels of sPD-L1 were significantly lower than intermediate-, high- and very high- risk group. Furthermore, in previous studies, it was reported that mPD-1/mPD-L1 can be induced by multiple inflammatory cytokines in cancer, including IFN-γ, IFN-α2, IL-2, IL-2α, IL-6, IL-7, IL-10, IL-15, IL-17, TNF-α, and we further tested them in our study. Correlation analysis showed that significant correlations were observed among different sICs as well as cytokines (Figure. 1). In addition, a positive correlation was found between TNF-α and sICs, including sPD-1, sPD-L1 and sST2 (P < 0.05). Conclusions: In conclusion, sPD-L1 was a negative independent prognostic factor for MDS patients at the time of diagnosis. High levels of sPD-L1 were presented in patients with lower hemoglobin and higher risk group, associating with disease progressing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Unique Clinical Features and Mutation Profile in De Novo Myelodysplastic Syndromes with MF-1 and a Proposed Model for Distinguishing MDS with MF-2/3 from TN-PMF Based on Clinical and Genetic Covariates

Unique Clinical Features and Mutation Profile in De Novo Myelodysplastic Syndromes with MF-1 and a Proposed Model for Distinguishing MDS with MF-2/3 from TN-PMF Based on Clinical and Genetic Covariates

De Novo myelodysplastic syndromes in patients 20–50 years old are enriched for adverse risk features

Data concerning the treatment approach and clinical outcomes in younger patients with myelodysplastic syndromes (MDS) are lacking. Furthermore, published results from genomic profiling in the young adult MDS population are few. We identified patients aged 20–50 at diagnosis evaluated for de novo MDS at our institution over a 32-year period. Clinical information and results from sequencing panels were extracted for analysis. 68 eligible patients were found, including 32% with multilineage dysplasia and 29% with excess blasts-2 WHO subtypes. Revised International Prognostic Scoring System for MDS (IPSS-R) categorization had 47% high/very high-risk, and this classification held prognostic significance. The median overall survival was 59 months, and most patients (75%) underwent allogeneic hematopoietic cell transplantation (alloHCT). Thirty-four patients had mutational profiling; the most commonly mutated gene was TP53 and most commonly altered gene category was epigenetic regulators. Younger patients with de novo MDS represented a unique subset with high-risk disease features (adverse cytogenetics, higher R-IPSS) frequently observed along with alterations in TP53 and genes related to epigenetic and transcription pathways.

Serum Level of IFN-a2 and Regulatory T-Cell Subsets Correlates with Disease Progression in De Novo Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a group of diseases with clonal proliferation of dysplastic bone marrow hematopoietic cells. Recent studies have established that the ‘immune signature’ is substantially different in low and high-risk MDS. Nevertheless, the exact mechanism by which the immune response of the disease progresses from an early stage to advanced MDS and acute myeloid leukemia (AML) remain poorly defined.In order to explore the key immune-related factors in the progression of MDS, a total of 56 patients with newly diagnosed MDS were enrolled in this prospective study, where patients were classified as 25 cases of low-risk group (the IPSS-R score, very good risk, good risk and intermediate risk) and 31 cases of high-risk group (the IPSS-R score, very poor risk, poor risk). Levels of 48 cytokines in patients' serum were determined using Luminex assays, and flow cytometry were used to measure the percentages and absolute counts of T regulatory cell (Treg) and its subsets: induced regulatory Treg (iTreg: CD45RO +CD3 +CD4 +CD25 +CD127low +) and naturally regulated Treg (nTreg: CD45RA +CD3 +CD4 +CD25 +CD127low +).Our results showed that there were significant differences in the expression of 19 cytokines between the high-risk group versus low-risk group (Figure 1A). MDS patients in high-risk group had lower serum levels of some interleukins with promoting or amplifying inflammation signals in directly or indirectly way, including IL-1a, IL-2, IL-4, IL-9, IL-12, IL-15 and IL-17ANGF; three growth factors, influencing the immune microenvironment by regulating the growth and differentiation of corresponding cells, including GM-CSF, PDGF-bb and b-NGF; three chemokines including GRO-a, IP-10 and MIP-1b,which regulates the migration of cells to their respective locations to play a pro-inflammatory role; two immunomodulatory cytokines including TNF-b and IFN-a2; leukemia inhibitory factor (LIF) as well as TNF-related apoptosis-inducing ligand (TRAIL) compared to the MDS patients in low-risk group(p<0.05). The higher serum levels of Eotaxin (p= 0.0493), facilitating proliferation and metastasis of tumor cells expressing the it's receptor, was identified in the higher-risk MDS patients. Although there is no significant difference of Treg cells in PB between the two groups, both the percentages and absolute counts of nTreg/iTreg was decreased in high-risk group of MDS patients (Figure 1B, p<0.05). After adjusting the Cox model according to the Revised International Prognostic Scoring System (IPSS-R), age and transfusion dependency, it was found that higher IFN-a2 level was correlated with longer progression-free survival (PFS) (Figure 1C, p=0.038, HR=0.89,95%CI: 0.797~0.994).These results suggested that the pro-inflammatory cytokines during the progression of MDS, the immune disorder of microenvironment and the transformation of nTreg to iTreg may be the key immunological mechanisms for the emergence of immune tolerance and transformation to AML. The serum level of IFN-a2, contributing to inhibit cancer cell proliferation and activate immune system, is an independent protective factor for disease progression or death in patients with MDS, which may be an effective indicator to predict the progression of MDS into higher risk category or transforming into AML. At the same time, it shows that some cytokines in serum are related to the progression and prognosis of MDS, which may provide a basis for early clinical intervention to improve the prognosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Factors Influencing Outcome in De Novo Myelodysplastic Syndromes Treated by Allogeneic Bone Marrow Transplantation: A Long-Term Study of 71 Patients

YELODYSPLASTIC syndromes (MDS) are a group M of clonal hematologic disorders characterized by progressive cytopenia. Some patients subsequently develop acute myeloid leukemia (AML), especially if they have an excess of marrow blast cells at diagnosis.',' Factors associated with poor survival include an advanced French-American-British (FAB) subtype or a high percentage of bone marrow (BM) blast cells, the presence of peripheral blood (PB) blast cells, cytopenia, an abnormal karyotype and advanced age. The combination of these different factors has been used by several authors to define scoring systems, which can segregate patients into subgroups with survival ranging from several months to several The median survival of patients with profound cytopenia or an excess of marrow blast cells does not exceed 2 years, even among young patients capable of tolerating intensive chemotherapy.6 In addition, when therapy-related, MDS and AML are rapidly life-threatening.' Intensive cytoreductive chemotherapy, low-dose cytarabine, differentiating agents such as retinoic acid, and growth factors have proven incapable of preventing death, although transient corrections of cytopenia have been reported.6.8-'0 Allogeneic BM transplantation (BMT) is the only treatment known to cure such patients, provided they are young (<50 years) and have a compatible donor. Several teams have published disease-free survival (DFS) rates of 35% to ~~ ~

Peripheral T-cell Lymphoma, NOS With Epstein-Barr Virus Positivity in an Elderly Patient With Myelodysplastic Syndrome: An Autopsy Case Report

Abstract Casestudy Epstein-Barr virus (EBV)-associated peripheral T-cell lymphomas are a group of aggressive neoplasms with a geographic predilection for South America and Asia, but are very rare in Western populations. Results We report a case of a 74-year-old Caucasian female who presented with pancytopenia and B symptoms with EBV-IgG detected on admission. Past medical history included: ITP, chronic urticaria, and recently diagnosed myelodysplastic syndrome (MDS) on bone marrow biopsy one month prior to admission. Excisional biopsies of an enlarged right neck lymph node (repeated within 6 months) and right axillary lymph node five years ago were negative for a lymphoproliferative disorder at the time. Repeated bone marrow biopsy, performed during the current admission, confirmed the diagnosis of MDS, with scattered T-cells without aberrant immunophenotype. Despite aggressive treatment from multiple specialties, the patient deteriorated and expired four weeks later from complications of MDS. At autopsy, there was diffuse lymphadenopathy involving the mediastinum, axilla, pelvis and peripancreatic fat. Lymph node sections demonstrated nodal architecture effacement by diffuse, vaguely nodular lymphoid infiltrates. Histologically, the infiltrates were composed of medium to large lymphocytes with round to slight irregular nuclei, rare Reed-Sternberg-like multinucleated cells, clumped chromatin, and indistinct nucleoli. Individual cell necrosis was abundant with mitotic figures readily identifiable. Immunohistochemistry revealed CD2+ CD3+ neoplastic T-cells that co-express MUM1 and a subset of CD30, while negative for CD4, CD5, CD8, CD56, ALK1, and TDT. EBV-encoded RNA in-situ hybridization was focally positive. The final postmortem diagnosis was peripheral T-cell lymphoma, not otherwise specified (NOS), with focal EBV positivity. Conclusion Co-existence of a de-novo MDS and non-Hodgkin lymphoma without any prior chemotherapeutic exposure is a highly unusual finding, although MDS-like presentations can occur with EBV-associated lymphomas. Peripheral T-cell lymphoma, NOS is an aggressive lymphoma and EBV positivity has been found correlated with a poor prognosis. This case demonstrates how postmortem examination remains an important tool in clinical- pathological correlation and highlights the potential pathogenetic role EBV plays in MDS and T-cell lymphoma.

De Novo Myelodysplastic Syndromes in Patients 20-50 Years Old Characterized By Frequent Mutations in TP53 and Transcription-Related Genes

De Novo Myelodysplastic Syndromes in Patients 20-50 Years Old Characterized By Frequent Mutations in TP53 and Transcription-Related Genes

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

De Novo and Therapy-Related Acute Myeloid Leukemia and Myelodysplastic Syndrome: Similarities and Differences in SNP-Array Detected Chromosomal Aberrations in Pre-Transplant Blood Samples

Germline and Acquired Genetic Variants in Myelodysplastic Syndromes in Young Adults without a Preexisting Disorder or Organ Dysfunction

Background and Aim:It is increasingly recognized that patients with a de novomyelodysplastic syndrome (MDS) onset as young adults, lacking any other feature of a congenital disorder, may share a pathogenic overlap due to the presence of both germline and somatic variants. Identifying an inherited pathogenic variant has important therapeutic implications beyond family counselling: adapting the selection of sibling donor, the use of highly cytotoxic therapy and the monitoring for other cancer development. However, most studies have focused on patients with suspected inherited disorders based on the presence of physical abnormalities and/or family history. In addition, a mixture of pediatric and adult cases is usually reported. The aim of this study is to characterize the germline and tumor variants in a group of adult MDS patients without accompanying congenital physical anomalies and or family antecedent of bone marrow failure.Methods: We included 72 patients from 15 Spanish centers with a diagnosis of MDS between 18 and 60 years old (y.o). Patients with a previously diagnosed or suspected (one physical anomaly or family history) congenital syndrome were excluded. Diagnoses were made in accordance with the WHO 2016 classification. Whole-exome sequencing (WES) libraries were prepared using SureSelectXT Target Enrichment and sequenced on a HiSeq4000 platform (IlluminaInc.). Mean number of reads per sample was 138,726,017 with a Phred Quality Score >30 in 95.05% of bases. Read mapping sequence alignment and variant calling were performed using Biomedical Workbench (Qiagen). WES was performed on 72 tumor and 32 paired germinal DNA (buccal swab). To identify potential germline-causal mutations, a selection tool was implemented incorporating 239 genes associated with cause or predisposition to bone marrow failure or cancer. Variants with an ExAC, TOPMed and/or European 1000 Genomes minor allele frequency ≥0.01 were discarded.Results: The median age at diagnosis was 49 y.o. The cohort was categorised into two groups, less or equal 50 y.o. (62.5%) and between 50 and 60 y.o. (37.5%). In the first group, the frequency according to the WHO classification were 12% MDS with single lineage dyplasia (MDS-SLD), 8% MDS with ring sideroblasts (MDS-RS), 11% MDS with multilineage dyplasia (MDS-MLD), 24% MDS with excess blasts(MD-EB), 4% MDS with isolated del(5q)(MDS-del5q), 4% MDS unclassifiable and 4% chronic myelomonocytic leukemia (CMML). Meanwhile, in the group with age more than 50 y.o., the subtypes were 3.7% MDS-SLD, 7.4% MDS-RS, 29.6% MDS-MLD, 40.7% MD-EB, 3.7% MDS-del5q, and 14.8% CMML.Patients less or equal 50 y.o. were stratified based on IPSS-R as very low (4%), low (64%), intermediate (20%), high (12%) and very high (0%); and the group of more than 50 y.o. as very low (14.8%), low (33.3%), intermediate (29.6%), high (11.1%) and very high (11.1%).The mean number of somatic mutations was 0.68 in patients with less or equal 50 y.o. and 1.37 in those between 50 and 60 y.o., p=0.033 (U Mann-Whitney); and regarding germline variants, the first group mean number was 2.44 (p25-75, 1-3) and the second group showed a mean of 1.85 (QI 25-75, 1-3), p= 0,331.In the whole cohort, germline variants were found in 62 out of 72 patients, with the following frequencies: ATR(N=5, 6.9%), followed by BARD1(N=5, 6.9%), ERCC6L2(N=4, 5.6%), MSH6(N=4, 5.6%), TCIRG1(N=4, 5.6%), NBEAL2(N=4, 5.6%), ASXL1(N=3, 4.2%), ATM(N=3, 4.2%), MPL(N=3, 4.2%), NF1(N=3, 4.2%), RECQL4(N=3, 4.2%), SAMD9L(N=3, 4.2%), WRN(N=3, 4.2%).Among germline variants, those reported previously as pathogenic or likely pathogenic, or involving genes associated with familial MDS/AML included: ERCC6L2(N=4, 5.6%), SAMD9L(N=3, 4.2%), and one case mutated for DDX41, FANCC, JAK2, MSH6, SETBP1, MUTYH, BRCA1and RECQL4. In the whole cohort, somatic variants were found in 38 patients, with the following frequencies: TP53(N=7, 9.7%), ASXL1(N=7, 9.7%), SETBP1(N=5, 6.9%), NF1(N=5, 6.9%), SRSF2(N=4, 5.5%).Conclusion:In this subset of young adults with de novo MDS without congenital anomalies and/or familial history suggesting the presence of an undiagnosed congenital syndrome, 18% of the cohort harbored a likely causative germline variant. In addition, we noted a predominance of variants affecting genes with a cancer predisposition limited to the hematopoietic system, rather than classical telomere, DNA damage genes with an established mendelian link. [Display omitted] DisclosuresDíez-Campelo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

The Role of FLT3-ITD Mutation on de Novo MDS in Chinese Population

BackgroundFLT3 mutations have been well-studied in acute myeloid leukemia (AML), and the detection of the FLT3 gene has become a clinical routine. However, it has not been fully analyzed in other hematologic malignancies, such as myelodysplastic syndromes (MDS). Materials and MethodsBetween 2010 and 2016, 304 adult patients with de novo MDS had the FLT3 sequence tested on their bone marrow sample. With 279 patients who had follow-up information, we also analyzed the impact of clinical and laboratory characteristics as well as FLT3 mutation status and treatment on prognosis. ResultsWe found that the transformation rate was 3 (42.9%) of 7 patients in the FLT3-ITD-positive group, compared with 31 (10.4%) of 297 among FLT3-ITD-negative patients (P = .033). The median progression-free survival of the FLT3-ITD mutated and wild-type groups were 43 days and 363.5 days, respectively (P < .0001). The median overall survival (OS) of the 2 groups were 218 days and 410.5 days, respectively (P < .0001). We also found that 5 factors had independent prognostic impact on OS: white blood cell counts, bone marrow blast percentage, cytogenetics, transplantation status, and FLT3-ITD mutation. Furthermore, compared with the transformation group, the non-progression group was younger (P = .034), with a lower platelet count (P = .022), a lower bone marrow blast percentage (P = .001), a lower FLT3-ITD incidence (P = .007), and a longer OS (P < .0001). ConclusionsWhen observed at the MDS stage, patients harboring FLT3-ITD mutations had higher AML transformation rate, quicker disease progression, and shorter survival than wild-type patients. Nevertheless, once the disease progressed to leukemia, the impact of FLT3-ITD mutations on prognosis was slight. In addition, the prognosis of secondary AML was very poor whether there was an FLT3-ITD mutation or not.

Comparable Outcomes of Therapy-Related and De Novo Myelodysplastic Syndrome after Allogeneic Hematopoietic Stem Cell Transplantation

Therapy-related myelodysplastic syndrome (t-MDS) is a well-known complication of conventional chemoradiotherapy used to treat a variety of hematologic malignancies as well as solid tumors. Because of poor prognosis of t-MDS when treated with conventional therapy, allogeneic hematopoietic stem cell transplantation (HSCT) is usually recommended with encouraging results reported.Herein, we retrospectively analyzed outcomes in of 309 adult patients with t-MDS (n =101) and de novo MDS (n=208) receiving allogeneic HSCT from 2005 to 2015 at MD Anderson Cancer Center and compared their outcomes adjusted for disease characteristics.The study cohort had median age of 59 (range, 18-77) at HSCT. Histological subtype at diagnosis was RAEB-1 and -2 in 120 (39%) and CMML in 30 (10%) patients. When cytogenetics were classified by monosomal karyotype (MK) defined by Breems et al., 87 patients (27%) had MK-positive (MKpos), 99 patients (32%) MK-negative (MKneg) and 100 patients (32%) good risk cytogenetics (including normal karyotype (n=88) and del(5q), del(12p), del(20q) (n=12)). Donors were matched related donor (MRD, n=177), matched unrelated donor (MUD, n=114) or haploidentical donor (n=18). Most patients received a myeloablative conditioning regimen (n=181, 59%). Hematopoietic stem cell comorbidity index (HCT-CI) was ≥3 in 144 (47%) patients.Among t-MDS and de novo MDS; median age, donor type and conditioning intensity were not different (Table 1). Compared with de novo MDS, t-MDS patients had more MKpos (20% vs. 46%, p<0.001) less good risk cytogenetics (38% vs. 10%, p<0.001) and more HCT-CI ≥3 (31% vs. 79%, p<0.001). The median time from diagnosis to HSCT was shorter in t-MDS (6 mo. vs. 9 mo., p<0.001).The median follow up of 139 survivors was 37 (range, 3-125) months. Outcome analyses were stratified for t-MDS and de novo MDS patients due to different disease characteristics observed.Univariate analyses showed that MKpos was associated with a poor prognosis for the cumulative incidence of progression both in t-MDS and de novo MDS compared with good cytogenetics (HR=4.3, p=0.05, HR=6.9, p<0.001, respectively) (Figure 1). BM blast count ≥3% at HSCT was also associated with increased incidence of progression in both groups (HR=1.5, p=0.2, HR=1.9, p=0.02, respectively). This enabled us to analyze t-MDS and de novo MDS together for multivariate regressions which revealed that MKpos patients with BM blast count ≥3% had the highest risk for disease progression compared with patients without MKpos and with BM blast count <3% (HR=5.8) (Table 2). Patients with MKpos but with BM blast count <3% also had higher incidence of progression (HR=3.9). The outcomes by risk groups for progression was comparable for t-MDS and de novo MDS (Data not presented).For progression free survival (PFS), univariate analyses revealed that MKpos had poor prognosis for both in t-MDS and de novo MDS compared with good-risk cytogenetics (HR=2.9, p=0.025 and HR=8.4, p<0.001, respectively). MKneg were also associated with inferior PFS compared with good-risk cytogenetics in both t-MDS and de novo MDS (HR=2.1, p=0.1 and HR=2.3, p=0.001, respectively). BM blast count ≥3% also decreased PFS both in t-MDS and de novo MDS for PFS (HR=1.6, p=0.07 and HR=1.8, p=0.004, respectively). When t-MDS and de novo MDS were analyzed together for multivariate regressions, we found that MKpos patients with BM blast count ≥3% had lowest PFS while patients good risk cytogenetics had the best outcomes independent of their BM blast count (Table 2). There was no impact of t-MDS on PFS and similar to progression, the outcomes were not inferior in t-MDS (Data not presented).In conclusion, although t-MDS has been felt to have a relatively poor prognosis after HSCT, our results suggest that t-MDS has comparable outcomes with de novo MDS after allogeneic HSCT when adjusted for disease characteristics. Patients with MKpos in t-MDS and de novo MDS have very high incidence of disease progression and low PFS and represent a target population for innovative transplant strategies. [Display omitted] [Display omitted] [Display omitted] DisclosuresCiurea:Cyto-Sen Therapeutics: Equity Ownership; Spectrum Pharmaceuticals: Other: Advisory Board.

Clonal Evolution of Gene Mutations in AML Transformation from Patients with De Novo Myelodysplastic Syndromes Carrying Spliceosome Mutations: An Analysis of 71 Paired Samples

Background and Purpose: The molecular pathogenesis of progression of myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (sAML) remains incompletely understood. We studied genemutations involving spliceosome in the transformation of MDS to sAML by comparing matched paired MDS/sAML bone marrow samples to determine the roles of SRSF2, U2AF1 SF3B1, and ZRSR2 mutations in the evolution of MDS to sAML.Patients and Methods: One hundred and forty-nine de novo MDS patients (2 RCUD, 5 RARS, 27 RCMD, 52 RAEB-1, and 63 RAEB-2) were examined forspliceosome mutations at initial diagnosis and 93 patients progressed to sAML with a median follow-up of 16.4 months. Seventy one of them had paired MDS/sAML bone marrow samples available for comparative analyses. Mutational analyses of spliceosome were performed by pyrosequencing with a detection sensitivity of 5% for U2AF1 (exons 2 and 6) and SRSF2 (P95 of exon 1) mutations and by direct sequencing for SF3B1 (exons13-16) and ZRSR2 (whole coding exons 1-11) mutations. Additional 29 gene mutations known to involve in myeloid neoplasms were also analyzed by direct sequencing or next-generation sequencing (NGS, Ion Torrent PGM) followed by Sanger sequencing validation; NGS was mainly used for mutation detection of epigenetic regulators and cohesin complex. The allele burden of targeted genes was determined by pyrosequencing and/or NGS.Results: The frequencies of U2AF1, SRSF2, SF3B1, and ZRSR2 mutations in the 149 MDS patients were 14.8% (22/149), 12.8% (19/149), 12.1% (17/141), and 7.5% (10/133), respectively. Together, spliceosome mutations occurred in 51.5% of MDS patients at initial diagnosis and were mutually exclusive. All the 5 RARS patients had SF3B1 mutations. Co-existed mutations with epigenetic regulators were detected in 36 patients (52.9%), with RUNX1 in 13 (19.1%), with cohesin complex including STAG2, SMC3, RAD21,or SMC1A, in 13 (19.1%), with signaling pathways including RAS, PTPN11, JAK2, or FLT3-TKD in 4 (5.9%), BCOR in 2, and one each with CEBPa and SETBP1. There were no differences in blood counts, percentage of blasts in bone marrow and blood, WHO subtype, cytogenetic risk group, and IPSS-R between spliceosome-mutated and -unmutated patients. Of the patients carrying spliceosome mutations, only SRSF2 mutation had prognostic impact on predicting a higher risk of sAML transformation (P = 0.025) and sAML-free survival (median 10.8 months, 95% CI 5.1-16.5 months) compared to SRSF2-unmutated patients (median 17 months, 95% CI 8.5-25.5 months, P = 0.050). Of the 71 paired MDS/sAML samples, 37 (52.1%) had spliceosome mutations at diagnosis; the mutational status and patterns remained unchanged in all the 37 matched sAML samples but allele burden was apparently increased in the sAML samples with SRSF2 (P = 0.017) or SF3B1 (P = 0.015) mutations. In addition, one ZRSR2 mutant clone and two SRSF2 mutant clones evolved during sAML progression. Acquisition of other mutated genes was found in 37 spliceosome-mutated patients at sAML phases, including RUNX1 in 5, N-RAS in 5, CEBPa in 4, K-RAS in 3, FLT3-ITD in 3, and one each with ASXL1, TET2, STAG2, WT1, PTPN11, CBL, FLT3-TKD, and C-FMS. Notably, of the 3 patients acquiring spliceosome mutations, none gained other mutated genes during sAML transformation.Clonal expansion of other mutated genes were observed in 4 cases with RUNX1, in 2 with TET2, and one each with N-RAS, CEBPa, ASXL1, SMC1A, and STAG2.Conclusions: Our results showed that spliceosome mutations occurred in more than half of de novo MDS patients at initial diagnosis. Clonal expansion, evolution, or unchanged allele burden of spliceosome mutations might occur during sAML transformation with frequent acquisition of additional mutated genes. SRSF2 mutation predicted a higher risk and more rapid sAML transformation. (Grants support: NHRI-EX103-10003NI, MOHW103-TD-B-111-09 and CMRPG3D1532) DisclosuresNo relevant conflicts of interest to declare.

Spectrum of the WHO Classification De Novo Myelodysplastic Syndrome: Experience from Southern Pakistan.

Myelodysplastic syndrome (MDS) is a clonal disorder of hemopoeitic stem cells, characterized by infective hematopoiesis, peripheral cytopenias along with hypercellularity of marrow and marked dysplastic features. Our aim was to study the spectrum of the WHO classification in adult Pakistani patients with MDS at disease presentation. This retrospective descriptive study was conducted at Liaquat National Hospital and Medical College, extending from January 2010 to December 2014. Patient data were retrieved from the maintained archives. Overall, 45 patients were diagnosed at our institution with de novo MDS during the study period. There were 28 males and 17 females. Age ranged between 18 and 95 years with a mean of 57.6±17.4 years. The male to female ratio was 1.7:1. According to the WHO classification, 53.3% had refractory cytopenia with multilineage dysplasia, 22.2% had refractory cytopenia with unilineage dysplasia, 4.4% each had refractory anemia with excess of blasts-1 and II and 15.5% had MDS unclassified. The main presenting complaints were generalized fatigue (60%), fever (33.3%), dyspnea (15.5%), bleeding (13.3%) and weight loss (11.1%). Physical examination revealed pallor in 37.7%, followed by petechial and purpuric rashes in 20% of patients. Hemoglobin was <10 g/dl in 41 (91.1%). Pancytopenia and bicytopenia were noted in 18 (40%) and 14 (31.1%) respectively. MDS in our patients presents at a relatively young age. Refractory c ytopenia with multilineage dysplasia was the dominant disease variant in our setting.

Phenotypic and Cytogenetic Characterization of Mesenchymal Stromal Cells in De Novo Myelodysplastic Syndromes.

Bone marrow (BM) mesenchymal stem/stromal cells (MSCs) are vital in hematopoiesis. Whether BM-MSCs alter their characteristics in Myelodysplastic Syndromes (MDS) is still controversial. We characterized MSCs of de novo MDS patients in Sri Lanka who have not been reported previously in the literature. We also analyzed MSCs derived from different MDS subtypes. MSCs were culture-expanded, characterized by flow cytometry, and induced towards osteogenic and adipogenic differentiation. Growth properties were determined using growth curves and population doubling times. Karyotyping and FISH were performed on MSCs. Cell morphology, differentiation potential, and CD marker expression of MDS-MSCs of all subtypes were comparable to those of control-MSCs. No significant growth differences were observed between control MSCs and MDS-MSCs of all subtypes (p > 0.05). 31% of MDS-MSCs had chromosomal aberrations (der(3),del(6q),del(7p), loss of chromosomes) whose BM karyotypes were normal. Highest percentage of karyotypic abnormalities was observed in RCMD-MSCs. Patients with abnormal BM karyotypes had no aberrant MSC clones. Results show that in spite of presence of genetically abnormal clones in MDS-MSC populations, in vitro phenotypic and growth characteristics of MSCs in MDS remain unchanged. Further, the occurrence of genetic abnormalities in BM-MSCs in MDS could be considered as an autonomous event from that of their hematopoietic counterparts.

Long Non-Coding RNA Induces De Novo Myelodysplastic Syndrome through Epigenetic Regulation

Long non-coding RNAs (lncRNAs) form the largest part of the mammalian non-coding transcriptome and control gene expression at various levels including chromatin modification, transcriptional and post-transcriptional processing. LncRNAs are implicated in initiation and progression of several cancers.Cancer-associated genomic regions are regions showing high frequency of cancer related abnormalities, such as loss of heterozygosity or amplifications. One such widely studied CAGR is the 8q24.21 genomic region. One SNP of particular importance present at this locus is rs6983267, with the G allele of the SNP conferring increased risk of colorectal, prostate, breast and bladder cancers. CCAT2 is a lncRNA that spans this highly conserved region. CCAT2 has been shown to play an important role in inducing chromosomal instability and supporting cell proliferation and cell cycle arrest. Despite advances in diagnosis of MDS patients, the underlying mechanisms that lead to spontaneous induction of MDS remains poorly understood. Here we attempted to elucidate the role of CCAT2 and its specific alleles (G/T) in regulation of cellular processes that drive spontaneous tumorigenesis using a genetically engineered mouse model. We generated transgenic mice for each CCAT2 allele using random integration approach in C57Bl6/N background, expressing CCAT2 in all tissues of mice.In this study, we identified that CCAT2 plays an important role in regulation of normal hematopoiesis. Constitutive in vivo overexpression of each CCAT2 transcript in the mice resulted in spontaneous induction of widespread pancytopenias. CCAT2(G/T) BM biopsies displayed severe myeloid or erythroid hyperplasia, and dysplastic megakaryocytic proliferation, along with enhanced proliferation and excessive apoptosis. Interestingly, we identified two distinct phenotypes in CCAT2(G/T) mice with equal prevalence of MDS or mixed MDS/MPN. This suggests that CCAT2 overexpression might affect regulation of hematopoietic stem cells, disturbing their self-renewal or maturation capacity, and subsequently resulting in BM failure. Percentage of HSPCs was significantly reduced in BM of MDS mice, with increased presence of immature erythroid blasts and granulocyte-macrophage progenitors suggesting a block in differentiation. HSPCs of CCAT2(G/T) mice also showed increased frequency of cytogenetic aberrations, including breaks and chromosomal fusions. However, these mice don't develop sAML, suggesting CCAT2 is critical in initiation of MDS. We further identified significantly higher CCAT2 expression in the MDS patients as compared to healthy volunteers. Patients with sAML had significantly lower expression of CCAT2 as compared to patients with only MDS.To determine the mechanism by which CCAT2 induces genomic instability and myelodysplasia, we screened for several genes that have been previously reported to induce myelodysplasia as potential targets of CCAT2. Interestingly, EZH2 was downregulated in the BMCs of CCAT2(G/T) mice compared to WT littermates. EZH2 downregulation was observed in both MDS only and MDS/MPD mice. In CCAT2(G/T) mice, EZH2 and H3K27Me3 reduction was observed in hematopoietic stem and progenitor cells (HSPCs) as well as lineage positive bulk cells,suggesting that CCAT2 might induce alteration in EZH2 levels in the HSC compartment. Interestingly, we identified miR-26a and miR-26b, that were already reported to target EZH2, were significantly overexpressed only in BM of CCAT2-G mice. These data suggests that CCAT2-G regulates EZH2 expression primarily through regulation of target miRNAs. On the other hand, we identified EZH2 to interact preferentially to CCAT2-T compared to WT or CCAT2-G transcript. These data confirmed that EZH2 preferentially binds to the CCAT2 in an allele-specific manner.In conclusion, deciphering the role of CCAT2 in spontaneously induced myelodysplasia and cytopenias will help us further characterize the poorly understood MDS/MPN phenotype. CCAT2 mice can serve as a robust model for studying initiation of de novo MDS/MPN that does not progress to secondary AML, and as a pre-clinical model for evaluation of new therapies for MDS. It has high translational potential as CCAT2 can be developed into a diagnostic and prognostic marker, as well as a novel intervention target for MDS therapy. DisclosuresNo relevant conflicts of interest to declare.

Myelodysplastic Syndromes in Pakistani Population-Analysis of 52 Cases and their Outcome on Best Available Treatment Options

The Myelodysplastic syndromes (MDS) are clonally evolved bone marrow disorders leading to the bone marrow failure, peripheral cytopenia and a propensity for progression to acute myeloid leukemia (AML). To observe the trend and outcome of MDS in Pakistani population, a retrospective data analysis was carried out in National Institute of Blood Diseases & Bone Marrow Transplantation, from January 2010 to December 2014. Fifty-two MDS or MDS/MPN cases were analyzed out of which39 (75%) were males. Median age at the time of diagnosis was 60 years. Secondary/t-MDS was observed in 2 (3.8%) cases while others were de-novo MDS. Low intensity treatment was offered to 16 (30.7%) cases out of which 5 (31.25%) cases responded to treatment. Full HLA matched related donor allogenic HSCT was unsuccessfully attempted in 2 (3.8 %) cases. At the time of analysis only 19cases were alive after a median follow up 10 months (range 2 months to 48 months). Median progression free survival was 1.9 months for very high risk, 6 Months for high risk, 12.8 months for intermediate risk and 39 Months for very low and low risk while median overall survival was 3 months for very high risk, 8.5 months for high risk and 18.7 months for intermediate risk. All patients were alive in very low and low risk category when study was concluded.