Topic: 32. Platelet disorders Background: ITP can be classified as primary or secondary based on the absence or presence of other causes or disorders associated with thrombocytopenia. Although these forms of ITP have been traditionally considered as distinct disease entities, comparative studies are rare and the differences and similarities between primary and secondary ITP have not been adequately elucidated thus far. Aims: To comparatively describe real world characteristics at diagnosis and evaluate disease outcome in patients with primary (Group-1) and secondary ITP (Group-2), using data from the national database (ITP registry) operated under the auspices of the Hellenic Society of Hematology. Methods: The Greek ITP registry recruits patients (n=1573) nationally through a network of 25 sites. In the present study, we retrospectively analyzed data from patients with primary and secondary ITP, aged over 18 years who were diagnosed from 1979 to 2022. Results: The number of evaluable patients was 580. Group-1 consisted of 464 (80%) and Group-2 of 116 (20%) patients. There were no differences in the median age, gender, the median platelet count or other hematological parameters between the 2 groups at diagnosis. Significantly fewer patients in Group-1 had comorbidities as compared to Group-2 (P=0.0206). Relevant medications were concurrently used at similar rates by the 2 patient groups. Bleeding manifestations were observed at a similar frequency between the 2 groups. Of those who were diagnosed as secondary ITP, 26.8% had infections, 24% had collagen disorders, 8.3% had Evans Syndrome, 5.5% had H.Pylori infection, 4.6% had Hashimoto thyroiditis and 3.6% antiphospholipid syndrome. In 3.7% of Group-2 patients ITP was drug-induced. Antiphospholipid antibody (Ab), platelet associated Ab, antinuclear Ab, HIV Ab and Rheumatoid Factor testing were performed more frequently in Group-2 patients. A significant higher proportion of Group-2 patients had a positive test for antinuclear antibodies, HCV Abs, antiphospholipid Abs and H.Pylori (P=0.0185, P=0.0004, P=0.0002 and P<0.0001, respectively). Fewer Group-1 patients received treatment at diagnosis (P=0.023). The choice of treatment at diagnosis did not differ between Group-1 and Group-2 patients with the exception of anti-CD20 which was administered more frequently in Group-1 patients (P=0.036). Overall response rates did not differ between the two groups. At 6 months post diagnosis significantly fewer Group-1 patients had bleeding manifestations and fewer Group-1 patients were treated with corticosteroids, IVIG, corticosteroids and IVIG and anti CD20 (P<0.0001, P=0.0144, P=0.0005 and P=0.039, respectively). At 6 months post diagnosis significantly fewer Group-1 patients had developed persistent ITP (p<0.005). Summary/Conclusion: The majority of adult Greek ITP patients suffered from primary ITP (Group-1). Secondary ITP (Group-2) was predominantly associated with collagen disorders and infections. Primary and secondary ITP patients did not differ in demographics, bleeding manifestations, and hematological parameters. However, patients with secondary ITP had a higher frequency of auto Abs. The form of ITP did not impact on the choice of initial treatment or the response rate but influenced the diagnostic work-up and the outcome of the disease, as suggested by the increased proportion of secondary ITP patients requiring treatment and developing persistent ITP at 6 months post diagnosis. Further investigation is warranted to probe more deeply into the differences and similarities of primary and secondary ITP in order to optimize management. Keywords: ITP, Immune thrombocytopenia (ITP)
Abstract Background As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine treated patients. Although epigenetic mechanisms are assumed, responding patients’ epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. Results Serial HbF measurements by high performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.019). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression free survival (PFS) (p = 0.032, HR = 0.19, CI:0.24–1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, rs=-0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site − 53 of HBG2 promoter (p = 0.039, rs=-0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, rs=0.482), a known HbF repressor. These changes were associated to blast cell clearance (pHBG2=0.011, rs=0.480/ pZBTB7A=0.026, rs=0.427) and showed prognostic value for PFS (pZBTB7A=0.037, HR = 1.14, CI:0.34–3.8). Conclusions Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in depth azacytidine epigenetic mechanism of action.
Acute Erythroid Leukemia (AEL) is a rare and aggressive subtype of Acute Myeloid Leukemia (AML). In 2022, the World Health Organization (WHO) defined AEL as a biopsy with ≥30% proerythroblasts and erythroid precursors that account for ≥80% of cellularity. The International Consensus Classification refers to this neoplasm as “AML with mutated TP53”. Classification entails ≥20% blasts in blood or bone marrow biopsy and a somatic TP53 mutation (VAF > 10%). This type of leukemia is typically associated with biallelic TP53 mutations and a complex karyotype, specifically 5q and 7q deletions. Transgenic mouse models have implicated several molecules in the pathogenesis of AEL, including transcriptional master regulator GATA1 (involved in erythroid differentiation), master oncogenes, and CDX4. Recent studies have also characterized AEL by epigenetic regulator mutations and transcriptome subgroups. AEL patients have overall poor clinical outcomes, mostly related to their poor response to the standard therapies, which include hypomethylating agents and intensive chemotherapy. Allogeneic bone marrow transplantation (AlloBMT) is the only potentially curative approach but requires deep remission, which is very challenging for these patients. Age, AlloBMT, and a history of antecedent myeloid neoplasms further affect the outcomes of these patients. In this review, we will summarize the diagnostic criteria of AEL, review the current insights into the biology of AEL, and describe the treatment options and outcomes of patients with this disease.
The unique heterogenous landscape of myelodysplastic syndromes/neoplasms (MDS) has resulted in continuous redefinition of disease sub-entities, in view of the novel translational research data that have clarified several areas of the pathogenesis and the progression of the disease. The new international classifications (WHO 2022, ICC 2022) have incorporated genomic data defining phenotypical alterations, that guide clinical management of specific patient subgroups. On the other hand, for over a decade, multiparameter flow cytometry (MFC) has proven its value as a complementary diagnostic tool for these diseases and although it has never been established as a mandatory test for the baseline evaluation of MDS patients in international guidelines, it is almost universally adopted in everyday clinical practice for the assessment of suspected cytopenias through simplified scoring systems or elaborate analytical strategies for the detection of immunophenotypical dysplastic features in every hematopoietic cell lineage in the bone marrow (BM). In this review, we explore the clinically meaningful interplay of MFC data and genetic profiles of MDS patients, to reveal the currently existing and the potential future role of each methodology for routine clinical practice, and the benefit of the patients. We reviewed the existing knowledge and recent advances in the field and discuss how an integrated approach could lead to patient re-stratification and guide personalized management.
Abstract Treatment decision and response assessment in myelodysplastic syndromes (MDS) can be enhanced by the implementation of advanced diagnostic and prognostic assays for the detection of multiple molecular features. Higher-risk (HR) MDS, ineligible for allogeneic hematopoietic stem cell transplantation (alloHSCT), require prompt therapeutic interventions such as treatment with hypomethylating agents (HMAs) to restore normal DNA methylation levels, mainly of oncosuppressor genes and consequently to delay disease progression and increase overall survival (OS). However, response assessment to HMA treatment relies on conventional methods with limited capacity to uncover a wide spectrum of molecular events. We studied bone marrow aspirates from twenty-one HR MDS patients pre- and post-HMA treatment and seven healthy controls. Genomic DNA was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 5’ methyl-cytosine (5mC), 5’ hydroxy-methyl cytosine (5hmC) levels detection, and global adenosine/thymidine ([dA]/[T]) ratio, to correlate differences during treatment course and at baseline state prior drug therapy. Results from methylation DNA sequencing (MeD-seq) from the same HR MDS cohort were also analyzed to identify targeted differentially methylated regions (DMRs). LC/MS-MS analysis revealed a significant hypomethylation status in responders (Rs) already established at baseline and a trend for further DNA methylation reduction post-HMA treatment. Non-responders (NRs) reached statistical significance for DNA hypomethylation only post-HMA treatment. MeD-seq confirmed results globally for both Rs and NRs and more specifically, identified DMRs associated with HMA treatment. Additionally, within statistically significant selected chromosomal bins, genes encoding for proteins and non-coding RNAs were highlighted with reversed methylation profiles between Rs and NRs. Dynamic DNA methylation changes in HR MDS patients undergoing HMA therapy demonstrated that response to treatment is associated only with few specific hypomethylated DMRs rather than presenting a global effect across genome. The 5hmC epigenetic mark was only rarely detected in Rs and NRs, in contrast to healthy controls. Global [dA]/[T] ratio was lower in both R and NR subgroups compared to controls suggesting high frequences of baseline transitions from 5mC to thymidine. Conclusively, LC-MS/MS methodology provided broad-based but rapid and cost-effective results on the molecular HR MDS background, potentially translatable into responsive phenotypes to HMA treatment.
Abstract Background As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients’ epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. Results Serial HbF measurements by high-performance liquid chromatography ( n = 20) showed induction of HbF only among responders ( p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) ( p = 0.032, HR = 0.19, CI 0.24–1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis ( n = 7) for FOG-1, RCOR-1 , ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology ( n = 28) revealed a strong inverse correlation between the degree of γ-globin gene ( HBG2) promoter methylation and baseline HbF levels ( p = 0.003, r s = − 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter ( p = 0.039, r s = − 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A ( p = 0.05, r s = 0.482), a known HbF repressor. These changes were associated to blast cell clearance ( p HBG2 = 0.011, r s = 0.480/ p ZBTB7A = 0.026, r s = 0.427) and showed prognostic value for PFS ( p ZBTB7A = 0.037, HR = 1.14, CI 0.34–3.8). Conclusions Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action. Graphical abstract
Myelodysplastic syndromes (MDS) consist of a group of hematological malignancies characterized by ineffective hematopoiesis, cytogenetic abnormalities, and often a high risk of transformation to acute myeloid leukemia (AML). So far, there have been only a very limited number of studies assessing the epigenetics component contributing to the pathophysiology of these disorders, but not a single study assessing this at a genome-wide level. Here, we implemented a generic high throughput epigenomics approach, using methylated DNA sequencing (MeD-seq) of LpnPI digested fragments to identify potential epigenomic targets associated with MDS subtypes. Our results highlighted that PCDHG and ZNF gene families harbor potential epigenomic targets, which have been shown to be differentially methylated in a variety of comparisons between different MDS subtypes. Specifically, CpG islands, transcription start sites and post-transcriptional start sites within ZNF124, ZNF497 and PCDHG family are differentially methylated with fold change above 3,5. Overall, these findings highlight important aspects of the epigenomic component of MDS syndromes pathogenesis and the pharmacoepigenomic basis to the hypomethylating agents drug treatment response, while this generic high throughput whole epigenome sequencing approach could be readily implemented to other genetic diseases with a strong epigenetic component.
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders with maturation and differentiation defects exhibiting morphological dysplasia in one or more hematopoietic cell lineages. They are associated with peripheral blood cytopenias and by increased risk for progression into acute myelogenous leukemia. Among their multifactorial pathogenesis, age-related epigenetic instability and the error-rate DNA methylation maintenance have been recognized as critical factors for both the initial steps of their pathogenesis and for disease progression. Although lower-risk MDS is associated with an inflammatory bone marrow microenvironment, higher-risk disease is delineated by immunosuppression and clonal expansion. “Epigenetics” is a multidimensional level of gene regulation that determines the specific gene networks expressed in tissues under physiological conditions and guides appropriate chromatin rearrangements upon influence of environmental stimulation. Regulation of this level consists of biochemical modifications in amino acid residues of the histone proteins’ N-terminal tails and their concomitant effects on chromatin structure, DNA methylation patterns in CpG dinucleotides and the tissue-specific non-coding RNAs repertoire, which are directed against various gene targets. The role of epigenetic modifications is widely recognized as pivotal both in gene expression control and differential molecular response to drug therapies in humans. Insights to the potential of synergistic cooperations of epigenetic mechanisms provide new avenues for treatment development to comfort human diseases with a known epigenetic shift, such as MDS. Hypomethylating agents (HMAs), such as epigenetic modulating drugs, have been widely used in the past years as first line treatment for elderly higher-risk MDS patients; however, just half of them respond to therapy and are benefited. Rational outcome predictors following epigenetic therapy in MDS and biomarkers associated with disease relapse are of high importance to improve our efforts in developing patient-tailored clinical approaches.
