Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). ...Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2-associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients.
Current estimates suggest that up to 10% of patients with myeloid neoplasms (MN) harbor variants associated with a germline predisposition. A pathogenic variant in the runt-related transcription ...factor 1 gene (RUNX1) is a frequent cause of germline predisposition to MN. RUNX1 variants detected in tumor tissue at a VAF close to 50% are potentially germline and causative of RUNX1 familial platelet disorder with associated myeloid malignancies. Previous studies have found germline RUNX1 variants in 3% of patients with acute myeloid leukemia; however, the frequency of germline RUNX1 variants in less advanced myeloid neoplasms has not been examined. We screened 590 patients suspected of MN, excluding myeloproliferative neoplasms, for germline variants in RUNX1. We found RUNX1 variants in 83 patients (14%) by targeted sequencing of tumor tissue. In 40 patients (6.8%), the VAF of RUNX1 was above 30%. In 32 of the 40 patients, skin biopsies were available and used for Sanger sequencing to assess the germline status. Two of the tested variants (6.3%) were confirmed as germline, and both variants were curated as variants of unknown significance. To further explore the pathogenicity of these variants, we implemented a novel CRISPR-Select functional genetic assay. The assay demonstrated a profound effect on proliferation in K562 cells for a known pathogenic variant but no effect for the two germline variants detected in the study. We therefore propose that both germline variants are classified as likely benign. In this study, we show that RUNX1 germline variants are rare in Danish patients with MN and use a novel assay for functional classification of germline RUNX1 variants.
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BFBNIB, FZAB, GIS, IJS, KILJ, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Abstract
Objectives
Flow cytometry (FC) is, together with morphology, genetics, and cytogenetics, used in the diagnostic assessment of cytopenia, as its value in evaluating bone marrow dysplasia been ...highlighted by several studies. However, despite the development of algorithms and guidelines, there is still a lack of standardization of the FC assessment of bone marrow dysplasia.
Methods
By combining FC, together with morphological analysis and cytogenetic/molecular assessment in a training cohort of 209 patients, we created a novel score, ProGraME, which includes four parameters, each from a different cell lineage (Progenitor cells, Granulocytes, Monocytes, Erythroid precursors), solely based on relevant population gating. Points for ProGraME were attained for: lymphoid precursors ≤5% of all CD34+ cells (1.5 point); a granulocyte‐to‐lymphocyte side‐scatter ratio ≤6 (1 point); a monocyte CD33‐CV% ≥ 63 (2 points), and an erythroid precursor CD36‐CV% ≥ 65 (2 points).
Results
Using a cutoff of ≥2 as suggestive of dysplasia, ProGraME showed a sensitivity of 91% and a specificity of 81% in the training cohort and 95% and 75%, respectively, in an independent validation cohort of 159 patients. In addition, ProGraME had a very high negative predictive value of 97.1% and 97.8% in the training and validation cohorts, respectively, offering a useful tool for excluding bone marrow dysplasia. Finally, among the 23 CCUS patients that scored positive for dysplasia with ProGraME in the training cohort, 16 of them (69%) carried high‐risk mutations, suggesting that FC might help identify early changes of dysplasia.
Conclusions
ProGraME can potentially optimize the FC diagnosis of low‐risk myelodysplasia without minimal requirements of flow analysis other than accurate population gating.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Objective
The aim of this study was to evaluate the diagnostic and prognostic role of multiparameter flow cytometry (FC) in patients with idiopathic cytopenia of undetermined significance (ICUS) and ...clonal cytopenia of undetermined significance (CCUS).
Methods
We performed FC using a standardized panel and two different diagnostic algorithms (Ogata, Wells) in a well‐characterized cohort of 79 patients with ICUS/CCUS and compared it with a retrospective blinded morphological evaluation and data from targeted next‐generation DNA sequencing of 20 myelodysplastic syndrome (MDS)‐related genes.
Results
Our data show that FC has low sensitivity in distinguishing CCUS from ICUS patients (40.5% for Ogata score and 59.5% for Wells score). The Wells score was suggestive of dysplasia in ICUS/CCUS patients with concurrent morphological signs of dysplasia in the bone marrow (following re‐evaluation by two hematopathologists) and in CCUS patients with a higher mutational burden. Eight patients with ICUS/CCUS from our cohort progressed to another myeloid malignancy (MDS, acute myeloid leukemia, or chronic myelomonocytic leukemia), all showing flow cytometric signs of dysplasia.
Conclusion
FC performs poorly in diagnosing CCUS versus ICUS. However, it can potentially provide prognostic information in cytopenic patients by identifying a subgroup of patients with a higher grade of dysplasia, higher mutational burden, and higher risk of progression and, together with mutational screening, also identify a group of patients who might require morphological reassessment of dysplastic changes in their bone marrow.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Immune Mechanisms in Myelodysplastic Syndrome Glenthøj, Andreas; Ørskov, Andreas Due; Hansen, Jakob Werner ...
International journal of molecular sciences,
06/2016, Volume:
17, Issue:
6
Journal Article
Peer reviewed
Open access
Myelodysplastic syndrome (MDS) is a spectrum of diseases, characterized by debilitating cytopenias and a propensity of developing acute myeloid leukemia. Comprehensive sequencing efforts have ...revealed a range of mutations characteristic, but not specific, of MDS. Epidemiologically, autoimmune diseases are common in patients with MDS, fueling hypotheses of common etiological mechanisms. Both innate and adaptive immune pathways are overly active in the hematopoietic niche of MDS. Although supportive care, growth factors, and hypomethylating agents are the mainstay of MDS treatment, some patients-especially younger low-risk patients with HLA-DR15 tissue type-demonstrate impressive response rates after immunosuppressive therapy. This is in contrast to higher-risk MDS patients, where several immune activating treatments, such as immune checkpoint inhibitors, are in the pipeline. Thus, the dual role of immune mechanisms in MDS is challenging, and rigorous translational studies are needed to establish the value of immune manipulation as a treatment of MDS.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Objective
We investigated mortality and long‐term development of malignant hematological disease, cancer, liver‐, renal‐, and rheumatic disease in patients with unexplained cytopenia (UC).
Methods
We ...screened all patients referred to the outpatient clinic at the Department of Hematology, Rigshospitalet, Copenhagen, with a suspected myeloid neoplasm from June 2009 to the end of 2012. Through registry linkage, we obtained information on hospital‐based ICD‐10 diagnoses and survival. We estimated cumulative incidences of disease and hazard ratios of all‐cause mortality using the Aalen‐Johansen estimator and Cox regression. We compared incidences and mortality with a control cohort.
Results
Among 1820 referrals, 221 had UC. The UC group had a 5‐year cumulative incidence of malignant hematological disease of 8.91% (CI 95%: 4.98‐12.84) compared to 0.93(CI 95%: 0.32‐1.55) in the matched controls. In addition, UC patients had higher incidences of cancer, liver, and rheumatic disease. Mortality was higher in UC patients compared to the matched controls with a HR of 1.43 P = 0.038, CI 95%: 1.02‐2.00 adjusted for comorbidity, sex, and age. Most of the mortality and morbidity were ascribed to patients 50 years or older.
Conclusions
Unexplained cytopenia patients had a higher incidence of malignant hematological‐, cancer‐, liver‐, and rheumatic disease and increased mortality compared to the general population.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Clonal hematopoiesis of indeterminate potential (CHIP) is common in the elderly and has been reported to associate with accelerated epigenetic age (AgeAccel), especially intrinsic (ie, cell‐type ...independent) AgeAccel and to a lesser degree extrinsic AgeAccel, which reflects the immune‐cell composition of the peripheral blood. We investigated the association between CHIP occurrence and AgeAccel in 154 Danish twin pairs aged 73–90 years (mean 79), using both individual‐level and intrapair analyses, the latter to control for shared genetic and environmental factors. Of 308 individuals, 116 carried a CHIP mutation. CHIP carriers had non‐significantly increased AgeAccel compared with non‐carriers; the strongest association was for the Intrinsic Epigenetic Age Acceleration (IEAA) estimator (CHIP carriers 1.4 years older, P = 0.052). In intrapair analyses, the extrinsic Hannum age estimator showed the strongest association (1.6 years older, P = 0.027). In mutation‐specific analyses, TET2 mutations were associated with the extrinsic Hannum age estimator in both individual‐level (3.0 years older, P = 0.003) and intrapair analyses (2.8 years older, P = 0.05). DNMT3A mutations were associated with IEAA in individual‐level (1.9 years older, P = 0.034) but not intrapair analysis (0.9 years, P = 0.41). Analyses of logit‐transformed variant allele frequency were generally consistent with these results. Together, these observations indicate that different factors may be driving the expansion of DNMT3A and TET2 clones, respectively. Finally, CHIP carriers accelerated in both the Hannum and the GrimAge age estimators did not have an increased mortality risk in our cohort followed for 22 years (HR = 1.02, P = 0.93), hence not replicating the stratification model proposed by Nachun et al.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Global hypomethylation has been linked to disease progression in several cancers, but has not been reported for Diffuse Large B Cell Lymphoma (DLBCL). This study aimed to assess global methylation in ...DLBCL and describe its prognostic value. Mean LINE1 methylation, a validated surrogate measure for global methylation, was measured in DNA from 67 tumor biopsies. Additionally, cell‐free circulating DNA (cfDNA) in plasma samples from 74 patients was tested to assess the feasibility of global hypomethylation as a biomarker in liquid biopsies. LINE1 methylation was assessed using a commercially available kit, based on pyrosequencing of PCR amplified bisulfite‐treated DNA. Global hypomethylation was detected in a subset of cases and was associated with poor overall survival in both tumor biopsies (P = .001) and cfDNA (P = .009). It was the strongest risk factor in multivariate analysis in both biopsies (HR: 10.65, CI: 2.03‐55.81, P = .005) and cfDNA (HR: 11.87, CI: 2.80‐50.20, P = .001), outperforming conventional clinical risk factors. Finally, hierarchical cluster analyses were performed for the cfDNA samples using previously published gene‐specific methylation data. This analysis shows that global hypomethylation co‐occurs with other epigenetic abnormalities, including DAPK1 promoter hypermethylation. In conclusion, we have shown that global hypomethylation is strongly associated with poor survival in DLBCL both when present in tumor biopsy DNA and when detected in plasma cfDNA, and has potential for clinical application as a prognostic biomarker.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The prognostic value of aberrant DNA methylation of cell-free circulating DNA in plasma has not previously been evaluated in diffuse large B cell lymphoma (DLBCL). The aim of this study was to ...investigate if aberrant promoter DNA methylation can be detected in plasma from DLBCL patients and to evaluate this as a prognostic marker. Furthermore, we wanted to follow possible changes in methylation levels during treatment. Seventy-four patients were enrolled in the study, of which 59 received rituximab and CHOP-like chemotherapy. Plasma samples were collected from all patients at the time of diagnosis and from 14 healthy individuals used as controls. In addition, plasma samples were collected during and after treatment for surviving patients. In total, 158 plasma samples were analyzed for DNA methylation in the promoter regions of DAPK (DAPK1), DBC1, MIR34A, and MIR34B/C using pyrosequencing.
Aberrant methylation levels at the time of diagnosis were detected in 19, 16, 8, and 10 % of the DLBCL plasma samples for DAPK1, DBC1, MIR34A, and MIR34B/C, respectively. DAPK1 methylation levels were significantly correlated with DBC1 and MIR34B/C methylation levels (P < 0.001). For the entire cohort, 5-year overall survival (OS) rates were significantly lower in the groups carrying aberrant DAPK1 (P = 0.004) and DBC1 (P = 0.044) methylation, respectively. DAPK1 methylation status were significantly correlated with stage (P = 0.015), as all patients with aberrant DAPK1 methylation were stages III and IV. Multivariate analysis identified DAPK1 as an independent prognostic factor for OS with a hazard ratio of 8.9 (95 % CI 2.7-29.3, P < 0.0007). Patients with DAPK1 methylated cell-free circulating DNA at time of diagnosis, who became long-term survivors, lost the aberrant methylation after treatment initiation. Conversely, patients that maintained or regained aberrant DAPK1 methylation died soon thereafter.
Aberrant promoter methylation of cell-free circulating DNA can be detected in plasma from DLBCL patients and hold promise as an easily accessible marker for evaluating response to treatment and for prognostication. In particular, aberrant DAPK1 methylation in plasma was an independent prognostic marker that may also be used to assess treatment response.
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