DNMT3A Mutations Research Articles

Molecular characteristics in Chinese with chronic lymphocytic leukemia by next-generation sequencing: A single-center retrospective analysis.

Although the prevalence of Asian chronic lymphocytic leukemia (CLL) patients is not as high as that of Caucasians, there are more atypical CLLs in Asia whose genetic characteristics and their clinical significance are distinct and remain unclear. A retrospective analysis of 85 CLL samples in our center was conducted from 2019 to 2022. We used next-generation sequencing with a 172 gene panel to explore the multi-gene mutational data and the mutational status of immunoglobulin heavy variable (IGHV) gene. MYD88 (20.0%) was the most frequently mutated gene, much higher than in Europe, followed in order by TP53 (18.8%), NOTCH1 (14.1%), IGLL5 (11.8%), and DNMT3A (8.2%). In addition, the incidence of ATM and SF3B1 mutations was relatively lower in our centre compared to Europe. Mutated (M)-IGHV patients were more likely to have a cooccurrence of MYD88 mutation, while complex karyotype and DNMT3A mutation were more common in the unmutated (U)-IGHV group. MYD88 mutated CLL was characterized by prevalence in young males in high-risk staging, with isolated 13q deletion and concomitant mutation of IGLL5. CLL patients with MYD88 and TP53 mutation showed an unfavorable prognosis. These results would be valuable in helping to understand the characteristics and significance of cytogenetic genetics in Chinese patients with CLL.

Abstract P3055: Identification Of Cell-intrinsic Effects Of Clonal Hematopoiesis In Heart Failure

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and associates with worse prognosis in patients with heart failure (HF). While, circulating immune cells derived from patients with HF harboring CHIP mutations show heightened proinflammatory signatures, it is unclear whether these signatures are derived from the relatively low number of cells harboring CHIP-driver mutations, or are indicators of a systemic pro-inflammatory activation associated with CHIP. Methods and Results: We assessed the cell-intrinsic effects of the most prevalent CHIP-driver gene (DNMT3A) mutant-carrying circulating immune cells in a total of 5 HF patients. Using a novel protocol to detect DNMT3A mutant cells on a single cell level (MutDetect-Seq), we show that CHIP mutations can be detected in all major immune cell types (e.g. T cells, monocytes, B cells, NK cells), with detected mutation similar to that predicted by traditional targeted DNA sequencing. DNMT3A mutant monocytes demonstrated the highest number of significantly upregulated genes relative to other cell types. DNMT3A mutant monocytes exhibit altered gene expression signatures associated with inflammation and phagolysosome function. Additionally, DNMT3A mutant carrying CD4+, but not CD8+ T cells, showed significant changes in gene expression patterns towards an activated and cytotoxic profile, which was validated in vitro. Finally, increased paracrine signaling pathways involved in amplifying inflammatory circuits are predicted between mutated and non-mutated monocytes, this was validated in vitro with further implications for cardiac fibroblast activation via aSMA and COL1A1 expression. Conclusion: This is the first study to decipher the genetic signature of DNMT3A-CHIP-driver mutation carrying circulating immune cells, thereby providing first evidence for the cell intrinsic effects of DNMT3A mutations on peripheral blood cell subsets in patients with HF. The altered gene expression profiles associated with inflammation and phagolysosome function of mutant monocyte, as well as skewing of mutated T cells towards effector cells provide novel insights into how CHIP may promote worse prognosis in heart failure patients.

Abstract P3070: Aortic Valve Calcification Is Accelerated By DNMT3A And TET2 Gene Driver Mutations Associated With Clonal Hematopoiesis

Background: Clonal hematopoiesis (CH) due to DNMT3A or TET2-driver mutations associates with heightened inflammation and a worse prognosis among patients with severe calcific aortic valve stenosis (AS). However, it is unknown what role CH plays in the pathogenesis of AS. AS is the most common age-related heart valve disease, with no medical therapy to halt progression. Methods and Results: Single-cell RNA-sequencing of immune cells of CAVD patients (n=13) with and without the most prevalent CH-mutations (DNMT3A and TET2), having a mean CH-driver gene variant allele frequency was 6.5% for DNMT3A and 17.9% for TET2. Monocytes of patients harboring DNMT3A and TET2 mutations shared 836 upregulated genes, which were associated with glycolytic, pro-inflammatory activation (CXCL10, IL38) along with paracrine pro-calcific factors (Oncostatin M (OSM), S100A9), signifying that CH might be causally involved in AS. Indeed, silencing of TET2 or DNMT3A in macrophages promotes M1-like polarization and increases release of pro-calcific mediators, particularly S100A9 and OSM. To assess whether CH-gene silenced macrophages may stimulate mesenchymal cells to secrete calcium, secreted factors from TET2 or DNMT3A-silenced macrophages were applied mesenchymal cells. Indeed, CH-gene silenced supernatants induced osteoblastic differentiation of mesenchymal cells, evidenced by increased Alizarin red calcium staining and elevated levels of ALP and RUNX2. This CH-mediated enhancement in osteoblastic transformation could be ablated by silencing of OSM. Finally, valves from atheroprone Ldlr-/- mice receiving Tet2-/- bone marrow transplants demonstrated increased total calcified area (p<0.05, 1.49-fold) along with increased numbers of calcification deposits (p<0.05, 2.33-fold) as shown by von Kossa staining, along with enhanced OSM and S100A9 levels. Conclusion: This is the first study to show that somatic CH-driver mutations in monocytes may accelerate AS. Moreover, calcification was ablated via silencing of OSM in CH-gene silenced macrophages. These data suggest Oncostatin M may be a therapeutically promising target in halting the development of AS in patients harboring CH mutations.

Molecular and clinical significance of FLT3, NPM1, DNMT3A and TP53 mutations in acute myeloid leukemia patients.

Acute myeloid leukemia (AML) is a type of blood cancer that affects the bone marrow and blood cells. AML is characterized by the rapid growth and accumulation of abnormal white blood cells, known as myeloblasts, which interfere with the production of normal blood cells. The main aim was to determine the relationship between these genetic alterations and the clinico-haematological parameters and prognostic factors with therapy for Iraqi patients with AML. We used Sanger Sequencing to detect the mutations in 76 AML patients. Clinical data of AML patients were retrospectively analysed to compare the prognosis of each gene mutation group. Somatic mutations were identified in 47.4% of the enrolled patients in a core set of pathogenic genes, including FLT3 (18 patients, 23.7%), DNMT3A (14, 18.4%), NPM1 (11, 14.5%) and TP53 (5, 6.8%). As multiple mutations frequently coexisted in the same patient, we classified patients into 10 further groups. Two novel mutations were detected in FLT3-ITD, with new accession numbers deposited into NCBI GenBank (OP807465 and OP807466). These two novel mutations were computationally analysed and predicted as disease-causing mutations. We found significant differences between patients with and without the detected mutations in disease progression after induction therapy (remission, failure and death; pv = < 0.001) and statistically significant differences were reported in total leukocyte count (pv = < 0.0001). These genes are among the most frequently mutated genes in AML patients. Understanding the molecular and clinical significance of these mutations is important for guiding treatment decisions and predicting patient outcomes.

Clinical features and prognostic significance of DNMT3A, FLT3, and NPM1 mutations in de novo acute myeloid leukemia patients.

Different co-mutation patterns are associated with varied clinical manifestations and prognosis. The purpose of this research was to explore the clinical characteristics and prognosis of individuals with AML who had DNMT3A, FLT3, and NPM1 mutations. A total of 259 newly diagnosed AML patients were investigated in this study, including 148 AMLFLT3mutDNMT3Awt , 48 AMLFLT3wtDNMT3Amut , and 63 AMLFLT3mutDNMT3Amut patients. Mutations were detected by targeted next-generation sequencing and Sanger sequencing. In addition, we utilized the publicly available data to analyze the expression profiles of AML. Correlation analysis showed NPM1 mutations were positively associated with FLT3-ITD and DNMT3A, but negatively with CEBPA and RUNX1 mutations. In the presence of both DNMT3A and FLT3 mutations, patients were associated with typical clinical manifestations such as heavy disease burden and old age. Patients with both FLT3 and DNMT3A mutations had lower complete remission rates and poorer clinical outcomes than those with FLT3 or DNMT3A mutation alone. Univariate analysis showed that age, response to treatment, DNMT3A R882 mutation, NPM1 mutation, and consolidation treatment options were associated with OS. According to multivariate analysis, only consolidation treatment options could be considered as an independent prognostic factor. In addition, the percentage of AMLFLT3mutDNMT3AmutNPM1mut patients in our study was about 5.9%. Interestingly, the expression profile of this subgroup was significantly related to HOX family and histone H1 family, and enriched pathways associated with transcriptional misregulation. We comprehensively evaluated the clinical and genetic characteristics, and expression profiles of AML patients with common mutations, and found that AML patients with triple mutations might be a distinct AML subtype, which should be redefined.

Utilization of Serial Next-Generation Sequencing Among Patients Receiving CPX-351 for Newly Diagnosed Acute Myeloid Leukemia

BackgroundThe phase III trial that led to the approval of CPX-351 for treating secondary acute myeloid leukemia (sAML) in 2017 did not study the effect of specific mutations on outcomes. MethodsThis retrospective study was done to evaluate the effect of next-generation sequencing (NGS) results at the time of best response and before allogeneic stem cell transplant (alloSCT) in patients treated with CPX-351 as frontline therapy for sAML between 2017 and 2021. ResultsThe most common mutations seen were DNMT3A (n = 17, 29.8%), SRSF2 (n = 13, 22.8%), RUNX1 (n = 13, 22.8%), TET2 (n = 9, 15.8%), ASXL1 (n = 9, 15.8%), and BCOR (n = 9, 15.8%). Median OS (mOS) for the entire cohort was 47 months. Though 64.7% patients cleared the DNMT3A mutation, only 44.4% and 22.2% of patients cleared the TET2 and ASXL1 mutations, respectively. The mOS for patients who cleared their mutations vs. for those who did not was not significantly longer (46 vs. 30 months; P = .991). The relapse-free survival (RFS) for patients who cleared mutations was numerically longer compared to those who had persistent mutations; however, this did not reach statistical significance (44 months vs. 26 months; P = .786). ConclusionThis is the first study reporting NGS at best response and before alloSCT and its effect on OS and RFS. We found that OS and RFS were numerically longer among patients who cleared mutations; however, this did not reach statistical significance. In addition, alloSCT led to improved RFS irrespective of mutational clearance.

DNMT3A mutation promotes leukemia development through NAM-NAD metabolic reprogramming

BackgroundDNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia (AML) with Arg882His (R882H) as the hotspot mutation. It has been reported that DNMT3A mutation plays a key role in leukemogenesis through hypomethylation of some target genes associated with cell growth and differentiation. In this study, we investigated the function of DNMT3A R882H in the malignant progression of AML by regulating metabolic reprogramming.MethodsUltra-High Performance Liquid Chromatography–High Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS) was used to detect metabolites in the serum of mice harboring Dnmt3a R878H mutation and the wild-type Dnmt3a. Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) and RNA sequencing (RNA-seq) were used to analyze the levels of DNA methylation and mRNA expression of genes in mouse Gr1+ bone marrow cells respectively. The TCGA and GO databases were used to analyze the differential genes between human samples carrying the DNMT3A R882 mutation and the wild-type DNMT3A. Co-immunoprecipitation and immunoblotting were used to illustrate the binding levels of Cyclins-CDKs and CDK inhibitors including CDKN1A and CDKN1B. Flow cytometry was used to analyze the cell differentiation, division, apoptosis and cell cycle. The effect of NAMPT inhibition on leukemia was evaluated by using in vivo fluorescence imaging in NOG mouse model bearing OCI-AML3 cells.ResultsDNMT3A mutation caused high expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage synthetic pathway, through DNA hypomethylation, and finally led to abnormal nicotinamide (NAM) metabolism and NAD synthesis. The NAM-NAD metabolic abnormalities caused accelerated cell cycle progression. Inhibition of NAMPT can reduce the binding degree between Cyclins-CDKs, and increase the binding interaction of the CDK inhibitors with Cyclins-CDKs complexes. Moreover, cells with high expression of NAMPT were more sensitive to the NAMPT inhibitor FK866 with a lower IC50. The inhibition of NAMPT can remarkably extend the survival time of tumor-bearing mice and reduce the infiltration of tumor cells.ConclusionsTaken together, our data showed that DNMT3A mutation caused NAMPT overexpression to induce the reprogramming of NAM-NAD metabolism and contribute to abnormal proliferation, which provided a potential direction for targeted therapy at the metabolic level in AML with DNMT3A mutation.

Clonal hematopoiesis with DNMT3A and PPM1D mutations impairs regeneration in autologous stem cell transplant recipients.

Clonal hematopoiesis (CH) is an age-related condition driven by stem and progenitor cells harboring recurrent mutations linked to myeloid neoplasms. Currently, potential effects on hematopoiesis, stem cell function and regenerative potential under stress conditions are unknown. We performed targeted DNA sequencing of 457 hematopoietic stem cell grafts collected for autologous stem cell transplantation (ASCT) in myeloma patients and correlated our findings with high-dimensional longitudinal clinical and laboratory data (26,510 data points for blood cell counts/serum values in 25 days around transplantation). We detected CHrelated mutations in 152 patients (33.3%). Since many patients (n=54) harbored multiple CH mutations in one or more genes, we applied a non-negative matrix factorization (NMF) clustering algorithm to identify genes that are commonly co-mutated in an unbiased approach. Patients with CH were assigned to one of three clusters (C1-C3) and compared to patients without CH (C0) in a gene specific manner. To study the dynamics of blood cell regeneration following ASCT, we developed a time-dependent linear mixed effect model to validate differences in blood cell count trajectories amongst different clusters. The results demonstrated that C2, composed of patients with DNMT3A and PPM1D single and co-mutated CH, correlated with reduced stem cell yields and delayed platelet count recovery following ASCT. Also, the benefit of maintenance therapy was particularly strong in C2 patients. Taken together, these data indicate an impaired regenerative potential of hematopoietic stem cell grafts harboring CH with DNMT3A and PPM1D mutations.

#3018 CARDIOVASCULAR DISEASE AND DETECTION OF CLONAL HEMATOPOIESIS OF INDETERMINATE POTENTIAL IN THE CHRONIC KIDNEY DISEASE POPULATION

Abstract Background and Aims Cardiovascular disease (CVD) is the main cause of morbidity and mortality in patients with chronic kidney disease (CKD). Novel determination of clonal expansion of hematopoietic stem cells carrying mutations in certain genes is called Clonal hematopoiesis of indeterminate potential (CHIP), it is well known the association with an increased risk of hematologic malignancies but in general population, CHIP has been associated with increased mortality and increased cardiovascular risk. The aim of our study was to analyze the influence of CHIP on the risk of CVD and heart disease in the population with CKD. Method 128 patients with different degrees of CKD were roll in our prospective study in between September 1, 2020 and January 31, 2021. All of them have followed up in Nephrology clinics and any patient have previous cardiovascular pathology. For detection of silent heart disease was realized measurement of troponin I and NT-Pro-BNP in the blood using a microparticle chemiluminescence assay and the degree of coronary calcification was calculated by the computed tomography (CT): Agatston method. All the patients were prospectively followed up for 18 months, recording the occurrence of major cardiovascular events. For detection of CHIP massive sequencing was performed with Ion Chef System On-demand technology using Ion AmpliSeqTM Kit for Chef DL8. The data analysis was done with the Torrent SuiteTM software and Varsome software, following the criteria of the American College of Medical Genetics (ACMG). Results 36.7% of the patients presented CHIP (pathogenic and probably pathogenic mutations) and 24.2% (only pathogenic mutations). TET2 was the most frequently affected gene (21.1%). The percentage of patients with CHIP MPL gene mutation was 12,5%, DNMT3 7%, JAK 1,6% and ASXL1 0,8%. 9 patients developed major cardiovascular events (MACE). Using univariate Cox regression analysis, the presence of MACE was related to global coronary calcification (OR 1.001, 95% CI 1.001-1.001, p = &amp;lt; 0.001), NT-ProBNP (OR 1.090, 95% CI 1.050-1.132, p = &amp;lt; 0.001) but there was no relation to smoking (OR 2.796, 95% CI 0.750-10.415, p = 0.125). Patients with CHIP did not have a higher risk of MACE, although patients with DNMT3A mutations did have a higher risk (HR 6.637, 95% CI 1.443-30.533, p = 0.015) independently of other variables. Using multivariate logistic regression analysis, the presence of CHIP was not related to coronary calcification (OR 0.387, 95% CI 0.142-1.058, p = 0.387), nor was it related to troponin I or NT-Pro-BNP. Conclusion The presence of CHIP was not associated with a greater risk of silent heart disease or cardiovascular events, although DNMT3a mutations, analyzed independently, were associated with a greater number of cardiovascular events in our CKD population.

The effect of haploidentical hematopoietic stem cell transplantation on comutations based on next-generation sequencing in adult acute myeloid leukemia patients with the FLT3-ITD mutation.

According to the 2022 European LeukemiaNet, all acute myeloid leukemia (AML) cases with FLT3-ITD mutations are now categorized as intermediate risk irrespective of the FLT3-ITD allelic ratio or concurrent presence of NPM1 mutation. However, whether other next-generation sequencing (NGS) comutation genes can add layers to FLT3-ITD and whether the poor outcomes of FLT3-ITD comutations can be overcome by haploidentical hematopoietic stem cell transplantation (haplo-HSCT) are unclear. This study aimed to investigate which comutations based on NGS at diagnosis affect the clinical prognosis of de novo AML patients with FLT3-ITD mutations and the effect of haplo-HSCT on comutations. We analyzed 95 de novo AML patients with FLT3-ITD mutations from January 2018 to August 2021 based on the NGS 99-gene platform. Forty-one other types of molecular mutations were detected. The most common cooccurring mutations were NPM1 (n=43, 45.3%) and DNMT3A (n=21, 22.1%). NPM1 mutation did not affect the clinical outcomes. Acute myeloid leukemia patients with FLT3-ITD and DNMT3A comutations had significantly worse 3-year Disease-free survival (DFS) (49.5% vs. 69.3%, P=0.01) and Overall survival (OS) rates (61.1% vs. 69.8%, P=0.54) than those without DNMT3A mutations, and survival was significantly more favorable after haplo-HSCT than that after chemotherapy (3-year DFS, 85.7% vs. 30.8%, P=0.006; 3-year OS, 85.7% vs. 43.1%, p=0.08). In multivariate analysis, DNMT3A mutation was a risk factor for DFS, while haplo-HSCT was a protective factor. In conclusion, DNMT3A mutation might be a poor prognostic factor in adult AML patients with FLT3-ITD mutations, and haplo-HSCT could overcome the poor prognosis of DNMT3A comutation.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: TFH lymphomas (TFHL) commonly harbor mutations in TET2, RHOA, DNMT3A and IDH2. While RHOA and IDH2 appear restricted to the neoplastic cells, TET2 and DNMT3A mutations occur in a significant proportion of cases in a hematopoietic progenitor cell and can also be detected in B or myeloid cells. Cell-free DNA (cfDNA) sequencing allows the detection of circulating tumor DNA in solid cancers or B-cell lymphomas, with predictive value, but few data exist on cfDNA sequencing in TFHL. Methods: In the frame of the ORACLE study, a phase 3 trial comparing oral azacitidine to investigator choice treatment in relapsed or refractory TFHL patients, we collected tumor biopsies and plasma in streck tube at inclusion, after 3 cycles, and at progression. Tumour and cfDNA were sequenced by NGS using 9 genes, amplicon-based libraries. Median sequencing depth was 2386X in tumor and 3519X in cfDNA. Results: Among patients with confirmed TFHL treated in LYSA centers, we collected 45 samples at inclusion, 16 after cycle 3, and 14 at progression. The median cfDNA concentration at each time point was respectively 7507 (IQR 3418–19414), 5182 (3438–12429), and 15750 (7316–26953) hEG/mL. Results of cfDNA at inclusion and tumor sequencing were compared in 43 patients. Common TET2 mutations were detected in the tumor of 36/43 (84%) and in cfDNA of 33/43 (77%) patients, with a median variant allele frequency (VAF) of 17.75% vs. 9.8% respectively. RHOAG17V was detected in 29/43 (67%, VAF 10%) and in 24/43 (59%, VAF 4.4%), DNMT3A in 13/43 (30%, VAF 17.9%) and 15/73 (35%, VAF 17.9%), and IDH2 in 12/43 (28%, VAF 5.9%) and 8/43 (19%, VAF 5.4%) of tumor biopsies and cfDNA samples respectively. Only one patient had detectable TET2, DNMT3A, IDH2, and RHOA mutations in the cfDNA and not in the tumor biopsy, corresponding to a tumor with low neoplastic cell content. By contrast, 3 patients had DNMT3A mutations, not affecting the R882 residue, detected in the cfDNA but not in the tumor likely corresponding to clonal hematopoiesis not related to the TFHL. We also compared 25 paired cfDNA samples collected at inclusion and after cycle 3 (including progression sample if occurring at cycle 3 or before). In all but one patient, we observed the persistence of TET2 and DNMT3A mutations, even in responding patients, confirming that these mutations are not restricted to neoplastic cells, and suggesting that treatment, especially with 5-azacitidine, does not affect the clonal hematopoiesis. By contrast, among the 14/25 patients with a detectable RHOA mutation in cfDNA at inclusion, progression-free survival was longer in the 4 patients with the disappearance of the RHOA mutations in cfDNA at cycle 3 than in the 10 others (median 29 vs. 3 months, p = 0.01). The research was funded by: BMS Force Hémato Keywords: Aggressive T-cell non-Hodgkin lymphoma, Liquid biopsy, Minimal residual disease No conflicts of interests pertinent to the abstract.

Efficacy and Safety of Decitabine Combined with Modified CAG Regimen in Patients Aged ≥ 70 Years with Newly Diagnosed Acute Myeloid Leukemia

To evaluate the clinical efficacy and safety of decitabine combined with modified CAG regimen (D-CAG regimen) in patients aged ≥70 years with newly diagnosed acute myeloid leukemia (AML). The clinical data of 59 AML patients (≥70 years old) who were newly diagnosed and treated in the Hematology Department of the First Affiliated Hospital of Nanjing Medical University from November 2010 to June 2021 were retrospectively analyzed. Among the 59 AML patients, 28 were males and 31 were females, with a median age of 74 (70-86) years. The complete remission (CR) rate was 69.4% (34/49), and the median duration of CR was 10.7 (0.6-125.4) months after 2 courses of D-CAG treatment. According to the British Medical Research Council (MRC) classification, there was only one patient in the favorable-risk group, and the CR rate was 71.8% (28/39) in the intermediate-risk group, and 55.6% (5/9) in the adverse-risk group, respectively. There was no statistical difference in the CR rate between the intermediate-risk and adverse-risk group. Referring to ELN 2017 genetic risk classification, CR rate was 88.2% (15/17) in the favorable-risk group, 45.5% (5/11) in the intermediate-risk group, and 66.7% (14/21) in the adverse-risk group. There was no significant difference in CR rate between the favorable-risk and adverse-risk categories, but both were significantly higher than that in the intermediate-risk group (P <0.05). Next-generation sequencing (NGS) analysis showed that 11 gene mutations with a frequency of more than 10%, including TET2 mutation (35.6%), ASXL1 mutation (30.5%), NPM1 mutation (28.8%), FLT3-ITD mutation (27.1%), DNMT3A mutation (22.0%), IDH1 mutation (15.3%), CEBPA single mutation (13.6%), TP53 mutation (13.6%), IDH2 mutation (11.9%), RUNX1 mutation (11.9%), and NRAS mutation (10.2%). There were no statistical differences in mutation frequency of these 11 genes between CR group and non-CR group. Compared with normal karyotypes, patients with complex karyotypes were more likely to develop TP53 mutations (P <0.001), while FLT3-ITD and DNMT3A mutations were more likely to occur in patients with normal karyotypes (P =0.04, P =0.047). The median follow-up, overall survival (OS), and event-free survival (EFS) of all the patients was 11.7 (1.5-128.2) months, 12.3 (1.5-128.2) months, and 8.5 (1.5-128.2) months, respectively. The median OS and EFS of CR patients were 19.8 and 13.3 months, respectively, which were significantly longer than 6.4 and 5.7 months in patients experiencing treatment failure (P < 0.001, P =0.009). In regard to genes with mutation frequency >10%, there were no statistical differences in CR rate, median OS, and median EFS between mutated and wild-type patients by Chi-square test and survival analysis. Univariate analysis showed that age, hemoglobin, lactate dehydrogenase, cytogenetics and CR were factors affecting prognosis, while multivariate analysis showed that only CR failure was an independent adverse prognostic factor for OS. The major adverse reactions to D-CAG regimen were grade 3-4 myelosuppression, pulmonary infection, and fever (infection focus was not identified). D-CAG regimen is safe and effective in the treatment of AML patients ≥70 years old, and can partially improve the prognosis of elderly and high-risk patients.

CLONAL ARCHITECTURE OF RELAPSED OR REFRACTORY FOLLICULAR HELPER T‐CELL LYMPHOMA: AN ANCILLARY STUDY OF THE ORACLE TRIAL, A LYSA STUDY

Follicular helper T-cell lymphoma (TFHL) results from the oncogenic transformation of a TFH cell, driven by mutations in genes involved in epigenetic regulation (TET2, DNMT3A, IDH2) and T-cell signaling (RHOA). Demonstration of TET2 and DNMT3A mutations in B cells, myeloid cells, or hematopoietic progenitor cells have suggested that TFHL can emerge from clonal hematopoiesis (CH) in a multi-step process. However, the frequency, the extension, and the relevance of such CH in TFHL oncogenesis are unclear. To describe and characterize the clonal architecture of refractory/relapsed (R/R) TFHL, we collected bone marrow samples of patients included in the ORACLE trial (NCT03593018), a phase 3 trial evaluating the oral 5-azacitidine (CC-486) compared to single-agent chemotherapy in patients with R/R TFHL. Bone marrow (BM) cells from 32 patients at inclusion were cultured in methylcellulose supplemented with standard growth factors. BFU-E, CFU-GM, and CFU-GEMM were counted and individually harvested. A total of 517 colonies derived from the 32 patients were then individually genotyped by next-generation sequencing (NGS) using a capture-based panel (limit of detection: 1%) covering 43 genes recurrently mutated in CH and/or TFHL, including TET2, DNMT3A, IDH2, and RHOA. Meanwhile, bulk BM cells from 29 patients were sequenced with the same NGS panel and then compared to the mutations found in cfDNA (31 patients) and tumor biopsies (28 patients). Overall, the frequency of CH and the phylogenetic link between the myeloid progenitors and the TFHL was evaluable in 30 TFHL patients. We successfully genotyped 471/517 (91%) colonies. CH defined by at least one somatic mutation detected in at least one myeloid colony or total BM cells was found in 29/30 (97%) patients. Among the 26/29 patients with mutated myeloid colonies, the median percentage of mutated colonies was 21% (IQR 7%–53%) and 7 had subclonal architecture with a median of 3 different clones. The 2 most frequent mutations were DNMT3A and TET2, detected in 24/30 (80%) and 18/30 (60%) patients' BM, respectively. We confirmed the absence of RHOAG17V and IDH2R172 mutations in BM cells of TFHL patients. While 17/18 patients with a detectable TET2 mutation in CH had a TET2 mutated TFHL, only 13/23 patients with a DNMT3A mutated CH had a DNMT3A mutated TFHL. We thus identified 3 groups of patients according to the distribution of mutations found in the BM and the LN lymph nodes: 16/30 (53.3%) patients had a TFHL clonally derived from CH (CH-related TFHL), 13 (43.3%) had TFHL, and clonally distinct CH (CH-unrelated TFHL), and one patient (3.3%) had undetectable CH. No significant difference in patient outcome was observed regarding the presence of CH-related or unrelated TFHL, but our results are limited by the small number of patients and treatment heterogeneity. Encore Abstract—previously submitted to EHA 2023 Keywords: aggressive T-cell non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Clinical features of SF3B1 mutation in patients with myelodysplastic syndrome with excess blasts

Objective: To exploring the clinical features of SF3B1-mutated myelodysplastic syndrome with excess blasts (MDS-EB) and analyzing the association between SF3B1 mutation, and efficacy and prognostic significance for patients with MDS-EB. Methods: This was a retrospective case series study. The clinical data of 266 patients with MDS-EB diagnosed in the First Affiliated Hospital of Zhengzhou University between April 2016 and November 2021 were analyzed. The observed indicators included blood routine counts, mutated genes, overall response rate (ORR), overall survival (OS), progression-free survival (PFS), and leukemia-free survival (LFS). The Kaplan-Meier method was used to depict the survival curves. The Log-rank test method was equally used to compare survival across groups and performed the Cox proportional hazard regression model for prognostic analysis. Results: In 266 patients with MDS-EB, 166 (62.4%) were men, and the median age was 57 (17-81) years. Moreover, there were included 26 and 240 patients in the SF3B1-mutated and SF3B1 wild-type groups. Patients in the SF3B1-mutated group were older [median age 65 (51, 69) years vs. 56 (46, 66) years, P=0.033], had higher white blood cell (WBC) counts [3.08 (2.35, 4.78) × 109/L vs. 2.13 (1.40, 3.77) × 109/L], platelet (PLT) counts [122.5 (50.5, 215.0) ×109/L vs. 49.0 (24.3, 100.8) × 109/L], absolute neutrophil counts (ANC) [1.83 (1.01, 2.88) × 109/L vs. 0.80 (0.41, 1.99) × 109/L]and occurrence of DNMT3A mutation [23.1% (6/26) vs. 6.7% (16/240)] (all P<0.05). The ORR were similar in both groups after 2 and 4 cycles of therapy (P=0.348, P=1.000). Moreover, the LFS (P=0.218), PFS (P=0.179) and OS (P=0.188) were similar across the groups. Univariate Cox analysis revealed that SF3B1 mutation did not affect the prognosis of patients with MDS-EB (OS: P=0.193; PFS: P=0.184). Conclusions: Patients with SF3B1 mutation were older, with greater WBC, PLT, and ANC, and SF3B1 mutation easily co-occurred with DNMT3A mutation. From this model, there were no significant differences in efficacy and survival of MDS-EB with or without SF3B1 mutation.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Background: Emerging evidence suggests the prognostic impact of the tumor microenvironment (TME) in peripheral T-cell lymphomas (PTCLs). To better understand PTCL pathobiology, we performed an integrative multi-omics study to explore the genetic subtypes and the TME signatures of PTCLs, especially nodal T-follicular helper T-cell lymphomas (nTFHLs) and PTCL, not otherwise specified (PTCL-NOS). Method: We performed whole-exome sequencing of 92 nTFHLs and 37 PTCL-NOSs, a total of 129 cases. Ninety-two nTFHLs included 85 angioimmunoblastic T-cell lymphomas (AITLs), four nPTCL with TFH phenotypes, two follicular T-cell lymphomas, and one unclassifiable nTFHL. Genetic drivers were integrated using non-negative matrix factorization consensus clustering (NMF). We estimated the proportion of immune cell fractions from bulk RNA sequencing data (n = 57) using CIBERSORTx and classified the TME signatures using hierarchical clustering. Results: NMF clustering analysis revealed three genetic subtypes within our cohort, except for 10 cases lacking recurrent genetic abnormalities (denoted as cluster 0). C2 was characterized by TP53 mutation, CDKN2A loss, PTEN loss, and chromosomal instability, corresponding to the previously described GATA3-PTCL subtype. C1 and C3 shared TFH-related genomic alterations, but C3 had significantly higher frequencies of chromosome (Chr) 5 gain, Chr 21 gain, RHOA, IDH2, and CD28 mutations. A higher frequency of rash and higher C-reactive protein levels than the other AITLs characterized C3-AITL. Pairwise association analysis of genomic alterations revealed that multiple TET2 mutations, rather than a single TET2 mutation, significantly co-occurred with IDH2 and RHOA mutations. A Bradley-Terry model estimated that the first TET2 and DNMT3A mutations occurred earlier, and the second or later TET2, RHOA, and IDH2 mutations occurred later. Compared to C1, significantly inferior survival was observed in C2 (hazard ratio (HR), 2.52; 95% CI, 1.37–4.63) and C3(HR, 2.14; 95% CI, 1.17–3.89; Figure A). The TME signatures were divided into TME1 (B-cells and follicular helper T-cells), TME2 (activated memory CD4 T-cells, M2 macrophages, and CD8 T-cells), and TME3 (naïve CD4 T-cells and activated mast cells). mTORC1 signaling was enriched in the TME2 signature compared to TME1 and TME3 signatures. The TME2 signature was significantly associated with shorter survival than the TME3 signature (HR: 3.4, 95% CI 1.6–7.5; Figure B). C2 had significantly more TME2 signature than C1 (64.3% vs. 7.7%), while C1 had significantly more TME3 signature than C2 (80.8% vs. 28.6%, p = 0.006; Figure C). Keywords: genomics, epigenomics, and other -omics, microenvironment Encore Abstract—previously submitted to EHA 2023 The research was funded by: Japan Society for the Promotion of Science KAKENHI grants (grant numbers: 19K23879, 22K19451, and 21H02945) and Japan Agency for Medical Research and Development (AMED) grants (20ck0106544 and 21ck0106644)

Loss of Dnmt3a impairs hematopoietic homeostasis and myeloid cell skewing via the PI3Kinase pathway.

Loss-of-function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of patients with acute myeloid leukemia (AML) with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early preleukemic event, which - when combined with other genetic lesions - result in full-blown leukemia. Here, we show that loss of Dnmt3a in hematopoietic stem and progenitor cells (HSC/Ps) results in myeloproliferation, which is associated with hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway. PI3Kα/β or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/β inhibitor treatment. In vivo RNA-Seq analysis on drug-treated Dnmt3a-/- HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment, and extracellular matrix compared with controls. Remarkably, drug-treated leukemic mice showed a reversal in the enhanced fetal liver HSC-like gene signature observed in vehicle-treated Dnmt3a-/- LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions, including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/β inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a potentially new target for treating DNMT3A mutation-driven myeloid malignancies.

Familial Clonal Hematopoiesis in a Long Telomere Syndrome

BackgroundTelomere shortening is a well-characterized cellular aging mechanism, and short telomere syndromes cause age-related disease. However, whether long telomere length is advantageous is poorly understood.MethodsWe examined the clinical and molecular features of aging and cancer in persons carrying heterozygous loss-of-function mutations in the telomere-related gene POT1 and noncarrier relatives.ResultsA total of 17 POT1 mutation carriers and 21 noncarrier relatives were initially included in the study, and a validation cohort of 6 additional mutation carriers was subsequently recruited. A majority of the POT1 mutation carriers with telomere length evaluated (9 of 13) had long telomeres (>99th percentile). POT1 mutation carriers had a range of benign and malignant neoplasms involving epithelial, mesenchymal, and neuronal tissues in addition to B- and T-cell lymphoma and myeloid cancers. Five of 18 POT1 mutation carriers (28%) had T-cell clonality, and 8 of 12 (67%) had clonal hematopoiesis of indeterminate potential. A predisposition to clonal hematopoiesis had an autosomal dominant pattern of inheritance, as well as penetrance that increased with age; somatic DNMT3A and JAK2 hotspot mutations were common. These and other somatic driver mutations probably arose in the first decades of life, and their lineages secondarily accumulated a higher mutation burden characterized by a clocklike signature. Successive generations showed genetic anticipation (i.e., an increasingly early onset of disease). In contrast to noncarrier relatives, who had the typical telomere shortening with age, POT1 mutation carriers maintained telomere length over the course of 2 years.ConclusionsPOT1 mutations associated with long telomere length conferred a predisposition to a familial clonal hematopoiesis syndrome that was associated with a range of benign and malignant solid neoplasms. The risk of these phenotypes was mediated by extended cellular longevity and by the capacity to maintain telomeres over time. (Funded by the National Institutes of Health and others.)

Molecular-defined clonal evolution in patients with classical myeloproliferative neoplasms.

Classical myeloproliferative neoplasms (MPNs) are characterized by distinct clinical phenotypes. The discovery of driver mutations in JAK2, CALR and MPL genes provided new insights into their pathogenesis. Next-generation sequencing (NGS) identified additional somatic mutations, most frequently in epigenetic modulator genes. In this study, a cohort of 95 MPN patients was genetically characterized using targeted NGS. Clonal hierarchies of detected mutations were subsequently analysed using colony forming progenitor assays derived from single cells to study mutation acquisition. Further, the hierarchy of mutations within distinct cell lineages was evaluated. NGS revealed mutations in three epigenetic modulator genes (TET2, DNMT3A, ASXL1) as most common co-mutations to the classical driver mutations. JAK2V617F as well as DNMT3A and TET2 mutations were detected as primary events in disease formation and most cases presented with a linear mutation pattern. Mutations appear mostly in the myeloid lineages but can also appear in lymphoid subpopulations. In one case with a double mutant MPL gene, mutations exclusively appeared in the monocyte lineage. Overall, this study confirms the mutational heterogeneity of classical MPNs and highlights the role of JAK2V617F and epigenetic modifier genes as early events in hematologic disease formation.

DNMT3AR882H accelerates angioimmunoblastic T-cell lymphoma in mice.

DNA methylation-related genes, including TET2, IDH2, and DNMT3A are highly frequently mutated in angioimmunoblastic T-cell lymphoma (AITL), an aggressive malignancy of T follicular helper (Tfh) cells associated with aberrant immune features. It has been shown that TET2 loss cooperates with RHOAG17V to promote AITL in mice but the functional role of DNMT3A mutations in AITL remains unclear. Here, we report that DNMT3AR882H, the most common mutation of DNMT3A in AITL, accelerates the development of Tet2-/-; RHOAG17V AITL in mice, indicated by the expansion of malignant Tfh cells and aberrant B cells, skin rash, and significantly shortened disease-free survival. To understand the underlying cellular and molecular mechanisms, we performed single-cell transcriptome analyses of lymph nodes of mice transplanted with Tet2-/-, Tet2-/-; RHOAG17V or DNMT3AR882H; Tet2-/-; RHOAG17V hematopoietic stem and progenitor cells. These single-cell landscapes reveal that DNMT3A mutation further activates Tfh cells and leads to rapid and terminal differentiation of B cells, probably through enhancing the interacting PD1/PD-L1, ICOS/ICOSL, CD28/CD86, and ICAM1/ITGAL pairs. Our study establishes the functional roles of DNMT3A mutation in AITL and sheds light on the molecular mechanisms of this disease.

Abstract A28: Mechanisms of adaptive immune evasion by pre-leukemic DNMT3A-mutated blood cell clones

Abstract Hematopoietic stem cells (HSC) frequently acquire gene mutations with age. An expansion of blood cells derived from a single HSC is termed clonal hematopoiesis (CH), a condition that often precedes acute myeloid leukemia (AML). CH is common in elderly individuals: by age 60, approximately 10% of individuals have a clonally expanded blood cell population with a leukemia-associated mutation. The most common mutations in CH (50% of all cases) inactivate the DNA methyltransferase DNMT3A, which regulates gene expression via methylation of CpG rich regions. These mutations provide a selective advantage over healthy HSCs during aging and inflammation. Our goal is to discover mechanisms that allow for clonal expansion of DNMT3A-driven CH. A better understanding of these mechanisms will inspire new therapeutic strategies to limit clonal expansion and increase survival. To evaluate the transcriptional consequences of DNMT3A mutations in myeloid cells, we analyzed our previously published single-cell RNA-sequencing data of DNMT3A-mutated AML patient samples. We found a significant enrichment of antigen presentation signatures in DNMT3A mutated cells along the myeloid differentiation trajectory. CD8+ T cells from these same patients had significantly increased signatures of T cell activation and T cell exhaustion. T cell exhaustion occurs after an activated T cell has been continuously exposed to antigens. Exhausted T cells lack most effector functions and the ability to properly surveil (i.e. recognize mutant peptides presented on the surface of mutated cells and kill the target upon recognition) and restrain mutant cells from expanding. We hypothesize that DNMT3A-driven CH leads to escape of immune surveillance via chronic antigen stimulation and eventual promotion of T cell exhaustion. This would allow for further expansion of the mutated cells and the transformation of CH into AML. To functionally test this hypothesis, we evaluated whether a mouse model of Dnmt3a-CH recapitulated our human data. Indeed, scRNA-seq data of mouse Dnmt3a-CH cells showed significantly increased antigen presentation signatures across the myeloid differentiation trajectory. Additionally, CD8+ T cells had increased signatures of activation. We next functionally tested whether Dnmt3a-mutated CH cells had enhanced antigen presentation by pulsing bone marrow cells derived from Dnmt3a-CH mice with DQ-Ovalbumin, a self-quenched exogenous protein that becomes fluorescent when endocytosed and digested in the phagolysosome. Dnmt3a-mutated HSCs and LSKs had significantly increased DQ-OVA uptake and processing. In conclusion, we found that DNMT3A-CH cells have increased antigen presentation and processing which corresponds with enhanced immunogenicity. These findings support a model in which T cells constrain the expansion of DNMT3A-mutated HSCs, until T cell exhaustion occurs, leading to further expansion of the mutated clone and an increased chance of transformation into AML. Citation Format: Kyle A Romine, Daniel Ssozi, Tina Mujica, Yoke Lee, Jennifer Trowbridge, Peter van Galen. Mechanisms of adaptive immune evasion by pre-leukemic DNMT3A-mutated blood cell clones [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A28.