Somatic Mutations In TET2 Research Articles

Genome sequencing in the management of myelodysplastic syndromes and related disorders.

Myeloid neoplasms originate from the clonal proliferation of hematopoietic stem cells, which is driven by the acquisition of somatic genetic mutations. Within these disorders, myelodysplastic syndromes (MDS) are specifically characterized by morphologic abnormalities (dysplasia) and impaired maturation of myeloid precursors (ineffective hematopoiesis), resulting in peripheral blood cytopenia. Several studies have advanced the field of MDS, with a few landmark papers leading to a paradigm shift, opening new avenues of research and enabling a molecular revolution. These seminal papers include the first description of the 5q- syndrome, the identification of somatic mutations of TET2 in myeloid neoplasms, the detection of common pathway mutations in the splicing machinery, and the discovery of clonal hematopoiesis. The somatic genomic landscape of MDS is now well-defined. Genes that are recurrently mutated include epigenetic regulators, as well as genes of RNA splicing machinery, transcription regulation, DNA repair control, cohesin complex, and signal transduction. Furthermore, several disorders with a germline genetic predisposition to MDS have been identified, collectively accounting for up to 15% of all MDS cases. Genomic profiling can significantly improve the diagnostic approach to MDS, allowing the identification of distinct nosologic entities such as SF3B1-mutant or TP53-mutant MDS. The Molecular International Prognostic Scoring System for MDS (IPSS-M) has already proven to be a valuable tool for individualized risk assessment and treatment decisions. In addition, the recently developed molecular taxonomy of MDS will likely facilitate the implementation of precision medicine approaches for these disorders. This will necessitate the establishment of specialized infrastructures within public health systems, involving close collaboration between healthcare institutions, academia, and the life sciences industry.

Aggressive Lymphoma after CD19 CAR T-Cell Therapy

SummaryThe development of a fatal, clonal, autonomously proliferating CD4−CD8− chimeric antigen receptor (CAR)+ peripheral T-cell lymphoma (PTCL) occurred 1 month after a patient received treatment with tisagenlecleucel for relapsed primary central nervous system lymphoma. The PTCL had a clonal T-cell receptor rearrangement, which was already detectable in the apheresis product for CAR T-cell manufacturing and 7 months earlier for autologous transplantation. Somatic DNMT3A and TET2 mutations in CD34+ stem cells and their progeny were detected in the PTCL, in the apheresis specimen that was obtained for CAR T-cell production, and in the autotransplant. The PTCL harbored an additional somatic TET2 mutation, which was already detectable in the CAR T-cell apheresis product and the final CAR T-cell product at very low frequencies, providing evidence that clonal hematopoiesis had contributed to lymphomagenesis.

Epstein-Barr virus-positive, primary cutaneous marginal zone lymphoma, with transformation: Case report and review of the literature.

We report a patient with EBV positive PCMZL with subsequent transformation to plasmablastic lymphoma and review the literature for transformed PCMZL to assess clinical and pathologic characteristics. In the case we describe, the patient presented with multifocal PCMZL, developed large B cell transformation with plasmacytic differentiation, followed by plasmablastic transformation (PBL), and ultimately died of disease progression despite multiple lines of therapy. Past history was significant for psoriatic arthritis (multiple prior lines of immunomodulatory therapy). The lymphomas and non-involved bone marrow share the same somatic DNMT3A and TET2 mutations, suggesting clonal relatedness and an association with clonal hematopoiesis (CH). Eighteen cases complied the cohort (seventeen cases from the literature and the case reported herein). Nearly half of the eighteen cases of PCMZL with transformation died of progressive disease (44%). Transformed cases were more commonly seen in patients with >2 sites at initial diagnosis. EBV was assessed in 5 patients, 3 were positive (all died of disease). Two patients with NGS studies demonstrated TET2 and DNMT3A mutations. Transformation of EBV positive PCMZL appears to be a poor prognostic indicator, with our reported case being the first well defined case transformed to PBL, suspected to arise from myeloid-CH.

Inflammation in myelodysplastic syndrome pathogenesis

Inflammation is a key driver of the progression of preleukemic myeloid conditions, such as clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS), to myelodysplastic syndromes (MDS). Inflammation is a critical mediator in the complex interplay of the genetic, epigenetic, and microenvironmental factors contributing to clonal evolution. Under inflammatory conditions, somatic mutations in TET2, DNMT3A, and ASXL1, the most frequently mutated genes in CHIP and CCUS, induce a competitive advantage to hematopoietic stem and progenitor cells, which leads to their clonal expansion in the bone marrow. Chronic inflammation also drives metabolic reprogramming and immune system deregulation, further promoting the expansion of malignant clones. This review underscores the urgent need to fully elucidate the role of inflammation in MDS initiation and highlights the potential of the therapeutical targeting of inflammatory pathways as an early intervention in MDS.

Colchicine prevents accelerated atherosclerosis in TET2-mutant clonal haematopoiesis.

Somatic mutations in the TET2 gene that lead to clonal haematopoiesis (CH) are associated with accelerated atherosclerosis development in mice and a higher risk of atherosclerotic disease in humans. Mechanistically, these observations have been linked to exacerbated vascular inflammation. This study aimed to evaluate whether colchicine, a widely available and inexpensive anti-inflammatory drug, prevents the accelerated atherosclerosis associated with TET2-mutant CH. In mice, TET2-mutant CH was modelled using bone marrow transplantations in atherosclerosis-prone Ldlr-/- mice. Haematopoietic chimeras carrying initially 10% Tet2-/- haematopoietic cells were fed a high-cholesterol diet and treated with colchicine or placebo. In humans, whole-exome sequencing data and clinical data from 37 181 participants in the Mass General Brigham Biobank and 437 236 participants in the UK Biobank were analysed to examine the potential modifying effect of colchicine prescription on the relationship between CH and myocardial infarction. Colchicine prevented accelerated atherosclerosis development in the mouse model of TET2-mutant CH, in parallel with suppression of interleukin-1β overproduction in conditions of TET2 loss of function. In humans, patients who were prescribed colchicine had attenuated associations between TET2 mutations and myocardial infarction. This interaction was not observed for other mutated genes. These results highlight the potential value of colchicine to mitigate the higher cardiovascular risk of carriers of somatic TET2 mutations in blood cells. These observations set the basis for the development of clinical trials that evaluate the efficacy of precision medicine approaches tailored to the effects of specific mutations linked to CH.

FGFR1 fusions as a novel molecular driver in rhabdomyosarcoma.

The wide application of RNA sequencing in clinical practice has allowed the discovery of novel fusion genes, which have contributed to a refined molecular classification of rhabdomyosarcoma (RMS). Most fusions in RMS result in aberrant transcription factors, such as PAX3/7::FOXO1 in alveolar RMS (ARMS) and fusions involving VGLL2 or NCOA2 in infantile spindle cell RMS. However, recurrent fusions driving oncogenic kinase activation have not been reported in RMS. Triggered by an index case of an unclassified RMS (overlapping features between ARMS and sclerosing RMS) with a novel FGFR1::ANK1 fusion, we reviewed our molecular files for cases harboring FGFR1-related fusions. One additional case with an FGFR1::TACC1 fusion was identified in a tumor resembling embryonal RMS (ERMS) with anaplasia, but with no pathogenic variants in TP53 or DICER1 on germline testing. Both cases occurred in males, aged 7 and 24, and in the pelvis. The 2nd case also harbored additional alterations, including somatic TP53 and TET2 mutations. Two additional RMS cases (one unclassified, one ERMS) with FGFR1 overexpression but lacking FGFR1 fusions were identified by RNA sequencing. These two cases and the FGFR1::TACC1-positive case clustered together with the ERMS group by RNAseq. This is the first report of RMS harboring recurrent FGFR1 fusions. However, it remains unclear if FGFR1 fusions define a novel subset of RMS or alternatively, whether this alteration can sporadically drive the pathogenesis of known RMS subtypes, such as ERMS. Additional larger series with integrated genomic and epigenetic datasets are needed for better subclassification, as the resulting oncogenic kinase activation underscores the potential for targeted therapy.

Association of Somatic TET2 Mutations With Giant Cell Arteritis.

Giant cell arteritis (GCA) is an age-related vasculitis. Prior studies have identified an association between GCA and hematologic malignancies (HMs). How the presence of somatic mutations that drive the development of HMs, or clonal hematopoiesis (CH), may influence clinical outcomes in GCA is not well understood. To examine an association between CH and GCA, we analyzed sequenced exomes of 470,960 UK Biobank (UKB) participants for the presence of CH and used multivariable Cox regression. To examine the clinical phenotype of GCA in patients with and without somatic mutations across the spectrum of CH to HM, we performed targeted sequencing of blood samples and electronic health record review on 114 patients with GCA seen at our institution. We then examined associations between specific clonal mutations and GCA disease manifestations. UKB participants with CH had a 1.48-fold increased risk of incident GCA compared to UKB participants without CH. GCA risk was highest among individuals with cytopenia (hazard ratio [HR] 2.98, P = 0.00178) and with TET2 mutation (HR 2.02, P = 0.00116). Mutations were detected in 27.2% of our institutional GCA cohort, three of whom had HM at GCA diagnosis. TET2 mutations were associated with vision loss in patients with GCA (odds ratio 4.33, P = 0.047). CH increases risk for development of GCA in a genotype-specific manner, with the greatest risk being conferred by the presence of mutations in TET2. Somatic TET2 mutations likewise increase the risk of GCA-associated vision loss. Integration of somatic genetic testing in GCA diagnostics may be warranted in the future.

The Prevalence and Mutational Spectrum of Clonal Hematopoiesis in Diverse Populations in New York City

Introduction: Clonal hematopoiesis (CH) is a pre-malignant condition characterized by over-representation of blood cells derived from a single stem-cell clone. CH is strongly associated with aging with 10-20% of people over the age of 70 in various series reported to harbor a detectable clone. The most commonly mutated genes in aging-related CH are DNMT3A & TET2 followed by ASXL1 as a distant third and a smaller frequency of mutations in the splicing genes SF3B1, SRSF2, PRPF8 and U2AF1. CH portends a strong underlying risk for development of Myelodysplastic Syndrome (MDS) and other hematologic neoplasms as well as future cardiovascular disease and all-cause mortality. While much is known about these pre-leukemic states that confer risk for developing AML, most of the published studies are based on predominantly white Europeans. There has also recently been an effort to provide genomic and clinical services to high-risk individuals (CH clinics) to predict their pre-leukemic risk, however almost all these services are unavailable to underserved, ethnic minority populations. We decided to query the rate and associations of CH in diverse populations of New York City (NYC). The few published studies investigating CH in diverse populations have used self-identified categorization of race and ethnicity. However, these groupings can be too general and inaccurate when investigating genetic variation, particularly in diverse and multi-ethnic populations. Here we present our CH analysis on the multi-ethnic All of Us (AoU) cohort (version 6) for NYC individuals. A population genetics approach was used to identify groups of individuals who share genetic segments identical by descent, which corresponded to several known communities. We estimated the frequency of CH among the populations identified through this approach. Methods & Results: Of a total of 8538 NYC individuals in the AoU cohort that had information on lifestyle (smoking), 393 (4.64%) had CH. We assigned these individuals to 11 genetic ancestry groups based on sharing of genomic segments identical by descent. Out of these 11 groups, 6 had >20 individuals with CH, so we focused on those groups for further analysis (Figure 1). We constructed a logistic regression model between CH status and ancestry taking into account age, age*age (to account for a non-linear effect), sex, smoking and mean BMI with individuals belonging to the ‘Non-Jewish European ancestry group’ as a reference. We found that Dominicans have a higher rate of CH in comparison to other ancestry groups (OR: 1.62; p-value: 0.018; Figure 2). Dominicans as an ancestry group independently showed statistically significant associations with cardiovascular diseases such as heart failure, cardiac arrythmia and cardiomegaly. Whether this association is related to elevated CH remains to be investigated. We also measured the enrichment of variants in individual CH genes in each ancestry group, keeping all other individuals as a reference. Our results showed that CH in individuals belonging to the African ancestry group, as well as the Ashkenazi Jewish group have significantly higher rates of somatic mutations in DNMT3A (OR: 1.82; p-value: 0.027) and TET2 (OR: 2.36; p-value 0.004), respectively, compared to the other populations. Conclusions: Leveraging population genetic-based inference of ancestry groups, we have demonstrated for the first time that different ancestries residing in NYC have a different risk and pattern of CH somatic mutations. In particular, we demonstrate that non-Jewish Europeans have lower risk of CH compared to all other groups, and Dominicans in particular have a significantly higher risk. Understanding the relative rates of CH across ancestries and their association with chronic diseases such as CVD and cancer will further refine recommendations directed towards lifestyle modifications and rational therapeutics that prevent the progression of CH & associated comorbidities.

Somatic TET2 Mutations Prime the Immune System for Response to Immune Checkpoint Blockade

The driver mutations causing Hematopoietic Stem Cell (HSC) expansion in Clonal Hematopoiesis (CH) are inherited by HSC progeny and in some settings alter the function of mutant immune cells. It is unclear how CH mutations impact immune cell function in response to immune checkpoint blockade (ICB), which is a critical cancer therapy for many solid tumours and hematological cancers. Understanding how CH influences ICB could provide a novel biomarker for patient stratification or personalized therapeutics and may offer insight into immunotherapy mechanisms, yielding novel drug targets to synergistically improve patient outcomes. Somatic mutations inactivating TET2, which is responsible for 5-methylcytosine hydroxylation and demethylation, are one of the most frequent drivers of CH. TET2 inactivation can enhance inflammation from monocytes/macrophages, CD4 + and CD8 + T cells, but its impact on ICB is unknown. Here we sought to define the impact of TET2-mutant CH (TET2-CH) on ICB response. First, we modelled TET2-CH by rescuing lethally irradiated C57BL6/j mice with Tet2-knockout (-KO), Tet2-heterozygous null (-het), or wild type (WT) bone marrow. Mice were subsequently implanted with the syngeneic colorectal adenocarcinoma line MC38 and treated with Programmed Cell Death Protein-1 (PD-1) targeted ICB or Isotype Control (control) antibodies. PD-1 ICB, but not control, treated tumors were significantly smaller in mice with Tet2-het or Tet2-KO versus WT hematopoiesis (Figure 1). There was a trend towards a dose-responsive effect, with more Tet2-het mice responding to PD-1 ICB than wt, and more Tet2-KO mice responding than Tet2-het or wt mice. Therefore, Tet2-mutant leukocytes promote response to PD-1 blockade. PD-1 ICB with targeted cell depletion revealed that the enhanced response in Tet2-het mice requires CD4 + T Cells, CD8 + T Cells and macrophages, but not NK cells. To understand the mechanism through which Tet2-inactivation promotes ICB responses we performed 10X single cell RNA-sequencing on 38,994 CD45 + tumor-infiltrating leukocytes (TILs) from Tet2-het and wt mice treated with PD-1 ICB or control. Tet2-het mutations dramatically changed the TIL landscape with PD-1 blockade by promoting the acquisition of cell fates that drive durable immunotherapy responses including CD8 + effector T cells, CD8 + memory T cells, CD4 + interferon-responsive cells, and macrophages expressing an inflammatory anti-tumour gene signature (Figure 2). In contrast, wt TILs from PD-1 treated tumours were significantly enriched for exhausted CD8 + T cells, CD4 + regulatory T cells, and immunosuppressive macrophages that can promote tumor growth (Figure 2). TIL DNA methylation analysis using Infinium Beadchip arrays revealed promoter hypermethylation in genes defining the immunosuppressive macrophage state. Collectively, this suggests that Tet2-CH reshapes TIL fate following ICB by promoting anti-tumor phenotypes and protecting cells from immunosuppressive states. To investigate the potential clinical relevance of our findings, we retrospectively tested for an association between TET2-CH and immunotherapy outcome in 569 ICB-treated solid tumor patients. CH mutations were detected in publicly-available exome-sequencing data at VAF >0.02 using Mutect2. While CH as a composite of all driver mutations did not correlate with outcome, in multivariate analysis TET2-CH was associated with 6-times greater odds of achieving 6 month clinical benefit in n=205 melanoma patients (95% CI 1.01-66.6). To explore whether TET2-CH might identify patients primed for ICB response, we screened for CH in the prospectively collected baseline PBMCs from 90 solid tumour patients treated in a Phase 2 trial of the PD-1 inhibitor Pembrolizumab (NCT02644369). While underpowered to draw conclusions, we noted that one patient, treated for the skin cancer Merkel Cell Carcinoma, was positive for TET2-CH and responded to ICB, and also survived longer than the cohort median and Merkel Cell Carcinoma median. In summary, our results show that TET2-CH promotes immunotherapy response by shifting TILs from immunosuppressive to anti-tumor fates. Mechanistically, this occurs through lineage-specific promoter hypermethylation and gene silencing. Further study of TET2-CH as a potential biomarker of ICB response is warranted and targeting TET2-regulated pathways may yield novel strategies to improve immunotherapy outcomes.

Colchicine prevents accelerated atherosclerosis development in TET2-mutant clonal hematopoiesis

Abstract Background Somatic mutations in the TET2 gene that lead to clonal hematopoiesis are associated with accelerated atherosclerosis development in mice and a higher risk of atherosclerotic cardiovascular disease events in humans. Mechanistically, these observations have been linked to exacerbated vascular inflammation. Purpose Here, we sought to evaluate whether colchicine, a widely available and inexpensive anti-inflammatory drug, prevents the accelerated atherosclerosis associated with TET2-mutant clonal hematopoiesis. Methods In mice, we mimicked TET2-mutant clonal hematopoiesis using a competitive bone marrow transplantation approach in atherosclerosis prone Ldlr/- mice. We generated hematopoietic chimeras carrying initially 10% Tet2-/- hematopoietic cells, which were fed a high-cholesterol diet and treated with colchicine or placebo. Atherosclerosis development was evaluated histologically. In humans, we analyzed whole exome sequencing data and electronic medical records from 50,291 participants in a hospital-based cohort to examine the potential modifying effect of colchicine prescription on the association between clonal hematopoiesis and myocardial infarction. Results Colchicine prevented the development of accelerated atherosclerosis in the mouse model of TET2-mutant clonal hematopoiesis, without affecting serum cholesterol levels or the expansion of TET2-deficient hematopoietic cells. In humans, consistent with experimental findings, patients who were prescribed colchicine had attenuated associations between TET2-mutant clonal hematopoiesis and myocardial infarction (ORno colchicine [95% CI]: 1.18 [0.93-1.48]; ORcolchicine [95% CI]: 0.78 [0.45-1.35]; Pinteraction=0.03). This interaction was not observed for carriers of other clonal hematopoiesis-related mutations, such as those affecting DNMT3A or ASXL1. Conclusions Our results highlight the potential value of colchicine as a targeted preventive care strategy to mitigate the higher cardiovascular risk of carriers of somatic TET2 mutations in blood cells. These observations set the basis for the development of clinical trials that evaluate the efficacy of precision medicine approaches tailored to the effects of specific somatic mutations linked to clonal hematopoiesis.

Biallelic TET2 mutations confer sensitivity to 5'-azacitidine in acute myeloid leukemia.

Precision medicine can significantly improve outcomes for patients with cancer, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here, we describe somatic biallelic TET2 mutations in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine but acutely sensitive to 5'-azacitidine (5'-Aza) hypomethylating monotherapy, resulting in long-term morphological remission. Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5'-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5'-Aza compared with cells with monoallelic mutations. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first-line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for patients with cancer.

Aging drives Tet2+/− clonal hematopoiesis via IL-1 signaling

AbstractClonal hematopoiesis of indeterminate potential (CHIP), also referred to as aging-related clonal hematopoiesis, is defined as an asymptomatic clonal expansion of mutant mature hematopoietic cells in ≥4% of blood leukocytes. CHIP associates with advanced age and increased risk for hematological malignancy, cardiovascular disease, and all-cause mortality. Loss-of-function somatic mutations in TET2 are frequent drivers of CHIP. However, the contribution of aging-associated cooperating cell-extrinsic drivers, like inflammation, remains underexplored. Using bone marrow (BM) transplantation and newly developed genetic mosaicism (HSC-SCL-Cre-ERT; Tet2+/flox; R26+/tm6[CAG-ZsGreen1]Hze) mouse models of Tet2+/−driven CHIP, we observed an association between increased Tet2+/− clonal expansion and higher BM levels of the inflammatory cytokine interleukin-1 (IL-1) upon aging. Administration of IL-1 to mice carrying CHIP led to an IL-1 receptor 1 (IL-1R1)–dependent expansion of Tet2+/− hematopoietic stem and progenitor cells (HSPCs) and mature blood cells. This expansion was caused by increased Tet2+/− HSPC cell cycle progression, increased multilineage differentiation, and higher repopulation capacity compared with their wild-type counterparts. In agreement, IL-1α–treated Tet2+/− hematopoietic stem cells showed increased DNA replication and repair transcriptomic signatures and reduced susceptibility to IL-1α–mediated downregulation of self-renewal genes. More important, genetic deletion of IL-1R1 in Tet2+/− HPSCs or pharmacologic inhibition of IL-1 signaling impaired Tet2+/− clonal expansion, establishing the IL-1 pathway as a relevant and therapeutically targetable driver of Tet2+/− CHIP progression during aging.

CHIP-associated mutant ASXL1 in blood cells promotes solid tumor progression.

Clonal hematopoiesis of indeterminate potential (CHIP) is an age‐associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid‐tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock‐in mice that express CHIP‐associated mutant Asxl1 (Asxl1‐MT), we showed that expression of Asxl1‐MT in T cells, but not in myeloid cells, promoted solid‐tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1‐MT–expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV‐PyMT. Intratumor analysis of the mammary tumors revealed the reduced T‐cell infiltration at tumor sites and programmed death receptor‐1 (PD‐1) upregulation in CD8+ T cells in MMTV‐PyMT/Asxl1‐MT mice. In addition, we found that Asxl1‐MT induced T‐cell dysregulation, including aberrant intrathymic T‐cell development, decreased CD4/CD8 ratio, and naïve‐memory imbalance in peripheral T cells. These results indicate that Asxl1‐MT perturbs T‐cell development and function, which contributes to creating a protumor microenvironment for solid tumors. Thus, our findings raise the possibility that ASXL1‐mutated blood cells exacerbate solid‐tumor progression in ASXL1‐CHIP carriers.

Clonal Hematopoiesis Is Associated with Increased Risk of Progression of Asymptomatic Waldenström Macroglobulinemia

▪IntroductionClonal hematopoiesis (CH) is associated with adverse outcomes in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma undergoing autologous stem cell transplantation. Still, its implications for patients with indolent NHL have not been well studied. Here, we report the prevalence of CH in patients with Waldenström Macroglobulinemia (WM) and its association with clinical outcomes.MethodsWe retrospectively reviewed clinical data of 602 patients with IgM monoclonal gammopathy of undetermined significance (MGUS), smoldering WM (SWM), and WM who had clinical next-generation sequencing (NGS) performed on bone marrow aspirates or peripheral blood obtained between October 2014 and February 2020 at the Dana-Farber Cancer Institute. An Illumina Truseq amplicon-based NGS assay of 95 genes recurrently mutated in myeloid and lymphoid neoplasms was utilized. Each specimen yielded ~2 million reads and ~1500X average coverage, with 90% of amplicons having >200X coverage. Pathogenic driver variants were identified based on mutation type, position, and frequency in published reports and public databases. To unambiguously differentiate CH mutations from those in the WM clone, CH was defined by the presence of somatic mutations in DNMT3A, TET2, or ASXL1 (DTA).ResultsThe cohort included 147 patients with MGUS or asymptomatic WM and 453 patients with symptomatic WM, with a median age of 66 years (range = 40-89) and 68 years (range = 33-93), respectively, at the time of first NGS assay. The prevalence of CH-DTA was 14% in symptomatic WM patients and did not differ significantly in MGUS (13%) or SWM (14%). Among precursor patients, there was an increased risk of progression to symptomatic WM for those with CH-DTA [7/20 patients with vs. 11/116 without CH-DTA progressed over a median of 54 months (P = 0.002)]. In symptomatic WM patients, CH-DTA was positively associated with older age (P < 0.001) at the time of NGS, with a median age of 72 vs. 67 years for patients with versus those without CH-DTA. CH-DTA was not associated with inferior overall survival (OS) with a relatively short median follow-up from diagnosis and NGS assay. OS was 6.7 years (95% CI = 6.1-7.6) for patients with CH-DTA and 2.5 years for patients without DH-DTA (95% CI = 2.2-2.8). The most common cause of death was disease progression, with no significant difference between those with or without CH-DTA. Patients with CH-DTA had an increased risk of cardiovascular disease (30% vs. 18%, P = 0.036).ConclusionsWe demonstrate that CH is common in WM patients and is associated with an increased risk of progression from precursor states but not with inferior survival. Further work is needed to determine how the presence of CH might promote progression to WM and whether it can be incorporated into future risk stratification models. Importantly, our data do not support changes in clinical management or alterations in therapy for patients with WM and coexistent CH and reinforce the need to interpret NGS results within their specific clinical context. DisclosuresSteensma: Novartis: Current Employment. Castillo: Abbvie: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy; Roche: Consultancy; TG Therapeutics: Research Funding. Treon: X4: Research Funding; Dana Farber Cancer Institute: Current Employment; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Self: Patents & Royalties: Holder of multiple patents related to testing and treatment of MYD88 and CXCR4 mutated B-cell malignancies; BMS: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy. Sperling: Adaptive: Consultancy.

Genomic Landscape of RUNX1-Familial Platelet Disorder with Myeloid Malignancies Reveals Rising Clonal Hematopoiesis

Familial platelet disorder with associated myeloid malignancies (FPDMM) is a rare autosomal dominant disease caused by germline RUNX1 mutations. FPDMM patients have defective megakaryocytic development, low platelet counts, prolonged bleeding times, and a life-long risk (20-50%) of developing hematological malignancies. FPDMM is a rare genetic disease in need of comprehensive clinical and genomic studies.In early 2019 we launched a longitudinal natural history study of patients with FPDMM at the NIH Clinical Center and by May 2021 we have enrolled 98 patients and 100 family controls from 55 unrelated families. Genomic data have been generated from 56 patients in 24 families, including whole exome sequencing (WES), RNA-seq, and single-nucleotide polymorphism (SNP) array. We have identified 21 different germline RUNX1 variants among these 24 families, which include lost-of-function mutations throughout the RUNX1 gene, but pathogenic/likely pathogenic missense mutations are mostly clustered in the runt-homology domain (RHD). As an important form of RUNX1 germline mutations, five splice site variants located between exon 4-5 and exon 5-6 were identified in 6 families, which led to the productions of novel transcript forms that are predicted to generate truncated RUNX1 proteins. Large deletions affecting the RUNX1 gene are also common, ranging from 50 Kb to 1.5Mb, which were detected in 8 of the 55 enrolled families. Besides RUNX1, copy number variation (CNV) analysis from both SNP array and WES showed limited CNV events in non-malignant FPDMM patients. In addition, fusion gene analysis did not detect any in-frame fusion gene in these patients, indicating a relatively stable chromosome status in FPDMM patients.Somatic mutation landscape shows that the overall mutation burden in non-malignant FPDMM patients is lower than AML or other cancer types. However, in 13 of the 44 non-malignant patients (30%), somatic mutations were detected in at least one of the reported clonal hematopoiesis of indeterminate potential (CHIP) genes, significantly higher than the general population (4.3%). Moreover, 85% of our patients who carried CHIP mutations are under 65 years of age; in the general population, only 10% of people above 65 years of age and 1% of people under 50 were reported to carry CHIP mutations. Among mutated genes related to clonal hematopoiesis, BCOR is the most frequently mutated gene (5/44) in our FPDMM cohort, which is not a common CHIP gene among the general population. Mutations in known CHIP genes including SF3B1, TET2, and DNMT3A were also found in more than one patient. In addition, sequencing of 5 patients who already developed myeloid malignancies detected somatic mutations in BCOR, TET2, NRAS, KRAS, CTCF, KMT2D, PHF6, and SUZ12. Besides reported CHIP genes or leukemia driver genes, 3 unrelated patients carried somatic mutations in the NFE2 gene, which is essential for regulating erythroid and megakaryocytic maturation and differentiation. Two of the NFE2 mutations are nonsense mutations, and the other is a missense mutation in the important functional domain. NFE2 somatic mutations may play important roles in developing malignancy because 2 of the 3 patients already developed myeloid malignancies. For multiple patients in our cohort, we have sequenced their DNA on multiple timepoints. We have observed patients with expanding clones carrying FKBP8, BCOR or FOXP1 mutations. We have also observed a patient with relatively stable clone(s) with somatic BCOR, DNMT3A, and RUNX1T1, who have been sampled over more than four years. We will follow these somatic mutations through sequencing longitudinally and correlate the findings with clinical observations to see if the dynamic changes of CHIP clones harboring the mutations give rise to MDS or leukemia.In summary, the genomic analysis of our new natural history study demonstrated diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in FPDMM patients. These findings indicate that monitoring the dynamic changes of these CHIP mutations prospectively will benefit patients' clinical management and help us understand possible mechanisms for the progression from FPDMM to myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

OAB-040: Clonal hematopoiesis is associated with increased risk of progression of asymptomatic Waldenström Macroglobulinemia

<h3>Background</h3> Clonal hematopoiesis (CH) is associated with adverse outcomes in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma undergoing autologous stem cell transplantation. Still, its implications for patients with indolent NHL have not been well studied. Here, we report the prevalence of CH in patients with Waldenström Macroglobulinemia (WM) and its association with clinical outcomes. <h3>Methods</h3> We retrospectively reviewed clinical data of 602 patients with IgM MGUS, smoldering WM and WM who had clinical next-generation sequencing (NGS) performed on bone marrow aspirates or peripheral blood obtained between October 2014 and February 2020 at the Dana-Farber Cancer Institute (DFCI). An Illumina Truseq amplicon-based NGS assay of 95 genes recurrently mutated in myeloid and lymphoid neoplasms was utilized. Each specimen yielded ~2 million reads and ~1500X average coverage with 90% of amplicons having >200X coverage. Pathogenic driver variants were identified based on mutation type, position, and frequency in published reports and public databases. In order to unambiguously differentiate CH mutations from those in the WM clone, CH was defined by the presence of somatic mutations in DNMT3A, TET2 or ASXL1 (CH-DTA). <h3>Results</h3> The cohort included 147 patients with MGUS or asymptomatic WM and 453 patients with symptomatic WM with a median age of 66 years (range: 40-89) and 68 years (range: 33-93), respectively, at time of first NGS assay. The prevalence of CH-DTA was 14% in symptomatic WM patients and did not differ significantly in MGUS and SWM (13% and 14%, respectively). Among precursor patients, there was an increased risk of progression to symptomatic WM in those with CH-DTA (7/20 patients with vs. 11/116 without CH-DTA progressed over a median of 54 months [p= 0.002]). In symptomatic WM patients CH-DTA was positively associated with older age (p<0.001) at time of NGS, with a median age of 72 vs. 67 years for patients with versus those without CH-DTA. CH-DTA was not associated with inferior OS with a relatively short median follow-up from diagnosis and NGS assay of 6.7 (95% CI: 6.1-7.6) and 2.5 (95% CI: 2.2-2.8) years, respectively. The most common cause of death was disease progression with no significant difference between those with or without CH-DTA. Patients with CH-DTA had an increased risk of cardiovascular disease (30% vs. 18%, p=0.036). <h3>Conclusion</h3> We demonstrate that CH is common in WM patients and is associated with increased risk of progression from precursor states but not with inferior survival. Further work is needed to determine how the presence of CH might promote progression to WM and whether it can be incorporated into future risk stratification models. Importantly, our data do not support changes in clinical management or alterations in therapy for patients with WM and coexistent CH and reinforce the need to interpret NGS results within their specific clinical context.

Clonal Architecture of TET2 Mutations Influences the Clinical Phenotype of Patients with Myeloid Neoplasms

Somatic TET2 mutations (TET2MT) are frequently found in myeloid neoplasia (MN) with a frequency that increases with age, consistent with the presence of subclinical TET2MT clones in asymptomatic older controls. Despite the prominent pathophysiologic role they play based on their prevalence; phenotype-genotype and prognostic associations have not been reliably established. We hypothesized that the diversity of TET2MT with regard to their configuration, genetic topography, and sub-clonal context, result in differences in biological, morphological, and clinical features.We studied blood and bone marrow samples from 4930 patients with MN using deep sequencing for common myeloid somatic mutations, including TET2 . This analysis identified 1205 TET2MT patients with 1781 distinct TET2MT, as many had more than one TET2MT. The most common TET2MT were frame shift (47%) and nonsense (34%) mutations, both gene truncating events. Truncations were subdivided into those occurring upstream enough to effectively delete the whole gene (46%) and those that only led to deletion of the catalytic domain. TET2MT were found in 17% of patients with MDS, 46% with MDS/myeloproliferative neoplasm (MDS/MPN), 19% with MPN, 21% with pAML and 24% with sAML. Across all MN's, single mutations were more common than biallelic or homozygous, except in MDS/MPN, where there were similar proportions of single and multiple mutant cases (22% single vs . 24% biallelic/homozygous TET2).TET2MT co-occurred most commonly with another TET2MT (43%), ASXL1 (21%), SRSF2 (18%), and NPM1 (13%). A similar distribution was seen in MDS, but with SF3B1 replacing NPM1 ; and in MDS/MPN, but with SF3B1 replaced by RUNX1 and CBL . In sAML the distribution of sub clonal lesions was similar to pAML, though with more mutations in NPM1 and FLT3ITD/TKD in pAML. Genes with the most significant differences in mutation rates between TETMTvs . TET2WT MDS/MPN patients were increased SRSF2 (34% vs . 15%, p<.001) and KRAS (8% vs . 3%, p<.001).The rank of TET2MT within the clonal hierarchy is heterogeneous. TET2MT were found to be ancestral hits in 40% of mutant cases. A second TET2MT was the most common secondary hit following an ancestral TET2 hit. Other common 2nd hits included mutations in SRSF2, ASXL1, DNMT3A, and SF3B1 . TET2MTirrespective of ancestral or secondary configurations had no impact on survival outcome. However, when comparing TET2MT clonal size, TET2 allelic burden negatively affected survival (p=.016) for all patients with MNs.Having ascertained that the majority of TET2 lesions are ancestral hits, we set to determine their impact on progression, extent of dysplasia, and proliferation (monocytosis, increase megakaryocytes fibrosis). We used logistic regression to assess odds ratios (OR) of a gene being associated with MDS vs. MDS/MPN. We then determined univariate Cox regression hazard ratios for each gene and used these to construct 2-dimensional regression plots (Figure 1). This plot shows that TET2MT as a sole ancestral hit implies a greater likelihood of dysplasia and less of progression to high risk (HR) disease as compared to low risk (LR). Subsequent secondary hits change the features of the disease as shown in Figure 1. A second (biallelic) hit in TET2 increased the odds for the disease being myeloproliferative (OR=1.17), a secondary U2AF1 hit increased the odds of progression (OR=2.64), and a sub clonal SRSF2 mutation increased both (OR=6.61, 3.89). RAS family mutations had a moderate effect on progression (OR=2.66, 1.71), but induced proliferative features (OR=8.77, 7.80), whereas SF3B1 mutations associated with dysplasia and LR (OR=0.26, 0.32). In comparison, all combined TET2 hits (irrespective of the secondary context) were plotted as a circle (Figure 1).In sum, our analyses demonstrate the genotypic/phenotypic impact of primary TET2 hits and how each subsequent mutation can affect the clinical phenotype of the disease. [Display omitted] DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

SAR insights into TET2 catalytic domain inhibition: Synthesis of 2-Hydroxy-4-Methylene-pentanedicarboxylates

The TET (Ten-Eleven Translocation) dioxygenase enzyme family comprising 3 members, TET1-3, play key roles in DNA demethylation. These processes regulate transcription programs that determine cell lineage, survival, proliferation, and differentiation. The impetus for our investigations described here is derived from the need to develop illuminating small molecule probes for TET enzymes with cellular activity and specificity. The studies were done so in the context of the importance of TET2 in the hematopoietic system and the preponderance of loss of function somatic TET2 mutations in myeloid diseases. We have identified that 2-hydroxy-4-methylene-pentanedicarboxylic acid 2a reversibly competes with the co-substrate α-KG in the TET2 catalytic domain and inhibits the dioxygenase activity with an IC50 = 11.0 ± 0.9 μM at 10 μM α-KG in a cell free system and binds in the TET2 catalytic domain with Kd = 0.3 ± 0.12 μM.

Frequent somatic TET2 mutations in chronic NK-LGL leukemia with distinct patterns of cytopenias

AbstractChronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher's exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch's t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.

Poor mobilization of autologous CD34+ peripheral blood stem cells in haematology patients undergoing autologous stem cell transplantation is associated with the presence of variants in genes implicated in clonal haematopoiesis of indeterminant potential.

Clonal haematopoiesis of indeterminant potential (CHIP) increases in frequency with age. The effect of CHIP on the mobilization of autologous CD34+ peripheral blood stem cells (PBSC) has not been reported. This study uses a DNA-based targeted candidate gene approach to identify the presence of somatic mutations in ASXL1, DNMT3A, JAK2, SF3B1, TET2 and TP53 in CD34+ haematopoietic progenitor cell-apheresis products of 96 patients who undergo PBSC mobilization for autologous stem cell transplantation (ASCT). Variants were identified in a significantly greater proportion of patients who experience poor CD34+ PBSC mobilization. A DNA-based targeted candidate gene array is able to predict poor CD34+ PBSC mobilization and may be deployed pre-emptively to minimize mobilization and graft failures.