Mutations In Epigenetic Regulators Research Articles

Epigenetic Modifications in Lymphoma and Their Role in the Classification of Lymphomas

The characterisation of the lymphoma epigenome has provided insight into mechanisms involved in lymphomagenesis. Multiple lymphoma subtypes demonstrate recurrent mutations in key epigenetic regulators that have been utilised to define clinicogenetic groups that can predict clinical behaviour in these heterogenous entities. The high frequency of mutations in epigenetic regulators provides rationale to incorporate these in the classification of some subtypes of lymphoma. In addition, their recurrent nature provides a rationale to target such mutations, or the relevant pathway, for treatment. In this review, we summarised the available literature on epigenetic dysregulation in lymphoma and how it has been utilised in diagnosis and classification.

Histopathologic, immunophenotypic, and mutational landscape of follicular lymphomas with plasmacytic differentiation

Follicular lymphomas with plasmacytic differentiation (FL-PCD) include two major subtypes: one with predominantly interfollicular PCD that usually harbors a BCL2 rearrangement (BCL2-R), and a second that has predominantly intrafollicular PCD and the frequent absence of a BCL2-R. It is proposed that these latter cases share some features with marginal zone lymphomas (MZL). To further explore this hypothesis in an expanded cohort of FL-PCD, a clinicopathologic investigation of 25 such cases was undertaken including an analysis of their mutational landscape. The 10 interfollicular FL-PCDs exhibited typical intrafollicular centrocytes/centroblasts (90%), CD10 expression (90%), full PCD including expression of CD138 by the plasma cells (PC) (100%), and PCs with class-switched immunoglobulin heavy chains (70%). These cases were BCL2-R positive (100%), BCL6-R positive in 30%, lacked extra BCL2 copies, and only 22% had extra copies of BCL6. Similar to classic FLs, 80% of interfollicular FL-PCDs harbored mutations in epigenetic regulators KMT2D (70%), CREBBP (40%), and/or EZH2 (30%). In contrast, only 45% of 11 intrafollicular FL-PCDs demonstrated typical intrafollicular centrocytes/centroblasts, 55% were CD10(-), 80% contained IgM+ PCs, and only 27% harbored BCL2-Rs. BCL6-Rs were identified in 27% of intrafollicular FL-PCD, while 60% showed extra copies of BCL2 and 50% extra copies of BCL6, consistent with complete or partial trisomies of chromosomes 18 and 3, respectively. Only 54% of intrafollicular FL-PCDs showed mutations in epigenetic regulators. Both subtypes showed mutational differences compared to classic FL, but only the interfollicular subtype showed differences from what is reported for nodal MZL. Four additional cases showed mixed intra- and interfollicular PCD. These results suggest that FL-PCD has some distinctive features and supports the existence of two major subtypes. The interfollicular PCD subtype shares many features with classic FL. The intrafollicular FL-PCDs are more heterogeneous, have differences from classic FL, and have a greater morphologic, immunophenotypic, and genetic overlap with MZL.

Loss of 5-hydroxymethylcytosine expression is near-universal in B-cell lymphomas with variable mutations in epigenetic regulators.

Not available.

Clinical and Molecular Characteristics Associated with Vitamin C Deficiency in Myeloid Malignancies; Real World Data from a Prospective Cohort

Background:Vitamin C is an essential water-soluble vitamin required for many redox reactions in our body and its deficiency causes scurvy, a well characterized disease with multiple hematological manifestations. Studies dating back to 1950's demonstrated that patients with myeloid neoplasms tend to have lower plasma levels of vitamin C than healthy controls. Recent studies have shown that as much as 80% of patients with hematological malignancies in a cohort from Denmark had low vitamin C levels. Myeloid neoplasms tend to harbor mutations in epigenetic regulators which play a role in DNA methylation. One such mutation commonly seen in myeloid neoplasms and clonal hematopoiesis of indeterminate potential (CHIP) is TET2 for which vitamin C serves as a cofactor. There is a scarcity of clinical data on patients with low vitamin C level in myeloid neoplasms. Our study investigated the rates of vitamin C deficiency and the disease clinical and genomic characteristics associated with it at our center.Methods:We retrospectively collected data from a prospectively maintained list of patients treated for myeloid neoplasms at a large tertiary cancer center on whom vitamin C levels where serially collected during the study period. We obtained multiple baseline characteristics at the time of diagnosis including cytogenetic and molecular mutational data. Baseline characteristics were defined using descriptive statistics. Categorical variables were compared using a Fisher's exact test and continuous variables were analyzed using Mann Whitney U test for statistical significance. Institutional review board approval was obtained for the study. Statistical analysis was done using R Studio version 1.4.1717.Results:A total of 50 patients with myeloid neoplasms were identified with vitamin C levels available at least once during the study period. Nine (18%) patients had a low vitamin C level (LOW) defined as less than 0.4 mg/dl as per the Mayo lab testing with a reference range between 0.4 to 2.0 mg/dl. Baseline characteristics of patients with low vitamin C level and patients with normal vitamin C level (NORMAL) are shown in Table 1. The median vitamin C level in the LOW group was 0.2 mg/dl and NORMAL group was 1 mg/dl (p <0.001). The median age at diagnosis for patients in the LOW cohort was 64 years compared to 72 years for patients with normal vitamin C level (p = 0.015). Twenty-two (53.6%) of patients were female in the NORMAL cohort while six patients (66.7%) were females in the LOW cohort (p=NS). In the vitamin C LOW group only 55% of the patients were white compared to 83% in the NORMAL group (p = 0.093). The majority of patients in the Vit C LOW group had acute myeloid leukemia (AML) 44.5%, compared to 9.8% in the group with normal vitamin C levels (p = 0.03). Median white blood cell count, platelet counts, peripheral blast count and bone marrow blast count were not statistically significant amongst the 2 groups. Majority of patients in both groups 56.1% (NORMAL) vs 77.8% (LOW) had normal cytogenetics at the time of diagnosis (p = 0.284). There was a higher tendency to harbor ASXL1 and IDH2 mutation in the cohort with LOW levels 44.5% (p = 0.09) and 22.2% (p value = 0.143) compared to 17% and 4.8% respectively in the NORMAL cohort.Conclusions:Our analysis of the baseline characteristics of patients with myeloid neoplasms with vitamin C levels reveals interesting findings including a lower age at diagnosis for patients with low vitamin C levels and higher proportion of patients with acute myeloid leukemia compared to the cohort with normal levels. We also noted a higher tendency for occurrence of certain molecular mutations including ASXL1 and IDH2 among the patients with low vitamin C level. With recent papers implicating the role of ASXL1 in leukaemogenesis these findings suggest the hypothesis that vitamin C deficiency could accelerate clonal evolution with a higher tendency to transform into acute leukemia at a lower age. Further multi-institutional studies are needed to understand the relevance of low vitamin C level in myeloid neoplasms and the role of therapeutic vitamin C supplementation to retard leukaemogenesis. [Display omitted] DisclosuresPatel: Celgene-BMS: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; PVI: Honoraria. Awan: Cardinal Health: Consultancy; Abbvie: Consultancy; Merck: Consultancy; Beigene: Consultancy; Johnson and Johnson: Consultancy; Astrazeneca: Consultancy; BMS: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Dava Oncology: Consultancy; Verastem: Consultancy; ADCT therapeutics: Consultancy; Incyte: Consultancy; MEI Pharma: Consultancy; Karyopharm: Consultancy; Kite pharma: Consultancy; Celgene: Consultancy; Gilead sciences: Consultancy; Pharmacyclics: Consultancy. Anderson: Celgene, BMS, Janssen, GSK, Karyopharm, Oncopeptides, Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Madanat: Blue Print Pharmaceutical: Honoraria; Stem line pharmaceutical: Honoraria; Onc Live: Honoraria; Geron Pharmaceutical: Consultancy.

Clinico-genomic profiling and clonal dynamic modeling of TP53-aberrant myelodysplastic syndrome and acute myeloid leukemia

TP53-aberrant myelodysplastic syndrome and acute myeloid leukemia have dismal outcomes. Here, we define the clinico-genomic landscape of TP53 disruptions in 40 patients and employ clonal dynamic modeling to map the mutational hierarchy against clinical outcomes. Most TP53 mutations (45.2%) localized to the L3 loop or LSH motif of the DNA-binding domain. TP53 disruptions had high co-occurrence with mutations in epigenetic regulators, spliceosome machinery, and cohesin complex and low co-occurrence with mutations in proliferative signaling genes. Ancestral and descendant TP53 mutations constituted measurable residual disease and fueled relapse. High mutant TP53 gene dosage predicted low durability of remission. The median overall survival (OS) was 280 days. Hypomethylating agent-based therapy served as an effective bridge to transplant, leading to improved median OS compared to patients who did not receive a transplant (14.7 vs. 5.1 months). OS was independent of the genomic location of TP53 disruption, which has implications for rational therapeutic design.

MDS-029: Prevalence of VEXAS Syndrome in MDS/CMML Patients with Systemic Inflammatory and Auto-Immune Disease

Context: Systemic inflammatory and auto-immune diseases (SIAD) are observed in 25% of patients with MDS or CMML, who harbor a high frequency of mutations in TET2, IDH1/2 and SRSF2 as we previously reported (Zhao et al, Leukemia 2021). Recently, the identification of mutations in UBA1 in patients with autoinflammatory disorders, some of whom have dysplastic bone marrow, defined novel disease called VEXAS syndrome (Beck et al, NEJM 2020). Objective: To explore the prevalence of UBA1 mutations in our initial cohort of MDS/CMML patients with SIAD (n=85) (Zhao et al, Leukemia 2021). Design and Patients: All male patients for whom material was available (n=33) were analyzed for the presence of UBA1 mutations. Primary Outcome: UBA1 mutations screening with Sanger sequencing. Results: Four out of 33 (12%) patients had UBA1 mutations. All of them had MDS without excess of blasts. Cytomorphologic review of bone marrow smears showed numerous vacuoles in precursors in all 4 cases. Karyotype was normal in 3/4 patients, and one patient had deletion 9q. One patient also had somatic mutations in DNMT3A (VAF 11%) and TP53 (VAF 2%), and another patient had a mutation of TET2 (VAF 18%). Three patients in our MDS/CMML SIAD cohort had relapsing polychondritis, and 2 of them had UBA1 mutations, while 6 patients from our initial SIAD cohort had a diagnosis of Sweet syndrome, including 2 with UBA1 mutations. Three out of 4mut patients had steroid refractory SIAD and were heavily treated with other immunosuppressive therapies including methotrexate, azathioprine or cytokine targeting agents. At last follow-up, none of the 4 patients with UBA1 mutations had MDS progression, and one of them had died from a stroke, 2.4 years after MDS diagnosis. Conclusions: Our retrospective study represents the first assessment of VEXAS syndrome among patients with myeloid malignancies associated with SIAD. The low prevalence of UBA1 mutations in our cohort suggests that other pathological mechanisms may drive inflammation in the large majority of MDS/CMML patients with associated SIAD, likely in relation with mutations in epigenetic regulators TET2/IDH and SRSF2 as we previously proposed.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior

AbstractGlycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma–associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell–like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell–like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell–specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN–expressing M2-polarized macrophages.

Cancer Genetics and Epigenetics in Cancer Risk Assesment

Compared to the normal tissues, cancer cells tend to have higher proliferation rate and often lost their ability to undergo apoptosis. In addition, cancer cells can separate themselves from their original tissue thus causing metastasis in other part of body. While undergoing program cell death, disordered cellular programming can happen. The main causes of this cellular programming anomaly are epigenetic and genetic alterations, which have been known as two separate mechanisms in carcinogenetic. A recent outcome of whole exome sequencing of thousands of human cancers has been the unexpected discovery of many inactivating mutations in genes that control the epigenome. These mutations have the potential to disturb the DNA methylation patterns, histone modifications, and nucleosome positioning, hence, the causing gene expression alternation. Genetic alteration of the epigenome therefore contributes to cancer just as epigenetic process can cause point mutations and disable DNA repair functions. Epigenetic mechanisms changes could cause genetic mutations, and genetic mutations in epigenetic regulators could cause epigenome changes. Knowing that epigenome play a major role in the hierarchy of gene control mechanisms suggests that mutations might have impact on multiple pathways related to cancer phenotype. This pinpoint the fact that recently, the way the genes are organized and controlled are suggested to be a relevant factor for human carcinogenesis.Keywords: cancer genetic, cancer epigenetic, oncogens, tumor suppressor genes, driver mutation, passenger mutation

Abstract 2093: Alternative splicing in hematopoietic stem cells is affected by the loss of DNMT3A

Abstract Myelodysplastic syndromes are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Survival ranges from months to years and bone marrow transplantation remains the only curative option. Large sequencing studies have revealed that somatic mutations in epigenetic regulators are one of the most commonly affected pathways in MDS. DNMT3A, a de novo methyltransferase is mutated in ~10-15% of MDS patients. Murine models of DNMT3A loss have shown that hematopoietic stem cells favor self-renewal over differentiation. Loss of function mutations in DNMT3A can lead to reduction in methylation resulting in differentially methylated regions (DMRs). The effects of altered DNA methylation at promoters and enhancers on gene expression in DNMT3A mutant cells are well characterized but do not completely explain the gene expression patterns in these cells. Previous studies have shown that DNA methylation within intragenic regions affects mRNA splicing through interactions with RNA polymerase II and DNA binding proteins. Therefore, our study aimed to determine whether altered methylation caused by DNMT3A loss affects mRNA splicing and gene expression. To address this, we used CRISPR/Cas9 to genetically knockout DNMT3A in K562 cells. Findings were further evaluated using lineage- c-kit+ hematopoietic stem and progenitor cells (HSPC) from a DMNT3A conditional knock in mouse model of the most common MDS-associated DNMT3A point mutation, R878H. Our central hypothesis is that aberrant methylation caused by loss of function mutations in DNMT3A leads to changes in RNA splicing resulting in differential isoform expression of genes involved in stem cell self-renewal and hematopoiesis. Using a combination of RNA sequencing and whole genome bisulfite sequencing we found increased alternative splicing in DNMT3A mutant cells compared to wildtype cells. Integration of methylation and splicing revealed that 15-20 percent of the alternative splicing events were associated with changes in methylation. We found that many of the genes aberrantly spliced were involved in cell cycle regulation, transcriptional regulation, and hematopoietic stem cell differentiation. In addition, isoform usage analysis revealed that genes involved in RNA processing were perturbed. We identified similar splicing patterns and affected pathways in murine HSPCs and K562 human cells. Notably, the type of splicing events changed with age. Our results show that DNMT3A mutant cells have distinct alternative splicing events and suggest an aberrant epigenome can cause dysregulated gene expression through splicing. These findings shed light onto additional mechanisms by which epigenetic regulator mutations lead to MDS development. Citation Format: LaShanale Wallace, Ana Leal-Cervantes, Jacquelyn Myers, Yiping Fan, Koral Campbell, Juan Martin Barajas, Aparna Calindi, Chandra Rolle, Baranda Hansen, Shondra Miller, Esther Obeng. Alternative splicing in hematopoietic stem cells is affected by the loss of DNMT3A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2093.

High Mobility Group A1 Chromatin Remodeling Proteins: Potential Therapeutic Targets Involved in Polycythemia Vera Transformation to Acute Leukemia in Humans and JAK2 V617F Transgenic Mouse Models

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although the mechanisms driving disease progression remain elusive. Polycythemia vera (PV) is the most common clinical subtype caused by acquired mutations of JAK2. While PV generally presents as an indolent process, ~25% of patients will progress to MF or AML and JAK2 mutations alone do not account for MPN transformation.HMGA1 / 2 genes encode oncogenic chromatin binding proteins that are overexpressed in acute leukemia and drive clonal expansion in murine leukemia models. In preliminary studies, we found up-regulation in gene expression of both HMGA1/2 in HSC with disease progression in MPN. We therefore sought to test the hypothesis that HMGA proteins are critical drivers of transformation and rational therapeutic targets to treat or prevent PV progression.Methods: We examined clonal evolution in HSC from 49 JAK2V617F -positive PV patients using standard and SNP-array karyotyping and a targeted resequencing panel of 163 genes linked to myeloid cancers. HSC clonal burden was assessed via JAK2V617F genotype analysis on single cells. We also measured HMGA1 / 2 in JAK2V617F -positive human AML cell lines from MPN patients (PV-AML cells), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F / MPLSV ; Blood 2015;126:484) using real-time quantitative RT-PCR. To elucidate the role of HMGA1/2, we silenced HMGA1 or HMGA2 via short hairpin RNA and assessed proliferation in human PV-AML cells. Finally, to investigate Hmga1 function in vivo, we crossed mice deficient in Hmga1 onto murine models of PV and PV-AML. We also compared leukemic engraftment in JAK2V617F -positive PV-AML cells with or without HMGA1/2 silencing.Results: Both HMGA1/2 mRNA were up-regulated in all JAK2V617F -positive contexts. In primary human PV CD34+ cells, HMGA1 and HMGA2 were increased by 7- and 100-fold, respectively, compared to controls. There was a dramatic up-regulation in both HMGA1/2 in HSC of patients who transformed from PV to MF or AML compared to chronic phase PV when analyzed in cross-sectional samples from different patients or prospectively in selected patients. Overexpression of HMGA1 /2 also correlated with clonal dominance of JAK2V617F -homozygous stem cells, and additional mutations of epigenetic regulators, including EZH2 and SETBP1 . Similarly, when assessed in unfractionated bone marrow or tumor samples in transgenic mouse models for PV and PV-AML, Hmga1 / 2 were overexpressed compared to control littermates, with highest levels in the PV-AML mouse model. Silencing HMGA1 or HMGA2 in human PV-AML cells (DAMI, SET-2) dramatically halted proliferation. Moreover, leukemic engraftment was prevented in PV-AML cells after silencing HMGA1 or HMGA2. In preliminary studies with the transgenic PV-AML murine model, Hmga1 deficiency ameliorates the fulminant phenotype of aggressive leukemia and early mortality. PV-AML mice survived a median of 5 weeks (range 3-10; n=40) whereas PV-AML in Hmga1 deficient backgrounds survived a median of 8 weeks (range 7-16; n=7). The leukemic burden was decreased in mice with Hmga1 deficiency.Conclusions:HMGA1 and HMGA2 are overexpressed in MPN, and higher levels associate with disease progression to MF and AML, in both human PV and in transgenic murine models of PV. Strikingly, silencing HMGA1 or HMGA2 halted proliferation and prevented leukemic engraftment in vivo . Preliminary data in Hmga1 deficient models suggest that Hmga1 is required for PV progression to AML. These data highlight a key role for HMGA proteins as critical drivers of PV transformation and suggest that HMGA1 /2 overexpression is a consequence of aberrant JAK/STAT signaling and epigenetic dysregulation. Our findings indicate that HMGA1 /2 overexpression may function as a necessary molecular switch for PV leukemic transformation. Therefore, HMGA proteins and their transcriptional pathways offer novel therapeutic targets aimed at the prevention of PV progression to MF and AML. DisclosuresNo relevant conflicts of interest to declare.

Clinical Correlates and Outcomes of Bone Marrow Failure and Myeloid Clonal Evolution in Patients with Germline GATA2 Haploinsufficiency

BackgroundGATA2 is a zinc finger transcription factor that plays a critical role in lymphatic development and hematopoiesis. Germline mutations in GATA2 are highly penetrant, resulting in an autosomal dominant spectrum of disorders characterized by familial MDS (myelodysplastic syndrome)/ AML (acute myeloid leukemia), B/NK/dendritic cell immunodeficiency, lymphedema, and HPV-driven warts (Mir, Cancer Medicine, 2015). Myeloid clonal evolution is seen in approximately 75% of individuals with GATA2 haploinsufficiency and is associated with the acquisition of somatic mutations. We conducted this study to assess clinical correlates and outcomes of bone marrow (BM) failure and myeloid clonal evolution in patients with germline GATA2 haploinsufficiency .MethodsSuccessive patients with germline GATA2 haploinsufficiency were identified from our institutional database. GATA2 mutations and deletions were confirmed using next generation (NGS) and Sanger sequencing (Mir, Cancer Medicine 2015). Time to clonal evolution was calculated from the time of earliest reported GATA2 -related clinical manifestation, to the development of clonal cytopenias of undetermined significance (CCUS), BM failure, or frank MDS/AML. In most patients, a myeloid relevant NGS panel was performed at the time of clonal evolution to identify the acquisition of somatic mutations. GVHD-free, relapse-free survival (GRFS) was calculated for hematopoietic stem cell transplant (HSCT) recipients (Holtan, Blood, 2015). Standard statistical measures were utilized.Clinical Features:Thirteen patients with GATA2 haploinsufficiency were identified, median age 31 years (range, 0.5 to 57), of which 39% (5) were male. Median follow-up time was 46.1 months. Seven individuals comprised a kinship with a c.1339A>C, p.S447R mutation in the C-terminal domain of GATA2 . Three individuals had mutations within the zinc-finger 2 (ZF2) domain, specifically c.1192C>T, p.R398W, c.1186C>G, p.R396G, and c.1187G>A, p.R396Q, which interfere with DNA binding (Chong, Leukemia, 2017). Three individuals had larger deletions of chromosome 3q involving GATA2 (Table 1, Figure 1). Of the 13 individuals, two were asymptomatic and without clinical manifestations (Table 1). The remaining individuals' earliest symptoms included recurrent infections or immunodeficiency (n=5), warts (n=3), and cytopenias (n=2), with 3 cases of MDS/AML at the time of diagnosis.Myeloid Clonal Evolution:Nine (69%) of 13 patients developed myeloid clonal evolution; CCUS -1, bone marrow failure - 2, MDS- 3, JAK2/CALR/MPL negative, accelerated myeloproliferation- 1, and AML - 2. Of the cases that did not present with clonal evolution, the median time to clonal evolution was approximately 144 months (range, 96 to 168) from the earliest manifestations of GATA2 haploinsufficiency. At the time of clonal evolution, NGS was performed in 6 out of 9 patients, with all 6 (100%) demonstrating truncating ASXL1 mutations. In the 2 AML patients, additional somatic mutations included; DNMT3A, ETV6, SRSF2, and SETBP1 . Karyotypic abnormalities were seen in 4 (44%) of 9 cases (table 1).Transplant Outcomes:Seven patients underwent allogeneic HSCT for MDS, AML, or refractory immunodeficiency; 5 with myeloablative and 2 with reduced-intensity conditioning. Two (29%) were from 9/10 HLA mismatched unrelated donors (M-MUD), 3 (43%) were from MUD, and 1 (14%) each was from a matched related donor and a haploidentical source, respectively. 86% developed acute GVHD, mostly grade I-II, with the exception of one patient who developed grade 4 GI GVHD resulting in death. Transplant outcomes were favorable, with a GFRS of 88.3% at one year, and a 3 year relapse free survival of 66.7%. The 6 patients who have not undergone HSCT are alive and are being closely observed, or are in the planning phase for HSCT (Table 1).ConclusionsGermline GATA2 haploinsufficiency is characterized by variable B/NK/dendritic cell immunodeficiency with an enhanced predisposition to myeloid neoplasms. Myeloid clonal evolution is seen >70% of cases, with almost all patients acquiring somatic truncating ASXL1 mutations at the time of evolution. In patients with AML, additional epigenetic regulator mutations are often seen. In our series, early allogeneic stem cell transplantation was associated with favorable outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular insights into the pathogenesis of follicular lymphoma

: Follicular lymphoma (FL) represents a group of B-cell neoplasms derived from germinal center (GC) B cells. A better understanding of the pathogenic mechanisms of FL is important for developing innovative therapies. The most common form of FL is primarily nodal and associated with the t(14;18). Recent advances obtained via genomic profiling have provided unprecedented insights into the pathogenesis of FL. Conventional FL evolves through multiple independent or convergent genetic pathways. The classical pathogenesis of t(14;18)-positive FL is a multi-stage and multi-hit process escalating along accumulation of genetic and epigenetic alterations, which starts from FL-like B cells, through premalignant lesions, into full-blown malignancy. Early precursor lesions have been recognized in the form of FL-like B cells in normal peripheral blood, and both in-situ follicular neoplasia (ISFN) and duodenal-type FL. In comparison, t(14;18)-negative FL is much more heterogeneous at the molecular level, and the underlying mechanisms are less well understood. Some variants of FL, while lacking upregulation of BCL2, share a common mutational profile with conventional FL, including mutations in epigenetic modifiers. These cases also show some clinical overlap with BCL2-positive FL, including mainly nodal involvement. Other forms of FL show more profound differences, both clinically and biologically. These emerge more clearly as separate entities and include FL, grade 3B, testicular FL (TFL), pediatric-type FL (PTFL), and primary cutaneous follicle center lymphoma (PCFCL). Mutations in epigenetic regulators and 1p36/TNFRSF14 abnormalities are highly recurrent and are seen across different subtypes of FL. Genetic profiling has offered important new insights, and will continue to impact the diagnostic approach, with changes in future classification schemes.

Epigenetic Treatment of Urothelial Carcinoma Cells Sensitizes to Cisplatin Chemotherapy and PARP Inhibitor Treatment.

Simple SummaryMuscle-invasive urothelial carcinoma of the bladder (UC) is treated with chemotherapies based on the DNA-damaging drug cisplatin, which only works temporarily due to the development of drug resistance. In this study, we show that it may be possible to overcome such resistances by treating the cancer cells with specific epigenetic drugs. We investigated the “epidrug” PLX51107 that inhibits the chromatin regulator BRD4 (Bromodomain Containing 4). PLX51107 inhibited cell growth, caused DNA damage, and blocked DNA repair response in UC cells. Concomitant application of PLX51107 with cisplatin or the drug talazoparib, interfering with DNA repair, caused cell death very efficiently. PLX51107 thus sensitizes UC cells to other drugs and may allow therapy with novel effective anti-tumor drugs like talazoparib that normally only work in a small proportion of patients with specific gene mutations. These results may help to improve current standard therapy and to develop new treatment options urgently required for UC patients.Muscle-invasive urothelial carcinoma (UC) is treated with cisplatin-based chemotherapy, which is only moderately efficient, mostly due to development of resistance. New therapy approaches are therefore urgently needed. Epigenetic alterations due to frequent mutations in epigenetic regulators contribute to development of the disease and to treatment resistance, and provide targets for novel drug combination therapies. Here, we determined the cytotoxic impact of the second-generation bromodomain protein inhibitor (BETi) PLX51107 on UC cell lines (UCC) and normal HBLAK control cells. PLX51107 inhibited proliferation, induced apoptosis, and acted synergistically with the histone deacetylase inhibitor romidepsin. While PLX51107 caused significant DNA damage, DNA damage signaling and DNA repair were impeded, a state defined as BRCAness. Accordingly, the drug strongly synergized with cisplatin more efficiently than romidepsin, and with the PARP inhibitor talazoparib to inhibit proliferation and induce cell death in UCC. Thus, a BETi can be used to “episensitize” UC cells to cytotoxic chemotherapy and inhibitors of DNA repair by inducing BRCAness in non BRCA1/2 mutated cancers. In clinical applications, the synergy between PLX51107 and other drugs should permit significant dosage reductions to minimize effects on normal tissues.

Genomic complexity is associated with epigenetic regulator mutations and poor prognosis in diffuse large B-cell lymphoma

ABSTRACT Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma with high mutation burdens but a low response rate to immune checkpoint inhibitors. In this study, we performed targeted next-generation sequencing and fluorescent multiplex immunohistochemistry, and investigated the clinical significance and immunological effect of mutation numbers in 424 DLBCL patients treated with standard immunochemotherapy. We found that KMT2D and TP53 nonsynonymous mutations (MUT) were significantly associated with increased nonsynonymous mutation numbers, and that high mutation numbers (MUThigh) were associated with significantly poorer clinical outcome in germinal center B-cell-like DLBCL with wild-type TP53. To understand the underlying mechanisms, we identified a gene-expression profiling signature and the association of MUThigh with decreased T cells in DLBCL patients with wild-type TP53. On the other hand, in overall cohort, MUThigh was associated with lower PD-1 expression in T cells and PD-L1 expression in macrophages, suggesting a positive role of MUThigh in immune responses. Analysis in a whole-exome sequencing dataset of 304 patients deposited by Chapuy et al. validated the correlation of MUT-KMT2D with genomic complexity and the significantly poorer survival associated with higher numbers of genomic single nucleotide variants in activated B-cell–like DLBCL with wild-type TP53. Together, these results suggest that KMT2D inactivation or epigenetic dysregulation has a role in driving DLBCL genomic instability, and that genomic complexity has adverse impact on clinical outcome in DLBCL patients with wild-type TP53 treated with standard immunochemotherapy. The oncoimmune data in this study have important implications for biomarker and therapeutic studies in DLBCL.

Single-Cell Sequencing Reveals Clonal Architecture of Myeloid Cancers.

Acute myeloid leukemia often had few clones, mostly with multiple epigenetic regulator mutations.

Phenogenomic heterogeneity of post-transplant plasmablastic lymphomas.

Plasmablastic lymphoma (PBL) is a rare and clinically aggressive neoplasm that typically occurs in immunocompromised individuals, including those infected with human immunodeficiency virus (HIV) and solid organ allograft recipients. Most prior studies have focused on delineating the clinico-pathological features and genetic attributes of HIVrelated PBL, in which MYC deregulation, Epstein-Barr virus (EBV) infection and, more recently, mutations in JAK/STAT, MAP kinase, and NOTCH pathway genes have been implicated in disease pathogenesis. The phenotypic spectrum of post-transplant (PT)-PBL is not well characterized and data on underlying genetic alterations are limited. This led us to perform comprehensive histopathological and immunophenotypic evaluation and targeted sequencing of 18 samples from 11 patients (8 males, 3 females; age range, 12-76 years) with PT-PBL; eight de novo and three preceded by other types of post-transplant lymphoproliferative disorders. Post-transplant PBL displayed morphological and immunophenotypic heterogeneity and some features overlapped those of plasmablastic myeloma. Six (55%) cases were EBV positive and five (45%) showed MYC rearrangement by fluorescence in situ hybridization. Recurrent mutations in epigenetic regulators (KMT2/MLL family, TET2) and DNA damage repair and response (TP53, mismatch repair genes, FANCA, ATRX), MAP kinase (KRAS, NRAS, HRAS, BRAF), JAK/STAT (STAT3, STAT6, SOCS1), NOTCH (NOTCH1, NOTCH3, SPEN), and immune surveillance (FAS, CD58) pathway genes were observed, with the mutational profiles of EBV+ and EBV– cases exhibiting both similarities and differences. Clinical outcomes also varied, with survival ranging from 0-15.9 years after diagnosis. Besides uncovering the biological heterogeneity of PT-PBL, our study highlights similarities and distinctions between PT-PBL and PBL occurring in other settings and reveals potentially targetable oncogenic pathways in subsets of the disease.

Follicular lymphoma t(14;18)-negative is genetically a heterogeneous disease

AbstractFifty-five cases of t(14;18)− follicular lymphoma (FL) were genetically characterized by targeted sequencing and copy number (CN) arrays. t(14;18)− FL predominated in women (M/F 1:2); patients often presented during early clinical stages (71%), and had excellent prognoses. Overall, t(14;18)− FL displayed CN alterations (CNAs) and gene mutations carried by conventional t(14;18)+ FL (cFL), but with different frequencies. The most frequently mutated gene was STAT6 (57%) followed by CREBBP (49%), TNFRSF14 (39%), and KMT2D (27%). t(14;18)− FL showed significantly more STAT6 mutations and lacked MYD88, NOTCH2, MEF2B, and MAP2K1 mutations compared with cFL, nodal marginal zone lymphoma (NMZL), and pediatric-type FL (PTFL). We identified 2 molecular clusters. Cluster A was characterized by TNFRSF14 mutations/1p36 alterations (96%) and frequent mutations in epigenetic regulators, with recurrent loss of 6q21-24 sharing many features with cFL. Cluster B showed few genetic alterations; however, a subgroup with STAT6 mutations concurrent with CREBBP mutations/16p alterations without TNFRSF14 and EZH2 mutations was noted (65%). These 2 molecular clusters did not distinguish cases by inguinal localization, growth pattern, or presence of STAT6 mutations. BCL6 rearrangements were demonstrated in 10 of 45 (22%) cases and did not cluster together. Cases with predominantly inguinal presentation (20 of 50; 40%) had a higher frequency of diffuse growth pattern, STAT6 mutations, CD23 expression, and a lower number of CNAs, in comparison with noninguinal cases (5.1 vs 9.1 alterations per case; P < .05). STAT6 mutations showed a positive correlation with CD23 expression (P < .001). In summary, t(14;18)− FL is genetically a heterogeneous disorder with features that differ from cFL, NMZL, and PTFL.

Overview of Histone Modification.

Epigenetics is the epi-information beyond the DNA sequence that can be inherited from parents to offspring. From years of studies, people have found that histone modifications, DNA methylation, and RNA-based mechanism are the main means of epigenetic control. In this chapter, we will focus on the general introductions of epigenetics, which is important in the regulation of chromatin structure and gene expression. With the development and expansion of high-throughput sequencing, various mutations of epigenetic regulators have been identified and proven to be the drivers of tumorigenesis. Epigenetic alterations are used to diagnose individual patients more accurately and specifically. Several drugs, which are targeting epigenetic changes, have been developed to treat patients regarding the awareness of precision medicine. Emerging researches are connecting the epigenetics and cancers together in the molecular mechanism exploration and the development of druggable targets.

Molecular Characterisation of Participants in the Phazar Trial Reveals Prognostic Impact of Mutations in Advanced-Phase-MPN

Molecular Characterisation of Participants in the Phazar Trial Reveals Prognostic Impact of Mutations in Advanced-Phase-MPN

Mechanisms of Therapeutic Response to Tipifarnib in a Mouse Model of Angioimmunoblastic T-Cell Lymphoma

Angioimmunoblastic T-cell lymphomas (AITLs) are aggressive non-Hodgkin lymphomas arising from malignant transformation of follicular helper T-cells (Tfh) and are associated with limited response to intensified chemotherapy and poor prognosis. The genomic landscape of AITL is characterized by loss of function mutations in epigenetic regulators, including TET2, and the highly prevalent RHOA G17V mutation identified in almost 70% of AITL cases (Palomero et al., 2014). Using a combination of loss of TET2 and expression of RHOA G17V in CD4+ T-cells, our group developed a new mouse model of AITL, which accurately recapitulates the features of the human disease (Cortes et al., 2018). Our model constitutes a relevant platform for studying AITL pathogenesis and developing experimental therapeutic approaches for the treatment of this disease. Tipifarnib is a farnesyltransferase inhibitor that has been linked to reduced cell proliferation and increased apoptosis in a variety of solid tumors and leukemias (Alsina et al., 2004; Kirschbaum et al., 2011). Interestingly, a phase II study of tipifarnib in patients with relapsed/refractory lymphoma indicated that, while tipifarnib had a modest anti-lymphoma activity within the whole cohort, it elicited an overall response rate of 31% within the Peripheral T-cell Lymphoma cases, particularly AITL (Witzig et al, 2011). However, the molecular bases for the therapeutic activity of tipifarnib in AITL are still unknown. To understand the role and the mechanisms of action of tipifarnib in AITL, we have used our TET2-/-RHOA G17V mouse AITL lymphoma model. Using this model, we first identified that treatment in vitro with increasing doses of tipifarnib showed limited effect on the viability of AITL lymphoma cells; however, when mouse lymphoma cells were co-cultured in the presence of splenocytes as supporting cells, tipifarnib induced a strong decrease in cell viability and proliferation, suggesting that tipifarnib might be exerting its therapeutic effects not only on the tumor cells, but also indirectly by regulating the tumor microenvironment. Indeed, tipifarnib treatment in a lymphoma model in vivo led to significantly decreased tumor load and substantial reduction of lymphoma cell infiltration in solid organs, demonstrating a consistent and strong anti-tumor effect of tipifarnib in AITL in vivo. Mechanistic analysis of signaling pathways regulated by tipifarnib indicated that tipifarnib induced downregulation of surface expression of the CXCR4 receptor in tumor cells and increased the circulating levels of its ligand, CXCL12, resulting in an effective increase of the CXCL12/CXCR4 ratio. In addition, our data revealed a significant tipifarnib-induced decrease in the levels of IFN-G, TNF-A, IL-10 and IL-17 cytokines. We also performed gene expression profiling in sorted AITL lymphoma cells and sorted stroma splenocytes from mice that had been treated with tipifarnib or vehicle control. Interestingly, most of the genes specifically affected by tipifarnib were expressed in stromal cells, where we detected an upregulation of Fas-L and, more notably, of multiple genes associated with NK cell function and differentiation (CD226, CD244a, Klre1, Xcl1 and Eomes). CYBERSORT analysis of hematopoietic cell populations indicated that treatment with tipifarnib in our mouse lymphoma model is associated with enrichment in NK signatures and a potential decrease of monocytes and neutrophils within the tumor microenvironment. In summary, we have demonstrated that tipifarnib has a strong anti-lymphoma effect on a mouse model of AITL and that this effect might be mediated by the recruitment and/or activation of different hematopoietic cell populations in the tumor microenvironment that can alter the survival and proliferation of lymphoma cells. Disclosures Palomero: Kura Onclology: Research Funding.