Gene Histone Modifications Research Articles

EBV‐positive HIV‐associated diffuse large B cell lymphomas are characterized by JAK/STAT (STAT3) pathway mutations and unique clinicopathologic features

Even in the era of highly active combination antiretroviral therapy (cART), patients with HIV have a disproportionate risk of developing aggressive lymphomas that are frequently Epstein-Barr virus (EBV)-related. Here, we investigate HIV-associated diffuse large B-cell lymphoma (HIV-DLBCL) and compare EBV-positive and EBV-negative cases. HIV-DLBCL were identified from two academic medical centres and characterised by immunohistochemistry, EBV status, fluorescence insitu hybridisation, cell of origin determination by gene expression profiling, and targeted deep sequencing using a custom mutation panel of 334 genes. We also applied the Lymphgen tool to determine the genetic subtype of each case. Thirty HIV-DLBCL were identified, with a median patient age of 46years and male predominance (5:1). Thirteen cases (48%) were EBV-positive and 14 (52%) EBV-negative. Nine of the 16 tested cases (56%) had MYC rearrangement, three (19%) had BCL6 (two of which were double hit MYC/BCL6) and none had BCL2 rearrangements. Using the Lymphgen tool, half of the cases (15) were classified as other. All HIV-DLBCL showed mutational abnormalities, the most frequent being TP53 (37%), MYC (30%), STAT3 (27%), HIST1H1E (23%), EP300 (20%), TET2 (20%), SOCS1 (17%) and SGK1 (17%). EBV-negative cases were mostly of germinal centre B-cell (GCB) origin (62%), showed more frequent mutations per case (a median of 13·5/case) and significant enrichment of TP53 (57% vs. 15%; P=0·046), SGK1 (36% vs. 0%; P=0·04), EP300 (43% vs. 0%; P=0·02) and histone-modifying gene (e.g. HIST1H1E, HIST1H1D, 79% vs. 31%; P=0·02) mutations. EBV-positive cases were mostly of non-GCB origin (70%), with fewer mutations per case (median 8/case; P=0·007), and these tumours were enriched for STAT3 mutations (P=0·10). EBV-positive cases had a higher frequency of MYC mutations but the difference was not significant (36% vs. 15%; P=0·38). EBV-association was more frequent in HIV-DLBCLs, arising in patients with lower CD4 counts at diagnosis (median 46·5 vs. 101, P=0·018). In the era of cART, approximately half of HIV-DLBCL are EBV-related. HIV-DLBCL are enriched for MYC rearrangements, MYC mutations and generally lack BCL2 rearrangements, regardless of EBV status. Among HIV-DLBCL, tumours that are EBV-negative and EBV-positive appear to have important differences, the latter arising in context of lower CD4 count, showing frequent non-GCB origin, lower mutation burden and recurrent STAT3 mutations.

North American Adult T Cell Leukemia Lymphoma (ATLL) Is Characterized By Distinct, Therapeutically Targetable Mutations in Epigenetic Modifiers

Introduction: We described the mutational spectrum of ATLL in North American patients at ASH 2016 and found that these patients have a heterogenous mutational spectrum (abstract #1758). We also noted some significant differences in comparison to Japanese ATLL cases (Kataoka et al. 2015 PMID: 26437031). In this abstract, we determine the mutational landscape of larger cohort of 27 cases and report on the presence of distinct variants in epigenetic and histone modifying genes.Methods: Twenty-seven North American primary ATLL patient samples (44% acute, 41% lymphomatous and 15% chronic/smoldering) and 7 Japanese cell lines were analyzed by targeted sequencing of 173 recurrently mutated genes in cancer (Genoptix) at a mean sequencing depth of 500X coverage.We then carried out proliferation assays with decitabine on 4 Japanese derived cell lines and 5 primary patient samples and belinostat on 1 cell line and 2 primary patient samples. We also did synergy studies with doxorubicin on 3 sensitive samples. R index was calculated for assessing synergy (Romanelli et al. 1998 PMID: 9523734).Results: We found a high number of epigenetic and histone modifying gene mutations (EP300, TET2, EZH2, MED12, PBRM1, DNMT3A, KMT2A, HIST1H1E, SPEN, IDH1 and ASXL1) in our patients, 15/27 patients (55.5%). Amongst these genes EP300 had the highest frequency of mutations and was present in 22% of patients. (Figure 1) In comparison, the Japanese cohort of 370 cases had 5.7% cases with EP300 mutations. (OR: 4.1; 95% CI: 1.5099 to 10.9492; p = 0.0055)The JAK/STAT pathway had significant number of mutations in the Japanese patients (25% had STAT3 mutations and 5% had JAK3 mutations) whereas in our North American cohort there were no STAT3 mutations and 1 patient had a JAK3 mutation (3.7%). The overall percentage of TP53 mutations was similar in both groups 17.8% in the Japanese and 25.9% in the North American patients. The presence of TP53 mutations was associated with a worse overall survival in our patients (p-value: 0.045) and has been described previously (Magalhaes et al. 2015 PMID: 25573202).EP300 is a histone acetyltransferase that acetylates histones and leads to activation of gene transcription. Inactivating mutations in EP300 can lead to aberrant gene silencing and can be associated with increased DNA methylation. A high prevalence of EP300 mutations prompted us to test the sensitivity of ATLL cell lines and primary samples to DNA methyltransferase inhibitor (Decitabine) and histone deacetylase inhibitor (Belinostat). Among the 4 cell lines tested, only the ATL43Tb(-) cell line with EP300 mutation was sensitive to the growth inhibitory effects of decitabine (IC50 < 1 uM) whereas the IC50 was not reached in resistant samples (three Japanese derived cell lines, ED40515(-) and ED41214(-) and Su9T01(-)) (Figure 2A). Since the cell lines were derived from Japanese ATLL, next, we established long term cultures of primary ATLL cells from North American cases and used them for functional studies. For primary samples, we observed that all samples (n=5) were sensitive to decitabine with IC50 < 1 uM. Furthermore, the three samples (EP300 mutant) were also sensitive to HDAC inhibitor Belinostat with an IC50 of upto 0.5 uM in all samples. Next, we tested the efficacy of combining decitabine with chemotherapy. The combination of doxorubicin and decitabine showed synergy with an R index >1 in three EP300 mutant samples tested Pt-5a, Pt-7a and ATL43Tb(-).Conclusions: Mutational analysis of the largest cohort of North American ATLL samples shows a distinct variant landscape with a high prevalence of mutations affecting epigenetic modifiers. EP300 is a histone acetyltransferase that is mutated in 22% of samples and is significantly higher in the North American cohort when compared to the Japanese cases.We also determined that majority of primary ATLL samples and EP300 mutant cell lines tested are sensitive to decitabine. The combination with doxorubicin shows synergy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In vitro characterization of human bone marrow mesenchymal stem cell-derived motor neurons induced by epigenetic modifiers

BackgroundMotor neurons (MNs) are distinct types of cells in the dorso-ventral axis of the spinal cord. These cells are developed in the presence of two main morphogens, including Sonic hedgehog (Shh) and retinoic acid (RA). On the other hand, human bone marrow mesenchymal stem cells (hBM-MSCs) are known as a multipotent type of cells with neural differentiation capacity. In this regard, the aim of this study was to quantitatively evaluate the expression of MN-related genes and the potent epigenetic regulatory genes involved in neurogenesis, including Enhancer of zeste homolog 2 (EZH-2) and P300, during hBM-MSC differentiation into MN-like cells, using RA and Shh. After isolating and inducing the cells with Shh and RA, the results were evaluated using immunocytochemistry and qRT-PCR.ResultsOur findings showed that the treated cells could express choline acetyltransferase (ChAT) and insulin gene enhancer binding protein-1 (Islet-1) antigens at the protein level, 2 weeks after induction. Moreover, at the second week after induction, the induced cells expressed MN-related genes (ChAT and ISLET-1) and epigenetic regulatory genes (EZH-2 and P300) at significant levels compared to the control (non-treated BM-MSCs) and to the induced cells at the first week (day 7). In addition, the expression of EZH-2, as a histone-modifying gene, was also significantly upregulated at the first week compared to the control. No significant upregulation was detected in the expression of motor neuron and pancreas homeobox 1 (MNX-1) in the treated groups compared to the control group.ConclusionWe concluded that epigenetic modifiers, P300 and EZH-2, are important mediators for regulating the process of motor neuron differentiation induced by RA and Shh.

Karyotypic complexity, TP53 pathogenic variants, and increased number of variants on Next-Generation Sequencing are associated with disease progression in a North American Adult T-Cell Leukemia/Lymphoma cohort.

Adult T-Cell Leukemia/Lymphoma (ATLL) is an aggressive T-cell malignancy without known characteristic cytogenetic abnormalities. Recurrent mutations in TP53, APC, and epigenetic and histone-modifying genes have been identified in North American ATLL. Their roles in disease progression are not yet fully elucidated. We studied the cytogenetic and Next-Generation Sequencing (NGS) findings of the North American ATLL cohort at our institution and compared the findings with Japanese and other North American cohorts. We also analyzed the genetic variants in TP53, APC, and histone-modifying genes and investigated the impact of their mutations on the number of mutations via NGS in ATLL. Cases with more than 6 chromosomal breaks (n=13) had significantly shorter overall survival compared to cases with fewer chromosomal breaks (n=7) (P=.0007). Cases with breaks on chromosome 3q (n=4) exhibited worse survival compared to the rest of the cases (n=16) (P=.012). Chromosomal abnormalities on 3q, 14q, 1q, 1p, and 17q are likely primary changes in ATLL based on frequency and association with prognosis. The average number of mutations via NGS was significantly higher in cases with mutations in TP53 (n=8) (P=.020) as well as APC (n=6) (P=.024) compared to cases without mutations in these genes. All TP53 variants were pathogenic missense and truncating mutations in COSMIC database. Cytogenetic and NGS methods are useful tools to monitor disease progression in indolent ATLL and assess prognosis in aggressive ATLL.

Deficiency of the X-inactivation escaping gene KDM5C in clear cell renal cell carcinoma promotes tumorigenicity by reprogramming glycogen metabolism and inhibiting ferroptosis.

Background: Clear cell renal cell carcinoma (ccRCC) is characterized by glycogen-laden, unexplained male predominance, and frequent mutations in the Von Hippel-Lindau (VHL) gene and histone modifier genes. Besides, poor survival rates of ccRCC patients seem to be associated with up-regulation of the pentose phosphate pathway (PPP). However, the mechanism underlying these features remains unclear.Methods: Whole exome sequencing was used to identify the gene mutation that implicated in the rewired glucose metabolism. RNA-seq analyses were performed to evaluate the function of KDM5C in ccRCC. Furthermore, heavy isotope tracer analysis and metabolites quantification assays were used to study how KDM5C affects intracellular metabolic flux. To provide more in vivo evidence, we generated the Kdm5c-/- mice by CRISPR-Cas9 mediated gene knockout and performed the xenografts with KDM5C overexpressing or depleted cell lines.Results: A histone demethylase gene KDM5C, which can escape from X-inactivation and is predominantly mutated in male ccRCC patients, was identified to harbor the frameshift mutation in the ccRCC cell line with the highest glycogen level, while the restoration of KDM5C significantly reduced the glycogen level. Transcriptome and metabolomic analysis linked KDM5C to metabolism-related biological processes. KDM5C specifically regulated the expression of several hypoxia-inducible factor (HIF)-related genes and Glucose-6-phosphate dehydrogenase (G6PD) that were involved in glycogenesis/glycogenolysis and PPP, respectively, mainly through the histone demethylase activity of KDM5C. Depletion of KDM5C increased the production of glycogen, which was then directed to glycogenolysis to generate glucose-6-phosphate (G6P) and subsequently PPP to produce nicotinamide adenine dinucleotide phosphate hydride (NADPH) and glutathione (GSH), thus conferring cells resistance to reactive oxygen species (ROS) and ferroptosis. KDM5C re-expression suppressed the glucose flux through PPP and re-sensitized cancer cells to ferroptosis. Notably, Kdm5c-knockout mice kidney tissues exhibited elevated glycogen level, reduced lipid peroxidation and displayed a transformation of renal cysts into hyperplastic lesions, implying a cancer-protective benefit of ferroptosis. Furthermore, KDM5C deficiency predicted the poor prognosis, and clinically relevant KDM5C mutants failed to suppress glycogen accumulation and promoted ferroptosis as wild type.Conclusion: This work revealed that a histone modifier gene inactive mutation reprogramed glycogen metabolism and helped to explain the long-standing puzzle of male predominance in human cancer. In addition, our findings may suggest the therapeutic value of targeting glycogen metabolism in ccRCC.

Dynamic Profiles and Transcriptional Preferences of Histone Modifications During Spermiogenesis.

During spermiogenesis, extensive histone modifications take place in developing haploid spermatids besides morphological alterations of the genetic material to form compact nuclei. Better understanding on the overall transcriptional dynamics and preferences of histones and enzymes involved in histone modifications may provide valuable information to dissect the epigenetic characteristics and unique chromatin status during spermiogenesis. Using single-cell RNA-Sequencing, the expression dynamics of histone variants, writers, erasers, and readers of histone acetylation and methylation, as well as histone phosphorylation, ubiquitination, and chaperones were assessed through transcriptome profiling during spermiogenesis. This approach provided an unprecedented panoramic perspective of the involving genes in epigenetic modifier/histone variant expression during spermiogenesis. Results reported here revealed the transcriptional ranks of histones, histone modifications, and their readers during spermiogenesis, emphasizing the unique preferences of epigenetic regulation in spermatids. These findings also highlighted the impact of spermatid metabolic preferences on epigenetic modifications. Despite the observed rising trend on transcription levels of all encoding genes and histone variants, the transcriptome profile of genes in histone modifications and their readers displayed a downward expression trend, suggesting that spermatid nuclei condensation is a progressive process that occurred in tandem with a gradual decrease in overall epigenetic activity during spermiogenesis.

Targeted sequencing reveals the somatic mutation landscape in a Swedish breast cancer cohort

Breast cancer (BC) is a genetically heterogeneous disease with high prevalence in Northern Europe. However, there has been no detailed investigation into the Scandinavian somatic landscape. Here, in a homogeneous Swedish cohort, we describe the somatic events underlying BC, leveraging a targeted next-generation sequencing approach. We designed a 20.5 Mb array targeting coding and regulatory regions of genes with a known role in BC (n = 765). The selected genes were either from human BC studies (n = 294) or from within canine mammary tumor associated regions (n = 471). A set of predominantly estrogen receptor positive tumors (ER + 85%) and their normal tissue counterparts (n= 61) were sequenced to ~ 140 × and 85 × mean target coverage, respectively. MuTect2 and VarScan2 were employed to detect single nucleotide variants (SNVs) and copy number aberrations (CNAs), while MutSigCV (SNVs) and GISTIC (CNAs) algorithms estimated the significance of recurrent somatic events. The significantly mutated genes (q ≤ 0.01) were PIK3CA (28% of patients), TP53 (21%) and CDH1 (11%). However, histone modifying genes contained the largest number of variants (KMT2C and ARID1A, together 28%). Mutations in KMT2C were mutually exclusive with PI3KCA mutations (p ≤ 0. 001) and half of these affect the formation of a functional PHD domain. The tumor suppressor CDK10 was deleted in 80% of the cohort while the oncogene MDM4 was amplified. Mutational signature analyses pointed towards APOBEC deaminase activity (COSMIC signature 2) and DNA mismatch repair (COSMIC signature 6). We noticed two significantly distinct patterns related to patient age; TP53 being more mutated in the younger group (29% vs 9% of patients) and CDH23 mutations were absent from the older group. The increased somatic mutation prevalence in the histone modifying genes KMT2C and ARID1A distinguishes the Swedish cohort from previous studies. KMT2C regulates enhancer activation and assists tumor proliferation in a hormone-rich environment, possibly pointing to a role in ER + BC, especially in older cases. Finally, age of onset appears to affect the mutational landscape suggesting that a larger age-diverse population incorporating more molecular subtypes should be studied to elucidate the underlying mechanisms.

Double-Hit Signature with TP53 Abnormalities Predicts Poor Survival in Patients with Germinal Center Type Diffuse Large B-Cell Lymphoma Treated with R-CHOP

Purpose: We performed detailed genomic analysis on 87 cases of de novo diffuse large B-cell lymphoma of germinal center type (GCB DLBCL) to identify characteristics that are associated with survival in those treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Experimental Design: The cases were extensively characterized by combining the results of immunohistochemistry, cell-of-origin gene expression profiling (Nanostring), double-hit gene expression profiling (DLBCL90), fluorescence in situ hybridization (FISH) cytogenetic analysis for double/triple-hit lymphoma, copy number analysis (CNA), and targeted deep sequencing using a custom mutation panel of 334 genes. Results: We identified four distinct biologic subgroups with different survivals, and with similarities to the genomic classifications from two large retrospective studies of DLBCL. Patients with the double-hit signature but no abnormalities of TP53, and those lacking EZH2 mutation and/or BCL2 translocation, had an excellent prognosis. However, patients with an EZB-like profile had an intermediate prognosis, whereas those with TP53 inactivation combined with the double-hit signature had an extremely poor prognosis. This latter finding was validated using two independent cohorts. Conclusions: We propose a practical schema to utilize genomic variables to risk-stratify patients with GCB DLBCL. This schema provides a promising new approach to identify high-risk patients for new and innovative therapies.

Bioinformatics and expression analysis of histone modification genes in grapevine predict their involvement in seed development, powdery mildew resistance, and hormonal signaling

BackgroundHistone modification genes (HMs) play potential roles in plant growth and development via influencing gene expression and chromatin structure. However, limited information is available about HMs genes in grapes (Vitis vinifera L.).ResultsHere, we described detailed genome-wide identification of HMs gene families in grapevine. We identified 117 HMs genes in grapevine and classified these genes into 11 subfamilies based on conserved domains and phylogenetic relationships with Arabidopsis. We described the genes in terms of their chromosomal locations and exon-intron distribution. Further, we investigated the evolutionary history, gene ontology (GO) analysis, and syntenic relationships between grapes and Arabidopsis. According to results 21% HMs genes are the result of duplication (tandem and segmental) events and all the duplicated genes have negative mode of selection. GO analysis predicted the presence of HMs proteins in cytoplasm, nucleus, and intracellular organelles. According to seed development expression profiling, many HMs grapevine genes were differentially expressed in seeded and seedless cultivars, suggesting their roles in seed development. Moreover, we checked the response of HMs genes against powdery mildew infection at different time points. Results have suggested the involvement of some genes in disease resistance regulation mechanism. Furthermore, the expression profiles of HMs genes were analyzed in response to different plant hormones (Abscisic acid, Jasmonic acid, Salicylic acid, and Ethylene) at different time points. All of the genes showed differential expression against one or more hormones.ConclusionVvHMs genes might have potential roles in grapevine including seed development, disease resistance, and hormonal signaling pathways. Our study provides first detailed genome-wide identification and expression profiling of HMs genes in grapevine.

Epigenetic Dysregulation in Advanced Kidney Cancer

Understanding the complex epigenome of advanced renal cell carcinoma may lead to novel epigenomic-based pharmaceutical strategies and identify new targets for therapeutic interventions. Epigenetic changes, such as DNA methylation and histone acetylation, modulate the activity of significant oncogenic signaling pathways by regulating gene expression. Such pathways include the WNT-β-catenin pathway, the von Hippel-Lindau-hypoxia-inducible factor pathway, and epithelial-mesenchymal transition pathway. Common genetic alterations in histone modifier genes in renal cell carcinoma may not only be responsible for the pathogenesis of this disease but also represent potential biomarkers of response to immunotherapies. Rational combinations strategies with histone deacetylase inhibitors are being tested in clinic trials. Renal cell carcinoma represents an ideal setting to dissect the epigenetic-driven changes in the tumor microenvironment that modulate the response to targeted therapies.

Tracking chronic myelomonocytic leukaemia diversity at the single cell level.

Chronic myelomonocytic leukaemia (CMML) is a prototypic myeloid neoplasm that associates features of myelodysplastic syndromes and myeloproliferative neosplasms. This rare and severe overlap disease is a very diverse clinical entity. Yet, genomic underpinnings of CMML combine a restricted number of somatic mutations in DNA methylation, histone modifier, splicing factor and signaling genes [1]. The discordance between mutational simplicity and clinical heterogeneity has suggested multiple explanations, from underestimation of genetic diversity to a prominent role of epigenomic and cell extrinsic factors [2, 3].

Abstract A18: Targeting epigenetic dysregulation in bladder cancer through inhibition of EZH2

Abstract Tumor genomic profiling has demonstrated that chromatin modifiers are frequently mutated in urothelial bladder cancers. The initial description of the mutational landscape of human bladder tumors indicated that 89% of cases had at least one alteration in histone-modifying genes and 64% had alterations in components of the SWI/SNF chromatin remodeling complex, suggesting that epigenetic dysregulation may play an important role in development and progression of bladder tumors. Among chromatin modifiers, ARID1A, a key component of the SWI/SNF remodeling complex, shows mutation rates of 25% in muscle-invasive bladder cancers. The chromatin modifier EZH2 functions in the suppression of gene expression. EZH2 is a histone lysine methyltransferase and the catalytic subunit of the PRC2 complex, which methylates histone H3 at lysine 27 (H3K27) to generate the transcriptionally repressive trimethylated modification state H3K27me3. EZH2 is frequently overexpressed in a wide variety of cancers, where it represses key tumor suppressors and genes involved in apoptosis and differentiation and, thus, often correlates with poor prognosis and survival. Given the role of EZH2 in cancer, we are currently developing the EZH2 inhibitor CPI-1205 in patients with castration-resistant prostate cancer. Recent publications have suggested that mutations in ARID1A create hypersensitive contexts for EZH2 inhibition in ovarian clear cell carcinomas. Given the high frequency of ARID1A mutations in bladder cancer, we were interested in whether a similar hypersensitive context exists for EZH2 inhibition in this indication. Long-term phenotypic growth assays in a panel of 21 bladder cancer cell lines treated with our second-generation EZH2 inhibitor CPI-0209 demonstrated preferential sensitivity in cell lines harboring ARID1A mutations. Sensitive cell lines showed increased subG1 populations compared to nonresponsive lines, indicating increased cell death in response to long-term EZH2 inhibition. Transcriptional profiling after CPI-0209 treatment in a panel of bladder cancer cell lines with and without ARID1A mutations showed widespread activation of EZH2 target gene expression, consistent with EZH2's transcriptionally repressive functions. These effects also translated to efficacy in vivo, where CPI-0209 treatment in bladder cell-line derived xenografts harboring ARID1A mutations led to dose-dependent tumor growth inhibition and tumor regression. Further, cotreatment of CPI-0209 with the chemotherapeutic agent cisplatin demonstrated combinatorial effects on cell viability in vitro and on in vivo tumor growth, suggesting EZH2 inhibition is also beneficial in the context of current chemotherapeutic agents in use for bladder cancers. These data support further investigation of CPI-0209 in clinical trials in bladder cancers with ARID1A mutations. Citation Format: Patricia J. Keller, Rosana Meyer, Emily Greenwald, Xinwei Han, Andrew R. Conery, Jennifer A. Mertz, Patrick Trojer. Targeting epigenetic dysregulation in bladder cancer through inhibition of EZH2 [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A18.

ICGEC: a comparative method for measuring epigenetic conservation of genes via the integrated signal from multiple histone modifications between cell types

BackgroundHistone post-translational modifications play crucial roles in epigenetic regulation of gene expression and are known to be associated with the phenotypic differences of different cell types. Therefore, it is of fundamental importance to dissect the genes and pathways involved in such a phenotypic variation at the level of epigenetics. However, the existing comparative approaches are largely based on the differences, especially the absolute difference in the levels of individual histone modifications of genes under contrasting conditions. Thus, a method for measuring the overall change in the epigenetic circumstance of each gene underpinned by multiple types of histone modifications between cell types is lacking.ResultsTo address this challenge, we developed ICGEC, a new method for estimating the degree of epigenetic conservation of genes between two cell lines. Different from existing comparative methods, ICGEC provides a reliable score for measuring the relative change in the epigenetic context of corresponding gene between two conditions and simultaneously produces a score for each histone mark. The application of ICGEC to the human embryonic stem cell line H1 and four H1-derived cell lines with available epigenomic data for the same 16 types of histone modifications indicated high robustness and reliability of ICGEC. Furthermore, the analysis of the epigenetically dynamic and conserved genes which were defined based on the ICGEC output results demonstrated that ICGEC can deepen our understanding of the biological processes of cell differentiation to overcome the limitations of traditional expression analysis. Specifically, the ICGEC-derived differentiation-direction-specific genes were shown to have putative functions that are well-matched with cell identity. Additionally, H3K79me1 and H3K27ac were found to be the main histone marks accounting for whether an epigenetically dynamic gene was differentially expressed between two cell lines.ConclusionsThe use of ICGEC creates a convenient and robust way to measure the overall epigenetic conservation of individual genes and marks between two conditions. Thus, it provides a basis for exploring the epigenotype-phenotype relationship. ICGEC can be deemed a state-of-the-art method tailored for comparative epigenomic analysis of changes in cell dynamics.

CEOP/IVE/GDP alternating regimen compared with CEOP as the first-line therapy for newly diagnosed patients with peripheral T cell lymphoma: results from a phase 2, multicenter, randomized, controlled clinical trial

BackgroundCyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP)/CHOP-like chemotherapy is widely used in peripheral T cell lymphoma (PTCL). Here we conducted a phase 2, multicenter, randomized, controlled trial, comparing the efficacy and safety of CEOP/IVE/GDP alternating regimen with CEOP in newly diagnosed PTCL.MethodsPTCL patients, except for anaplastic large cell lymphoma-anaplastic lymphoma kinase positive, were 1:1 randomly assigned to receive CEOP/IVE/GDP (CEOP, cyclophosphamide 750 mg/m2, epirubicin 70 mg/m2, vincristine 1.4 mg/m2 [maximum 2 mg] on day 1, and prednisone 60 mg/m2 [maximum 100 mg] on days 1–5 every 21 days, at the first and fourth cycle; IVE, ifosfamide 2000 mg/m2 on days 1–3, epirubicin 70 mg/m2 on day 1, and etoposide 100 mg/m2 on days 1–3 every 21 days, at the second and fifth cycle; and GDP, gemcitabine 1000 mg/m2 on days 1 and 8, cisplatin 25 mg/m2 on days 1–3, and dexamethasone 40 mg on days 1–4 every 21 days, at the third and sixth cycle) and CEOP (every 21 days for 6 cycles). Analysis of efficacy and safety was of the intent-to-treatment population. The primary endpoint was a complete response rate at the end of treatment. Meanwhile, whole exome sequencing and targeted sequencing were performed in 62 patients with available tumor samples to explore prognostic biomarkers in this cohort as an exploratory post hoc analysis.ResultsAmong 106 patients, 53 each were enrolled to CEOP/IVE/GDP and CEOP. With 51 evaluable patients each in two groups, a complete response rate of the CEOP/IVE/GDP group was similar to that of the CEOP group (37.3% vs. 31.4%, p = 0.532). There was no difference in median progression-free survival (PFS; 15.4 months vs. 9.2 months, p = 0.122) or overall survival (OS; 24.3 months vs. 21.9 months, p = 0.178). Grade 3–4 hematological and non-hematological adverse events were comparable. Histone modification genes were most frequently mutated (25/62, 40.3%), namely KMT2D, KMT2A, SETD2, EP300, and CREBBP. Multivariate analysis indicated that CREBBP and IDH2 mutations were independent factors predicting poor PFS and OS (all p < 0.001), while KMT2D predicting poor PFS (p = 0.002).ConclusionsCEOP/IVE/GDP alternating regimen showed no remission or survival advantage to standard chemotherapy. Future clinical trials should aim to develop alternative regimen targeting disease biology as demonstrated by recurrent mutations in epigenetic factors.Trial registrationThe study was registered on ClinicalTrial.gov (NCT02533700) on August 27, 2015.

Abstract P4-05-03: Mutational analysis of triple-negative breast cancer (TNBC): CALGB 40603 (Alliance)

Abstract Background: Triple-negative breast cancer is an aggressive disease with limited treatment options beyond chemotherapy. In CALGB 40603, adding either carboplatin (Cb) or bevacizumab (Bev) to standard neoadjuvant chemotherapy (NACT) increased pathologic complete response (pCR) rates in TNBC and the subset of Basal-like breast cancers. Transcriptomic studies have previously found that evidence of immune activation was significantly associated with pCR. We are now examining frequency and pCR impact of germline and somatic mutations as secondary analyses. Methods: 281 pretreatment tumors and matched blood samples were sequenced using a 1,123 cancer-associated gene panel. Germline and somatic mutations were determined and somatic mutations were further evaluated and filtered using a previously identified list of likely false positives (Bailey, Cell 2019) and RNA expression of the variant. We examined mutation frequency and association of mutations with overall in-breast pCR (result available for 274 of our cohort) and for benefit of addition of Cb or Bev on pCR. Results: The pCR rate in patients with DNA sequencing was 53.6% in TNBC (147 pCR, 127 non-pCR) and 53.3% in Basal-like (131 pCR, 115 non-pCR). Total number of somatic mutations per sample from our targeted cancer panel ranged from one to 1,041, with a median of 18 mutations per patient. Ten samples had more than 100 mutations, of which six had POLE or POLD1 mutations. Focusing on non-silent coding mutations reduced the number of variants in the entire cohort from 10,483 to 4,275. A second filter was used to remove likely false positives. Mutations in TP53 were identified in 90% of samples. Only two other genes - KMT2C (12%) and MACF1 (11%) - were mutated in more than 10% of the samples. Mutations were also identified in NF1 (8%), PIK3CA (8%), PTEN (7%), FAT1 (7%), and RB1 (7%). Somatic alterations in BRCA1 and BRCA2 were observed in 6% and 5% of samples, respectfully. We also found that 40% of the tumors had one or more mutations in a set of 18 histone modification genes. However, these mutations were not associated with pCR in the entire cohort or in the Basal-like subset (n=246). The mutations were also not associated with response to Cb or Bev. The germline was also analyzed for inherited variants. We identified 35 (12%) patients with pathogenic or likely pathogenic variants in BRCA1 (n=23), BRCA2 (n=6), PALB2 (n=3), ATM (n=2), and CHEK2 (n=1). Germline variants were not associated with pCR in the entire cohort (n=33) or Basal subset (n=32), but there was a trend toward higher pCR in germline carriers (n=15) versus non-germline carriers (n=123) within the Bev-treated cohort (80% vs 54%, p=0.09). The pCR rate in patients with a pathogenic germline or somatic BRCA1/2 mutation (n=55) was similar to that for the overall study population (53% pCR rate), as was the increase in pCR seen with the addition of Cb (61% vs. 41% pCR rate) (Sikov, JCO 2015). Conclusions: Analysis of recurrently mutated genes in TNBC among patients treated in CALGB 40603 revealed high rates of mutation and very high TP53 mutation frequency but failed to identify mutations significantly associated with pCR rate. Integrated analyses with copy number, immune and other RNA features are underway. Support for the project came from U10CA180821, U10CA180882; BCRF, Susan G. Komen, and Genentech. https://acknowledgments.alliancefound.org ClinicalTrials.gov Identifier: NCT00861705 Citation Format: Katherine A Hoadley, Bradford C. Powell, Dona Kanavy, David Marron, Lisle E. Mose, Terry Hyslop, Donald A. Berry, Olwen Hahn, Sara M. Tolaney, William M. Sikov, Charles M. Perou, Lisa A. Carey. Mutational analysis of triple-negative breast cancer (TNBC): CALGB 40603 (Alliance) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-03.

Molecular therapeutics for anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) represents one of the most lethal human cancers and although this tumor type is rare, ATC accounts for the majority of deaths from thyroid cancer. Due to the rarity of ATC, a comprehensive genomic characterization of this tumor type has been challenging, and thus the development of new therapies has been lacking. To date, there is only one mutation-driven targeted therapy for BRAF-mutant ATC. Recent genomic studies have used next generation sequencing to define the genetic landscape of ATC in order to identify new therapeutic targets. Together, these studies have confirmed the role of oncogenic mutations of MAPK pathway as key drivers of differentiated thyroid cancer (BRAF, RAS), and that additional genetic alterations in the PI3K pathway, TP53, and the TERT promoter are necessary for anaplastic transformation. Recent novel findings have linked the high mutational burden associated with ATC with mutations in the Mismatch Repair (MMR) pathway and overactivity of the AID/APOBEC family of cytidine deaminases. Additional novel mutations include cell cycle genes, SWI/SNF chromatin remodeling complex, and histone modification genes. Mutations in RAC1 were also identified in ATC, which have important implications for BRAF-directed therapies. In this review, we summarize these novel findings and the new genetic landscape of ATC. We further discuss the development of therapies targeting these pathways that are being tested in clinical and preclinical studies.

Monomorphic epitheliotropic intestinal T-cell lymphoma: a clinicopathological analysis of twelve cases

Objective: To investigate the clinicopathological features, diagnosis and differential diagnosis of monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL). Methods: A total of 12 specimens were collected, which were surgically resected and verified as MEITL by postoperative pathology, immumohistochemical staining and gene rearrangement at the First Affiliated Hospital of Nanjing Medical University from 2012 to 2018, and all of these had complete clinical and pathological data. The MEITL cases were reviewed to compare the clinicopathological characteristics, including morphologic and immunophenotypic features and followed up by telephone and clinic visit. Results: All the cases were diagnosed with MEITL. There were 8 males and 4 females. Male to female ratio was 2∶1, at a median age of 54 years. The sites of involvement included jejunum (4 cases), ileum (5 cases), duodenum (1 case), ileocecal junction (1 case) and rectum (1 case). The neoplastic cells were monotonous of small to intermediate cells in size with round to slightly irregular nuclei in 11 cases. The immunophenotyping showed that CD3 (12/12), CD8 (11/12), CD43 (11/12), CD56 (11/12), TIA-1 (12/12) were positive; CD5 (12/12), Gran B (9/12), and perforin (7/12) were negative. Two cases aberrantly expressed the B-cell marker CD20. A high proliferation index was demonstrated by Ki-67 immunostaining. In situ hybridization for EBER was all negative(12/12). The whole exome sequencing(WES) mutational landscape of MEITL was remarkably homogeneous, showing significantly enriched clusters among histone modifier genes, JAK-STAT and MAPK-signal pathways. Histonelysine N-methytransferase SETD2 gene was mutated in 2/4 tumors. All the patients analyzed harbored at least one mutation in the JAK-STAT signal pathway, including STAT5B (2/4), JAK3 (3/4) and STAT5A (2/4). Furthermore, frequent alterations (TP53) were observed in the MAPK pathway in 3/4 of MEITL cases. The CNV analysis derived from WES data identified multiple regions of frequent gains and losses. In particular, gains in 1q, 7q and 9q, and recurrent losses involving 7p and 8p were observed. Conclusions: MEITL is a rare and aggressive type of extranodal T-cell lymphoma. The differential diagnosis of MEITL includes EATL, extranodal NT/T-cell lymphoma and other types of PTCL. Diagnosis should be correlated to clinical symptoms while the final diagnosis is mainly based on the pathological features, immunophenotypes and genetic testing.

Changes in the expression of genes involved in DNA methylation and histone modification in response to daily food availability times in zebra finches: epigenetic implications.

We hypothesised that daily food availability times serve as an 'epigenetic' factor and affect reproductive physiology in continuously reproducing species. This we tested by measuring mRNA expression of genes coding for enzymes involved in DNA methylation-demethylation (dnmt, tet) and histone modification (hat1, hdac) in the hypothalamus, liver and gonads of male and female zebra finches that were paired for a year under 12h light:12h dark conditions with food availability restricted to 4h in the morning (morning FA group) or evening (evening FA group), with controls provided with food ad libitum The overall hypothalamic and hepatic expression patterns of hat1 and hdac were similar but those of dnmt and tet were different between males and females. Irrespective of the timing of food availability, both hat1 and hdac mRNA levels were increased in the hypothalamus, but not in the liver, in which hat1 mRNA levels were increased in the morning FA group. While hypothalamic tet levels were higher in evening FA males, hepatic tet levels were higher in morning FA birds (tet1, only males). Gonadal expression levels similarly varied and showed sex differences. Histone-modifying genes did not show food availability effects, except for elevated testicular hdac3 levels. Similarly, testicular dnmt3b and tet2 mRNA levels were increased and decreased in morning and evening FA groups, respectively, whereas ovarian dnmt1 and tet2 levels were reduced in morning FA and tet1 levels were reduced in evening FA groups. The present results suggest that an enforced daily feeding schedule in the long term could serve as a conditioning environment that shapes overall hypothalamic regulation, and liver and gonadal function at the epigenetic level in diurnal vertebrates.

Epigenetics in Multiple Sclerosis.

Multiple sclerosis (MS) is an aggravating autoimmune disease that cripples young patients slowly with physical, sensory and cognitive deficits. The break of self-tolerance to neuronal antigens is the key to the pathogenesis of MS, with autoreactive T cells causing demyelination that subsequently leads to inflammation-mediated neurodegenerative events in the central nervous system. The exact etiology of MS remains elusive; however, the interplay of genetic and environmental factors contributes to disease development and progression. Given that genetic variation only accounts for a fraction of risk for MS, extrinsic risk factors including smoking, infection and lack of vitamin D or sunshine, which cause changes in gene expression, contribute to disease development through epigenetic regulation. To date, there is a growing body of scientific evidence to support the important roles of epigenetic processes in MS. In this chapter, the three main layers of epigenetic regulatory mechanisms, namely DNA methylation, histone modification and microRNA-mediated gene regulation, will be discussed, with a particular focus on the role of epigenetics on dysregulated immune responses and neurodegenerative events in MS. Also, the potential for epigenetic modifiers as biomarkers and therapeutics for MS will be reviewed.

TCDD-induced multi- and transgenerational changes in the methylome of male zebrafish gonads.

The legacy endocrine disrupting chemical and aryl hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is produced as a byproduct of industrial processes and causes adverse health effects ranging from skin irritation to cancer. TCDD endpoints are also observed in subsequent, unexposed generations; however, the mechanisms of these multi- and transgenerational effects are unknown. We hypothesized an epigenetic mechanism, specifically DNA methylation for the transgenerational, male-mediated reproductive effects of developmental TCDD exposure. Using whole genome bisulfite sequencing, we evaluated DNA methylation changes in three generations of zebrafish, the first of which was exposed to TCDD during sexual development at 50 ppt for 1 h at both 3- and 7-week post-fertilization. We discovered that TCDD induces multi- and transgenerational methylomic changes in testicular tissue from zebrafish with decreased reproductive capacity, but most significantly in the indirectly exposed F1 generation. In comparing differentially methylated genes to concurrent transcriptomic changes, we identified several genes and pathways through which transgenerational effects of low level TCDD exposure are likely inherited. These include significant differential methylation of genes involved in reproduction, endocrine function, xenobiotic metabolism, and epigenetic processing. Notably, a number of histone modification genes were both differentially methylated and expressed in all generations, and many differentially methylated genes overlapped between multiple generations. Collectively, our results suggest that DNA methylation is a promising mechanism to explain male-mediated transgenerational reproductive effects of TCDD exposure in zebrafish, and these effects are likely inherited through integration of multiple epigenetic pathways.