PcG proteins mediate heritable transcriptional silencing by generating and recognizing covalent histone modifications. One conserved PcG complex, PRC2, is composed of several proteins including the histone methyltransferase (HMTase) Ezh2, the WD-repeat protein Eed, and the Zn-finger protein Suz12. Ezh2 methylates histone H3 on lysine 27 (H3K27), which serves as an epigenetic mark mediating silencing. H3K27 can be mono-, di-, or trimethylated (1mH3K27, 2mH3K27, and 3mH3K27, respectively). Hence, either PRC2 must be regulated so as to add one methyl group to certain nucleosomes but two or three to others, or distinct complexes must be responsible for 1m-, 2m-, and 3mH3K27. Consistent with the latter possibility, 2mH3K27 and 3mH3K27, but not 1mH3K27, are absent in Suz12-/- embryos, which lack both Suz12 and Ezh2 protein. Mammalian proteins required for 1mH3K27 have not been identified. Here, we demonstrate that unlike Suz12 and Ezh2, Eed is required not only for 2m- and 3mH3K27 but also global 1mH3K27. These results provide a functionally important distinction between PRC2 complex components and implicate Eed in PRC2-independent histone methylation.

Ionizing radiation causes acute radiation syndrome, which leads to hematopoietic, gastrointestinal, and cerebrovascular injuries. We investigated a population of mice that recovered from high-dose radiation to live normal life spans. These “elite-survivors” harbored distinct gut microbiota that developed after radiation and protected against radiation-induced damage and death in both germ-free and conventionally housed recipients. Elevated abundances of members of the bacterial taxa Lachnospiraceae and Enterococcaceae were associated with postradiation restoration of hematopoiesis and gastrointestinal repair. These bacteria were also found to be more abundant in leukemia patients undergoing radiotherapy, who also displayed milder gastrointestinal dysfunction. In our study in mice, metabolomics revealed increased fecal concentrations of microbially derived propionate and tryptophan metabolites in elite-survivors. The administration of these metabolites caused long-term radioprotection, mitigation of hematopoietic and gastrointestinal syndromes, and a reduction in proinflammatory responses.

Epigenetic regulation is essential for temporal, tissue-specific and parent-of-origin-dependent gene expression. It has recently been found that the mouse Polycomb group (PcG) gene Eed (embryonic ectoderm development) acts to maintain repression of the imprinted X chromosome. Here, we investigated whether Eed is also required for regulation of autosomal imprinted loci. Expression analyses showed that transcripts from the silent alleles of a subset of paternally repressed genes were present in Eed -/-embryos. Parent-of-origin methylation was preserved in these embryos, but we observed changes in the methylation status of specific CpGs in differentially methylated regions (DMRs) at affected but not at unaffected loci. These data identify Eed as a member of a new class of trans-acting factors that regulate parent-of-origin expression at imprinted loci.A subset of the mouse and human genomes is expressed from only one allele in a parent-oforigin-specific manner. This subset includes imprinted X-chromosome inactivation and autosomal imprinted loci. It has been proposed that this epigenetic regulation is accomplished through covalent modifications of both the DNA and the N-terminal tails of core histones in nucleosomes 1,2 . There are more than 60 identified autosomal imprinted genes, about half of which are paternally repressed and half maternally repressed. Most imprinted genes that have been examined contain at least one DMR located in the 5′ promoter region or in the body of the gene itself 3 . Recently, several proteins (DNA methyltransferases, CpG methyl binding proteins, chromatin insulators) have been identified as trans-acting factors involved in the epigenetic regulation of these loci 4 . Many of these factors either possess or associate with proteins that possess DNA methyltransferase activity. Additionally, recent studies have shown correlations between covalent histone modifications and the transcriptional status of imprinted alleles 5 . In particular, methylation of histone H3 has been associated with the inactive X chromosome 6 .PcG protein complexes are thought to maintain long-term gene silencing during development through alterations of local chromatin structure 7 . In both Drosophila and mammals, recent reports have shown that the Eed/Ezh2 PcG complex contains histone methyltransferase (HMT) activity, methylating histone H3, and that mutations in the SET domain of the Ezh2 fly homolog, E(Z), abolish the enzymatic activity of the complex in vitro [8][9][10] . The Eed/Ezh2 complex has also been shown to interact with histone deacetylases (HDACs; ref. 11). These

Background Diff-Quik stained fine-needle aspiration (FNA) smears and touch preparations from biopsies represent alternative specimens for molecular testing when cell block or biopsy material is insufficient. We describe the use of these samples for targeted next-generation sequencing (NGS) of primary and metastatic lung adenocarcinoma and report DNA quality and success rates of FNA smears compared with other specimens from one year of clinical use. Methods A validation set of 10 slides from 9 patients with prior clinical EGFR Sanger sequencing and KRAS pyrosequencing (5 KRAS +/EGFR−, 4 KRAS/EGFR−) underwent DNA extraction, quality assessment, and targeted NGS. Subseqently, lung adenocarcinoma specimens submitted for NGS solid tumor mutation panel testing in one calendar year (60 biopsies, 57 resections, 33 FNA cell blocks, 12 FNA smears, 10 body fluid cell blocks) were reviewed for specimen adequacy, sequencing success, and DNA quality. Results All 10 validation samples met the DNA quality threshold (ΔCT threshold <8, range −2.2 to 4.9) and yielded 0.5 to 22 μg of DNA. KRAS and EGFR mutation status from FNA smears by NGS were concordant with previous clinical testing for all 10 samples. In the one year review, FNA smears were 100% successful, suggesting performance equivalent to or better than established specimen types, including FNA cell blocks. DNA quality by ΔCT was significantly better from FNA smears than from biopsies, resections, and FNA cell blocks. Conclusions We conclude that FNA smears of lung adenocarcinomas are a high quality alternative specimen for a targeted NGS panel with a high success rate in clinical practice.

Burkitt lymphoma (BL) is the most common paediatric cancer in sub-Saharan Africa (SSA). Anthracyline-based treatment is standard in resource-rich settings, but has not been described in SSA. Children ≤ 18 years of age with newly diagnosed BL were prospectively enrolled from June 2013 to May 2015 in Malawi. Staging and supportive care were standardized, as was treatment with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) for six cycles. Among 73 children with BL, median age was 9.2 years (interquartile range 7.7–11.8), 48 (66%) were male and two were positive for human immunodeficiency virus. Twelve (16%) had stage I/II disease, 36 (49%) stage III and 25 (34%) stage IV. Grade 3/4 neutropenia occurred in 17 (25%), and grade 3/4 anaemia in 29 (42%) of 69 evaluable children. Eighteen-month overall survival was 29% (95% confidence interval [CI] 18–41%) overall. Mortality was associated with age >9 years [hazard ratio [HR] 2.13, 95% CI 1.15–3.94], female gender (HR 2.12, 95% CI 1.12–4.03), stage (HR 1.52 per unit, 95% CI 1.07–2.17), lactate dehydrogenase (HR 1.03 per 100 iu/l, 95% CI 1.01–1.05), albumin (HR 0. 96 per g/l, 95% CI 0.93–0.99) and performance status (HR 0.78 per 10-point increase, 95% CI 0.69–0.89). CHOP did not improve outcomes in paediatric BL compared to less intensive regimens in Malawi.

Our multidisciplinary approach can be a model for strong pathology services that provide direct, real-time support to clinical care and research in SSA.

Context.— The advent of molecular tools capable of subclassifying eosinophilia has changed the diagnostic and clinical approach to what was classically called hypereosinophilic syndrome. Objectives.— To review the etiologies of eosinophilia and to describe the current diagnostic approach to this abnormality. Data Sources.— Literature review. Conclusions.— Eosinophilia is a common, hematologic abnormality with diverse etiologies. The underlying causes can be broadly divided into reactive, clonal, and idiopathic. Classically, many cases of eosinophilia were grouped together into the umbrella category of hypereosinophilic syndrome, a clinical diagnosis of exclusion. In recent years, an improved mechanistic understanding of many eosinophilias has revolutionized the way these disorders are understood, diagnosed, and treated. As a result, specific diagnoses can now be assigned in many cases that were previously defined as hypereosinophilic syndrome. Most notably, chromosomal rearrangements, such as FIP1L1-PDGFRA fusions caused by internal deletions in chromosome 4, are now known to be associated with many chronic eosinophilic leukemias. When present, these specific molecular abnormalities predict response to directed therapies. Although an improved molecular understanding is revolutionizing the treatment of patients with rare causes of eosinophilia, it has also complicated the approach to evaluating and treating eosinophilia. Here, we review causes of eosinophilia and present a framework by which the practicing pathologist may approach this diagnostic dilemma. Finally, we consider recent cases as clinical examples of eosinophilia from a single institution, demonstrating the diversity of etiologies that must be considered.

Clonal hematopoiesis mutations are commonly reported on unpaired NGS testing. It is important to recognize clonal hematopoiesis as a possible cause of misattribution of mutation origin when applying NGS findings to a patient's care..