Nathan D. Montgomery

The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation.

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.

Genome imprinting regulated by the mouse Polycomb group protein Eed

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.

FNA smears as a potential source of DNA for targeted next-generation sequencing of lung adenocarcinomas

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. This study describes the use of these samples for targeted next‐generation sequencing (NGS) of primary and metastatic lung adenocarcinoma and reports the DNA quality and success rates of FNA smears versus other specimens from 1 year of clinical use.

Diagnostic Complexities of Eosinophilia

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. CONCLUSION 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.

Outcomes for paediatric Burkitt lymphoma treated with anthracycline-based therapy in Malawi

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.

Knockout of Epstein-Barr Virus BPLF1 Retards B-Cell Transformation and Lymphoma Formation in Humanized Mice

ABSTRACT BPLF1 of Epstein-Barr virus (EBV) is classified as a late lytic cycle protein but is also found in the viral tegument, suggesting its potential involvement at both initial and late stages of viral infection. BPLF1 possesses both deubiquitinating and deneddylating activity located in its N-terminal domain and is involved in processes that affect viral infectivity, viral DNA replication, DNA repair, and immune evasion. A recently constructed EBV BPLF1-knockout (KO) virus was used in conjunction with a humanized mouse model that can be infected with EBV, enabling the first characterization of BPLF1 function in vivo. Results demonstrate that the BPLF1-knockout virus is approximately 90% less infectious than wild-type (WT) virus. Transformation of human B cells, a hallmark of EBV infection, was delayed and reduced with BPLF1-knockout virus. Humanized mice infected with EBV BPLF1-knockout virus showed less weight loss and survived longer than mice infected with equivalent infectious units of WT virus. Additionally, splenic tumors formed in 100% of mice infected with WT EBV but in only 25% of mice infected with BPLF1-KO virus. Morphological features of spleens containing tumors were similar to those in EBV-induced posttransplant lymphoproliferative disease (PTLD) and were almost identical to cases seen in human diffuse large B-cell lymphoma. The presence of EBV genomes was detected in all mice that developed tumors. The results implicate BPLF1 in human B-cell transformation and tumor formation in humanized mice. IMPORTANCE Epstein-Barr virus infects approximately 90% of the worlds population and is the causative agent of infectious mononucleosis. EBV also causes aggressive lymphomas in individuals with acquired and innate immune disorders and is strongly associated with diffuse large B-cell lymphomas, classical Hodgkin lymphoma, Burkitt lymphoma, and nasopharyngeal carcinoma (NPC). Typically, EBV initially infects epithelial cells in the oropharynx, followed by a lifelong persistent latent infection in B-cells, which may develop into lymphomas in immunocompromised individuals. This work is the first of its kind in evaluating the effects of EBVs BPLF1 in terms of pathogenesis and lymphomagenesis in humanized mice and implicates BPLF1 in B-cell transformation and tumor development. Currently, there is no efficacious treatment for EBV, and therapeutic targeting of BPLF1 may lead to a new path to treatment for immunocompromised individuals or transplant recipients infected with EBV. Epstein-Barr virus infects approximately 90% of the worlds population and is the causative agent of infectious mononucleosis. EBV also causes aggressive lymphomas in individuals with acquired and innate immune disorders and is strongly associated with diffuse large B-cell lymphomas, classical Hodgkin lymphoma, Burkitt lymphoma, and nasopharyngeal carcinoma (NPC). Typically, EBV initially infects epithelial cells in the oropharynx, followed by a lifelong persistent latent infection in B-cells, which may develop into lymphomas in immunocompromised individuals. This work is the first of its kind in evaluating the effects of EBVs BPLF1 in terms of pathogenesis and lymphomagenesis in humanized mice and implicates BPLF1 in B-cell transformation and tumor development. Currently, there is no efficacious treatment for EBV, and therapeutic targeting of BPLF1 may lead to a new path to treatment for immunocompromised individuals or transplant recipients infected with EBV.

Hodgkin lymphoma, HIV, and Epstein–Barr virus in Malawi: Longitudinal results from the Kamuzu Central Hospital Lymphoma study

Contemporary descriptions of classical Hodgkin lymphoma (cHL) are lacking from sub‐Saharan Africa where human immunodeficiency virus (HIV) and Epstein–Barr virus (EBV) are prevalent.

CHOP Chemotherapy for Aggressive Non-Hodgkin Lymphoma with and without HIV in the Antiretroviral Therapy Era in Malawi.

There are no prospective studies of aggressive non-Hodgkin lymphoma (NHL) treated with CHOP in sub-Saharan Africa. We enrolled adults with aggressive NHL in Malawi between June 2013 and May 2015. Chemotherapy and supportive care were standardized, and HIV+ patients received antiretroviral therapy (ART). Thirty-seven of 58 patients (64%) were HIV+. Median age was 47 years (IQR 39–56), and 35 (60%) were male. Thirty-five patients (60%) had stage III/IV, 43 (74%) B symptoms, and 28 (48%) performance status ≥2. B-cell NHL predominated among HIV+ patients, and all T-cell NHL occurred among HIV- individuals. Thirty-one HIV+ patients (84%) were on ART for a median 9.9 months (IQR 1.1–31.7) before NHL diagnosis, median CD4 was 121 cells/μL (IQR 61–244), and 43% had suppressed HIV RNA. HIV+ patients received a similar number of CHOP cycles compared to HIV- patients, but more frequently developed grade 3/4 neutropenia (84% vs 31%, p = 0.001), resulting in modestly lower cyclophosphamide and doxorubicin doses with longer intervals between cycles. Twelve-month overall survival (OS) was 45% (95% CI 31–57%). T-cell NHL (HR 3.90, p = 0.017), hemoglobin (HR 0.82 per g/dL, p = 0.017), albumin (HR 0.57 per g/dL, p = 0.019), and IPI (HR 2.02 per unit, p<0.001) were associated with mortality. HIV was not associated with mortality, and findings were similar among patients with diffuse large B-cell lymphoma. Twenty-three deaths were from NHL (12 HIV+, 11 HIV-), and 12 from CHOP (9 HIV+, 3 HIV-). CHOP can be safe, effective, and feasible for aggressive NHL in Malawi with and without HIV.

Loss of the histone pre-mRNA processing factor stem-loop binding protein in Drosophila causes genomic instability and impaired cellular proliferation.

Background Metazoan replication-dependent histone mRNAs terminate in a conserved stem-loop structure rather than a polyA tail. Formation of this unique mRNA 3′ end requires Stem-loop Binding Protein (SLBP), which directly binds histone pre-mRNA and stimulates 3′ end processing. The 3′ end stem-loop is necessary for all aspects of histone mRNA metabolism, including replication coupling, but its importance to organism fitness and genome maintenance in vivo have not been characterized. Methodology/Principal Findings In Drosophila, disruption of the Slbp gene prevents normal histone pre-mRNA processing and causes histone pre-mRNAs to utilize the canonical 3′ end processing pathway, resulting in polyadenylated histone mRNAs that are no longer properly regulated. Here we show that Slbp mutants display genomic instability, including loss of heterozygosity (LOH), increased presence of chromosome breaks, tetraploidy, and changes in position effect variegation (PEV). During imaginal disc growth, Slbp mutant cells show defects in S phase and proliferate more slowly than control cells. Conclusions/Significance These data are consistent with a model in which changing the 3′ end of histone mRNA disrupts normal replication-coupled histone mRNA biosynthesis and alters chromatin assembly, resulting in genomic instability, inhibition of cell proliferation, and impaired development.

Plasma Epstein-Barr virus DNA for pediatric Burkitt lymphoma diagnosis, prognosis, and response assessment in Malawi.

Point‐of‐care tools are needed in sub‐Saharan Africa (SSA) to improve pediatric Burkitt lymphoma (BL) diagnosis and treatment. We evaluated plasma Epstein–Barr virus (pEBV) DNA as a pediatric BL biomarker in Malawi. Prospectively enrolled children with BL were compared to classical Hodgkin lymphoma (cHL) and nonlymphoma diagnoses. Pediatric BL patients received standardized chemotherapy and supportive care. pEBV DNA was measured at baseline, mid‐treatment, and treatment completion. Of 121 assessed children, pEBV DNA was detected in 76/88 (86%) with BL, 16/17 (94%) with cHL, and 2/16 (12%) with nonlymphoma, with proportions higher in BL versus nonlymphoma (p < 0.001) and similar in BL versus cHL (p = 0.69). If detected, median pEBV DNA was 6.1 log10copies/mL for BL, 4.8 log10copies/mL for cHL, and 3.4 log10copies/mL for nonlymphoma, with higher levels in BL versus cHL (p = 0.029), and a trend toward higher levels in BL versus nonlymphoma (p = 0.062). pEBV DNA declined during treatment in the cohort overall and increased in several children before clinical relapse. Twelve‐month overall survival was 40% in the cohort overall, and for children with baseline pEBV detected, survival was worse if baseline pEBV DNA was ≥6 log10copies/mL versus <6 log10copies/mL (p = 0.0002), and also if pEBV DNA was persistently detectable at mid‐treatment versus undetectable (p = 0.041). Among children with baseline pEBV DNA detected, viremia was the only significant risk factor for death by 12 months in multivariate analyses (adjusted hazard ratio 1.35 per log10copies/mL, 95% CI 1.04–1.75, p = 0.023). Quantitative pEBV DNA has potential utility for diagnosis, prognosis, and response assessment for pediatric BL in SSA.