Russell J.H. Ryan

Genetic and Epigenetic Fine-Mapping of Causal Autoimmune Disease Variants

Genome-wide association studies have identified loci underlying human diseases, but the causal nucleotide changes and mechanisms remain largely unknown. Here we developed a fine-mapping algorithm to identify candidate causal variants for 21 autoimmune diseases from genotyping data. We integrated these predictions with transcription and cis-regulatory element annotations, derived by mapping RNA and chromatin in primary immune cells, including resting and stimulated CD4+ T-cell subsets, regulatory T cells, CD8+ T cells, B cells, and monocytes. We find that ∼90% of causal variants are non-coding, with ∼60% mapping to immune-cell enhancers, many of which gain histone acetylation and transcribe enhancer-associated RNA upon immune stimulation. Causal variants tend to occur near binding sites for master regulators of immune differentiation and stimulus-dependent gene activation, but only 10–20% directly alter recognizable transcription factor binding motifs. Rather, most non-coding risk variants, including those that alter gene expression, affect non-canonical sequence determinants not well-explained by current gene regulatory models.

Origins of tumor-associated macrophages and neutrophils

Tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) can control cancer growth and exist in almost all solid neoplasms. The cells are known to descend from immature monocytic and granulocytic cells, respectively, which are produced in the bone marrow. However, the spleen is also a recently identified reservoir of monocytes, which can play a significant role in the inflammatory response that follows acute injury. Here, we evaluated the role of the splenic reservoir in a genetic mouse model of lung adenocarcinoma driven by activation of oncogenic Kras and inactivation of p53. We found that high numbers of TAM and TAN precursors physically relocated from the spleen to the tumor stroma, and that recruitment of tumor-promoting spleen-derived TAMs required signaling of the chemokine receptor CCR2. Also, removal of the spleen, either before or after tumor initiation, reduced TAM and TAN responses significantly and delayed tumor growth. The mechanism by which the spleen was able to maintain its reservoir capacity throughout tumor progression involved, in part, local accumulation in the splenic red pulp of typically rare extramedullary hematopoietic stem and progenitor cells, notably granulocyte and macrophage progenitors, which produced CD11b+ Ly-6Chi monocytic and CD11b+ Ly-6Ghi granulocytic cells locally. Splenic granulocyte and macrophage progenitors and their descendants were likewise identified in clinical specimens. The present study sheds light on the origins of TAMs and TANs, and positions the spleen as an important extramedullary site, which can continuously supply growing tumors with these cells.

EZH2 Codon 641 Mutations are Common in BCL2- Rearranged Germinal Center B Cell Lymphomas

Mutations at codon 641 of EZH2 are recurrent in germinal center B cell lymphomas, and the most common variants lead to altered EZH2 enzymatic activity and enhanced tri-methylation of histone H3 at lysine 27, a repressive chromatin modification. As an initial step toward screening patients for cancer genotype-directed therapy, we developed a screening assay for EZH2 codon 641 mutations amenable for testing formalin-fixed clinical specimens, based on the sensitive SNaPshot single nucleotide extension technology. We detected EZH2 mutations in 12/55 (22%) follicular lymphomas (FL), 5/35 (14%) diffuse large B cell lymphomas with a germinal center immunophenotype (GCB-DLBCL), and 2/11 (18%) high grade B cell lymphomas with concurrent rearrangements of BCL2 and MYC. No EZH2 mutations were detected in cases of Burkitt lymphoma (0/23). EZH2 mutations were frequently associated with the presence of BCL2 rearrangement (BCL2-R) in both the FL (28% of BCL-R cases versus 0% of BCL2-WT cases, p<0.05) and GCB-DLBCL groups (33% of BCL2-R cases versus 4% of BCL2-WT cases, p<0.04), and across all lymphoma types excluding BL (27% of BCL2-R cases versus 3% of BCL2-WT cases, p<0.003). We confirmed gain-of-function activity for all previously reported EZH2 codon 641 mutation variants. Our findings suggest that EZH2 mutations constitute an additional genetic “hit” in many BCL2-rearranged germinal center B cell lymphomas. Our work may be helpful in the selection of lymphoma patients for future trials of pharmacologic agents targeting EZH2 and EZH2-regulated pathways.

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes

While the pro‐differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch‐dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte‐derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation‐related genes, while it is not required for induction of ‘canonical’ Notch targets like p21WAF1/Cip1, Hes1 and Hey1. Down‐modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras‐induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

Genetic Events That Shape the Cancer Epigenome

Mutations in chromatin-related genes in human tumors support a role for epigenetic mechanisms in driving cancer. Since the discovery of the first recurrent mutations in oncogenes and tumor suppressor genes, it has been clear that cancer is, in large part, a genetic disease. Yet nearly every human neoplasm retains a phenotype reflective of its tissue of origin, thus underscoring the centrality of epigenetics in cancer biology. Indeed, there is increasing recognition that transmissible epigenetic changes—chemical modifications to the genome or its scaffold that do not involve a change in the nucleotide sequence—may be acquired de novo, and that these “epimutations” may also contribute to carcinogenesis. Aberrations of DNA methylation have epitomized this concept, largely because of the direct mechanism by which hypermethylation of a DNA locus can be faithfully transmitted through cell division. Localized hypermethylation of silenced gene promoters and global DNA hypomethylation are characteristic features of many human tumors (1, 2). However, the idea that histone modifications and other chromatin features also mediate epimutations in tumors has been more controversial, in part due to the obscurity of models for direct epigenetic transmission (3). The recent flurry of reported mutations in chromatin-related genes in human tumors indicates the need to reassess the perceived roles for chromatin and epigenetic mechanisms in cancer biology.

A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.

Extranodal Marginal Zone Lymphoma of Mucosa- Associated Lymphoid Tissue With Amyloid Deposition A Clinicopathologic Case Series

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) lymphoma is a mature B-cell neoplasm that typically follows an indolent clinical course. Amyloid deposition associated with MALT lymphoma is uncommon. We describe the clinical and pathologic features of 20 cases of MALT lymphoma and associated amyloid deposition across diverse primary sites. Frozen section immunofluorescence performed on 4 cases suggests that these deposits are a localized form of AL amyloid. Clinical follow-up was available for 15 patients. Amyloid deposits distant from the initial site occurred in 5 cases, always at sites also involved by the underlying lymphoma. No definitive evidence of systemic amyloidosis affecting the heart, kidneys, or liver was present in any patient. Given the generally indolent clinical behavior of MALT lymphomas with associated amyloid, we do not recommend extensive follow-up testing for systemic amyloidosis or more aggressive therapy than would be indicated for other MALT lymphomas of similar clinical stage.

Mast cell sarcoma: a rare and potentially under-recognized diagnostic entity with specific therapeutic implications

Mast cell sarcoma is a rare, aggressive neoplasm composed of cytologically malignant mast cells presenting as a solitary mass. Previous descriptions of mast cell sarcoma have been limited to single case reports, and the pathologic features of this entity are not well known. Here, we report three new cases of mast cell sarcoma and review previously reported cases. Mast cell sarcoma has a characteristic morphology of medium-sized to large epithelioid cells, including bizarre multinucleated cells, and does not closely resemble either normal mast cells or the spindle cells of systemic mastocytosis. One of our three cases arose in a patient with a remote history of infantile cutaneous mastocytosis, an association also noted in one previous case report. None of our three cases were correctly diagnosed as mast cell neoplasms on initial pathological evaluation, suggesting that this entity may be under-recognized. Molecular testing of mast cell sarcoma has not thus far detected the imatinib-resistant KIT D816V mutation, suggesting that recognition of these cases may facilitate specific targeted therapy.

Case records of the Massachusetts General Hospital. Case 11-2013. A 4-year-old boy with fever and abdominal pain.

From the Departments of Pediatric Surgery (D.P.R.), Pediatrics (A.M.F.), Radiology (M.D.S.), and Pathology (R.J.H.R.), Massachusetts General Hospital; and the Departments of Surgery (D.P.R.), Pediatrics (A.M.F.), Radiology (M.D.S.), and Pathology (R.J.H.R.), Harvard Medical School — both in Boston.

Case 24-2012: A 38-year-old man with abdominal pain and altered mental status

Dr. Gabriel A. Brooks (Medical Oncology): A 38-year-old man was admitted to this hospital because of abdominal pain, altered mental status, hypercalcemia, and thrombocytopenia. The patient had been well until 11 days before admission, when weakness developed. The next day, nausea, abdominal pain, and fatigue developed and he had multiple episodes of nonbilious, nonbloody emesis. Nine days before admission, he went to another hospital. He reported being in distress because of nausea; the vital signs were reportedly normal, the epigastrium was mildly tender, and the remainder of the examination was normal. Results of laboratory tests are shown in Table 1. The electrocardiogram reportedly showed sinus rhythm at 46 beats per minute and early repolarization. The patient was admitted to the other hospital, and normal saline and pamidronate were administered intravenously, with improvement in the symptoms and blood calcium level. Results of urinalysis were normal. Ultrasonography of the kidneys revealed normal size, a slight increase in the echotexture, and mild ascites. He was discharged on the fourth day, taking potassium phosphate tablets, with plans for additional outpatient evaluation. That evening after dinner, dull and constant lower abdominal pain developed and prevented the patient from sleeping. The next morning, he returned to the other hospital because of the pain, which he rated at 8 on a scale of 0 to 10, with 10 indicating the most severe pain. He reported no fever, chills, nausea, vomiting, diarrhea, dizziness, chest pain, or dyspnea. On examination, the vital signs were normal. There was moderate cervical and inguinal lymphadenopathy (≤1.5 cm in diameter) and mild abdominal tenderness in the right lower quadrant, without rebound or guarding; the remainder of the examination was normal. Laboratory-test results are shown in Table 1. Computed tomography (CT) of the abdomen, with the administration of contrast material, reportedly showed diffuse heterogeneous attenuation in the liver, borderline splenomegaly (span, 14 cm), and a small amount of ascites, without masses or evidence of appendicitis. Intravenous fluids, potassium phosphate, pantoprazole, ibuprofen, tramadol, and morphine were administered. On the second day, testing for hepatitis B surface antigen and antibodies to