ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients.

BACKGROUND: Breast malignancies are the predominant cancer-related cause of death in women. New methods of diagnosis, prognosis and treatment are necessary. Previously, we identified the breast cancer cell surface protein ADAM8 as a marker of poor survival, and a driver of Triple-Negative Breast Cancer (TNBC) growth and spread. Immunohistochemistry (IHC) with a research-only anti-ADAM8 antibody revealed 34.0% of TNBCs (17/50) expressed ADAM8. To identify those patients who could benefit from future ADAM8-based interventions, new clinical tests are needed. Here, we report on the preclinical development of a highly specific IHC assay for detection of ADAM8-positive breast tumors. METHODS: Formalin-fixed paraffin-embedded sections of ADAM8-positive breast cell lines and patient-derived xenograft tumors were used in IHC to identify a lead antibody, appropriate staining conditions and controls. Patient breast cancer samples (n = 490) were used to validate the assay. Cox proportional hazards models assessed association between survival and ADAM8 expression. RESULTS: ADAM8 staining conditions were optimized, a lead anti-human ADAM8 monoclonal IHC antibody (ADP2) identified, and a breast staining/scoring control cell line microarray (CCM) generated expressing a range of ADAM8 levels. Assay specificity, reproducibility, and appropriateness of the CCM for scoring tumor samples were demonstrated. Consistent with earlier findings, 36.1% (22/61) of patient TNBCs expressed ADAM8. Overall, 33.9% (166/490) of the breast cancer population was ADAM8-positive, including Hormone Receptor (HR) and Human Epidermal Growth Factor Receptor-2 (HER2) positive cancers, which were tested for the first time. For the most prevalent HR-positive/HER2-negative subtype, high ADAM8 expression identified patients at risk of poor survival. CONCLUSIONS: Our studies show ADAM8 is widely expressed in breast cancer and provide support for both a diagnostic and prognostic value of the ADP2 IHC assay. As ADAM8 has been implicated in multiple solid malignancies, continued development of this assay may have broad impact on cancer management.

Clinicopathological spectrums and prognosis of primary appendiceal adenocarcinoma, goblet cell adenocarcinoma, and low-grade appendiceal mucinous neoplasms.

Primary appendiceal adenocarcinoma (APCA), goblet cell adenocarcinoma (GCA), and low/high-grade appendiceal mucinous neoplasms (LAMN/HAMN) are distinct entities with overlapping clinical presentation and histomorphology, leading to diagnostic challenges. We retrospectively reviewed our archived cases between 2010 and 2018 for diagnosis reappraisal and comparative analysis using updated terminology and modern parameters. A total of 87 cases (22 APCA, 40 GCA, and 25 LAMN pT≥3) were included. The entire cohort had 49 women and 38 men with a median age of 59.9 (range 26-88) years. There were no statistically significant differences in age and sex among the three groups. Clinically, patients with GCA were more likely to present with acute appendicitis (65%) and more likely to have appendectomy as initial surgery (68%). Both APCA and GCA were more likely to involve the proximal appendix while LAMN was more likely to involve the distal appendix (p<0.05). All APCAs were associated with mucosal precursor lesions, most commonly tubular, tubulovillous, or villous adenoma, flat LAMN/HAMN-pTis mucinous epithelium, or mixed, which correlated with distinct histomorphology, tumour differentiation, and stage. Although polypoid precursor lesions were rare in GCA, a significant proportion of GCA showed crypt atypia associated with neoplastic cells. Immunohistochemically, APCA had more frequent ß-catenin nuclear positivity and loss of SATB2 expression (p<0.05). KRAS mutation was more common in APCA than in GCA (8/11 vs 1/7, p<0.01). We further validated the three-tiered grading system (G1, G2, G3) in GCA, which correlated well with tumour stage and patient survival. APCA had worse progression-free and disease-specific survivals than GCA and LAMN (pT≥3) with the latter being relatively indolent even when perforated with peritoneal spread. Our study is the first comprehensive comparison between all three appendiceal neoplasms. We also describe a spectrum of previously under-recognised crypt atypia in GCA, which should trigger a diligent search for GCA if present.

Fine-needle aspiration specimens of 3 cases of intra-abdominal Rosai-Dorfman disease with comprehensive review of the literature.

INTRODUCTION: Rosai-Dorfman disease (RDD) is a rare usually self-limited non-Langerhans cell histiocytosis of unknown etiology. Nodal and extranodal RDD appear to represent distinct conditions with different molecular alterations and prognosis. They also pose different diagnostic challenges on biopsies and fine-needle aspiration (FNA) cytology. The aim of this study was to report on 3 cases of intra-abdominal RDD and perform an extensive review of the literature on FNA findings of RDD. MATERIALS AND METHODS: We reviewed FNA specimens from cases diagnosed histologically or cytologically as RDD during the past 10 years. We searched the PubMed and Google Scholar databases for cases of RDD sampled by FNA. RESULTS: We identified 3 cases of intra-abdominal RDD, involving the kidney, periportal lymph node, and pancreas. FNA of the latter was hypocellular with fibrosis and was nondiagnostic. FNA of the first 2 yielded hypercellular smears that were diagnosed as RDD due to the identification of emperipolesis occurring in large uni- or binucleated histiocytes with large nuclei, fine chromatin, and prominent nucleoli in smears and cell-block sections. Immunohistochemistry showed positive staining for S100 and CD68 and negative staining for CD1a. The large histiocytes with emperipolesis were more difficult to identify histologically and their demonstration required immunohistochemical stains. CONCLUSION: Our experience and an extensive review of the literature suggest that extranodal RDD can be diagnosed on FNA, and that the recognition of histiocytes with emperipolesis may be less challenging cytologically than histologically. The fibrosis frequently seen in extranodal RDD may lead to nondiagnostic aspirates, however.

Becalming Type 17 Inflammation in Ulcerative Colitis.

Genome-wide association studies in ulcerative colitis point to a role for FcγRIIA, a receptor for IgG. Castro-Dopico et al. (2019) find a profound induction of anti-commensal IgG in the colonic mucosa of UC patients and outline a pathway whereby FcγR activation by IgG triggers IL-1ß production, type 17 immunity, and the exacerbation of inflammation.

Colonic epithelial cell diversity in health and inflammatory bowel disease.

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.

Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells.

BACKGROUND: Patients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia. OBJECTIVES: Because regulation of the phosphoinositide 3-kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]). METHODS: Patients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse. RESULTS: Autoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T-cell reduction, and changes in T- and B-cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse. CONCLUSION: Heterozygous loss of function of PTEN in human subjects has a significant effect on T- and B-cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T-cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency.

Active adenoviral vascular penetration by targeted formation of heterocellular endothelial-epithelial syncytia.

The endothelium imposes a structural barrier to the extravasation of systemically delivered oncolytic adenovirus (Ad). Here, we introduced a transendothelial route of delivery in order to increase tumor accumulation of virus particles (vp) beyond that resulting from convection-dependent extravasation alone. This was achieved by engineering an Ad encoding a syncytium-forming protein, gibbon ape leukemia virus (GALV) fusogenic membrane glycoprotein (FMG). The expression of GALV was regulated by a hybrid viral enhancer-human promoter construct comprising the human cytomegalovirus (CMV) immediate-early enhancer and the minimal human endothelial receptor tyrosine kinase promoter ("eTie1"). Endothelial cell-selectivity of the resulting Ad-eTie1-GALV vector was demonstrated by measuring GALV mRNA transcript levels. Furthermore, Ad-eTie1-GALV selectively induced fusion between infected endothelial cells and uninfected epithelial cells in vitro and in vivo, allowing transendothelial virus penetration. Heterofusion of infected endothelium to human embryonic kidney 293 (HEK 293) cells, in mixed in vitro cultures or in murine xenograft models, permitted fusion-dependent transactivation of the replication-deficient Ad-eTie1-GALV, due to enabled access to viral E1 proteins derived from the HEK 293 cytoplasm. These data provide evidence to support our proposed use of GALV to promote Ad penetration through tumor-associated vasculature, an approach that may substantially improve the efficiency of systemic delivery of oncolytic viruses to disseminated tumors.

Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells.

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

Toward more effective gene delivery.

A report on the symposium 'In vivo barriers to gene delivery', Cold Spring Harbor, USA, 26-29 November 2007.