MLH1/PMS2-deficient Endometrial Carcinomas in a Universally Screened Population: MLH1 Hypermethylation and Germline Mutation Status.

MLH1/PMS2 loss due to epigenetic hypermethylation of the MLH1 promoter is the most common cause of mismatch repair deficiency in endometrial carcinoma, and typically provides reassurance against an associated germline mutation. To further characterize the genetic features of MLH1/PMS2-deficient endometrial cancers, the departmental database was searched for cases with dual MLH1/PMS2 loss and retained MSH2/6 expression which underwent MLH1 hypermethylation testing. Genetic testing results were obtained when available. One hundred seventeen endometrial cancers met inclusion criteria: 100 (85%) were MLH1-hypermethylated, 3 (3%) were low-level/borderline, 7 (6%) were nonmethylated, and 7 (6%) were insufficient for testing. Sixteen cases (12 MLH1-hypermethylated, 3 nonmethylated, and 1 insufficient for testing) underwent germline testing, 6 of which (37.5%) demonstrated germline variants of unknown significance (VUS) (MSH6, PMS2, POLD1, BRIP1, RAD51D, CHEK2) but no known deleterious mutations. Notably, however, the patients harboring the MSH6 and PMS2 germline VUS had clinical features concerning for Lynch syndrome. One nonmethylated, germline-normal case underwent somatic tumor testing, and demonstrated a somatic MLH1 mutation. In summary, MLH1-hypermethylation accounts for the vast majority of MLH1/PMS2-deficient cancers in a universally screened population, although MLH1 somatic and germline mutations can occur. Occasionally, patients with MLH1-hypermethlated tumors also bear germline VUS in other mismatch repair genes as well as genes implicated in other hereditary cancer syndromes, but their clinical relevance is unclear. Family and personal cancer histories must always be evaluated to determine the need for germline testing in women with loss of MLH1/PMS2, even in the setting of hypermethylation.

Artificial Intelligence-based Analytics for Diagnosis of Small Bowel Enteropathies and Black Box Feature Detection.

OBJECTIVES: Striking histopathological overlap between distinct but related conditions poses a disease diagnostic challenge. There is a major clinical need to develop computational methods enabling clinicians to translate heterogeneous biomedical images into accurate and quantitative diagnostics. This need is particularly salient with small bowel enteropathies; environmental enteropathy (EE) and celiac disease (CD). We built upon our preliminary analysis by developing an artificial intelligence (AI)-based image analysis platform utilizing deep learning convolutional neural networks (CNNs) for these enteropathies. METHODS: Data for the secondary analysis was obtained from three primary studies at different sites. The image analysis platform for EE and CD was developed using CNNs including one with multizoom architecture. Gradient-weighted class activation mappings (Grad-CAMs) were used to visualize the models' decision-making process for classifying each disease. A team of medical experts simultaneously reviewed the stain color normalized images done for bias reduction and Grad-CAMs to confirm structural preservation and biomedical relevance, respectively. RESULTS: Four hundred and sixty-one high-resolution biopsy images from 150 children were acquired. Median age (interquartile range) was 37.5 (19.0-121.5) months with a roughly equal sex distribution; 77 males (51.3%). ResNet50 and shallow CNN demonstrated 98% and 96% case-detection accuracy, respectively, which increased to 98.3% with an ensemble. Grad-CAMs demonstrated models' ability to learn different microscopic morphological features for EE, CD, and controls. CONCLUSIONS: Our AI-based image analysis platform demonstrated high classification accuracy for small bowel enteropathies which was capable of identifying biologically relevant microscopic features and emulating human pathologist decision-making process. Grad-CAMs illuminated the otherwise "black box" of deep learning in medicine, allowing for increased physician confidence in adopting these new technologies in clinical practice.

Study of Environmental Enteropathy and Malnutrition (SEEM) in Pakistan: protocols for biopsy based biomarker discovery and validation.

BACKGROUND: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority. METHODS: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community. DISCUSSION: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals. TRIAL REGISTRATION: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .

Sustained activation of the HER1-ERK1/2-RSK signaling pathway controls myoepithelial cell fate in human mammary tissue.

Human mammary glands arise from multipotent progenitor cells, which likely respond both to cell-autonomous and to extrinsic cues. However, the identity of these cues and how they might act remain unclear. We analyzed HER1 ligand effects on mammary morphogenesis using a three-dimensional organoid model generated from human breast tissue that recapitulates both qualitatively and quantitatively the normal ductal network in situ. Strikingly, different HER1 ligands generate distinct patterns of cell fate. Epidermal growth factor (EGF) causes a massive expansion of the myoepithelial lineage. Amphiregulin, in contrast, enables normal ductal development. These differences cannot be ascribed to preferential apoptosis or proliferation of differentiated cell populations, but are dependent on HER1 signal intensity. Inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) effector RSK prevents the EGF-induced myoepithelial expansion. Notably, mouse mammary organoids are much less responsive to HER1 ligands. Little is known about the myoepithelial lineage or about growth factor effects on mammary progenitor differentiation, and our studies provide an important window into human mammary development that reveals unexpected differences from the mouse model.

Mucinous Differentiation With Tumor Infiltrating Lymphocytes Is a Feature of Sporadically Methylated Endometrial Carcinomas.

Defects in the DNA mismatch-repair system are identified in ∼25%-30% of endometrial carcinomas (ECs). Whereas some ECs are due to germline Lynch syndrome (LS)-associated mutations, the majority demonstrate sporadic MLH1 promoter hypermethylation (MLH1hm). MLH1hm characterizes a unique subset of colorectal cancers with a relatively poor prognosis; however, the morphology and behavior of sporadically methylated ECs (SMECs) are less well understood. We herein review the clinicopathologic features of 34 SMECs diagnosed at The University of Virginia and compare them with LS-associated and Lynch-like endometrial cancers. Most SMECs were centered in the uterine fundus (74%) and displayed Grade 1 (35%) or Grade 2 (41%) endometrioid morphology. Mucinous differentiation was present in 50%, and 65% showed tumor-infiltrating lymphocytes. Cases with undifferentiated components or sarcomatous differentiation were relatively rare (9%). Most SMECs presented at stage 1 or 2 (91%). When compared with LS and Lynch-like endometrial cancers, SMECs occurred ∼8 yr later and were significantly more likely to show conventional endometrioid morphology (P=0.04), mucinous differentiation (P=0.002), and tumor-infiltrating lymphocytes (P=0.002). These findings suggest that SMECs constitute a clinicopathologically distinct subset of mismatch-repair-deficient/high-level microsatellite instability tumors. Future studies are needed to further understand the implications of MLH1hm on the prognosis and treatment of endometrial cancer.

Duodenal quantitative mucosal morphometry in children with environmental enteric dysfunction: a cross-sectional multicountry analysis.

BACKGROUND: Environmental enteric dysfunction (EED), a chronic inflammatory condition of the small intestine, is an important driver of childhood malnutrition globally. Quantifying intestinal morphology in EED allows for exploration of its association with functional and disease outcomes. OBJECTIVES: We sought to define morphometric characteristics of childhood EED and determine whether morphology features were associated with disease pathophysiology. METHODS: Morphometric measurements and histology were assessed on duodenal biopsy slides for this cross-sectional study from children with EED in Bangladesh, Pakistan, and Zambia (n = 69), and those with no pathologic abnormality (NPA; n = 8) or celiac disease (n = 18) in North America. Immunohistochemistry was also conducted on 46, 8, and 18 biopsy slides, respectively. Linear mixed-effects regression models were used to reveal morphometric differences between EED compared with NPA or celiac disease and identify associations between morphometry and histology or immunohistochemistry among children with EED. RESULTS: In duodenal biopsies, median EED villus height (248 µm), crypt depth (299 µm), and villus:crypt (V:C) ratio (0.9) values ranged between those of NPA (396 µm villus height; 246 µm crypt depth; 1.6 V:C ratio) and celiac disease (208 µm villus height; 365 µm crypt depth; 0.5 V:C ratio). Among EED biopsy slides, morphometric assessments were not associated with histologic parameters or immunohistochemical markers, other than pathologist-determined subjective semiquantitative villus architecture. CONCLUSIONS: Morphometric analysis of duodenal biopsy slides across geographies identified morphologic features of EED, specifically short villi, elongated crypts, and a smaller V:C ratio relative to NPA slides, although not as severe as in celiac slides. Morphometry did not explain other EED features, suggesting that EED histopathologic processes may be operating independently of morphology. Although acknowledging the challenges with obtaining relevant tissue, these data form the basis for further assessments of the role of morphometry in EED.

Multiplexed immunohistochemical evaluation of small bowel inflammatory and epithelial parameters in environmental enteric dysfunction.

BACKGROUND: Environmental enteric dysfunction (EED) is characterized by reduced absorptive capacity and barrier function of the small intestine, leading to poor ponderal and linear childhood growth. OBJECTIVES: To further define gene expression patterns that are associated with EED to uncover new pathophysiology of this disorder. METHODS: Duodenal biopsies from cohorts of children with EED from Bangladesh, Pakistan and Zambia were analyzed by immunohistochemistry (IHC) to interrogate gene products that distinguished differentiation and various biochemical pathways in immune and epithelial cells, some identified by prior bulk RNA sequence analyses. Immunohistochemical staining was digitally quantified from scanned images and compared to cohorts of North American children with celiac disease (gluten-sensitive enteropathy) or with no known enteric disease and no pathologic abnormality (NPA) detected in their clinical biopsies. RESULTS: After multivariable statistical analysis, we identified statistically significant (P < 0.05, 2-tailed t-test) elevated signals representing cluster of differentiation 45 (80%; 95% confidence interval [CI]: 24%, 127%), lipocalin 2 (659%; 95% CI: 198%, 1838%), and regenerating family 1 beta (221%; 95% CI: 47%, 600%) and lower signals corresponding to granzyme B (-74%; 95% CI: -82%, -62%), and sucrase isomaltase (-58%; 95% CI: -75%, -29%) in EED biopsies compared with NPA biopsies. Computerized algorithms also detected statistically significant elevation in intraepithelial lymphocytes (49%; 95% CI: 9%, 105%) and proliferation of leukocytes (267%; 95% CI: 92%, 601%) in EED biopsies compared with NPA biopsies. CONCLUSIONS: Our results support a model of chronic epithelial stress that decreases epithelial differentiation and absorptive function. The close association of several IHC parameters with manual histologic scoring suggests that automated digital quantification of IHC panels complements traditional histomorphologic assessment in EED.

Histopathology underlying environmental enteric dysfunction in a cohort study of undernourished children in Bangladesh, Pakistan, and Zambia compared with United States children.

BACKGROUND: Environmental enteric dysfunction (EED) is an asymptomatic intestinal disorder associated with growth impairment, delayed neurocognitive development, and impaired oral vaccine responses. OBJECTIVES: We set out to develop and validate a histopathologic scoring system on duodenal biopsies from a cohort study of children with growth failure in Bangladesh, Pakistan, and Zambia ("EED") with reference to biopsies from United States children with no clinically reported histologic pathology (referred to hereafter as "normal") or celiac disease. METHODS: Five gastrointestinal pathologists evaluated 745 hematoxylin and eosin slide images from 291 children with EED (mean age: 1.6 y) and 66 United States children (mean age: 6.8 y). Histomorphologic features (i.e., villus/crypt architecture, goblet cells, epithelial and lamina propria acute/chronic inflammation, Brunner's glands, Paneth cells, epithelial detachment, enterocyte injury, and foveolar metaplasia) were used to score each histopathologic slide. Generalized estimating equations were used to determine differences between EED, normal, and celiac disease, and receiver operating characteristic curves were used to assess predictive value. RESULTS: Biopsies from the duodenal bulb showed higher intramucosal Brunner's gland scores and lower intraepithelial lymphocyte scores than from the second or third parts of the duodenum (D2/3), so only D2/3 were included in the final analysis. Although 7 parameters differed significantly between EED and normal biopsies in regression models, only 5 (blunted villus architecture, increased intraepithelial lymphocytosis, goblet cell depletion, Paneth cell depletion, and reduced intramucosal Brunner's glands) were required to create a total score percentage (TSP-5) that correctly identified EED against normal biopsies (AUC: 0.992; 95% CI: 0.983, 0.998). Geographic comparisons showed more severe goblet cell depletion in Bangladesh and more marked intraepithelial lymphocytosis in Pakistan. CONCLUSIONS: This scoring system involving 5 histologic parameters demonstrates very high discrimination between EED and normal biopsies, indicating that this scoring system can be applied with confidence to studies of intestinal biopsies in EED.

Clinically significant copy number alterations and complex rearrangements of MYB and NFIB in head and neck adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) of the head and neck is a malignant tumor with poor long-term prognosis. Besides the recently identified MYB-NFIB fusion oncogene generated by a t(6;9) translocation, little is known about other genetic alterations in ACC. Using high-resolution, array-based comparative genomic hybridization, and massively paired-end sequencing, we explored genomic alterations in 40 frozen ACCs. Eighty-six percent of the tumors expressed MYB-NFIB fusion transcripts and 97% overexpressed MYB mRNA, indicating that MYB activation is a hallmark of ACC. Thirty-five recurrent copy number alterations (CNAs) were detected, including losses involving 12q, 6q, 9p, 11q, 14q, 1p, and 5q and gains involving 1q, 9p, and 22q. Grade III tumors had on average a significantly higher number of CNAs/tumor compared to Grade I and II tumors (P = 0.007). Losses of 1p, 6q, and 15q were associated with high-grade tumors, whereas losses of 14q were exclusively seen in Grade I tumors. The t(6;9) rearrangements were associated with a complex pattern of breakpoints, deletions, insertions, inversions, and for 9p also gains. Analyses of fusion-negative ACCs using high-resolution arrays and massively paired-end sequencing revealed that MYB may also be deregulated by other mechanisms in addition to gene fusion. Our studies also identified several down-regulated candidate tumor suppressor genes (CTNNBIP1, CASP9, PRDM2, and SFN) in 1p36.33-p35.3 that may be of clinical significance in high-grade tumors. Further, studies of these and other potential target genes may lead to the identification of novel driver genes in ACC.

Somatic Sequencing and Microsatellite Instability Results From Mismatch Repair-deficient Endometrial Carcinoma Patients Without Lynch Syndrome ("Lynch-like" tumors): Implications for Heritable Cancer Screening, Molecular Prognostication, and Immunotherapeutic Vulnerability.

Immunostaining of endometrial carcinomas for mismatch repair (MMR) protein loss is standard-of-care for Lynch syndrome screening, but also identifies MMR-deficient tumors without germline pathogenic variants. While the majority show MLH1 hypermethylation ( MLH1hm ), somatic MMR pathogenic variants are increasingly recognized drivers of immunohistochemistry-germline discordance. Because MMR abnormalities with both germline and somatic origins have prognostic significance and impart susceptibility to immune checkpoint inhibitors, it is important to understand how frequently tumors with MMR immunohistochemical loss and normal germline testing ("Lynch-like" tumors) have underlying somatic MMR pathogenic variants. Somatic tumor sequencing±microsatellite instability (MSI) testing was performed on 18 endometrial cancers with MMR immunohistochemical loss but negative MMR germline results and negative MLH1hm where relevant. Tumor sequencing and MSI testing were successful in 94%. Where successful, 80% were MSI-high and 94% had a molecular correlate for the initial immunohistochemical interpretation. The single case without an identified somatic pathogenic variant was MSI-low and initially showed loss of MSH6 by immunohistochemistry but with extremely limited internal control staining. On review, MSH6 immunohistochemistry was reclassified as equivocal, and repeat staining revealed improved control expression with intact MSH6. Following reclassification of this case, 100% tumors with MMR deficiency by immunohistochemistry had at least 1 confirmed somatic MMR pathogenic variant, and 86% were MSI-high. These results demonstrate that when correctly interpreted immunohistochemistry is a strong surrogate for somatic MMR pathogenic variants and support its use as the frontline MMR biomarker in endometrial cancer for heritable screening, molecular prognostic classification, and immunotherapeutic biomarker testing purposes.

The Cooperative Human Tissue Network of the National Cancer Institute: Supporting Cancer Research for 35 Years.

The Cooperative Human Tissue Network was created by the NCI in 1987 to support a coordinated national effort to collect and distribute high quality, pathologist-validated human tissues for cancer research. Since then, the network has expanded to provide different types of tissue samples, blood and body fluid samples, immunohistologic and molecular sample preparations, tissue microarrays, and clinical datasets inclusive of biomarkers and molecular testing. From inception through the end of 2021, the network has distributed 1,375,041 biospecimens. It served 889 active investigators in 2021. The network has also taken steps to begin to optimize the representation of diverse communities among the distributed biospecimens. In this article, the authors review the 35-year history of this network, describe changes to the program over the last 15 years, and provide operational and scientific highlights from each of the divisions. Readers will learn how to engage with the network and about the continued evolution of the program for the future.

Inverse agonists of retinoic acid receptor/retinoid X receptor signaling as lineage-specific antitumor agents against human adenoid cystic carcinoma.

BACKGROUND: Adenoid cystic carcinoma (ACC) is a lethal malignancy of exocrine glands, characterized by the coexistence within tumor tissues of 2 distinct populations of cancer cells, phenotypically similar to the myoepithelial and ductal lineages of normal salivary epithelia. The developmental relationship linking these 2 cell types, and their differential vulnerability to antitumor treatments, remains unknown. METHODS: Using single-cell RNA sequencing, we identified cell-surface markers (CD49f, KIT) that enabled the differential purification of myoepithelial-like (CD49fhigh/KITneg) and ductal-like (CD49flow/KIT+) cells from patient-derived xenografts (PDXs) of human ACCs. Using prospective xenotransplantation experiments, we compared the tumor-initiating capacity of the 2 cell types and tested whether one could differentiate into the other. Finally, we searched for signaling pathways with differential activation between the 2 cell types and tested their role as lineage-specific therapeutic targets. RESULTS: Myoepithelial-like cells displayed higher tumorigenicity than ductal-like cells and acted as their progenitors. Myoepithelial-like and ductal-like cells displayed differential expression of genes encoding for suppressors and activators of retinoic acid signaling, respectively. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) signaling (all-trans retinoic acid, bexarotene) promoted myoepithelial-to-ductal differentiation, whereas suppression of RAR/RXR signaling with a dominant-negative RAR construct abrogated it. Inverse agonists of RAR/RXR signaling (BMS493, AGN193109) displayed selective toxicity against ductal-like cells and in vivo antitumor activity against PDX models of human ACC. CONCLUSIONS: In human ACCs, myoepithelial-like cells act as progenitors of ductal-like cells, and myoepithelial-to-ductal differentiation is promoted by RAR/RXR signaling. Suppression of RAR/RXR signaling is lethal to ductal-like cells and represents a new therapeutic approach against human ACCs.

A 20-gene model for molecular nodal staging of bladder cancer: development and prospective assessment.

BACKGROUND: Neoadjuvant chemotherapy before cystectomy confers a survival benefit in bladder cancer, but it has not been widely adopted since most patients do not benefit and we are at present unable to predict those that do. Since the most important predictor of recurrence after cystectomy is pathologically positive nodes, our aim was to assess techniques that define this stage for the selection of patients for neoadjuvant chemotherapy. METHODS: We developed a gene expression model (GEM) to predict the pathological node status in primary tumour tissue from three independent cohorts of patients who were clinically node negative. From a subset of transcripts detected faithfully by microarrays from both paired frozen and formalin-fixed tissues (32 pairs), we developed both the GEM and cutoffs that identified patient strata with raised risk of nodal involvement by use of two separate training cohorts (90 and 66 patients). We then assessed the GEM and cutoffs to predict node-positive disease in tissues from a phase 3 trial cohort (AUO-AB-05/95; 185 patients). FINDINGS: We developed a 20-gene GEM with an area under the curve of 0·67 (95% CI 0·60-0·75) for prediction of nodal disease at cystectomy in AUO-AB-05/95. The cutoff system identified patients with high relative risk (1·74, 95% CI 1·03-2·93) and low relative risk (0·70, 95% CI 0·51-0·96) of node-positive disease. Multivariate logistic regression showed the GEM predictor was independent of age, sex, pathological stage, and lymphovascular space invasion (coefficient 9·81, 95% CI 1·64-18·00; p=0·019). INTERPRETATION: Selecting patients for neoadjuvant chemotherapy on the basis of risk of node-positive disease has the potential to benefit high-risk patients while sparing other patients toxic effects and delay to cystectomy. FUNDING: US National Cancer Institute (R01CA143971).

Proteogenomic analysis of lung adenocarcinoma reveals tumor heterogeneity, survival determinants, and therapeutically relevant pathways.

We present a deep proteogenomic profiling study of 87 lung adenocarcinoma (LUAD) tumors from the United States, integrating whole-genome sequencing, transcriptome sequencing, proteomics and phosphoproteomics by mass spectrometry, and reverse-phase protein arrays. We identify three subtypes from somatic genome signature analysis, including a transition-high subtype enriched with never smokers, a transversion-high subtype enriched with current smokers, and a structurally altered subtype enriched with former smokers, TP53 alterations, and genome-wide structural alterations. We show that within-tumor correlations of RNA and protein expression associate with tumor purity and immune cell profiles. We detect and independently validate expression signatures of RNA and protein that predict patient survival. Additionally, among co-measured genes, we found that protein expression is more often associated with patient survival than RNA. Finally, integrative analysis characterizes three expression subtypes with divergent mutations, proteomic regulatory networks, and therapeutic vulnerabilities. This proteogenomic characterization provides a foundation for molecularly informed medicine in LUAD.

Loss of VOPP1 overexpression in squamous carcinoma cells induces apoptosis through oxidative cellular injury.

The vesicular overexpressed in cancer prosurvival protein 1 (VOPP1) gene product (previously known as GASP and ECOP) has a poorly characterized functional role in cancer cells, although its expression levels are known to be elevated in many cancer types. To determine the role that VOPP1 has in human squamous cell carcinoma (SCC), a series of siRNA-mediated expression knockdown experiments were performed in carcinoma-derived model systems with confirmed endogenous VOPP1 overexpression (three SCC-derived cell lines: SCC-9, FaDu, and H2170, as well as the cervical adenocarcinoma HeLa cell line, which has been examined in relevant previous reports). The data indicate that VOPP1 knockdown induces cell death at 72 h post-transfection and this is caused by the induction of apoptosis via the intrinsic pathway. Analysis of microarray gene expression profiling showed that genes whose expression was affected by VOPP1 knockdown exhibited enrichment in annotations of oxidative stress and mitochondrial dysfunction. Reporters of reactive oxygen species (ROS) and mitochondrial membrane potential show that ROS levels become elevated and mitochondrial dysfunction occurs with VOPP1 knockdown at time points before the activation of effector caspases and cell death seen at later time points. Furthermore, the introduction of the antioxidant N-acetyl cysteine was able to abrogate the induction of apoptosis observed with VOPP1 knockdown in a dose-responsive manner. Reporter constructs for NF-κB-mediated transcription are not affected in SCC cell lines by VOPP1 knockdown. Taken together, these data support the hypothesis that VOPP1 overexpression in cancer participates in the control of the intracellular redox state, and that its loss leads to oxidative cellular injury leading to cell death by the intrinsic apoptotic pathway.

Development and characterization of xenograft model systems for adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is one of the most common malignancies to arise in human salivary glands, and it also arises in the glandular tissue of other organ systems. To address the paucity of experimental model systems for this tumor type, we have undertaken a program of transplanting tissue samples of human ACC into immunodeficient nu/nu mice to create xenograft model systems. In 17 of 23 attempts (74%), xenograft tumors were successfully grown. In all cases, the histologic appearance of the donating tumor was recapitulated in the subsequent xenograft. Characterization of a subset of xenograft models by immunohistochemical biomarkers and by RNA transcript microarray analysis showed good fidelity in the recapitulation of gene expression patterns in the xenograft tumors compared with the human donor tumors. As ACC is known to frequently contain a t(6;9) translocation that fuses the MYB and NFIB genes, fluorescence in situ hybridization (FISH) of 12 ACC xenograft models was performed that assayed MYB locus break-apart and MYB-NFIB locus fusion. Of 12 xenograft models, 11 (92%) revealed MYB locus rearrangement and 10 (83%) showed evidence of fusion of the MYB and NFIB loci. The two related xenograft models (derived from primary and metastatic tumors, respectively, of the same human subject) were karyotyped, showing a t(1;6) translocation, suggesting MYB translocation to a novel fusion partner gene. Overall, our results indicate that ACC is amenable to xenografting and that ACC xenograft models recapitulate the molecular and morphologic characteristics of human tumors, suggesting utility as valid experimental and preclinical model systems for this disease.

Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, acinar cell damage, and systemic inflammation in acute pancreatitis.

In acute pancreatitis, endoplasmic reticulum (ER) stress prompts an accumulation of malfolded proteins inside the ER, initiating the unfolded protein response (UPR). Because the ER chaperone tauroursodeoxycholic acid (TUDCA) is known to inhibit the UPR in vitro, this study examined the in vivo effects of TUDCA in an acute experimental pancreatitis model. Acute pancreatitis was induced in Wistar rats using caerulein, with or without prior TUDCA treatment. UPR components were analyzed, including chaperone binding protein (BiP), phosphorylated protein kinase-like ER kinase (pPERK), X-box binding protein (XBP)-1, phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CCAAT/enhancer binding protein homologues protein, and caspase 12 and 3 activation. In addition, pancreatitis biomarkers were measured, such as serum amylase, trypsin activation, edema formation, histology, and the inflammatory reaction in pancreatic and lung tissue. TUDCA treatment reduced intracellular trypsin activation, edema formation, and cell damage, while leaving amylase levels unaltered. The activation of myeloperoxidase was clearly reduced in pancreas and lung. Furthermore, TUDCA prevented caerulein-induced BiP upregulation, reduced XBP-1 splicing, and caspase 12 and 3 activation. It accelerated the downregulation of pJNK. In controls without pancreatitis, TUDCA showed cytoprotective effects including pPERK signaling and activation of downstream targets. We concluded that ER stress responses activated in acute pancreatitis are grossly attenuated by TUDCA. The chaperone reduced the UPR and inhibited ER stress-associated proapoptotic pathways. TUDCA has a cytoprotective potential in the exocrine pancreas. These data hint at new perspectives for an employment of chemical chaperones, such as TUDCA, in prevention of acute pancreatitis.

Analysis of MYB expression and MYB-NFIB gene fusions in adenoid cystic carcinoma and other salivary neoplasms.

Recent studies have shown that the recurrent t(6;9)(q22-23;p23-24) translocation in adenoid cystic carcinoma results in a novel fusion of the MYB proto-oncogene with the transcription factor gene NFIB. To determine the frequency of this finding, we used RT-PCR assays of the MYB and MYB-NFIB fusion transcripts, and immunohistochemistry for the MYB protein, to study adenoid cystic carcinomas and other epithelial tumors of the salivary glands, and head and neck region. MYB-NFIB fusion transcript was detected in 25 of 29 (86%) frozen adenoid cystic carcinoma tumor samples, and in 14 of 32 (44%) formalin-fixed paraffin-embedded adenoid cystic carcinoma tumor specimens. In contrast, the MYB-NFIB fusion was not expressed in non-adenoid cystic carcinoma neoplasms of the head and neck, confirming the high specificity of the MYB-NFIB fusion. Adenoid cystic carcinomas from various anatomic sites, including salivary gland, sinonasal cavity, tracheobronchial tree, larynx, breast, and vulva were repeatedly fusion-positive, indicating that adenoid cystic carcinomas located in different anatomic sites not only have important morphologic features in common, but also probably evolve through activation of the same molecular pathways. Studies of the expression of MYB revealed that 89% of the tumors, including both fusion-positive and fusion-negative cases, overexpressed MYB RNA. Similarly, 82% of adenoid cystic carcinomas stained positive for MYB protein, compared with 14% of non-adenoid cystic carcinoma neoplasms, indicating that MYB immunostaining may be useful for the diagnosis of adenoid cystic carcinoma, but that neoplasms sometimes in the differential diagnosis are also labeled. The latter are, however, fusion-negative. In summary, our studies show that MYB activation through gene fusion or other mechanisms is a major oncogenic event in adenoid cystic carcinoma occurring at various anatomic sites. In addition to being a diagnostically useful biomarker for adenoid cystic carcinoma, MYB and its downstream effectors are also novel potential therapeutic targets.

Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) still remains a lethal malignancy benefiting from the identification of the new target for early detection and/or development of new therapeutic regimens based on a better understanding of the biological mechanism for treatment. The overexpression of Her2 and Her3 receptors have been identified in various solid tumors, but its prognostic relevance in HNSCC remains controversial. METHODS: Three hundred eighty-seven primary HNSCCs, 20 matching metasis and 17 recurrent HNSCCs were arrayed into tissue microarrays. The relationships between Her2 and Her3 protein expression and clinicopathological parameters/survival of HNSCC patients were analyzed with immunohistochemistry. RESULTS: Her3 is detected as either a cytoplasmic or a membranous dominant expression pattern whereas Her2 expression showed uniform membranous form. In primary tumor tissues, high membranous Her2 expression level was found in 104 (26.9%) cases while positive membranous and cytoplasmic Her3 expression was observed in 34 (8.8%) and 300 (77.5%) samples, respectively. Membranous Her2 expression was significantly associated with histological grade (P = 0.021), as grade 2 tumors showed the highest positive expression. Membranous Her3 over-expression was significantly prevalent in metastatic tissues compared to primary tumors (P = 0.003). Survival analysis indicates that membranous Her3 expression is significantly associated with worse overall survival (P = 0.027) and is an independent prognostic factor in multivariate analysis (hazard ratio, 1.51; 95% confidence interval, 1.01-2.23; P = 0.040). CONCLUSIONS: These results suggest that membranous Her3 expression is strongly associated with poor prognosis of patients with HNSCC and is a potential candidate molecule for targeted therapy.

Fully covered self-expanding metal stents placed temporarily in the bile duct: safety profile and histologic classification in a porcine model.

BACKGROUND: Fully covered Self-Expanding metal stents (FCSEMS) have been shown efficacious in palliating malignant biliary obstructions. There is little data analyzing mucosal response to their temporary placement in the bile duct. METHODS: Ten mini pigs underwent endoscopic placement of a FCSEMS (Wallflex, Boston Scientific). FCSEMS were kept in place for three months. At the end of the 3 months, FCSEMS were removed endoscopically. Five pigs were euthanized and their bile ducts harvested. The other five were kept alive for another month post removal. A single pathologist, created a scoring system (to determine degree of inflammation, fibrosis, and epithelial injury), examined all specimens in a blinded fashion. RESULTS: Four FCSEMS spontaneously migrated in the duodenum. On post mortem examination, mild mucosal thickness was noted in three bile duct specimens while superficial inflammation of the bile duct was noted in five animals. Histologic examination of the bile duct revealed focal acute inflammation in both groups. For the 5 animals euthanized immediately after stent removal, there was a tendency to have superficial mucosal erosion and fibrosis. In contrast, increased chronic inflammation was more commonly seen in the animals 1 month post stent removal, with all animals in this group showing moderate degrees of mononuclear inflammatory cell mucosal infiltrates. No severe inflammatory or fibrotic duct injury was observed in any of the study animals, with degree of injury graded as mild to moderate. CONCLUSION: FCSEMS appear to induce minimal tissue overgrowth or fibrosis post placement. Ease of removability and no significant histologic injury are advantages noted with FCSEMS., however, further studies are needed to evaluate treating benign biliary strictures with FCSEMS in humans.