Smad4 restricts injury-provoked biliary proliferation and carcinogenesis.

Cholangiocarcinoma (CCA) is a deadly and heterogeneous type of cancer characterized by a spectrum of epidemiologic associations as well as genetic and epigenetic alterations. We seek to understand how these features inter-relate in the earliest phase of cancer development and through the course of disease progression. For this, we studied murine models of liver injury integrating the most commonly occurring gene mutations of CCA - including Kras, Tp53, Arid1a and Smad4 - as well as murine hepatobiliary cancer models and derived primary cell lines based on these mutations. Among commonly mutated genes in CCA, we found that Smad4 functions uniquely to restrict reactive cholangiocyte expansion to liver injury through restraint of the proliferative response. Inactivation of Smad4 accelerates carcinogenesis, provoking pre-neoplastic biliary lesions and CCA development in an injury setting. Expression analyses of Smad4-perturbed reactive cholangiocytes and CCA lines demonstrated shared enriched pathways, including cell-cycle regulation, MYC signaling and oxidative phosphorylation, suggesting that Smad4 may act via these mechanisms to regulate cholangiocyte proliferation and progression to CCA. Overall, we showed that TGFß/SMAD4 signaling serves as a critical barrier restraining cholangiocyte expansion and malignant transformation in states of biliary injury.

Arid1a mutation suppresses TGF-ß signaling and induces cholangiocarcinoma.

Activating KRAS mutations and functional loss of members of the SWI/SNF complex, including ARID1A, are found together in the primary liver tumor cholangiocarcinoma (CC). How these mutations cooperate to promote CC has not been established. Using murine models of hepatocyte and biliary-specific lineage tracing, we show that Kras and Arid1a mutations drive the formation of CC and tumor precursors from the biliary compartment, which are accelerated by liver inflammation. Using cultured cells, we find that Arid1a loss causes cellular proliferation, escape from cell-cycle control, senescence, and widespread changes in chromatin structure. Notably, we show that the biliary proliferative response elicited by Kras/Arid1a cooperation and tissue injury in CC is caused by failed engagement of the TGF-ß-Smad4 tumor suppressor pathway. We thus identify an ARID1A-TGF-ß-Smad4 axis as essential in limiting the biliary epithelial response to oncogenic insults, while its loss leads to biliary pre-neoplasia and CC.

Tumor Size, Not Small Vessel Invasion, Predicts Survival in Patients With Hepatocellular Carcinoma.

OBJECTIVES: The 8th edition American Joint Committee on Cancer (AJCC) staging system for hepatocellular carcinoma (HCC) has been criticized for failing to stratify patients. We aimed to reassess and modify the tumor staging criteria for HCC. METHODS: Three independent study cohorts were collected and analyzed. RESULTS: The initial cohort consists of 103 patients with HCC. By Kaplan-Meier survival analysis, the 8th edition failed to distinguish between T1b and T2. Only tumor size and large vessel invasion, but not small vessel invasion or other histopathologic parameters, predicted HCC survival. We modified the T staging criteria by eliminating small vessel invasion while emphasizing tumor size in the middle categories (T2 and T3), which achieved more even distribution of cases and significantly improved risk stratifications (P < .001). This modification was then validated in a cohort of 250 consecutive patients from Mount Sinai Hospital and an online Surveillance, Epidemiology, and End Results data set comprising 9,685 patients, which showed similar results. Small vessel invasion was not an independent prognostic factor in either validation cohort. CONCLUSIONS: Our study showed that tumor size, but not small vessel invasion, predicts survival in patients with HCC. We suggest incorporating our modified T staging criteria in future AJCC revisions.

Interobserver agreement in the diagnosis of anal dysplasia: comparison between gastrointestinal and gynaecological pathologists and utility of consensus conferences.

AIMS: Management of anal dysplasia relies upon the accurate diagnosis of anal biopsy specimens. As institutions move towards subspecialty signout (SSSO), decisions must be made regarding whether to assign anal biopsies to the gastrointestinal (GI) or gynaecological (GYN) pathology service. MATERIALS AND RESULTS: We identified 200 archival tissue biopsies of anal mucosa and circulated them among three GI pathologists and three GYN pathologists. Each pathologist separately scored each biopsy as normal, atypical, low-grade squamous intra-epithelial lesion (LSIL) or high-grade squamous intra-epithelial lesion (HSIL). Every case that was called HSIL by at least one pathologist was stained with p16 immunostain and a 'gold standard' interpretation of whether or not a case represented HSIL was made. The GI pathologists agreed on 97 (49%) cases prior to consensus; the GYN pathologists agreed on 33 (17%). The sensitivities of the three GI pathologists in detecting HSIL against the 'gold standard' were 47, 100 and 21% and for the GYN pathologists the sensitivities were 74, 89 and 84%; the sensitivities of the GI and GYN consensus diagnoses were 74% each. The specificities of the three GI pathologists in detecting HSIL were 99, 90 and 100% and for the GYN pathologists the specificities were 99, 92 and 91%; the specificities of both the GI and GYN consensus diagnoses were 100%. CONCLUSIONS: A mild to moderate degree of interobserver variability exists in the diagnosis of anal dysplasia among pathologists. Our study indicates the utility of some form of consensus conference, as overall agreement among GI pathologists and among GYN pathologists improved following in-person consensus.

Congenital hepatic fibrosis and its mimics: a clinicopathologic study of 19 cases at a single institution.

BACKGROUND: Congenital hepatic fibrosis (CHF) is a rare inherited form of ductal plate malformation associated with polycystic kidney disease. The diagnosis requires histopathologic confirmation, but can be challenging to distinguish from other undefined fibrocystic liver diseases. We aimed to describe the clinicopathologic features of congenital hepatic fibrosis (CHF), with comparisons to other entities that may clinically and/or histologically mimic CHF. METHODS: Nineteen cases that carried a clinical and/or histologic impression of CHF were identified at our institution, of which the histology was reassessed and reappraised into two categories: CHF (n=13) and mimics (n=6). The clinicopathologic features between the two groups were analyzed and compared. RESULTS: The CHF group was further sub-classified into those with clinical suspicion (CHF-c, n=8) and those as incidental histology findings (CHF-i, n=5). Patients of CHF-i were much older than CHF-c or mimics (P<0.05). Male and female were equally affected. Six of 8 CHF-c (66.7%) had concurrent kidney diseases, including 5 polycystic kidney diseases. Five of 6 mimics (83.3%) had various kidney diseases, including nephronophthisis, Alport syndrome, renal agenesis, and nephrolithiasis. None of the CHF-i patients had kidney disease, but 3 were associated with hepatic carcinomas. Histology analysis demonstrated characteristic triads (bile duct abnormalities, portal vein hypoplasia, and fibrosis) in all CHF cases. One mimic had paucity of intrahepatic bile ducts, while the other 5 mimics showed abnormal portal veins and nodular regenerative hyperplasia consistent with hepatoportal sclerosis (HPS). CONCLUSIONS: Our study demonstrates classic histology triad of CHF despite a wide spectrum of clinical presentations. HPS is unexpectedly a clinical mimicker of CHF, which can be distinguished histologically.

Clinicopathologic Features of Varicella Zoster Virus Infection of the Upper Gastrointestinal Tract.

Reactivation of latent varicella zoster virus (VZV) may be limited to a dermatome or involve multiple organs, including the gastrointestinal tract. Although gastrointestinal manifestations of disseminated zoster have been likened to those of herpes simplex virus (HSV), histologic features of VZV-related injury to the tubular gut are not well-documented. We performed this study to describe the clinicopathologic features of VZV-related gastrointestinal injury. We identified 6 such patients with VZV infection. All involved the upper gastrointestinal tract, affecting the esophagus (n=3), stomach (n=2), or both (n=1). All patients were immunocompromised adults with hematologic malignancies (n=5) or a heart transplant (n=1); 3 with hematologic malignancies had received stem cell transplants. Five patients had cutaneous and gastrointestinal zoster; 1 had gastrointestinal disease alone. When compared with 14 HSV-related esophagitis controls, there were several notable differences. VZV caused hemorrhagic ulcers with nodularity or erythema, whereas HSV produced round, shallow ulcers on a background of nearly normal mucosa (P=0.01). VZV-related ulcers featured fibrin-rich, pauci-inflammatory exudates compared with the macrophage-rich exudates of HSV (P=0.003). The cytopathic changes of VZV were present at all levels of the squamous epithelium, especially in a peripapillary distribution. In contrast, HSV inclusions were located in the superficial layers (P=0.003) and detached keratinocytes. Unlike HSV, VZV involved the stomach, producing hemorrhage accompanied by striking apoptosis in the deep glands. We conclude that VZV produces unique patterns of gastrointestinal injury that facilitate its diagnosis. Recognition of gastrointestinal VZV infection is important because it heralds potentially life-threatening disseminated disease.

Gastrin Staining in Inflamed Stomach Biopsies Labeled as "Antral" Rarely Detects Atrophic Gastritis.

OBJECTIVES: Autoimmune metaplastic atrophic gastritis (AMAG) is an underrecognized entity, especially in its early stage. This study assessed whether the use of gastrin immunohistochemistry would increase sensitivity for diagnosing early AMAG. METHODS: Three-hundred gastric biopsies were prospectively stained for gastrin by immunohistochemistry. Inclusion criteria included well-oriented gastric mucosa with mucus glands and minimal plasma cell infiltrate not suspected to represent pyloric metaplasia. Patient age, sex, designated location of biopsy, presence or absence of intestinal metaplasia, and clinical information were not criteria. Any case with absence of gastrin-positive endocrine cells reflexed to chromogranin immunohistochemistry. Maloriented biopsies or cases with current Helicobacter infection were excluded. RESULTS: The 298-patient study cohort comprised 222 females (mean age, 47 years; range, 16-80 years) and 76 males (mean age, 49 years; range, 7-80 years). Biopsies were designated as "antral/antral nodules" (61%), and the rest were labeled "gastric/random stomach" (39%). Nine cases (3%) exhibited absence of gastrin-positive endocrine cells; one of those showed endocrine cell hyperplasia by chromogranin staining. CONCLUSIONS: Pathologists should be aware of the histologic features of early AMAG and meticulously analyze tissue regardless of specimen labeling. Gastrin immunostain is a supplemental diagnostic tool when encountering inflamed antral-appearing specimens.

Effects of subspecialty signout and group consensus on the diagnosis of microscopic colitis.

Microscopic colitis (MC) includes lymphocytic colitis (LC) and collagenous colitis (CC). Microscopic changes are required to establish these diagnoses. While criteria exist, interobserver variability has been reported previously. This has not been evaluated in the context of subspecialty signout (SSSO) or a consensus conference. We identified 133 colon biopsies diagnosed as LC, CC, MC, or normal but with mild changes insufficient for MC. All predated the introduction of SSSO at our institution. They were independently reviewed by three gastrointestinal (GI) pathologists. Cases lacking independent consensus were reviewed by the same pathologists in consensus conference to establish a final diagnosis. Individual diagnoses were compared with the consensus diagnoses, and consensus diagnoses were compared with original diagnoses made by GI and non-GI pathologists. Consensus diagnoses were normal (n = 34), LC (n = 57), and CC (n = 42). "Normal" was the diagnosis most commonly agreed upon independently (27/34 cases, P = 0.0073 versus LC, P = 0.0172 versus CC). The reviewing pathologists independently agreed with 80%, 80%, and 94% of consensus diagnoses (κ = 0.70, 0.69, and 0.91). The group consensus agreed with the diagnoses in 49 of 58 (84%) cases originally signed out by non-GI pathologists (κ = 0.77) and in 44 of 57 (77%) cases originally signed out by GI pathologists (κ = 0.63). Good interobserver agreement exists for MC, though whether GI subspecialty training improves agreement remains unclear. Group consensus may aid in diagnosis of difficult/borderline MC cases.

Florid Vascular Proliferation of the Small Bowel and Colon, a Potential Masquerader of Malignancy: Report of Three Cases.

Vascular abnormalities and lesions of the small bowel and colon are rare. A florid vascular proliferation (FVP) associated with colon obstruction and intussusception has been described and can mimic malignant vascular tumors such as angiosarcoma. In this article, we report a case of colonic FVP associated with colon obstruction, a case of small bowel FVP associated with a Meckel's diverticulum, and a case of small bowel FVP with intussusception. All cases occurred in older adults (mean 73 years of age, range 62-80 years of age). FVP grossly appeared as a mass-like lesion in one small bowel case, while the other cases did not demonstrate a grossly identifiable mass. Histologically, all cases demonstrated a transmural vascular proliferation with plump endothelial cells. No significant cytologic atypia was seen, and mitotic figures were rare. No recurrence was seen in all cases with an average follow-up of 22 months. It is important to be aware of this entity as it appears to be a nonneoplastic reactive process, unlike some of its histologic mimics.

Impact of Specimen Type and Specimen Number on HER2 Status in Gastroesophageal Junction and Gastric Adenocarcinoma.

OBJECTIVES: Human epidermal growth factor receptor 2 (HER2) is expressed in some gastric and gastroesophageal junction adenocarcinomas. There were two goals: assess the impact of specimen type on HER2 status and evaluate HER2 concordance with multiple specimens. METHODS: All cases were evaluated by immunohistochemistry (IHC) for HER2 and interpreted using established criteria. Fluorescence in situ hybridization (FISH) was performed on a subset. RESULTS: Of 460 specimens, 83.9% were IHC negative, 5.4% were equivocal, and 10.7% were IHC positive. Of those with FISH testing, 78.5% were FISH negative, and 21.5% were FISH positive. IHC-FISH concordance for biopsy specimens, resections, and metastases was 82%, 84%, and 86%, respectively. With one vs two vs three or more specimens, the HER2-positive rate increased from 10.5% to 18.1% to 24.1%, respectively. CONCLUSIONS: HER2 testing may be performed on biopsy specimens with a relatively high concordance rate with resection specimens, and if multiple samples are analyzed from a single patient, the HER2-positive rate increases over twofold.

ARID1A, a SWI/SNF subunit, is critical to acinar cell homeostasis and regeneration and is a barrier to transformation and epithelial-mesenchymal transition in the pancreas.

OBJECTIVE: Here, we evaluate the contribution of AT-rich interaction domain-containing protein 1A (ARID1A), the most frequently mutated member of the SWItch/sucrose non-fermentable (SWI/SNF) complex, in pancreatic homeostasis and pancreatic ductal adenocarcinoma (PDAC) pathogenesis using mouse models. DESIGN: Mice with a targeted deletion of Arid1a in the pancreas by itself and in the context of two common genetic alterations in PDAC, Kras and p53, were followed longitudinally. Pancreases were examined and analysed for proliferation, response to injury and tumourigenesis. Cancer cell lines derived from these models were analysed for clonogenic, migratory, invasive and transcriptomic changes. RESULTS: Arid1a deletion in the pancreas results in progressive acinar-to-ductal metaplasia (ADM), loss of acinar mass, diminished acinar regeneration in response to injury and ductal cell expansion. Mutant Kras cooperates with homozygous deletion of Arid1a, leading to intraductal papillary mucinous neoplasm (IPMN). Arid1a loss in the context of mutant Kras and p53 leads to shorter tumour latency, with the resulting tumours being poorly differentiated. Cancer cell lines derived from Arid1a-mutant tumours are more mesenchymal, migratory, invasive and capable of anchorage-independent growth; gene expression analysis showed activation of epithelial-mesenchymal transition (EMT) and stem cell identity pathways that are partially dependent on Arid1a loss for dysregulation. CONCLUSIONS: ARID1A plays a key role in pancreatic acinar homeostasis and response to injury. Furthermore, ARID1A restrains oncogenic KRAS-driven formation of premalignant proliferative IPMN. Arid1a-deficient PDACs are poorly differentiated and have mesenchymal features conferring migratory/invasive and stem-like properties.

Kras and Tp53 Mutations Cause Cholangiocyte- and Hepatocyte-Derived Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a primary liver cancer epidemiologically linked with liver injury, which has poorly understood incipient stages and lacks early diagnostics and effective therapies. While iCCA is conventionally thought to arise from the biliary tract, studies have suggested that both hepatocytes and biliary cells (cholangiocytes) may give rise to iCCA. Consistent with the plasticity of these cell lineages, primary liver carcinomas exhibit a phenotypic range from hepatocellular carcinoma (HCC) to iCCA, with intermediates along this spectrum. Here, we generated mouse models to examine the consequence of targeting mutant Kras and Tp53, common alterations in human iCCA, to different adult liver cell types. Selective induction of these mutations in the SOX9+ population, predominantly consisting of mature cholangiocytes, resulted in iCCA emerging from premalignant biliary intraepithelial neoplasia (BilIN). In contrast, adult hepatocytes were relatively refractory to these mutations and formed rare HCC. In this context, injury accelerated hepatocyte-derived tumorigenesis and promoted a phenotypic switch to iCCA. BilIN precursor lesions were absent in the hepatocyte-derived iCCA models, pointing toward distinct and direct emergence of a malignant cholangiocytic phenotype from injured, oncogenically primed hepatocytes. Tp53 loss enhanced the reprogramming of hepatocytes to cholangiocytes, which may represent a mechanism facilitating formation of hepatocyte-derived iCCA. Overall, our work shows iCCA driven by Kras and Tp53 may originate from both mature cholangiocytes and hepatocytes, and factors such as chronic liver injury and underlying genetic mutations determine the path of progression and resulting cancer phenotype.Significance: The histopathogenesis of biliary tract cancer, driven by Tp53 and Kras mutations, can be differentially impacted by the cell of origin within the mature liver as well by major epidemiologic risk factors. Cancer Res; 78(16); 4445-51. ©2018 AACR.

IMP3 Immunohistochemical Expression Remains Consistent Among All Grades of Gastrointestinal Neuroendocrine Tumors.

Insulin-like growth factor-II messenger RNA-binding protein-3 (IMP3), is an oncofetal protein whose aberrant expression has previously been detected in multiple malignant neoplasms. Pulmonary neuroendocrine carcinomas demonstrate increased expression compared with pulmonary carcinoid tumors, but this relationship has not been studied in gastrointestinal neuroendocrine tumors (GINETs). This study examined IMP3 expression in GINETs, with a focus on correlation with established grading criteria. Fifty-four GINETs were immunohistochemically studied using a monoclonal antibody against IMP3. Using established World Health Organization criteria, the cases were stratified by grade and included 31 grade 1 neuroendocrine tumors (G1 GINETs), 15 grade 2 neuroendocrine tumors (G2 GINETs), and 8 neuroendocrine carcinomas (GINECs). The majority (51/54, 94.4%) of GINETs demonstrated IMP3 staining. Thirty cases (55.6%) showed IMP3 cytoplasmic/membranous staining in 60% or greater of tumor cells, with moderate to strong staining in nearly all of these cases (29/30; 96.7%). Of the remaining 24 cases, 3 cases showed no staining, whereas 17 (81%) demonstrated weak staining. When stratified by grade, there was no statistically significant difference in IMP3 staining among the 3 grades of GINETs; of the G1 GINETs, 14 (45.2%) demonstrated staining in at least 60% of tumor cells, compared with 10 (66.7%) G2 GINETs and 6 (75%) GINECs. Hindgut neoplasms of any grade were the most likely to show significant IMP3 staining. Unlike what has been demonstrated in neuroendocrine neoplasms in the lungs, GINETs appear to have a consistent IMP3 expression profile among all tumors grades, which may be reflective of their unique tumor biology.

Accuracy of vascular invasion reporting in hepatocellular carcinoma before and after implementation of subspecialty surgical pathology sign-out.

CONTEXT: Liver cancers (including hepatocellular carcinoma [HCC] and cholangiocarcinoma) are the fifth most common cause of cancer death. The most powerful independent histologic predictor of overall survival after transplantation for HCC is the presence of microscopic vascular invasion (VI). AIMS: Given that VI is known to have somewhat high interobserver variability in both HCC and other tumors, we hypothesized that pathologists with special interest and training in liver pathology would be more likely to identify and report VI in HCC than would general surgical pathologists. SETTINGS AND DESIGN: We searched our departmental surgical pathology archives for transplant hepatectomies performed for HCC. SUBJECTS AND METHODS: We identified 143 such cases with available sign-out reports and hematoxylin and eosin-stained slides. STATISTICAL ANALYSIS USED: Kappa results (level of agreement) were calculated. RESULTS: Before surgical pathology subspecialty sign-out (SSSO) implementation, 49 of 88 HCC cases were reported as negative for VI; on rereview, 20 of these had VI. After SSSO implementation, 39 of 55 cases were reported as negative for VI; on our review, 8 of these had VI. Kappa (agreement) between general SO and subspecialty rereview was 0.562 (95% confidence interval [CI] = 0.411-0.714) "weak agreement." Kappa (agreement) between SSSO and rereview by select liver pathologists was 0.693 (95% CI = 0.505-0.880) "moderate agreement." CONCLUSIONS: Our study is one of only a few so far that have suggested improved accuracy of certain parameters under SSSO.

Immunohistochemistry as a surrogate for molecular subtyping of gastric adenocarcinoma.

The Cancer Genome Atlas Research Network recently classified gastric adenocarcinoma into 4 molecular subtypes: Epstein-Barr virus-positive tumors, microsatellite-unstable tumors, tumors with chromosomal instability, and genomically stable tumors. We theorized that immunohistochemistry might be useful in similar categorization and that that HER2 expression might relate to subtype. We stained 104 gastric adenocarcinomas for MLH1, p53, and EBER in situ hybridization. We grouped them based on staining pattern and compared the groups. Cases were categorized as follows: group 1 (EBER positive), 7 cases (7%); group 2 (MLH1 deficient), 17 cases (16%); group 3 (aberrant p53 staining, EBER negative, retained MLH1), 40 cases (38%); group 4 (unremarkable staining), 40 cases (38%). This distribution was comparable to that found by the Research Network after accounting for the TP53 mutation rate in the chromosomal instability group. Group 1 patients had significantly longer follow-up times (median, 70 months versus 13 months for other groups; P = .0324). No group 2 cases overexpressed HER2. In group 3, 3 of 40 cases were HER2 immunohistochemistry positive, but 7 of 27 were HER2 positive by fluorescence in situ hybridization. Staining offers an efficient, reasonably accurate alternative for molecular subtyping of gastric adenocarcinoma, although some cases with chromosomal instability cannot be identified. These findings have potential prognostic and therapeutic implications.

A microRNA biomarker of hepatocellular carcinoma recurrence following liver transplantation accounting for within-patient heterogeneity.

BACKGROUND: Liver cancer, of which hepatocellular carcinoma (HCC) is by far the most common type, is the second most deadly cancer (746,000 deaths in 2012). Currently, the only curative treatment for HCC is surgery to remove the malignancy (resection) or to remove the entire diseased liver followed by transplantation of healthy liver tissue. Given the shortage of healthy livers, it is crucial to provide transplants to patients that have the best chance of long-term survival. Currently, transplantation is determined via the Milan criteria-patients within Milan (single tumor < 5 cm or 2-3 tumors < 3 cm with no extrahepatic spread nor intrahepatic vascular invasion) are typically eligible for transplantation. However, combining microRNA expression profiling with the Milan criteria can improve prediction of recurrence. HCC often presents with multiple distinct tumor foci arising from local spread of a primary tumor or from the oncogenic predisposition of the diseased liver. Substantial genomic heterogeneity between tumor foci within a single patient has been reported; therefore, biomarker development must account for the possibility of highly heterogeneous genomic profiles from the same individual. METHODS: MicroRNA profiling was performed on 180 HCC tumor samples from 89 patients who underwent liver transplantation at the University of Rochester Medical Center. The primary outcome was recurrence-free survival time, and patients were observed for 3 years post-transplantation. RESULTS: MicroRNA expression profiles were used to develop a biomarker that distinguishes HCC patients at greater risk of recurrence post-transplantation. Unsupervised clustering uncovered two distinct subgroups with vast differences in standard transplantation selection criteria and recurrence-free survival times. These subgroups were subsequently used to identify microRNAs strongly associated with HCC recurrence. Our results show that reduced expression of five specific microRNAs is significantly associated with HCC recurrence post-transplantation. CONCLUSIONS: MicroRNA profiling of distinct tumor foci, coupled with methods that address within-subject tumor heterogeneity, has the potential to significantly improve prediction of HCC recurrence post-transplantation. The development of a clinically applicable HCC biomarker would inform treatment options for patients and contribute to liver transplant selection criteria for practitioners.

MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors.

UNLABELLED: Vascular invasion provides a direct route for tumor metastasis. The degree to which microRNA (miRNA) expression plays a role in tumor vascular invasion is unclear. Here, we report that miR-494 is up-regulated in human hepatocellular carcinoma (HCC) tumors with vascular invasion and can promote HCC cell invasiveness by gene inactivation of multiple invasion-suppressor miRNAs. Our results show that ten eleven translocation (TET) methylcytosine dioxygenase, predominantly TET1 in HCC cells, is a direct target of miR-494. The reduced 5'-hydroxymethylcytosine levels observed in the proximal cytosine-phosphate-guanine (CpG) regions of multiple invasion-suppressor miRNA genes are strongly associated with their transcriptional repression upon miR-494 overexpression, whereas enforced DNA demethylation can abolish the repression. Furthermore, TET1 knockdown shows a similar effect as miR-494 overexpression. Conversely, miR-494 inhibition or enforced TET1 expression is able to restore invasion-suppressor miRNAs and inhibit miR-494-mediated HCC cell invasion. CONCLUSIONS: miR-494 can trigger gene silencing of multiple invasion-suppressor miRNAs by inhibiting genomic DNA demethylation by direct targeting of TET1, thereby leading to tumor vascular invasion.

Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression.

Mutations in p53 and RAS potently cooperate in oncogenic transformation, and correspondingly, these genetic alterations frequently coexist in pancreatic ductal adenocarcinoma (PDA) and other human cancers. Previously, we identified a set of genes synergistically activated by combined RAS and p53 mutations as frequent downstream mediators of tumorigenesis. Here, we show that the synergistically activated gene Plac8 is critical for pancreatic cancer growth. Silencing of Plac8 in cell lines suppresses tumor formation by blocking autophagy, a process essential for maintaining metabolic homeostasis in PDA, and genetic inactivation in an engineered mouse model inhibits PDA progression. We show that Plac8 is a critical regulator of the autophagic machinery, localizing to the lysosomal compartment and facilitating lysosome-autophagosome fusion. Plac8 thus provides a mechanistic link between primary oncogenic mutations and the induction of autophagy, a central mechanism of metabolic reprogramming, during PDA progression.

Tissue pattern recognition error rates and tumor heterogeneity in gastric cancer.

The anatomic pathology discipline is slowly moving toward a digital workflow, where pathologists will evaluate whole-slide images on a computer monitor rather than glass slides through a microscope. One of the driving factors in this workflow is computer-assisted scoring, which depends on appropriate selection of regions of interest. With advances in tissue pattern recognition techniques, a more precise region of the tissue can be evaluated, no longer bound by the pathologist's patience in manually outlining target tissue areas. Pathologists use entire tissues from which to determine a score in a region of interest when making manual immunohistochemistry assessments. Tissue pattern recognition theoretically offers this same advantage; however, error rates exist in any tissue pattern recognition program, and these error rates contribute to errors in the overall score. To provide a real-world example of tissue pattern recognition, 11 HER2-stained upper gastrointestinal malignancies with high heterogeneity were evaluated. HER2 scoring of gastric cancer was chosen due to its increasing importance in gastrointestinal disease. A method is introduced for quantifying the error rates of tissue pattern recognition. The trade-off between fully sampling tumor with a given tissue pattern recognition error rate versus randomly sampling a limited number of fields of view with higher target accuracy was modeled with a Monte-Carlo simulation. Under most scenarios, stereological methods of sampling-limited fields of view outperformed whole-slide tissue pattern recognition approaches for accurate immunohistochemistry analysis. The importance of educating pathologists in the use of statistical sampling is discussed, along with the emerging role of hybrid whole-tissue imaging and stereological approaches.

Kras(G12D) and p53 mutation cause primary intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (IHCC) is a primary cancer of the liver with an increasing incidence and poor prognosis. Preclinical studies of the etiology and treatment of this disease are hampered by the relatively small number of available IHCC cell lines or genetically faithful animal models. Here we report the development of a genetically engineered mouse model of IHCC that incorporates two of the most common mutations in human IHCC, activating mutations of Kras (Kras(G12D)) and deletion of p53. Tissue-specific activation of Kras(G12D) alone resulted in the development of invasive IHCC with low penetrance and long latency. Latency was shortened by combining Kras(G12D) activation with heterozygous or homozygous deletion of p53 (mean survival of 56 weeks vs. 19 weeks, respectively), which also resulted in widespread local and distant metastasis. Serial analysis showed that the murine models closely recapitulated the multistage histopathologic progression of the human disease, including the development of stroma-rich tumors and the premalignant biliary lesions, intraductal papillary biliary neoplasms (IPBN), and Von Meyenburg complexes (VMC; also known as biliary hamartomas). These findings establish a new genetically and histopathologically faithful model of IHCC and lend experimental support to the hypothesis that IPBN and VMC are precursors to invasive cancers.