Liver transplant for primary biliary tract neuroendocrine tumor in a nine-year-old girl.

BACKGROUND: Neuroendocrine tumors (NETs) are rare epithelial neoplasms that arise most commonly from the gastrointestinal tract. In pediatrics, the most common site of origin is in the appendix, with the liver being the most common site of metastasis. Neuroendocrine tumors arising from the biliary tract are extremely rare. METHODS: We describe a case of a nine-year-old girl who presented with obstructive cholestasis and was found to have multiple liver masses identified on biopsy as well-differentiated neuroendocrine tumor with an unknown primary tumor site. RESULT: The patient underwent extensive investigation to identify a primary tumor site, including endoscopy, endoscopic ultrasound, and capsule endoscopy. The patient ultimately underwent definitive management with liver transplant, and on explant was discovered to have multiple well-differentiated neuroendocrine tumors, WHO Grade 1, with extensive infiltration into the submucosa of bile duct, consistent with primary biliary tract neuroendocrine tumor. CONCLUSION: Identifying the site of the primary tumor in NETs found within the liver can be challenging. To determine if an extrahepatic primary tumor exists, workup should include endoscopy, EUS, and capsule endoscopy. Children with well-differentiated hepatic NETs, with no identifiable primary tumor, and an unresectable tumor, are considered favorable candidates for liver transplantation.

Liver pathology in pregnancy.

Liver dysfunction occurs in up to 3% of pregnancies and can be due to pregnancy-associated liver injury, exacerbation of pre-existing liver disease, or co-incident with pregnancy. The most common form of pregnancy-associated liver injury is intrahepatic cholestasis of pregnancy (ICP). This condition is typically benign and self-limited, but is associated with fetal morbidity and mortality with high levels of serum bile acids. Acute fatty liver of pregnancy (AFLP) and the hypertensive disorders of pregnancy (including pre-eclampsia, eclampsia, and hemolysis, elevated liver enzymes, and low platelets [HELLP] syndrome) are more commonly associated with maternal and fetal complications and may necessitate expedient delivery. Histologically, ICP shows nonspecific features of cholestasis, while AFLP and the hypertensive disorders have more characteristic histologic findings. While not a true liver disease, hyperemesis gravidarum can cause elevated liver enzymes. Pregnant patients are at increased risk of developing severe hepatitis E and herpesvirus infections, Budd-Chiari syndrome, and gallstones, and they may also experience worsening of known chronic liver disease. Mass lesions in pregnancy including hemangiomas, focal nodular hyperplasia, and hepatocellular adenomas and carcinomas can present unique challenges for diagnosis and management. This review will explore the pathophysiology, presentation, histologic features, and management of these conditions.

Three-dimensional analysis of extrahepatic cholangiocarcinoma and tumor budding.

Advances in tissue clearing and microscopy make it possible to study human diseases in three dimensions (3D). High-grade tumor budding is known to be associated with poor prognosis in various cancers; however, little is known about the 3D architecture of tumor budding. Using tissue clearing, we analyzed the 3D structure of tumor budding and E-cadherin expression in 31 extrahepatic cholangiocarcinomas. A total of 31 thick slabs (up to 5 mm) were harvested from surgically resected tumor tissue, including 27 hilar and 4 distal cholangiocarcinomas. Twenty-eight cases were adenocarcinoma, and three were undifferentiated carcinoma. After clearing, the tissues were immunolabeled with antibodies to cytokeratin 19 and to E-cadherin, and then visualized using light-sheet and confocal laser scanning microscopy. Tumor budding was evaluated in hematoxylin and eosin-stained sections (2D) using standard pathological criteria. Of the 31 cancers, 13 showed low-grade tumor budding and 18 showed high-grade tumor budding. First, 3D analysis revealed that the neoplastic cells in tumor buds of adenocarcinoma were typically not individual islands of cells, but rather tips of attenuated protrusions connected to the main tumor. Second, adenocarcinomas with low-grade tumor budding were composed predominantly of tubules that only focally form cords at the periphery. By contrast, adenocarcinomas with high-grade tumor budding predominantly formed cords in both centers and peripheries of the tumors. Third, adenocarcinoma with low-grade tumor budding was characterized by a few short protrusions with few branches, whereas adenocarcinoma with high-grade tumor budding was characterized by longer protrusions with more branching. Finally, immunolabeling of E-cadherin was stronger in the center of the adenocarcinoma but decreased at the tips of protrusions. E-cadherin loss was more extensive in the protrusions of high-grade tumor budding than in the protrusions of low-grade tumor budding. Our findings suggest that tumor buds as seen in 2D are, in fact, cross-sections of attenuated but contiguous protrusions extending from the main tumor. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Three-dimensional visualization of cleared human pancreas cancer reveals that sustained epithelial-to-mesenchymal transition is not required for venous invasion.

Venous invasion is three times more common in pancreatic cancer than it is in other major cancers of the gastrointestinal tract, and venous invasion may explain why pancreatic cancer is so deadly. To characterize the patterns of venous invasion in pancreatic cancer, 52 thick slabs (up to 5 mm) of tissue were harvested from 52 surgically resected human ductal adenocarcinomas, cleared with a modified iDISCO method, and labeled with fluorescent-conjugated antibodies to cytokeratin 19, desmin, CD31, p53 and/or e-cadherin. Labeled three-dimensional (3D) pancreas cancer tissues were visualized with confocal laser scanning or light sheet microscopy. Multiple foci of venous and even arterial invasion were visualized. Venous invasion was detected more often in 3D (88%, 30/34 cases) than in conventional 2D slide evaluation (75%, 25/34 cases, P < 0.001). 3D visualization revealed pancreatic cancer cells crossing the walls of veins at multiple points, often at points where preexisting capillary structures bridge the blood vessels. The neoplastic cells often retained a ductal morphology (cohesive cells forming tubes) as they progressed from a stromal to intravenous location. Although immunolabeling with antibodies to e-cadherin revealed focal loss of expression at the leading edges of the cancers, the neoplastic cells within veins expressed e-cadherin and formed well-oriented glands. We conclude that venous invasion is almost universal in pancreatic cancer, suggesting that even surgically resectable PDAC has access to the venous spaces and thus the ability to disseminate widely. Furthermore, we observe that sustained epithelial-mesenchymal transition is not required for venous invasion in pancreatic cancer.

The immunomodulatory potential of natural compounds in tumor-bearing mice and humans.

Cancer is considered a fetal disease caused by uncontrolled proliferation and progression of abnormal cells. The most efficient cancer therapies suppress tumor growth, prevent progression and metastasis, and are minimally toxic to normal cells. Natural compounds have shown a variety of chemo-protective effects alone or in combination with standard cancer therapies. Along with better understanding of the dynamic interactions between our immune system and cancer development, nutritional immunology-the use of natural compounds as immunomodulators in cancer patients-has begun to emerge. Cancer cells evolve strategies that target many aspects of the immune system to escape or even edit immune surveillance. Therefore, the immunesuppressive tumor microenvironment is a major obstacle in the development of cancer therapies. Because interaction between the tumor microenvironment and the immune system is a complex topic, this review focuses mainly on human clinical trials and animal studies, and it highlights specific immune cells and their cytokines that have been modulated by natural compounds, including carotenoids, curcumin, resveratrol, EGCG, and ß-glucans. These natural compounds have shown promising immune-modulating effects, such as inhibiting myeloid-derived suppressor cells and enhancing natural killer and cytolytic T cells, in tumor-bearing animal models, but their efficacy in cancer patients remains to be determined.

Effects of Hepatic Ischemia-Reperfusion Injuries and NRF2 on Transcriptional Activities of Bile Transporters in Rats.

BACKGROUND: The effect of hepatic ischemia-reperfusion injury (IRI) on bile transporter (BT) gene expression is unknown. We hypothesized that abnormal expression of BTs during hepatic IRI is dependent on nuclear factor erythroid 2-related factor 2 (NRF2), which contributes to the cholestasis after reperfusion. METHODS: Sham surgery and short (60 min) or long (90 min) periods of warm ischemia time (WIT) with or without reperfusion for 24 h were applied to wild-type Sprague-Dawley rats and Nrf2 knockout rats (n = 5 per group). At each stage of IRI, the serum levels of aminotransferase, total bilirubin, and bile acids were measured. In addition, hepatic tissue was sampled to determine the histologic score of IRI (Suzuki score), measure adenosine triphosphate (ATP), and identify the quantitative real-time polymerase chain reactions of BTs (Oatp1, Ntcp, Mrp2, Bsep, and Mrp3). RESULTS: In short periods of WIT, BT expression increased during the ischemia stage and returned to the baseline after reperfusion. However, in long periods of WIT, BT expression did not increase after ischemia and decreased further after reperfusion. Short WIT did not increase BT expression in Nrf2 knockout animals. The level of BT expression was correlated with the Suzuki score, the serum levels of aminotransferase, bilirubin, and bile acids, and tissue ATP level. Stepwise multiple regression analysis derived equations to predict the Suzuki score (R2 = 76.8, P < 0.001), serum total bilirubin (R2 = 61.2, P < 0.001), and tissue ATP (R2 = 61.1, P < 0.001). CONCLUSIONS: Short WIT induces the transcriptional activities of BT, whereas long WIT depresses them, and the effect was blunted by Nrf2 knockout status. BT expression can be considered a surrogate marker for hepatic IRI.

Loss of FFAR2 promotes colon cancer by epigenetic dysregulation of inflammation suppressors.

Free fatty acid receptor 2 (FFAR2, also named GPR43), is activated by short-chain fatty acids (SCFAs), such as butyrate, that are produced when gut bacteria ferment dietary fiber. FFAR2 has been suggested to regulate colonic inflammation, which is a major risk factor for the development of colon cancer and is also linked to epigenetic dysregulation in colon carcinogenesis. The current study assessed whether FFAR2, acting as an epigenetic regulator, protects against colon carcinogenesis. To mimic the mild inflammation that promotes human colon cancer, we treated mice with dextran sodium sulfate (DSS) overnight, which avoids excessive inflammation but induces mild inflammation that promotes colon carcinogenesis in the ApcMin/+ and the azoxymethane (AOM)-treated mice. Our results showed that FFAR2 deficiency promotes the development of colon adenoma in the ApcMin/+ /DSS mice and the progression of adenoma to adenocarcinoma in the AOM/DSS mice. FFAR2's downstream cAMP-PKA-CREB pathway was enhanced, leading to overexpression of histone deacetylases (HDACs) in the FFAR2-deficient mice. ChIP-qPCR analysis revealed differential binding of H3K27me3 and H3K4me3 histone marks onto the promoter regions of inflammation suppressors (e.g., sfrp1, dkk3, socs1), resulting in decreased expression of these genes in the FFAR2-deficient mice. Also, more neutrophils infiltrated into tumors and colon lamina propria of the FFAR2-deficient mice. Depletion of neutrophils blocked the progression of colon tumors. In addition, FFAR2 is required for butyrate to suppress HDAC expression and hypermethylation of inflammation suppressors. Therefore, our results suggest that FFAR2 is an epigenetic tumor suppressor that acts at multiple stages of colon carcinogenesis.

Clinicopathological Distinction of Low-AFP-Secreting vs. High-AFP-Secreting Hepatocellular Carcinomas.

Ilntroduction and aims. We aimed to investigate the clinical and pathological differences between low-AFP-secreting (AFP < 20 ng/mL) and high-AFP-secreting (AFP ≥ 20 ng/mL) hepatocellular carcinomas in patients who undergo liver transplant (LT). MATERIAL AND METHODS: We evaluated 145 patients who underwent deceased donor LT for HCC from January 1, 2005 until August 1, 2015 at the Johns Hopkins Hospital. RESULTS: Median pre-LT AFP in the entire cohort was 13 ng/mL (IQR 6-59). Using serum AFP cutoff of 20 ng/mL, 61 (42%) patients had high-AFP-secreting tumors and 84 (58%) had low-AFP-secreting tumors. Patients with high-AFP-secreting tumors had larger lesions (3 cm vs. 2.4 cm, p = 0.024), and were more likely to have microvascular-invasion (36.1% vs. 20.2%, p = 0.02) and poor-differentiation (18% vs. 4.8%, p = 0.01), and tumor recurrence following LT (28% vs. 6%, p < 0.001). The 1-year, 3-year, and 5-year recurrence-free survival for patients in the low-AFP-secreting group compared to the high-AFP-secreting group were 100%, 92%, 92% vs. 81.3%, 71.3%, 68.5% respectively (p = 0.0003). CONCLUSION: AFP is a suboptimal predictor of tumor recurrence following liver transplant in HCC patients. However, it can have some value in distinguishing more aggressive forms of HCC (high-AFP-secreting) that are associated with higher tumor recurrence. Novel tumor biomarkers are needed that can enhance predicting tumor recurrence following LT based on tumor biology.

Could Aspirin and Diets High in Fiber Act Synergistically to Reduce the Risk of Colon Cancer in Humans?

Early inhibition of inflammation suppresses the carcinogenic process. Aspirin is the most commonly used non-steroid anti-inflammatory drugs (NSAIDs), and it irreversibly inhibits cyclooxygenase-1 and -2 (COX1, COX2). Multiple randomized clinical trials have demonstrated that aspirin offers substantial protection from colon cancer mortality. The lower aspirin doses causing only minimal gastrointestinal disturbance, ideal for long-term use, can achieve only partial and transitory inhibition of COX2. Aspirin's principal metabolite, salicylic acid, is also found in fruits and vegetables that inhibit COX2. Other phytochemicals such as curcumin, resveratrol, and anthocyanins also inhibit COX2. Such dietary components are good candidates for combination with aspirin because they have little or no toxicity. However, obstacles to using phytochemicals for chemoprevention, including bioavailability and translational potential, must be resolved. The bell/U-shaped dose-response curves seen with vitamin D and resveratrol might apply to other phytochemicals, shedding doubt on 'more is better'. Solutions include: (1) using special delivery systems (e.g., nanoparticles) to retain phytochemicals; (2) developing robust pharmacodynamic biomarkers to determine efficacy in humans; and (3) selecting pharmacokinetic doses relevant to humans when performing preclinical experiments. The combination of aspirin and phytochemicals is an attractive low-cost and low-toxicity approach to colon cancer prevention that warrants testing, particularly in high-risk individuals.

Loss of free fatty acid receptor 2 enhances colonic adenoma development and reduces the chemopreventive effects of black raspberries in ApcMin/+ mice.

We previously showed that black raspberries (BRBs) have beneficial effects in human colorectal cancer and a mouse model of colorectal cancer (ApcMin/+). The current study investigated the role of free fatty acid receptor 2 (FFAR2) in colon carcinogenesis and whether the FFAR2 signaling pathway contributes to BRB-mediated chemoprevention in mice. FFAR2 (also named GPR43) is a member of the G-protein-coupled receptor family that is expressed in leukocytes and colon. ApcMin/+ and ApcMin/+-FFAR2-/- mice were given a control diet or the control diet supplemented with 5% BRBs for 8 weeks. FFAR2 deficiency promoted colonic polyp development, with 100% incidence and increased polyp number and size. The ApcMin/+ mice developed colonic tubular adenoma, whereas the ApcMin/+-FFAR2-/- mice developed colonic tubular adenoma with high-grade dysplasia. FFAR2 deficiency also enhanced the cAMP-PKA-CREB-HDAC pathway, downstream of FFAR2 signaling, and increased activation of the Wnt pathway, and raised the percentage of GR-1+ neutrophils in colonic lamina propria (LP) and increased infiltration of GR-1+ neutrophils into colonic polyps. BRBs suppressed colonic polyp development and inhibited the cAMP-PKA-CREB-HDAC and Wnt pathways in the ApcMin/+ mice but not the ApcMin/+-FFAR2-/- mice. They also increased the percentage of GR-1+ neutrophils and cytokine secretion in colonic LP and decreased the infiltration of GR-1+ neutrophils and IL-1ß expression in colon polyps of ApcMin/+ mice but not ApcMin/+-FFAR2-/- mice. These results suggest that loss of FFAR2 drives colon tumorigenesis and that BRBs require functional FFAR2 to be chemopreventive. BRBs have the potential to modulate the host immune system, thereby enhancing the antitumor immune microenvironment.

Systemic Metabolite Changes in Wild-type C57BL/6 Mice Fed Black Raspberries.

INTRODUCTION: Freeze-dried black raspberries (BRBs) elicit chemopreventive effects against colorectal cancer in humans and in rodents. The objective of this study was to investigate potential BRB-caused metabolite changes using wild-type (WT) C57BL/6 mice. METHODS AND RESULTS: WT mice were fed either control diet or control diet supplemented with 5% BRBs for 8 wk. A nontargeted metabolomic analysis was conducted on colonic mucosa, liver, and fecal specimens collected from both diet groups. BRBs significantly changed the levels of 41 colonic mucosa metabolites, 40 liver metabolites, and 34 fecal metabolites compared to control diet-fed mice. BRBs reduced 34 lipid metabolites in colonic mucosa and increased levels of amino acids in liver. One metabolite, 3-[3-(sulfooxy) phenyl] propanoic acid, might be a useful biomarker of BRB consumption. In addition, BRB powder was found to contain 30-fold higher levels of linolenate compared to control diets. Consistently, multiple omega-3 polyunsaturated fatty acids (ω-3 PUFAs), including stearidonate, docosapentaenoate (ω-3 DPA), eicosapentaenoate (EPA), and docosahexaenoate (DHA), were significantly elevated in livers of BRB-fed mice. CONCLUSION: The data from the current study suggest that BRBs produce systemic metabolite changes in multiple tissue matrices, supporting our hypothesis that BRBs may serve as both a chemopreventive agent and a beneficial dietary supplement.

Cholesteryl Ester Storage Disease: An underdiagnosed cause of cirrhosis in adults.

Cholesteryl Ester Storage Disease (CESD), is a rare multisystem autosomal recessive disorder and belongs to the broad family of lysosomal storage disorders. It can present anytime from infancy and childhood to even adulthood. The clinical manifestations are generally severe in infants and with milder forms in adults. One of the prominent sites of involvement is liver. Due to low awareness of this condition among physicians including surgical pathologists, majority of the liver biopsies, especially from the adults are often misdiagnosed as non-alcoholic fatty liver disease/non-alcoholic steatohepatitis or cryptogenic cirrhosis. Given the recent availability of safe and effective enzyme replacement therapy that can alter the natural course of CESD, the pathologists signing out adult and pediatric liver biopsies should be aware of this entity, thus contributing to timely patient management. This review discusses the clinical features, pathogenesis, diagnostic approach, differential diagnosis and management of CESD in adults.

Diagnostic phrasing is independently correlated with the decision to treat for graft-versus-host disease: retrospective review of colon biopsies with rare apoptosis.

AIMS: The risks of immunosuppression and the non-specific nature of rare crypt apoptosis has led to debate over the lower threshold for histological diagnosis of colonic graft-versus-host disease (GVHD). A recent study proposed the diagnostic category of indeterminate for GVHD (iGVHD) for cases with six or fewer apoptotic bodies per 10 crypts. Our aim was to assess colon biopsies with iGVHD histology to determine whether the diagnosis was retrospectively predictive of the decision to treat, and to correlate these findings with endoscopic and clinical findings. METHODS AND RESULTS: A retrospective search was performed for colonic biopsies taken to evaluate for GVHD from 2008 to 2014. Biopsies were blindly reviewed for the maximum number of apoptotic bodies per 10 contiguous crypts, evidence of crypt dropout, and ulceration. Clinical information was collected through chart review. One hundred and twenty-two biopsies from 84 transplant patients were included. Forty-seven cases met the histological criteria for iGVHD. Patients with an original diagnosis of iGVHD were more likely to be managed conservatively than those with a diagnosis of grade 1 GVHD (25% versus 0%). Eight symptomatic patients reclassified as iGVHD had resolution of symptoms without increased immunosuppression. A clinicopathologically similar group of 10 patients with iGVHD histology, normal or subtle endoscopic findings and no evidence of GVHD at other organ sites were treated with increased immunosuppression. On multivariate analysis, the original diagnostic category was the most significant predictor of the decision to treat. CONCLUSION: The use of the diagnostic category iGVHD alerts clinicians to the presence of minimal crypt apoptosis, and allows treatment based on clinical judgement.

Management of Difficult Cases of Autoimmune Hepatitis.

Autoimmune hepatitis (AIH) is a complex autoimmune disease characterized by immune-mediated destruction of hepatic parenchyma which can result in cirrhosis, liver failure, and death. Current American Association for the Study of Liver Diseases (AASLD) and European Association for the Study of Liver (EASL) guidelines recommend corticosteroids alone or in combination with azathioprine as first-line treatment strategies. However, a significant proportion of patients may not be able to tolerate or achieve complete biochemical response with these options. In this article, we discuss approaches to these patients and other challenging AIH patient groups such as the asymptomatic, pregnant, elderly, and liver transplant recipients.

Black raspberries suppress colonic adenoma development in ApcMin/+ mice: relation to metabolite profiles.

Freeze-dried black raspberries (BRBs) have demonstrated chemopreventive effects in a dietary intervention trial with human colorectal cancer patients. The aim of this study was to investigate BRB-caused metabolite changes using the Apc(Min/+) mouse as a model of human colorectal cancer. Wild-type (WT) mice were fed control diet, and Apc(Min/+) mice were fed either control diet or control diet supplemented with 5% BRBs for 8 weeks. Colonic and intestinal polyp size and number were measured. A non-targeted metabolomic analysis was conducted on colonic mucosa, liver and fecal specimens. Eight weeks of BRB treatment significantly decreased intestinal and colonic polyp number and size in Apc(Min/+) mice. The apc gene mutation significantly changed 52 metabolites in colonic mucosa associated with increased amino acid and decreased lipid metabolites, as well as 39 liver and 8 fecal metabolites. BRBs significantly reversed 23 apc-regulated metabolites, including 13 colonic mucosa, 8 liver and 2 fecal metabolites that were involved in amino acid, glutathione, lipid and nucleotide metabolism. Of these, changes in eight metabolites were linearly correlated with decreased colonic polyp number and size in BRB-treated Apc(Min/+) mice. Elevated levels of putrescine and linolenate in Apc(Min/+) mice were significantly decreased by BRBs. Ornithine decarboxylase expression, the key enzyme in putrescine generation, was fully suppressed by BRBs. These results suggest that BRBs produced beneficial effects against colonic adenoma development in Apc(Min/+) mice and modulated multiple metabolic pathways. The metabolite changes produced by BRBs might potentially reflect the BRB-mediated chemopreventive effects in colorectal cancer patients.

Differences in the degree of cerulein-induced chronic pancreatitis in C57BL/6 mouse substrains lead to new insights in identification of potential risk factors in the development of chronic pancreatitis.

A frequently used experimental model of chronic pancreatitis (CP) recapitulating human disease is repeated injection of cerulein into mice. C57BL/6 is the most commonly used inbred mouse strain for biomedical research, but widespread demand has led to generation of several substrains with subtly different phenotypes. In this study, two common substrains, C57BL/6J and C57BL/6NHsd, exhibited different degrees of CP, with C57BL/6J being more susceptible to repetitive cerulein-induced CP as assessed by pancreatic atrophy, pancreatic morphological changes, and fibrosis. We hypothesized that the deficiency of nicotinamide nucleotide transhydrogenase (NNT) protein in C57BL/6J is responsible for the more severe C57BL/6J phenotype but the parameters of CP in NNT-expressing transgenic mice generated on a C57BL6/J background do not differ with those of wild-type C57BL/6J. The highly similar genetic backgrounds but different CP phenotypes of these two substrains presents a unique opportunity to discover genes important in pathogenesis of CP. We therefore performed whole mouse genome Affymetrix microarray analysis of pancreatic gene expression of C57BL/6J and C57BL/6NHsd before and after induction of CP. Genes with differentially regulated expression between the two substrains that might be candidates in CP progression included Mmp7, Pcolce2, Itih4, Wdfy1, and Vtn. We also identified several genes associated with development of CP in both substrains, including RIKEN cDNA 1810009J06 gene (trypsinogen 5), Ccl8, and Ccl6.