Marc-Oliver Riener

Serotonin promotes tumor growth in human hepatocellular cancer

In addition to its function as a neurotransmitter and vascular active molecule, serotonin is also a mitogen for hepatocytes and promotes liver regeneration. A possible role in hepatocellular cancer has not yet been investigated. Human hepatocellular cancer cell lines Huh7 and HepG2 were used to assess the function of serotonin in these cell lines. Characteristics of autophagy were detected with transmission electron microscopy, immunoblots of microtubule‐associated protein light chain 3(LC3) and p62 (sequestosome 1). Immunoblots of the mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4E‐BP1 were used to investigate signaling pathways of serotonin. Two different animal models served as principle of proof of in vitro findings. Clinical relevance of the experimental findings was evaluated with a tissue microarray from 168 patients with hepatocellular carcinoma. Serotonin promotes tumor growth and survival in starved hepatocellular carcinoma cells. During starvation hepatocellular carcinoma cells exhibited characteristics of autophagy, which disappeared in serotonin‐treated cells. Rapamycin, an inhibitor of mTOR, is known to induce autophagy. Serotonin could override rapamycin by an mTOR‐independent pathway and activate common downstream signals such as p70S6K and 4E‐BP1. In two tumor models of the mouse, inhibition of serotonin signaling consistently impaired tumor growth. Human biopsies revealed expression of the serotonin receptor HTR2B, correlating with downstream signals, e.g., phosphorylated p70S6K and proliferation. Conclusion: This study provides evidence that serotonin is involved in tumor growth of hepatocellular cancer by activating downstream targets of mTOR, and therefore serotonin‐related pathways might represent a new treatment strategy. (HEPATOLOGY 2010.)

Golgi phosphoprotein 2 (GOLPH2) expression in liver tumors and its value as a serum marker in hepatocellular carcinomas.

Hepatocellular carcinomas (HCCs) and bile duct carcinomas (BDCs) have a poor prognosis. Therefore, surveillance strategies including sensitive and specific serum markers for early detection are needed. Recently, Golgi Phosphoprotein 2 (GOLPH2) has been proposed as a serum marker for HCC, but GOLPH2 expression data in liver tissues was not available. Using tissue microarrays and immunohistochemistry, we semiquantitatively analyzed GOLPH2 protein expression in patients with HCC (n = 170), benign liver tumors (n = 22), BDC (n = 114) and normal liver tissue (n = 105). A newly designed sandwich enzyme‐linked immunoassay (ELISA) was used to analyze GOLPH2 levels in the sera of patients with HCC (n = 62), hepatitis C virus (HCV) (n = 29), BDC (n = 10), and healthy control persons (n = 12). By immunohistochemistry 121/170 (71%) of HCC showed strong GOLPH2 expression, which was significantly associated with a higher tumor grade (P = 0.01). A total of 97/114 (85%) BDCs showed a strong GOLPH2 expression which proved to be an independent prognostic factor for overall survival (P < 0.05). Serum levels of GOLPH2 measured by ELISA were significantly elevated in patients with HCC with underlying HCV infection (median 18 mg/L, P < 0.05) and patients with BDC (median = 14.5 mg/L, P < 0.01) in comparison to healthy controls (median 4 mg/L). Conclusion: GOLPH2 protein is highly expressed in tissues of HCC and BDC. GOLPH2 protein levels are detectable and quantifiable in sera by ELISA. In patients with hepatitis C, serial ELISA measurements in the course of the disease appear to be a promising complementary serum marker in the surveillance of HCC. GOLPH2 should be further evaluated as a serum tumor marker in BDC on a larger scale. (HEPATOLOGY 2009.)

Rare PIK3CA hotspot mutations in carcinomas of the biliary tract.

Somatic mutations of the PIK3CA gene, which encodes the p110α catalytic subunit of phosphatidylinositol 3‐kinase (PI3K), are frequent in various cancer types. The majority of mutations cluster at hotspots within exons 9 and 20, which encode the helical and kinase domains of p110α. PIK3CA mutations in bile duct and gallbladder carcinomas have not been reported yet. In this study, we analysed 118 carcinomas of the biliary tract and the liver (45 intra‐ and extrahepatic cholangiocarcinomas (CCA), 23 gallbladder carcinomas, 50 hepatocellular carcinomas) for PIK3CA hotspot mutations using polymerase chain reaction and direct DNA sequencing. PIK3CA missense mutations were found in one of 11 intrahepatic CCA (E545K, 9%), one of 23 gallbladder carcinomas (E542K, 4%), and one of 50 hepatocellular carcinomas (H1047R, 2%). All three mutations represent hotspot mutations, which also occur in other cancer types. PI3K pathway activation in hepato‐biliary carcinomas was analyzed using immunohistochemistry for the downstream targets eIF4‐E and phosphorylated 4E‐BP1 on tissue microarrays. eIF4‐E expression was found in 3/13 intrahepatic CCA (23%), 9/38 extrahepatic CCA (24%), 12/34 gallbladder carcinomas (35%), and 9/61 hepatocellular carcinomas (15%). 4E‐BP1 phosphorylation was observed in 1/13 intrahepatic CCA (8%), 8/38 extrahepatic CCA (21%), 15/34 gallbladder carcinomas (44%), and 16/61 hepatocellular carcinomas (26%). These results indicate that somatic PIK3CA mutations contribute to the frequent activation of the PI3K/AKT pathway in carcinomas of the biliary tract and liver.

Periostin is up-regulated in high grade and high stage prostate cancer

BackgroundExpression of periostin is an indicator of epithelial-mesenchymal transition in cancer but a detailed analysis of periostin expression in prostate cancer has not been conducted so far.MethodsHere, we evaluated periostin expression in prostate cancer cells and peritumoural stroma immunohistochemically in two independent prostate cancer cohorts, including a training cohort (n = 93) and a test cohort (n = 325). Metastatic prostate cancers (n = 20), hormone refractory prostate cancers (n = 19) and benign prostatic tissues (n = 38) were also analyzed.ResultsIn total, strong epithelial periostin expression was detectable in 142 of 418 (34.0%) of prostate carcinomas and in 11 of 38 benign prostate glands (28.9%). Increased periostin expression in carcinoma cells was significantly associated with high Gleason score (p < 0.01) and advanced tumour stage (p < 0.05) in the test cohort. Whereas periostin expression was weak or absent in the stroma around normal prostate glands, strong periostin expression in tumour stroma was found in most primary and metastatic prostate cancers. High stromal periostin expression was associated with higher Gleason scores (p < 0.001). There was a relationship between stromal periostin expression and shortened PSA relapse free survival times in the training cohort (p < 0.05).ConclusionsOur data indicate that periostin up-regulation is related to increased tumour aggressiveness in prostate cancer and might be a promising target for therapeutical interventions in primary and metastatic prostate cancer.

Intraductal papillary neoplasms of the bile duct: stepwise progression to carcinoma involves common molecular pathways

Intraductal papillary neoplasms of the bile duct are still poorly characterized regarding (1) their molecular alterations during the development to invasive carcinomas, (2) their subtype stratification and (3) their biological behavior. We performed a multicenter study that analyzed these issues in a large European cohort. Intraductal papillary neoplasms of the bile duct from 45 patients were graded and subtyped using mucin markers and CDX2. In addition, tumors were analyzed for common oncogenic pathways, and the findings were correlated with subtype and grade. Data were compared with those from 22 extra- and intrahepatic cholangiocarcinomas. Intraductal papillary neoplasms showed a development from preinvasive low- to high-grade intraepithelial neoplasia to invasive carcinoma. Molecular and immunohistochemical analysis revealed mutated KRAS, overexpression of TP53 and loss of p16 in low-grade intraepithelial neoplasia, whereas loss of SMAD4 was found in late phases of tumor development. Alterations of HER2, EGFR, β-catenin and GNAS were rare events. Among the subtypes, pancreato-biliary (36%) and intestinal (29%) were the most common, followed by gastric (18%) and oncocytic (13%) subtypes. Patients with intraductal papillary neoplasm of the bile duct showed a slightly better overall survival than patients with cholangiocarcinoma (hazard ratio (cholangiocarcinoma versus intraductal papillary neoplasm of the bile duct): 1.40; 95% confidence interval: 0.46–4.30; P=0.552). The development of biliary intraductal papillary neoplasms of the bile duct follows an adenoma-carcinoma sequence that correlates with the stepwise activation of common oncogenic pathways. Further large trials are needed to investigate and verify the finding of a better prognosis of intraductal papillary neoplasms compared with conventional cholangiocarcinoma.

IMP3 expression in lesions of the biliary tract: a marker for high-grade dysplasia and an independent prognostic factor in bile duct carcinomas

The oncofetal protein IMP3 (insulin-like growth factor II mRNA binding protein 3) is expressed during embryogenesis and carcinogenesis. Various tumor types have been analyzed for IMP3 expression, which was exclusively found in tumor cells and correlated with increased tumor aggressiveness and reduced overall survival. To our knowledge, IMP3 expression has not been investigated in bile duct carcinomas. Using large tissue sections from resection specimens of the extrahepatic biliary tract, we analyzed IMP3 in normal bile ducts (n = 36), bile ducts with acute inflammation and reactive epithelial changes (n = 26), low-grade dysplasia (n = 9), and high-grade dysplasia (n = 11). Furthermore, IMP3 expression was assessed in bile duct carcinoma (n = 115) using clinically well-characterized tissue microarrays. The findings were correlated with clinical-pathologic parameters including survival. High-grade dysplasia was strongly positive for IMP3 in all cases studied compared with no or weak expression in normal, inflamed, and low-grade dysplastic bile ducts. Of the bile duct carcinomas 58.3% (67/115) were strongly positive for IMP3, which was associated with a higher proliferation rate (P = .004) and p53 positivity (P = .022). Patients with strong IMP3 expression had significantly reduced overall survival (P = .037) similarly to the subgroup of pT3 carcinomas (P = .007). In multivariate analysis, IMP3 expression was an independent prognostic factor for overall survival (P = .040, RR = 1.809). This comprehensive study shows that IMP3 is an independent prognostic biomarker in bile duct carcinoma. In addition, it may be a marker for high-grade dysplasia in the extrahepatic biliary tract.

Frequent expression of the novel cancer testis antigen MAGE-C2/CT-10 in hepatocellular carcinoma

Cancer testis (CT) antigens are attractive targets for immunotherapy in cancer patients. Immunohistochemistry was used to study the expression of the CT antigens MAGE‐C2/CT‐10, MAGE‐C1/CT‐7, GAGE, MAGE‐A4 and NY‐ESO‐1 in 146 hepatocellular carcinomas, 13 intrahepatic cholangiocarcinomas, 37 extrahepatic cholangiocarcinomas and 32 gallbladder carcinomas. Immunopositivity was correlated with clinicopathological parameters, MHC Class 1 expression, intratumoral CD4+, CD8+ and FOXP3+ T cells and CD163+ antigen‐presenting cells. Of the 146 hepatocellular carcinomas, 34% were positive for MAGE‐C2/CT‐10, 12% for MAGE‐C1/CT‐7, 11% for GAGE and 2% for NY‐ESO‐1, respectively. MHC Class 1 coexpression was identified in almost all CT antigen‐positive tumors. The number of intratumoral FOXP3+ regulatory T cells was increased in CT antigen‐positive hepatocellular carcinomas (p < 0.004), suggesting inhibition of immune response in such tumors. Furthermore, MAGE‐C1/CT‐7 and GAGE positivity was correlated with reduced overall survival in patients with hepatocellular carcinoma (p = 0.03 and 0.01, respectively). Four (13%) gallbladder carcinomas stained positive for MAGE‐C2/CT‐10, of which 1 tumor (3%) was also positive for NY‐ESO‐1 and GAGE. CT antigens were not expressed in intra‐ and extrahepatic cholangiocarcinomas. Our results suggest that MAGE‐C2/CT‐10 may be a good candidate for peptide vaccination in patients with hepatocellular carcinoma.

Expression of the extracellular matrix protein periostin in liver tumours and bile duct carcinomas

Riener M‐O, Fritzsche F R, Soll C, Pestalozzi B C, Probst‐Hensch N, Clavien P‐Alain, Jochum W, Soltermann A, Moch H & Kristiansen G
(2010) Histopathology56, 600–606

Cell adhesion molecules P-cadherin and CD24 are markers for carcinoma and dysplasia in the biliary tract ☆

P-cadherin (CDH3) and CD24 are cell adhesion molecules that control morphogenic processes, cell motility, and invasive growth of tumor cells. The aim of our study was to investigate P-cadherin and CD24 expression in carcinomas and dysplastic lesions of the biliary tract and to evaluate the potential diagnostic usefulness of these cell adhesion molecules. Using immunohistochemistry on tissue microarrays, we analyzed P-cadherin, CD24, and p53 expression in 117 carcinomas of the biliary tract (19 intrahepatic cholangiocarcinomas, 59 extrahepatic cholangiocarcinomas, and 39 gallbladder carcinomas) and correlated our findings with clinicopathologic parameters. We found P-cadherin positivity in 37% of intrahepatic cholangiocarcinomas, 73% of extrahepatic cholangiocarcinomas, and 64% of gallbladder carcinomas, respectively. CD24 reactivity was observed in 21% of intrahepatic cholangiocarcinomas, 58% of extrahepatic cholangiocarcinomas, and 42% of gallbladder carcinomas. Nuclear p53 expression was found in 37% of intrahepatic cholangiocarcinomas, 46% of extrahepatic cholangiocarcinomas, and 45% of gallbladder carcinomas. We also studied P-cadherin, CD24, and p53 expression in normal (n = 30), inflamed (n = 22), and dysplastic (n = 21) biliary epithelium of extrahepatic bile ducts. Dysplastic biliary epithelium was positive for P-cadherin in 91%, for CD24 in 71%, and for p53 in 24% of lesions, respectively. In contrast, normal and inflamed epithelia were negative for all 3 proteins. We conclude that P-cadherin and CD24 are expressed in carcinomas of the biliary tract with high frequency and at an early stage of carcinogenesis. Therefore, they may be useful markers for early detection and as targets for therapy of cholangiocarcinoma.

Diagnostic Value of Immunohistochemical IMP3 Expression in Core Needle Biopsies of Pancreatic Ductal Adenocarcinoma

The oncofetal protein, insulin-like growth factor II messenger ribonucleic acid-binding protein 3 (IMP3), has been analyzed in many different tumors. Various studies have found that IMP3 is a marker for malignancy and is correlated with increased tumor aggressiveness and reduced overall survival. The diagnosis of pancreatic ductal adenocarcinoma (PDAC) in core needle biopsies can be challenging, and immunohistochemical markers are needed. We studied IMP3 expression in 177 core needle biopsies of the pancreas, including 112 PDACs, 55 cases with chronic sclerosing pancreatitis, and 10 biopsies with tumor-free pancreatic tissue without inflammation. An additional 18 biopsies of PDAC metastases (16 liver biopsies and 2 lymph node biopsies) were analyzed. To study IMP3 expression in large tissue sections, 45 pancreatic resection specimens (26 with PDAC and 19 with chronic sclerosing pancreatitis) were investigated. In contrast to normal or inflamed pancreatic tissue, which was negative in 47 of 65 (72.3%) cases and weakly positive in 15 of 65 (23.1%) cases, strong IMP3 expression was found in 99 of 112 (88.4%) PDACs. Therefore, sensitivity and specificity of IMP3 expression in the differential diagnosis of PDAC and chronic sclerosing pancreatitis using core needle biopsies were found to be 88.4% and 94.6%, respectively. These results were confirmed in the pancreas resection specimens. Furthermore, strong IMP3 expression was found in 17 of 18 (94.4%) of the PDAC metastases that were analyzed. Our study shows that IMP3 is an easy to use and potentially new immunohistochemical marker for the diagnosis of PDAC in core needle biopsies.