53BP1 expression is a modifier of the prognostic value of lymph node ratio and CA 19-9 in pancreatic adenocarcinoma.

BACKGROUND: 53BP1 binds to the tumor suppressor p53 and has a key role in DNA damage response and repair. Low 53BP1 expression has been associated with decreased survival in breast cancer and has been shown to interact with several prognostic factors in non-small cell lung cancer. The role of 53BP1 in pancreatic ductal adenocarcinoma (PDAC) has yet to be determined. We aimed to investigate whether 53BP1 levels interact with established prognostic factors in PDAC. METHODS: 106 patients for whom there was tissue available at time of surgical resection for PDAC were included. A tissue microarray was constructed using surgical specimens, stained with antibodies to 53BP1, and scored for expression intensity. Univariate and multivariate statistical analyses were performed to investigate the association between 53BP1 and patient survival with known prognostic factors for survival. RESULTS: The association of 53BP1 with several established prognostic factors was examined, including stage, tumor grade, surgical margin, peripancreatic extension, lymph node ratio (LNR), and CA 19-9. We found that 53BP1 modified the effects of known prognostic variables including LNR and CA 19-9 on survival outcomes. When 53BP1 intensity was low, increased LNR was associated with decreased OS (HR 4.84, 95% CI (2.26, 10.37), p<0.001) and high CA19-9 was associated with decreased OS (HR 1.72, 95% CI (1.18, 2.51), p=0.005). When 53BP1 intensity was high, LNR and CA19-9 were no longer associated with OS (p=0.958 and p=0.606, respectively). CONCLUSIONS: In this study, 53BP1, a key player in DNA damage response and repair, was found to modify the prognostic value of two established prognostic factors, LNR and CA 19-9, suggesting 53BP1 may alter tumor behavior and ultimately impact how we interpret the value of other prognostic factors.

Alcohol induces liver neoplasia in a novel alcohol-preferring rat model.

BACKGROUND: Alcohol is a significant risk factor for the development of hepatocellular carcinoma (HCC). To date, no rodent model has demonstrated the formation of hepatic neoplasia in the setting of chronic alcohol consumption alone. METHODS: We investigated whether rats selectively bred for high alcohol preference (P rats), allowed free access to water, or water and 10% (v/v) alcohol, for 6, 12, or 18 months, develop hepatic neoplasia. RESULTS: At necropsy, liver tumor incidence and multiplicity were significantly increased in 18-month alcohol-consuming versus water-consuming P rats. These data were confirmed histologically by glutathione-S-transferase pi-class (GSTp) staining. Phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK) staining was also increased in the sinusoidal lining cells within livers of alcohol-consuming versus water only P rats. In addition, cytochrome p450IIE1 (CYP2E1) mRNA, protein expression/activity, and intrahepatic oxidative stress were significantly increased in alcohol-consuming P rat livers versus water only. In contrast, acetaldehyde dehydrogenase expression decreased in alcohol-consuming versus water only P rats. No significant difference in alcohol dehydrogenase expression was detected. CONCLUSIONS: These data demonstrate that chronic alcohol consumption is associated with hepatic neoplasia, MAPK/ERK activation, increased CYP2E1 activity, and intrahepatic oxidative stress in P rats. As these rats are well characterized as a model of alcoholism, these findings identify a novel rodent model of alcohol or "alcoholism"-induced liver neoplasia.

Resistance to mitogen-activated protein kinase kinase (MEK) inhibitors correlates with up-regulation of the MEK/extracellular signal-regulated kinase pathway in hepatocellular carcinoma cells.

The extracellular signal-regulated (ERK), mitogen-activated protein kinase (p42/p44 MAPK) pathway is up-regulated in hepatocellular carcinoma (HCC). Molecular targeting of this critical mitogenic pathway may have therapeutic potential for the treatment of HCC; however, chemoresistance to long-term therapy may develop. In the present study, we employed small-molecule MAPK kinase (MEK) inhibitors, including U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] and PD184161 (Neoplasia 8:1-8, 2006), in HepG2 and Hep3B human HCC cell lines to identify potential mechanism(s) of resistance. U0126 dose-dependently suppressed ERK phosphorylation at both 1- and 24-h time points in HepG2 cells, previously shown to be sensitive to growth inhibition by U0126. In contrast, ERK phosphorylation was only decreased at the 1-h time point but not at 24 h in the more resistant Hep3B cells. It is interesting that the lack of prolonged phospho-ERK suppression was associated with MEK hyperphosphorylation in Hep3B cells. Several MEK/ERK pathway intermediates were up-regulated in Hep3B cells; furthermore, transfection of Raf-1 small interfering RNA to suppress MEK/ERK pathway activation sensitized Hep3B cells to U0126. MEK inhibitor resistance was independent of p53 or hepatitis Bx protein status. Finally, we showed that combining two chemically distinct MEK inhibitors enhanced growth inhibition and apoptosis compared with the single agents. Taken together, these results suggest that up-regulated expression or activity of the MEK/ERK pathway contributes to MEK inhibitor resistance in HCC cells. Our findings also provide preclinical evidence suggesting that the status of the MEK/ERK pathway in patients may predict response to MEK/ERK-targeted therapeutics.

Sulindac prevents carcinogen-induced intrahepatic cholangiocarcinoma formation in vivo.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) incidence and mortality are increasing in the United States and worldwide. ICC etiologies involve chronic inflammation. We hypothesize that the nonsteroidal anti-inflammatory agent sulindac may prevent ICC by targeting cyclooxygenase-1 and -2 (COX-1, -2) as well as COX-independent pathways. MATERIALS AND METHODS: ICC was induced with the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) in Syrian golden hamsters. Cholangiocarcinogenesis was accelerated by a choline-deficient diet and administration of DL-ethionine and L-methionine. Hamsters were gavaged twice daily for 10 wk with vehicle or sulindac 25, 50, or 75 mg/kg/dose. Harvested livers underwent gross and histopathological examinations. Tissues were analyzed by immunostaining, Western blot, and enzyme-linked immunosorbent assay (ELISA). RESULTS: ICC incidence and multiplicity were decreased in sulindac treatment groups versus control (P < 0.05). In addition, ICC and nontumor lesion sizes decreased in treatment versus control animals. Proliferative indices (Ki-67 immunostaining) decreased and apoptosis (ApopTag immunostaining) increased in treatment versus control (P < 0.05). No changes in COX-1 and -2 protein levels were detected by Western blot. Furthermore, prostaglandin E(2) (PGE(2)) levels were unchanged in treatment and control serum and liver tissues (P > 0.05), suggesting that the antitumor effects of sulindac are mediated by COX-independent mechanisms. Nuclear p65 (activated NF-kappaB) immunostaining decreased (P < 0.05), and protein levels of the NF-kB inhibitor IkappaB-alpha increased in treatment versus control groups. p65 ELISA of liver extracts confirmed decreased NF-kappaB binding activity in sulindac-treated versus control animals (P < 0.05). CONCLUSION: Sulindac effectively prevents experimental cholangiocarcinogenesis, in part by inhibiting the NF-kappaB pathway.

Targeting MEK is effective chemoprevention of hepatocellular carcinoma in TGF-alpha-transgenic mice.

Hepatocellular carcinoma (HCC) causes 600,000 mortalities per year worldwide. Previous studies from our lab provide evidence for altered mitogen-activated protein kinase and extracellular signal-regulated kinase kinase (MEK) signaling in HCC pathogenesis. We hypothesized that pharmacologic targeting of MEK may prevent HCC. Transforming growth factor-alpha-transgenic mice (CD1-MT42) exposed to diethylnitrosamine were randomized to 20 (trial I) or 35 (trial II) weeks of MEK inhibitor PD0325901 (1, 10 mg/kg) or control via orogastric gavage. Ten HCC (44%) formed in trial I controls versus 0 in treatment arms (p<0.05). Fourteen HCC (50%) formed in trial II controls versus 1 (9%) in treatment arms (p<0.05). Mean HCC volume was 578 mm3 in control versus 46 mm3 in the single tumor formed in trial II. In trial I, foci of altered hepatocytes (FAH) formed in 78% of control versus 40% and 0% (1 and 10 mg/kg PD0325901) in treatment arms (p<0.05). In trial II, incidence of FAH was 80% in control versus 20% and 50% (1 and 10 mg/kg PD0325901) in treatment arms (p<0.05). Hepatocyte expression of phosphorylated extracellular signal-regulated kinase dose-dependently decreased in trial I but remained the same in trial II. Control and treated HCC demonstrated similar proliferation rates, but apoptosis appeared increased with treatment. MEK targeting is effective HCC chemoprevention, perhaps by lowering the apoptotic threshold.

Pattern of breast cancer susceptibility gene 1 expression is a potential prognostic biomarker in resectable pancreatic ductal adenocarcinoma.

OBJECTIVES: The tumor-suppressor breast cancer susceptibility gene 1 (BRCA1) is a nuclear-cytoplasmic shuttling protein that when in the nucleus is required for DNA repair whereas when in the cytoplasm is important in activating cell death processes. Although BRCA1 mutations have been shown to be associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC), its role in disease progression is yet to be determined. We hypothesized that BRCA1 expression pattern could be used as a prognostic biomarker. METHODS: Sixty-seven patients who underwent resections for PDAC were included. A tissue microarray was constructed, stained with antibodies to BRCA1, and scored for intensity and subcellular location. Univariate and multivariate statistical analyses were performed. RESULTS: An increase in cytosolic BRCA1 distribution was associated with higher pathologic stage (P = 0.006). Nuclear-cytosolic BRCA1 distribution was associated with a decrease in recurrence-free survival with a hazards ratio of 1.4 (P = 0.059). Decreased BRCA1 intensity was associated with higher pathologic stage (P = 0.027), but BRCA1 intensity was not associated with overall survival or recurrence-free survival. CONCLUSIONS: Our results demonstrate a possible association of BRCA1 expression pattern with pathologic stage, implying a potential role of BRCA1 in PDAC development and progression.

Lymph node ratio and preoperative CA 19-9 levels predict overall survival and recurrence-free survival in patients with resected pancreatic adenocarcinoma.

AIM: Clinicopathologic factors predicting overall survival (OS) would help identify a subset to benefit from adjuvant therapy. METHODS: One hundred and sixty-nine patients patients from 1984 to 2009 with curative resections for pancreatic adenocarcinoma were included. Tumors were staged by American Joint Committee on Cancer 7th edition criteria. Univariate and multivariable analyses were performed using Kaplan-Meier methodology or Cox proportional hazard models. Log-rank tests were performed. Statistical inferences were assessed by two-sided 5% significance level. RESULTS: Median age was 67.1 (57.2-73.0) years with equal gender distribution. Tumors were in the head (89.3%) or body/tail (10.7%). On univariate analysis, adjuvant therapy, lymph node (LN) ratio, histologic grade, negative margin status, absence of peripancreatic extension, and T stage were associated with improved OS. Adjuvant therapy, LN ratio, histologic grade, number of nodes examined, negative LN status, and absence of peripancreatic extension were associated with improved recurrence-free survival (RFS). On multivariable analysis, LN ratio and carbohydrate antigen (CA) 19-9 levels were associated with OS. LN ratio was associated with RFS. CONCLUSION: The LN ratio and CA 19-9 levels are independent prognostic factors following curative resections of pancreatic cancer.

Neuroendocrine and squamous colonic composite carcinoma: case report with molecular analysis.

Composite colorectal carcinomas are rare. There are a modest number of cases in the medical literature, with even fewer cases describing composite carcinoma with neuroendocrine and squamous components. There are to our knowledge no reports of composite carcinoma molecular alterations. We present a case of composite carcinoma of the splenic flexure in a 33 year-old Caucasian male to investigate the presence and prognostic significance of molecular alterations in rare colonic carcinoma subtypes. Formalin-fixed paraffin-embedded (FFPE) tissue was hematoxylin and eosin- and mucicarmine-stained according to protocol, and immuno-stained with cytokeratin (CK)7, CK20, CDX2, AE1/AE3, chromogranin-A and synaptophysin. DNA was extracted from FFPE tissues and molecular analyses were performed according to lab-developed methods, followed by capillary electrophoresis. Hematoxylin and eosin staining showed admixed neuroendocrine and keratinized squamous cells. Positive nuclear CDX2 expression confirmed intestinal derivation. CK7 and CK20 were negative. Neuroendocrine cells stained positively for synaptophysin and AE1/AE3 and negatively for chromogranin and mucicarmine. Hepatic metastases showed a similar immunohistochemical profile. Molecular analysis revealed a G13D KRAS mutation. BRAF mutational testing was negative and microsatellite instability was not detected. The patient had rapid disease progression on chemotherapy and died 60 d after presentation. Although the G13D KRAS mutation normally predicts an intermediate outcome, the aggressive tumor behavior suggests other modifying factors in rare types of colonic carcinomas.

Intestinal malrotation in an extremely preterm very low birthweight infant.

A case of a very low birthweight premature infant with a clinical presentation of necrotising enterocolitis that was found to have malrotation and midgut volvulus at autopsy is presented.