Teresa A. Mettler

Association of MicroRNA Expression in Hepatocellular Carcinomas with Hepatitis Infection, Cirrhosis, and Patient Survival

Purpose: MicroRNA (miRNA) is a new class of small, noncoding RNA. The purpose of this study was to determine if miRNAs are differentially expressed in hepatocellular carcinoma (HCC). Experimental Design: More than 200 precursor and mature miRNAs were profiled by real-time PCR in 43 and 28 pairs of HCC and adjacent benign liver, respectively, and in normal liver specimens. Results: Several miRNAs including miR-199a, miR-21, and miR-301 were differentially expressed in the tumor compared with adjacent benign liver. A large number of mature and precursor miRNAs were up-regulated in the adjacent benign liver specimens that were both cirrhotic and hepatitis-positive compared with the uninfected, noncirrhotic specimens (P < 0.01). Interestingly, all of the miRNAs in this comparison had increased expression and none were decreased. The expression of 95 randomly selected mRNAs was not significantly altered in the cirrhotic and hepatitis-positive specimens, suggesting a preferential increase in the transcription of miRNA. Comparing the miRNA expression in the HCC tumors with patients survival time revealed two groups of patients; those with predominantly lower miRNA expression and poor survival and those with predominantly higher miRNA expression and good survival (P < 0.05). A set of 19 miRNAs significantly correlated with disease outcome. A number of biological processes including cell division, mitosis, and G1-S transition were predicted to be targets of the 19 miRNAs in this group. Conclusion: We show that a global increase in the transcription of miRNA genes occurs in cirrhotic and hepatitis-positive livers and that miRNA expression may prognosticate disease outcome in HCC.

Utility of serum immunoglobulin G4 in distinguishing immunoglobulin G4-associated cholangitis from cholangiocarcinoma.

Elevated serum immunoglobulin G4 (sIgG4) is a feature of autoimmune pancreatitis (AIP) and IgG4‐associated cholangitis (IAC); a >2‐fold increase in sIgG4 is considered highly specific for these disorders. Many patients with IAC present with biliary strictures and obstructive jaundice, making cholangiocarcinoma (CCA) an important differential diagnosis. We determined the value of sIgG4 in distinguishing IAC from CCA. sIgG4 levels were measured in a test cohort of 126 CCA and 50 IAC patients. The results were confirmed in a validation cohort of 161 CCA and 47 IAC patients. Of the 126 CCA patients in the test cohort, 17 (13.5%) had elevated sIgG4 (>140 mg/dL) and four (3.2%) had a >2‐fold (>280 mg/dL) increase. Primary sclerosing cholangitis (PSC) was present in 31/126 CCA patients, of whom seven (22.6%) had elevated sIgG4 and two (6.5%) had a >2‐fold elevation. Of the 50 IAC patients, 39 (78.0%) had elevated sIgG4 and 25 (50.0%) had a >2‐fold increase. The results in the validation cohort were consistent with those of the test cohort. Conclusion: Although elevated sIgG4 levels are characteristic of IAC, some patients with CCA, particularly with PSC, have elevated sIgG4 levels, including a small percentage with a more than a 2‐fold increase in sIgG4. Therefore, sIgG4 elevation alone does not exclude the diagnosis of CCA. Depending on the prevalence of the two diagnoses, the use of a 2‐fold cutoff for sIgG4 may not reliably distinguish IAC from CCA. At a cutoff of 4 times the upper limit of normal, sIgG4 is 100% specific for IAC. (HEPATOLOGY 2011;)

Cirrhosis Is Present in Most Patients With Hepatitis B and Hepatocellular Carcinoma

BACKGROUND & AIMS There are few data available about the prevalence or effects of cirrhosis in patients with hepatocellular carcinoma (HCC) from viral hepatitis. We compared patients with HCC and hepatitis B virus (HBV) or hepatitis C virus (HCV) infections to determine the proportions of cirrhosis in each group, virologic and tumor characteristics, and overall survival. METHODS This analysis included patients with HBV (n = 64) or HCV (n = 118) infection who were diagnosed with HCC at the Mayo Clinic in Rochester, Minnesota from 1994-2008; groups were matched for age and sex. The diagnosis of cirrhosis was based on histology and, if histologic information was insufficient or unavailable, clinical indicators that included ascites or varices, thrombocytopenia or splenomegaly, and radiographic configuration of cirrhosis. Virologic characteristics, tumor stage, and patient survival were also assessed. RESULTS The prevalence of histologic cirrhosis was 88% among patients with HBV infection and 93% among those with HCV infection (P = .46). When the most inclusive criteria for cirrhosis were applied, cirrhosis was present in 94% of patients with HBV and 97% with HCV (P = .24). Among HCV patients, 5.2% were negative for HCV RNA after antiviral treatment; 63.4% of HBV patients had HBV DNA <2000 IU/mL with or without treatment. Patients with HBV tended to have less surveillance and more advanced stages of HCC, without differences in survival from those with HCV infection (P = .75). CONCLUSIONS Most patients with HCC and chronic viral hepatitis had evidence of cirrhosis, including those with HBV infection and those without active viral replication.

Continuation of metformin use after a diagnosis of cirrhosis significantly improves survival of patients with diabetes

The risks and benefits of metformin use in patients with cirrhosis with diabetes are debated. Although data on a protective effect of metformin against liver cancer development have been reported, metformin is frequently discontinued once cirrhosis is diagnosed because of concerns about an increased risk of adverse effects of metformin in patients with liver impairment. This study investigated whether continuation of metformin after cirrhosis diagnosis improves survival of patients with diabetes. Diabetic patients diagnosed with cirrhosis between 2000 and 2010 who were on metformin at the time of cirrhosis diagnosis were identified (n = 250). Data were retrospectively abstracted from the medical record. Survival of patients who continued versus discontinued metformin after cirrhosis diagnosis was compared using the log‐rank test. Hazard ratio (HR) and 95% confidence interval (CI) were calculated using Coxs proportional hazards analysis. Overall, 172 patients continued metformin whereas 78 discontinued metformin. Patients who continued metformin had a significantly longer median survival than those who discontinued metformin (11.8 vs. 5.6 years overall, P < 0.0001; 11.8 vs. 6.0 years for Child A patients, P = 0.006; and 7.7 vs. 3.5 years for Child B/C patients, P = 0.04, respectively). After adjusting for other variables, continuation of metformin remained an independent predictor of better survival, with an HR of 0.43 (95% CI: 0.24‐0.78; P = 0.005). No patients developed metformin‐associated lactic acidosis during follow‐up. Conclusion: Continuation of metformin after cirrhosis diagnosis reduced the risk of death by 57%. Metformin should therefore be continued in diabetic patients with cirrhosis if there is no specific contraindication. (Hepatology 2014;60:2007–2015)

Risk factors for intrahepatic cholangiocarcinoma: Association between metformin use and reduced cancer risk

The associations between diabetes, smoking, obesity, and intrahepatic cholangiocarcinoma (ICC) risk remain inconclusive. Metformin is purportedly associated with a reduced risk for various cancers. This case‐control study evaluated risk factors for ICC and explored the effects of metformin on ICC risk in a clinic/hospital‐based cohort. ICC patients observed at the Mayo Clinic (Rochester, MN) between January 2000 and May 2010 were identified. Age, sex, ethnicity, and residential area‐matched controls were selected from among Mayo Clinic Biobank participants. The associations between potential factors and ICC risk were determined. Six hundred and twelve cases and 594 controls were identified. Factors associated with increased ICC risk included biliary tract diseases (adjusted odds ratio [AOR]: 81.8; 95% confidence interval [CI]: 11.2‐598.8; P < 0.001), cirrhosis (AOR, 8.0; 95% CI: 1.8‐36.5; P = 0.007), diabetes (AOR, 3.6; 95% CI: 2.3‐5.5; P < 0.001), and smoking (AOR, 1.6; 95% CI: 1.3‐2.1; P < 0.001). Compared to diabetic patients not treated with metformin, the odds ratio (OR) for ICC for diabetic patients treated with metformin was significantly decreased (OR, 0.4; 95% CI: 0.2‐0.9; P = 0.04). Obesity and metabolic syndrome were not associated with ICC. Conclusion: This study confirmed diabetes and smoking as independent risk factors for ICC. A novel finding was that treatment with metformin was significantly associated with a 60% reduction in ICC risk in diabetic patients. (HEPATOLOGY 2013)

The JNK inhibitor SP600129 enhances apoptosis of HCC cells induced by the tumor suppressor WWOX.

BACKGROUND/AIMS The FRA16D fragile site gene WWOX is a tumor suppressor that participates in p53-mediated apoptosis. The c-jun N-terminal kinase JNK1 interacts with WWOX and inhibits apoptosis. We investigated the function of WWOX in human hepatocellular carcinoma (HCC) and the effect of JNK inhibition on WWOX-mediated apoptosis. METHODS Allelic imbalance on chromosome 16 was analyzed in 73 HCCs using 53 microsatellite markers. WWOX mRNA in HCC cell lines and primary HCCs was measured by real-time RT-PCR. Effects of WWOX on proliferation and apoptosis and the interaction between WWOX and JNK inhibition were examined. RESULTS Loss on chromosome 16 occurred in 34 of 73 HCCs. Of 11 HCC cell lines, 2 had low, 7 intermediate, and 2 had high WWOX mRNA. Of 51 primary tumors, 23 had low WWOX mRNA. Forced expression of WWOX in SNU387 cells decreased FGF2-mediated proliferation and enhanced apoptosis induced by staurosporine and the JNK inhibitor SP600129. Conversely, knockdown of WWOX in SNU449 cells using shRNA targeting WWOX increased proliferation and resistance to SP600129-induced apoptosis. CONCLUSIONS WWOX induces apoptosis and inhibits human HCC cell growth through a mechanism enhanced by JNK inhibition.

Viral Hepatitis Among Somali Immigrants in Minnesota: Association of Hepatitis C With Hepatocellular Carcinoma

OBJECTIVE To study the frequencies of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, and their associations with hepatocellular carcinoma (HCC) in immigrant Somalis seen at Mayo Clinic in Rochester, Minnesota. PATIENTS AND METHODS We determined the frequencies of HBV and HCV infection and HCC in immigrant Somalis seen at Mayo Clinic from July 1, 1996, through October 31, 2009. Non-Somali Olmsted County residents served as controls. RESULTS For Somali males and females, age-adjusted proportions (per 1000 population) were 209 and 123 for hepatitis B surface antigen (HBsAg), 644 and 541 for hepatitis B core antibody (HBcAb), and 99 and 66 for anti-HCV. The comparative proportions in non-Somalis were 20 and 9 for HBsAg, 126 and 97 for HBcAb, and 32 and 17 for anti-HCV. Hepatitis C virus RNA confirmed that 68 of 73 Somalis (93.2%) and 261 of 282 non-Somalis (92.6%) with positive anti-HCV test results had active HCV infection. Of 30 Somali patients with HCC, 22 (73.3%) tested anti-HCV positive (odds ratio [OR], 31.3; 95% confidence interval [CI], 13.0-75.5; P<.001; compared with anti-HCV-negative Somalis), 5 (16.7%) were HBsAg positive (OR, 1.4; 95% CI, 0.5-3.7; P=.53), and 18 (60.0%) were HBcAb positive (OR, 1.8; 95% CI, 0.8-4.2; P=.16). Viral hepatitis was diagnosed coincident with HCC in 9 of 20 patients (45.0%) with HCV-associated HCCs. Only 4 of 24 cases of HCC (16.7%) were detected during surveillance. CONCLUSION Both HBV and HCV occurred frequently in this sample of Somali immigrants. However, HCV was the major risk factor for HCC. Screening Somali immigrants for HCV infection may enhance the prevention, early detection, and optimal treatment of HCC.

MYH Y165C and G382D mutations in hepatocellular carcinoma and cholangiocarcinoma patients

Purpose: Production of reactive oxygen species (ROS) during chronic inflammation has been implicated in the progression of liver diseases and carcinogenesis. Subjects with inflammatory liver disease and one non-functional allele of the base excision repair gene, MYH, may be more susceptible to progression to cancer due to MYH haploinsufficiency in repairing oxidative damage caused by ROS. Here, we investigated the association of two common germline MYH mutations in patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma. Methods: DNA from patients with HCC (n=48) or cholangiocarcinoma (n=84) compared to non-cancerous controls (n=308) were genotyped for the Y165C and G382D mutations in MYH. Results: There was no significant difference in MYH mutation carrier status between patients with HCC (1/48), cholangiocarcinoma (3/84), and non-cancerous controls (4/308). Conclusions: Patients with HCC or cholangiocarcinoma do not have an increased incidence of monoallelic MYH mutations pre-disposing them to disease.

Liver transplantation after radioembolization in a patient with unresectable HCC

Background. A 58-year-old white man who was being followed by his hepatologist for nonalcoholic steatohepatitis-related liver cirrhosis and portal hypertension and who had been found to have a biopsy-proven hepatocellular carcinoma (HCC) on routine screening, self-referred to our center for a second opinion on the management of his HCC.Investigations. Laboratory investigations, CT scan of the abdomen and chest, bone scan and technetium macroaggregated albumin scan.Diagnosis. The patient had unresectable HCC.Management. The patient underwent two treatments with Yttrium-90 glass microspheres, which were performed as outpatient procedures 1 month and 3 months after diagnosis. He underwent orthotopic liver transplantation (OLT) 1 year after the initial diagnosis of HCC. The post-OLT immunoregimen included OKT3 plus rituximab and high-dose steroids. On discharge from hospital he was on immunosuppressive treatment with tacrolimus. He had de novo autoimmune hepatitis 6 months post-OLT, which was treated with a short course of low-dose steroids and addition of mycophenolate mofetil.

Association between variants in inflammation and cancer‐associated genes and risk and survival of cholangiocarcinoma

Genetic risk factors for cholangiocarcinoma (CCA) remain poorly understood. We assessed the effect of single‐nucleotide polymorphisms (SNPs) of genes modulating inflammation or carcinogenesis on CCA risk and survival. We conducted a case‐control, candidate gene association study of 370 CCA patients and 740 age‐, sex‐, and residential area‐matched healthy controls. Eighteen functional or putatively functional SNPs in nine genes were genotyped. The log‐additive genotype effects of SNPs on CCA risk and survival were determined using logistic regression and the log‐rank test, respectively. Initial analysis identified significant associations between SNP rs2143417 and rs689466 in cyclooxygenase 2 (COX‐2) and CCA risk, after adjusting for multiple comparisons (cutoff of P = 0.0028). However, these findings were not replicated in another independent cohort of 212 CCA cases and 424 matched controls. No significant association was found between any SNP and survival of CCA patients. Although COX‐2 has been shown to contribute to cholangiocarcinogenesis, the COX‐2 SNPs tested were not associated with risk of CCA. This study shows a lack of association between variants of genes related to inflammation and carcinogenesis and CCA risk and survival. Other factors than these genetic variants may play more important roles in CCA risk and survival.