Abby B. Siegel

Epigenome-wide studies in hepatocellular carcinoma (HCC) have identified numerous genes with aberrant DNA methylation. However, methods for triaging functional candidate genes as useful biomarkers for epidemiological study have not yet been developed. We conducted targeted next-generation bisulfite sequencing (bis-seq) to investigate associations of DNA methylation and mRNA expression in HCC. Integrative analyses of epigenetic profiles with DNA copy number analysis were used to pinpoint functional genes regulated mainly by altered DNA methylation. Significant differences between HCC tumor and adjacent non-tumor tissue were observed for 28 bis-seq amplicons, with methylation differences varying from 12% to 43%. Available mRNA expression data in Oncomine were evaluated. Two candidate genes (GRASP and TSPYL5) were significantly under-expressed in HCC tumors in comparison with precursor and normal liver tissues. The expression levels in tumor tissues were, respectively, 1.828 and − 0.148, significantly lower than those in both precursor and normal liver tissue. Validations in an additional 42 paired tissues showed consistent under-expression in tumor tissue for GRASP (−7.49) and TSPYL5 (−9.71). A highly consistent DNA hypermethylation and mRNA repression pattern was obtained for both GRASP (69%) and TSPYL5 (73%), suggesting that their biological function is regulated by DNA methylation. Another two genes (RGS17 and NR2E1) at Chr6q showed significantly decreased DNA methylation in tumors with loss of DNA copy number compared to those without, suggesting alternative roles of DNA copy number losses and hypermethylation in the regulation of RGS17 and NR2E1. These results suggest that integrative analyses of epigenomic and genomic data provide an efficient way to filter functional biomarkers for future epidemiological studies in human cancers.

Liver transplantation offers excellent long-term survival for hepatocellular carcinoma (HCC) patients who fall within established criteria. For those outside such criteria, or with high-risk pathologic features in the explant, HCC recurrence rates are higher. We conducted a multicenter phase I trial of sorafenib in liver transplantation patients with high-risk HCC. Subjects had HCC outside the Milan criteria (pre- or post-transplant), poorly differentiated tumors, or vascular invasion. We used a standard 3+3 phase I design with a planned duration of treatment of 24 weeks. Correlative studies included the number of circulating endothelial cells (CECs), plasma biomarkers, and tumor expression of p-Erk, p-Akt, and c-Met in tissue micro-arrays. We enrolled 14 patients with a median age of 63 years. Of these, 93% were men and 71% had underlying hepatitis C virus (HCV) and 21% had HBV. The maximum tolerated dose of sorafenib was 200 mg BID. Grade 3-4 toxicities seen in >10% of subjects included leukopenia (21%), elevated gamma-glutamyl transferase (21%), hypertension (14%), hand-foot syndrome (14%) and diarrhea (14%). Over a median follow-up of 953 days, one patient died and four recurred. The mean CEC number at baseline was 21 cells/4 ml for those who recurred, and 80 cells/4 ml for those who did not (p=0.10). Mean soluble vascular endothelial growth factor receptor-2 levels decreased after 1 month on sorafenib (p=0.09), but did not correlate with recurrence. There was a trend for tumor c-Met expression to correlate with increased risk of recurrence. Post-transplant sorafenib was found to be feasible and tolerable at 200 mg PO BID. The effect of post-transplant sorafenib on recurrence-free survival is potentially promising but needs further validation in a larger study.

OBJECTIVES:Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. CA19-9 is a glycoprotein that predicts poor prognosis in pancreatic and biliary malignancies. We evaluated it as a prognostic biomarker for patients with HCC.METHODS:We prospectively enrolled 145

Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. The rise in metabolic syndrome has contributed to this trend. Adipokines, such as adiponectin, are associated with prognosis in several cancers, but have not been well studied in HCC. Methods: We prospectively enrolled 140 patients with newly diagnosed or recurrent HCC with Child-Pugh (CP) class A or B cirrhosis. We examined associations between serum adipokines, clinicopathological features of HCC, and time to death. We also examined a subset of tumors with available pathology for tissue adiponectin receptor (AR) expression by immunohistochemistry. Results: The median age of subjects was 62 years; 79% were men, 59% had underlying hepatitis C, and 36% were diabetic. Adiponectin remained a significant predictor of time to death (hazard ratio 1.90; 95% confidence interval 1.05-3.45; p = 0.03) in a multivariable adjusted model that included age, alcohol history, CP class, stage, and serum E-fetoprotein level. Cytoplasmic AR expression (AR1 and AR2) in tumors trended higher in those with higher serum adiponectin levels and in those with diabetes mellitus, but the association was not statistically significant. Conclusions: In this hypothesis-generating study, we found the serum adiponectin level to be an independent predictor of overall survival in a diverse cohort of HCC patients. Impact: Understanding how adipokines affect the HCC outcome may help develop novel treatment and prevention strategies. i 2014 S. Karger AG, Basel

Background. More than 50% of US adults, and an even larger proportion of cancer patients, use dietary supplements. Since many supplements require hepatic metabolism, they may be particularly likely to cause toxicities in patients with hepatocellular carcinoma (HCC). However, little is known about supplement use in patients with HCC. Methods. From 2008 to 2012, we gave newly diagnosed HCC patients at our institution a standardized questionnaire about dietary supplement use, demographic factors, and clinical characteristics. We then followed patients for four years or until time to death to examine the relationship with supplement use. Results. Of 146 patients, 71% had used vitamins and 45% herbal supplements. Most commonly used supplements were antioxidants (51%), multivitamins (46%), vitamin D (25%), and milk thistle (23%). People in mid-higher income brackets were more likely to use herbal supplements (19% of those earning <$30 000, 50% of those earning $30 000-60 000, and 34% of those earning >$60 000 used supplements). Hepatitis C (HCV) patients were more likely to use milk thistle than those without HCV (30% vs 13%, P = .03), and patients with hepatitis B (HBV) were more likely than non-HBV patients to use vitamin C (32% vs 14%, P = .01). Supplement use was not associated with overall survival. Conclusions. Like cancer patients in other studies, the majority of our HCC patients used dietary supplements. Supplement use was not associated with overall survival but should be studied in larger patient samples.

Background: Chronic inflammation has been associated with colorectal cancer. Prediagnostic levels of C-reactive protein (CRP), a highly sensitive marker of inflammation, have been weakly associated with increased colorectal cancer incidence, but few data are available examining its relationship with colorectal cancer mortality. Methods: In the Third National Health and Nutrition Examination Survey (NHANES III), 65% of the 15,924 adult participants had CRP levels ≤0.21 mg/dL. Using this as the reference group, we calculated hazard ratios (HR) for higher CRP categories and colorectal cancer mortality, and compared them with HRs for other mortality causes. Results: Over a median follow-up period of 14.2 years, there were 92 deaths from colorectal cancer. Compared with the reference group, multivariable adjusted HRs for colorectal cancer mortality were 2.66 [95% confidence interval (CI), 1.36–5.20] for CRP levels 0.22–0.50 mg/dL; 3.40 (95% CI, 1.48–7.77) for levels 0.51–1.00 mg/dL; and 3.96 (95% CI, 1.64–9.52) for levels >1.00 mg/dL. Estimates for colorectal cancer mortality did not change appreciably after excluding deaths within the first 3 years or by limiting follow-up to 5 or 10 years. Conclusions: In a large representative study of U.S. adults, we observed strong dose–response associations between CRP levels and colorectal cancer mortality. Impact: Further evaluation of CRP may help identify high-risk groups for colorectal cancer screening and those who might benefit most from prophylactic anti-inflammatory therapy. Cancer Epidemiol Biomarkers Prev; 23(8); 1609–18. ©2014 AACR.

Background: Observational studies have suggested that antioxidant nutrients may reduce cancer and overall mortality risks. However, most randomized trials have failed to show survival benefits. Examining nonlinear associations between antioxidant levels and health outcomes may help to explain these discrepant findings. Methods: We evaluated all-cause, cancer, and cardiovascular mortality risks associated with quintiles (Q1–Q5) of serum antioxidant (vitamins C and E, β-carotene, and selenium) and vitamin A levels, in 16,008 adult participants of The Third National Health and Nutrition Examination Survey (NHANES III; 1988–1994). Results: Over a median follow-up period of 14.2 years, there were 4,225 deaths, including 891 from cancer and 1,891 from cardiovascular disease. We observed a dose–response decrease in cancer and overall mortality risks with higher vitamin C levels. In contrast, for vitamin A, risk of cancer death decreased from Q1–Q2, with no further decline in risk at higher levels. For vitamin E, having levels in Q4 was associated with the lowest cancer mortality risk. Both vitamin A and E had U-shaped associations with all-cause mortality. Cancer mortality risks decreased from Q1–Q2 for β-carotene and from Q1–Q4 for selenium. However, for β-carotene and selenium, overall mortality risks decreased from Q1–Q2 but then did not change significantly with higher levels. Conclusions: Antioxidant supplement use should be studied in the context of overall mortality and other competing mortality risks. Impact: These data suggest the need for novel intervention studies where doses of these agents are individualized based on their serum levels, and possibly, markers of oxidative stress and systemic inflammatory response. Cancer Epidemiol Biomarkers Prev; 22(12); 2202–11. ©2013 AACR.

Purpose. To determine the maximum tolerated dose per day of silybin phosphatidylcholine (Siliphos) in patients with advanced hepatocellular carcinoma (HCC) and hepatic dysfunction. Experimental Design. Patients with advanced HCC not eligible for other therapies based on poor hepatic function were enrolled in a phase I study of silybin phosphatidylcholine. A standard phase I design was used with 4 planned cohorts, dose escalating from 2, 4, 8, to 12 g per day in divided doses for 12 weeks. Results. Three participants enrolled in this single institution trial. All enrolled subjects consumed 2 g per day of study agent in divided doses. Serum concentrations of silibinin and silibinin glucuronide increased within 1 to 3 weeks. In all 3 patients, liver function abnormalities and tumor marker α-fetoprotein progressed, but after day 56 the third patient showed some improvement in liver function abnormalities and inflammatory biomarkers. All 3 participants died within 23 to 69 days of enrolling into the trial, likely from hepatic failure, but it could not be ruled out that deaths were possibly due to the study drug. Conclusion. Short-term administration of silybin phosphatidylcholine in patients with advanced HCC resulted in detectable increases in silibinin and its metabolite, silibinin glucuronide. The maximum tolerated dose could not be established. Since patients died soon after enrollment, this patient population may have been too ill to benefit from an intervention designed to improve liver function tests.

For many cancers, features of the metabolic syndrome, such as diabetes and obesity, have been associated with both increased risk of cancer development and poor outcomes. Methods We examined a large retrospective cohort of 342 consecutive patients who underwent liver transplantation for hepatocellular carcinoma between January 1999 and July 2010 at our institution. We evaluated the relationship between diabetes, obesity, hepatocellular carcinoma (HCC) recurrence, and overall survival. Results We found that a body mass index (BMI) higher than 30 was an independent predictor of poor overall survival in a multivariable Cox model, approximately doubling the risk of death after transplantation. A BMI higher than 30 was also a predictor of recurrent HCC, although this was of borderline statistical significance (hazard ratio for recurrence, 1.9; 95% confidence interval, 0.9–4.1). We also found increased BMI to be an independent predictor of microvascular invasion within HCC tumors, lending a possible explanation to these results. Those with diabetes showed worsened overall survival compared with those without diabetes in univariate but not multivariable analysis, possibly related to longer wait times. Conclusions Our findings suggest a relationship between higher BMI, tumor vascular invasion, increased recurrence, and worsened overall survival. These findings may help explain why those with high BMI have worse outcomes from their cancers. A better understanding of the role of obesity and diabetes in patients with cancer should help develop better predictors of outcome and improved treatment options for patients with HCC.