Alison A. Evans

Progress in Chemoprevention Drug Development: The Promise of Molecular Biomarkers for Prevention of Intraepithelial Neoplasia and Cancer—A Plan to Move Forward

This article reviews progress in chemopreventive drug development, especially data and concepts that are new since the 2002 AACR report on treatment and prevention of intraepithelial neoplasia. Molecular biomarker expressions involved in mechanisms of carcinogenesis and genetic progression models of intraepithelial neoplasia are discussed and analyzed for how they can inform mechanism-based, molecularly targeted drug development as well as risk stratification, cohort selection, and end-point selection for clinical trials. We outline the concept of augmenting the risk, mechanistic, and disease data from histopathologic intraepithelial neoplasia assessments with molecular biomarker data. Updates of work in 10 clinical target organ sites include new data on molecular progression, significant completed trials, new agents of interest, and promising directions for future clinical studies. This overview concludes with strategies for accelerating chemopreventive drug development, such as integrating the best science into chemopreventive strategies and regulatory policy, providing incentives for industry to accelerate preventive drugs, fostering multisector cooperation in sharing clinical samples and data, and creating public-private partnerships to foster new regulatory policies and public education.

Past HBV Viral Load as Predictor of Mortality and Morbidity from HCC and Chronic Liver Disease in a Prospective Study

BACKGROUND AND AIMS:In a prospective cohort study with 11 yr of follow-up, we assessed the relationship between past hepatitis B virus (HBV) viral load and mortality. Surviving cohort members were evaluated for current liver disease.METHODS:We measured HBV viral load by real-time polymerase chain reaction on stored samples from cohort entry (1992–1993) in 2,763 hepatitis B surface antigen (HBsAg)-positive adults. Major end points were death from hepatocellular carcinoma (HCC) or chronic liver disease (CLD). There were 447 deaths. In the 1,683 survivors, we assessed severity of liver disease on a return visit in 2003. Viral load was divided into three categories: undetected (<1.6 × 103 copies/mL); low titer (<105 copies/mL); and high titer (≥105 copies/mL).RESULTS:For HCC, there was a significant increase in mortality across viral load categories (ptrend < 0.001). Compared to the HBV undetected category, the relative risk (RR) for HCC mortality in the low viral load group was 1.7 (95% confidence interval [CI] 0.5–5.7) and 11.2 (3.6–35.0) in the high viral load group. For CLD mortality, the RRs were 1.5 (0.2–12.1) and 15.2 (2.1–109.8), respectively (ptrend < 0.001). The RR associated with high viral load did not change with increased follow-up time. In surviving cohort members evaluated for liver disease in 2003, there was also a significant association of viral load with disease severity.CONCLUSION:In this prospective study, viral load is associated with increased mortality from HCC and CLD in HBV-infected subjects. Viral load may be a useful prognostic tool in HBV infection, and interventions aimed at its reduction should be explored.

Gender differences in genetic susceptibility for lung cancer

In contrast to men, the incidence of lung cancer among women has increased over the past decade. The basis for this increase among female smokers remains unknown. Surgical patients with a diagnosis of lung cancer and control subjects without a history of malignancy completed a smoking questionnaire and donated a blood sample. DNA was extracted from peripheral mononuclear cells and genotyped for polymorphisms in cytochrome P450 1A1 (CYP1A1) (exon 7) and glutathione S-transferase M1 (GSTM1) (null). No gender differences in either age at diagnosis or histological subtype were observed among lung cancer patients. In both patients (n = 180) and controls (n = 163), females smoked significantly less than males. The pack-year history associated with adenocarcinoma was smaller than that for squamous cell carcinoma. No significant association was observed between the GSTM1 null genotype and cancer risk. However, women had a larger cancer risk than men (odds ratio 4.98 vs. 1.37) if they possessed the mutant CYP1A1 genotype. Female cancer patients were significantly more likely than female controls to have both the CYP1A1 mutation and GSTM1 null genotype. The combined variant genotypes conferred an odds ratio of 6.54 for lung cancer in women versus 2.36 for men, independent of age or smoking history. These data suggest that polymorphisms in CYP1A1 and GSTM1 contribute to the increased risk of females for lung cancer.

Underestimation of chronic hepatitis B virus infection in the United States of America

Dear Sir, In the December 2006 issue of Journal of Viral Hepatitis, Drs. Gish and Gadano made the important point that the current prevalence estimate of 1.25 million individuals in the USA with chronic hepatitis B virus (HBV) infection should be ‘corrected by taking into account HBV prevalence in immigrant populations and current estimates may be as high as 2 million’ [1]. The Hepatitis B Foundation (HBF) agrees with this statement, that the prevalence of chronic HBV infection in the United States is, in fact, significantly greater than the currently accepted estimate. We have performed our own calculation of the chronic hepatitis B prevalence estimate including the high prevalence rates of hepatitis B in immigrant populations. As a result, we estimate the current burden of chronic hepatitis B in the USA to be approximately 2 million people. We reason that an underestimation of the true number of infected individuals in the USA has occurred mostly because the highest-risk populations are under-represented in surveillance studies, and a large percentage of chronically infected individuals remain undiagnosed. Estimates of the number of HBV infections in the USA have been based on serosurvey data from the National Health and Nutrition Examination Surveys for the years 1976–1994 (NHANES II, III), which did not include significant numbers of Asians and Pacific Islanders (API, the ethnic groups most affected by hepatitis B), making it difficult to obtain accurate prevalence estimates [2]. Also, the large influx of foreign born individuals from countries with moderate or high incidence of hepatitis B increases the limitation of the NHANES estimate. Using Census data (2005 American Community Survey) and current prevalence estimates for chronic hepatitis B, we calculate that in APIs alone, there are approximately 832 433 individuals chronically infected with hepatitis B (Table 1). This number was reached using hepatitis B prevalence estimates of 8.9% for foreign and 1.4% for US born APIs [3], based on HBV survey data of API pregnant women. These estimates are conservative in comparison with recent studies, which have found the prevalence among foreign-born APIs to be between 10–15% [4, 5]. Table 1 Estimate of hepatitis B prevalence in the USA, based on census data and prevalence estimates, by ethnicity We calculated a chronic HBV burden of 1 157 137 individuals in the Caucasian, African American, and ‘other’ ethnicities, with a prevalence estimate of 0.42% in these groups (based on NHANES III data [6]) (Table 1). Adding our estimate for the API population (832 433) brings the total burden estimate to 1 989 570. This represents an increase of almost 800 000 individuals from the currently accepted HBV prevalence estimate, and it does not account for undocumented APIs, which could increase the number of chronically infected by another 100 000 (there are an estimated 1 500 000 undocumented APIs in the USA [7]). This calculation also does not include other high-risk groups, such as the incarcerated and homeless populations, which would increase the burden even further. While these calculations have limitations, they are provocative, in that they highlight the strong likelihood that chronic hepatitis B remains an under-recognized disease in the USA, one that deserves greater priority from public health leaders, research institutions and clinicians. The nation needs to support public health surveillance systems that yield reliable state and local data and can be used to calculate more accurate estimates of chronic hepatitis B. As the readers of JVH are aware, chronic hepatitis B infection leads to serious liver disease and early death in up to 25% of individuals [8], and the direct and indirect financial burden of chronic hepatitis B reaches

Spontaneous Seroconversion in Hepatitis B e Antigen-Positive Chronic Hepatitis B: Implications for Interferon Therapy

1 billion each year [9]. It is a leading cause of primary liver cancer (hepatocellular carcinoma), which is rising in both incidence and mortality in the USA, and now ranks 8th among leading causes of cancer death for Americans [10]. In the API population, liver cancer ranks 3rd among causes of cancer death [11]. With the number of new infections remaining steady despite the availability of a vaccine, and the significant rise of primary liver cancer in the USA, it is imperative that hepatitis B be prioritized as an important public health concern. An accurate assessment of the true prevalence of chronic hepatitis B that includes updated estimates from high-risk, undercounted populations, is a crucial first step towards significantly reducing the burden of chronic hepatitis B in this country. The Hepatitis B Foundation calls for action to be taken to allow all individuals in the USA who are chronically infected with hepatitis B to be counted.

A Proteomic Approach for the Discovery of Early Detection Markers of Hepatocellular Carcinoma

This study compared rates of spontaneous hepatitis B e antigen (HBeAg)-positive to -negative seroconversion in chronic carriers of hepatitis B virus (HBV) with rates reported during interferon-alpha therapy. Four hundred fifty-four Asian-American HBeAg-positive HBV carriers, followed for 1-10 years, were tested approximately every 6 months for HBeAg. Patients with alanine aminotransferase levels > or = 50 IU/mL at entry had 1067.3 seroconversions/10(5) person-months in the 5- to 19-year age group, 1753.3 in the 20- to 34-year group, and 1257.2 in the 35- to 50-year group. Published data indicate that 30% of children and 33% of adults seroconvert during interferon-alpha treatment and follow-up. In our study population, spontaneous seroconversion occurred in 15% of children (95% confidence interval [CI], 8%-27%), 23% of adults 20-34 years (95% CI, 15%-34%), and 17% of adults 35-50 years (95% CI, 10%-28%) during the same interval. The high rate of spontaneous seroconversion should be weighed in decisions to treat HBV carriers with interferon-alpha.

Blood Folate Levels and Risk of Liver Damage and Hepatocellular Carcinoma in a Prospective High-Risk Cohort

Individuals chronically infected with hepatitis B or C virus (HBV, HCV) are at high risk for the development of hepatocellular carcinoma (HCC), with disease progression occurring relentlessly over many years. The diagnosis of HCC usually occurs at late stages in the disease when there are few effective treatment options and the prognosis for patients with HCC is very poor. The long latency period, together with clearly identified at risk populations, provide opportunities for earlier detection that will allow more timely and effective treatment of this devastating cancer. We are using a proteomic approach to test the hypothesis that changes in the amount of certain serum polypeptides, or changes in their post-translational modifications, can be used to predict the onset of HCC. Advances in the standardization of two dimensional gel electrophoresis (2DE) coupled with computerized image analysis now permit the reproducible resolution of thousands of polypeptides per run. Serum polypeptides from individuals at different stages in the disease continuum are being resolved by 2DE to identify those that change with disease progression. Polypeptides found by this method can be further characterized by mass spectrometry. In addition, the potential for changes in the glycan structure of certain polypeptides to serve as a marker for disease progression can be explored. The proteomic approach is expected to liberate us from the need to “cherry pick” or guess the best biomarkers and let the data tell us which are the best indicators of disease. Information may also be gleaned about the pathobiology of the disease process.

Toenail selenium and risk of hepatocellular carcinoma mortality in Haimen City, China

Background: Studies in experimental animals suggest that low folate levels may play a role in liver damage and hepatocarcinogenesis. To examine this association in humans, folate levels in blood and risk for subsequent liver damage and hepatocellular carcinoma (HCC) were assessed in a population at high risk of liver cancer in China. Methods: Four hundred fifteen hepatitis B surface antigen–positive participants of the Haimen City Cohort were prospectively followed between 1998 and 2002. Serum and RBC folate levels were determined at baseline. Alanine aminotransferase (ALT) and hepatitis B virus DNA levels were measured semiannually. Logistic regression modeling was used to examine the presence of hepatitis B virus DNA and HCC, whereas linear regression with a log-link function was used to examine ALT levels. Results: There was a statistically significant inverse association between serum folate level and ALT level. ALT levels decreased with each quartile increase in serum folate (adjusted odds ratio, 0.86; 95% confidence interval, 0.76-0.97 for the highest compared with the lowest quartile; Ptrend = 0.002). After exclusion of three persons with prevalent HCC, 20 (4.9%) of the 412 study participants developed HCC during follow-up, with a median time between enrollment and HCC diagnosis of 2.66 years (interquartile range, 1.8-4.1). When comparing persons in the lowest quartile RBC folate to persons in all other quartiles, the analysis found that higher RBC folate levels were associated with reduced risk of hepatocarcinogenesis (odds ratio, 0.33, 95% confidence interval, 0.13-0.86; Ptrend = 0.02). Conclusions: This study suggests that increased folate levels in humans may be inversely associated with the development of liver damage and HCC. (Cancer Epidemiol Biomarkers Prev 2007;6(6):1279–82)

Methylation of the CpG Sites Only on the Sense Strand of the APC Gene Is Specific for Hepatocellular Carcinoma

Selenium (Se) is an essential trace mineral with known anticarcinogenic properties in humans. However, few studies have examined the association between Se nutrient status and risk of liver cancer. We conducted a nested case‐control study comparing the Se content in toenail clippings of 166 individuals (154 men, 12 women) with hepatocellular carcinoma (HCC) to 394 healthy controls (360 men, 34 women) in Haimen City, China, where HCC is a leading cause of mortality. Toenail Se concentration was measured by inductively coupled plasma‐optical emission spectroscopy. Median toenail Se was lower for HCC cases than controls (p = 0.03). Adjusted odds ratios and 95% confidence intervals for HCC mortality by increasing quartile of toenail Se were 1.00 (reference), 0.58 (0.32–1.03), 0.83 (0.48–1.42) and 0.50 (0.28–0.90), with a marginally significant trend in risk observed (p for trend = 0.06). This inverse association appeared stronger among those who did not consume alcohol and among women. Future studies are needed to examine the interrelationship between Se, viral hepatitis infection and HCC in order to better understand the etiologic mechanisms involved and evaluate the true chemopreventive potential of Se compounds for liver diseases.

Dichlorodiphenyltrichloroethane and risk of hepatocellular carcinoma

Hypermethylation of the promoter of the tumor suppressor gene, adenomatous polyposis coli (APC), occurs in various malignancies, including hepatocellular carcinoma (HCC). However, reports on the specificity of the methylation of the APC gene for HCC have varied. To gain insight into how these variations occur, bisulfite PCR sequencing was performed to analyze the methylation status of both sense and antisense strands of the APC gene in samples of HCC tissue, matched adjacent non-HCC liver tissue, hepatitis, cirrhosis, and normal liver tissues. DNA derived from fetal liver and 12 nonhepatic normal tissue was also examined. These experiments revealed liver-specific, antisense strand-biased CpG methylation of the APC gene and suggested that, although methylation of the antisense strand of the APC gene exists in normal liver and other non-HCC disease liver tissue, methylation of the sense strand of the APC gene occurs predominantly in HCC. To determine the effect of the DNA strand on the specificity of the methylated APC gene as a biomarker for HCC detection, quantitative methylation-specific PCR assays for sense and antisense strand DNA were developed and performed on DNA isolated from HCC (n = 58), matched adjacent non-HCC (n = 58), cirrhosis (n = 41), and hepatitis (n = 39). Receiver operating characteristic curves were constructed. With the cutoff value set at the limit of detection, the specificity of sense and antisense strand methylation was 84% and 43%, respectively, and sensitivity was 67.2% and 72.4%, respectively. This result demonstrated that the identity of the methylated DNA strand impacted the specificity of APC for HCC detection. Interestingly, methylation of the sense strand of APC occurred in 40% of HCCs from patients with serum AFP levels less than 20 ng/mL, suggesting a potential role for APC as a biomarker to complement AFP in HCC screening.