C. K. Leow

High frequency of P16INK4A gene alterations in hepatocellular carcinoma

The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. In order to investigate the role of p16 gene in the tumorigenesis of hepatocellular carcinoma (HCC), 48 cases of HCC were analysed for p16 alterations by: methylation-specific PCR (MSP) to determine the methylation status of the p16 promoter region; comparative multiplex PCR to detect homozygous deletion; PCR – SSCP and DNA sequencing analysis to identify mutation of the p16 gene. We found high frequency of hypermethylation of the 5′ CpG island of the p16 gene in 30 of 48 cases (62.5%) of HCC tumors. Moreover, homozygous deletion at p16 region were present in five of 48 cases (10.4%); and missense mutation were detected in three of 48 cases (6.3%). The overall frequency of p16 alterations, including homozygous deletion, mutation and hypermethylation, in HCC tumors was 70.8% (34 of 48 cases). These findings suggest that: (a) the inactivation of the p16 is a frequent event in HCC; (b) the p16 gene is inactivated by multiple mechanisms including homozygous deletion, promoter hypermethylation and point mutation; (c) the most common somatic alteration of the p16 gene in HCC is de novo hypermethylation of the 5′ CpG island; and (d) in contrast to other studies, high frequency of genomic alterations are not uncommon in the 9p21 of the p16 gene. Our results strongly suggest that the p16 gene plays an important role in the pathogenesis of HCC.

Cholecystectomy or Gallbladder In Situ After Endoscopic Sphincterotomy and Bile Duct Stone Removal in Chinese Patients

Background & Aims: In patients with stones in their bile ducts and gallbladders, cholecystectomy is generally recommended after endoscopic sphincterotomy and clearance of bile duct stones. However, only approximately 10% of patients with gallbladders left in situ will return with further biliary complications. Expectant management is alternately advocated. In this study, we compared the treatment strategies of laparoscopic cholecystectomy and gallbladders left in situ. Methods: We randomized patients (>60 years of age) after endoscopic sphincterotomy and clearance of their bile duct stones to receive early laparoscopic cholecystectomy or expectant management. The primary outcome was further biliary complications. Other outcome measures included adverse events after cholecystectomy and late deaths from all causes. Results: One hundred seventy-eight patients entered into the trial (89 in each group); 82 of 89 patients who were randomized to receive laparoscopic cholecystectomy underwent the procedure. Conversion to open surgery was needed in 16 of 82 patients (20%). Postoperative complications occurred in 8 patients (9%). Analysis was by intention to treat. With a median follow-up of approximately 5 years, 6 patients (7%) in the cholecystectomy group returned with further biliary events (cholangitis, n = 5; biliary pain, n = 1). Among those with gallbladders in situ, 21 (24%) returned with further biliary events (cholangitis, n = 13; acute cholecystitis, n = 5; biliary pain, n = 2; and jaundice, n = 1; log rank, P = .001). Late deaths were similar between groups (cholecystectomy, n = 19; gallbladder in situ, n = 11; P = .12). Conclusions: In the Chinese, cholecystectomy after endoscopic treatment of bile duct stones reduces recurrent biliary events and should be recommended.

Hepatocellular carcinoma: From bedside to proteomics

Hepatocellular carcinoma (HCC or hepatoma) is the most common primary cancer of the liver. It is responsible for approximately one million deaths each year, mainly in underdeveloped and developing countries. The aetiological factors identified in the development of HCC included persistent infection by hepatitis B and hepatitis C viruses, and exposure to aflatoxins. Although immunization can protect individuals from being infected by the hepatitis B virus, the early detection of HCC in those who have been infected by the virus remains a challenge. Thus most HCCs present late and are not suitable for curative treatment. Hence there is a tremendous interest and urgency to identify novel HCC diagnostic marker(s) for early detection, and tumour specific disease associated proteins as potential therapeutic targets in the treatment of HCC. Screening for these HCC proteins has been facilitated by proteomics, a key technology in the global analysis of protein expression and understanding gene function. Present and earlier proteome analyses of HCC have used predominantly experimental in vitro systems. The protein expression profiles of several hepatoma cell lines such as HepG2, Huh7, SK‐Hep1, and Hep3B have been compared with normal liver, and nontransformed cell lines (Chang and WRL‐68), while a comprehensive proteome analysis to create a protein database was carried out for the cell line HCC‐M. In the future, proteome analyses utilizing tumour tissues, which reflect the pathological state of HCC more closely, will be undertaken. This work will complement the gene expression studies of HCC which are already underway. Efforts have also been directed at the proteome analysis of hepatic stellate cells, as these cells play an important role in liver fibrosis. Since liver fibrosis is reversible but not cirrhosis, it is of considerable importance to identify therapeutic targets that can slow its progression.

Identification of discriminators of hepatoma by gene expression profiling using a minimal dataset approach

The severity of hepatocellular carcinoma (HCC) and the lack of good diagnostic markers and treatment strategies have rendered the disease a major challenge. Previous microarray analyses of HCC were restricted to the selected tissue sample sets without validation on an independent series of tissue samples. We describe an approach to the identification of a composite discriminator cassette by intersecting different microarray datasets. We studied the global transcriptional profiles of matched HCC tumor and nontumor liver samples from 37 patients using cDNA (cDNA) microarrays. Application of nonparametric Wilcoxon statistical analyses (P < 1 × 10−6) and the criteria of 1.5‐fold differential gene expression change resulted in the identification of 218 genes, including BMI‐1, ERBB3, and those involved in the ubiquitin‐proteasome pathway. Elevated ERBB2 and epidermal growth factor receptor (EGFR) expression levels were detected in ERBB3‐expressing tumors, suggesting the presence of ERBB3 cognate partners. Comparison of our dataset with an earlier study of approximately 150 tissue sets identified multiple overlapping discriminator markers, suggesting good concordance of data despite differences in patient populations and technology platforms. These overlapping discriminator markers could distinguish HCC tumor from nontumor liver samples with reasonable precision and the features were unlikely to appear by chance, as measured by Monte Carlo simulations. More significantly, validation of the discriminator cassettes on an independent set of 58 liver biopsy specimens yielded greater than 93% prediction accuracy. In conclusion, these data indicate the robustness of expression profiling in marker discovery using limited patient tissue specimens as well as identify novel genes that are highly likely to be excellent markers for HCC diagnosis and treatment. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;39:944–953.)

Liver resection after irinotecan, 5-fluorouracil, and folinic acid for patients with unresectable colorectal liver metastases : a multicenter phase II study by the Cancer Therapeutic Research Group

The main objectives of this study were to assess the use of irinotecan, 5-fluorouracil (5-FU), and leucovorin (FA) as neoadjuvant chemotherapy for patients with unresectable colorectal liver metastases and to determine the response rate and proportion of patients that could be downstaged to resectable tumors. Forty patients were treated with irinotecan (180 mg/m2 over 30 min) on d 1, FA (200 mg/m2 over 30 min) followed by 5-FU (400 mg/m2 bolus and continuous infusion of 600 mg/m2 over 22 h) on d 1 and 2 every 2 wk. The overall response rate was 55% (95% CI: 39.5–70.4%). The progression-free survival was 12.1 mo (95% CI: 11.4–14.8 mo). The median overall survival was 20 mo (95% CI: 17.7–26.6 mo). Four patients (10%) have undergone liver resection after a median of eight cycles. Those patients remained alive with a median follow up period of 33 mo. The principal grade 3–4 toxicity was neutropenia in 20 patients (50%). We conclude that the regimen of irinotecan/5-FU/FA was highly active in patients with colorectal cancer and liver metastases with limited toxicity. In a subgroup of patients with initial inoperable liver metastases, this regimen was able to downstage the disease to an operable stage.

Frequent allelic loss on chromosome 9 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and highly associated with chronic virus‐B or ‐C infection and cirrhosis. Molecular studies have shown high frequency of loss of heterozygosity (LOH) in some specific chromosome regions, but LOH on chromosome 9 in HCC has not been thoroughly investigated. In our investigation of chromosome 9 with 19 polymerase‐chain‐reaction (PCR)‐based polymorphic microsatellite markers, 30 of 48 HCC tissue samples (63%) had LOH, and a distinct common deletion region and a region of loss were identified. The first region was located at the 9p21 region and the minimal deletion region was located between loci D9S1747 and D9S1748. This is a region of approximately 200 kb which includes the p16 tumor‐suppressor gene. A region of loss was located on 9p13 to 9q33. The putative tumor‐suppressor gene for nevoid‐basal‐cell‐carcinoma syndrome (NBCCS) at 9q22.3 resides within this region. In addition to LOH, 4 HCC cases showed possible homozygous deletions at 9p21 with markers D9S1748, D9S1752 and D9S171 by multiplex PCR analysis. In 3 cases, the minimal region of possible homozygous deletion was approximately 300 kb and was defined between markers D9S1747 and D9S1752. Since this deletion region includes both the p15 and the p16 tumor‐suppressor genes, these genes were possibly inactivated by homozygous deletion in HCC. In addition, a second region of possible homozygous deletion was present on the centromeric side of 9p21. However, these changes are not associated with age, gender, size or tumor‐cell differentiation. Our data also suggest that inactivation of the p16 and the p15 genes and the possibility of other unknown tumor‐suppressor genes located on these defined deleted regions of chromosome 9 may be involved in the pathogenesis of HCC. Int. J. Cancer 81:319–324, 1999.

Liver fibrogenesis: effects of transforming growth factor β and tamoxifen, and possible treatment modalities

Background Liver fibrosis and subsequent cirrhosis continues to be a major health problem. Recent understanding of the pathophysiology of the process will, hopefully, give an insight into the possible treatment of the condition. Methods We have reviewed the research in the area of liver fibrosis and the roles of transforming growth factor β (TGFβ) from the literature. The relationship between liver fibrosis and tamoxifen was also reviewed. Results and discussion The pathophysiology of liver fibrogenesis is reviewed. TGFβ is the key-player in the initiation and perpetuation of liver fibrogenesis. Tamoxifen, on the other hand, enhances the TGFβ-mediated process of fibrogenesis. Possible therapeutic interventions, based on the pathophysiological pathways of liver fibrosis, are discussed.

Evacuation of the Pleural Cavity with an Infant Feeding Catheter Following en Bloc Resection of Hepatocellular Carcinoma and Involved Diaphragm-an Institutional Experience

En bloc resection of hepatocellular carcinoma and the involved diaphragm will, towards the end of operation, require evacuation of the pleural cavity, usually with a chest drain. We describe our method and experience of evacuating the pleural cavity, at the time of diaphragmatic repair, with an infant feeding catheter without the need of a chest drain. We have found the method safe and efficacious.