Mikael E. Craanen

Selective gene delivery toward gastric and esophageal adenocarcinoma cells via EpCAM-targeted adenoviral vectors

Application of recombinant adenoviral vectors for cancer gene therapy is currently limited due to lack of specificity for tumor cells. For gastric and esophageal adenocarcinoma, we present here that the relative abundant expression of the primary adenovirus receptor, coxsackie/adenovirus receptor (CAR), on normal epithelium compared to carcinoma favors the transduction of the epithelium. As such, to achieve specific transduction of cancer cells, targeting approaches are required that ablate the binding of the virus to CAR and redirect the virus to tumor-specific receptors. By immunohistochemistry and reverse transcriptase polymerase chain reaction assays, we demonstrate a marked difference in expression of the human epithelial cell adhesion molecule (EpCAM) between normal and (pre)malignant lesions of the stomach and esophagus. Based on this, we explored the feasibility of using EpCAM to achieve gastric and esophageal adenocarcinoma selective gene transfer. Adenoviral vectors redirected to EpCAM using bispecific antibodies against the adenovirus fiber-knob protein and EpCAM specifically infected gastric and esophageal cancer cell lines. Using primary human cells, an improved ratio of tumor transduction over normal epithelium transduction was accomplished by the EpCAM-targeted vectors. This study thus indicates that EpCAM-targeted adenoviral vectors may be useful for gastric and esophageal cancer–specific gene therapy in patients. Cancer Gene Therapy (2001) 8, 342–351

Epstein-Barr virus in gastric carcinomas and gastric stump carcinomas: a late event in gastric carcinogenesis

Background: To determine at what stage during gastric carcinogenesis Epstein-Barr virus (EBV) enters the gastric epithelial cells, the presence of EBV was investigated in two pathogenetically related but distinct forms of adenocarcinoma of the stomach—gastric carcinoma of the intact stomach (GCIS) and gastric stump carcinoma (GSC)—and their presumed precursor lesions. Patients and methods: Eleven patients with EBV positive GCIS and eight patients with EBV positive GSC, demonstrated by the highly sensitive EBV encoded RNA 1/2 (EBER1/2) RNA in situ hybridisation (RISH) technique, were studied. Paraffin wax embedded tissue available from preoperative gastric biopsies and tumour adjacent tissue from the resection specimens containing normal gastric mucosa, inflamed gastric mucosa, and preneoplastic lesions (intestinal metaplasia and dysplasia) was investigated by EBER1/2 RISH, in addition to EBV nuclear antigen 1 (EBNA-1) and latent membrane protein 1 (LMP-1) immunohistochemistry (IHC). Results: In both GCIS and GSC and their precursor lesions EBER1/2 transcripts were restricted to the carcinoma cells. In addition, positivity of EBNA-1 IHC was also restricted to the tumour cells. IHC for LMP-1 was negative in all cases tested. Conclusions: The absence of EBER1/2 transcripts in preneoplastic gastric lesions (intestinal metaplasia and dysplasia) and their presence in two distinct types of gastric carcinoma strongly suggest that EBV can only infect neoplastic gastric cells and thus is a late event in gastric carcinogenesis.

Loss of E-cadherin expression in early gastric cancer.

Reduction or loss of E‐cadherin expression was examined in early gastric carcinomas and precursor lesions with the following aims: (1) to assess overall E‐cadherin expression in various stages of gastric carcinogenesis; (2) to correlate E‐cadherin expression with the Lauren type, the grade of differentiation and the type of growth pattern of the tumours; and (3) to correlate E‐cadherin expression with lymph node metastasis and overall prognosis.

Helicobacter pylori and Epstein-Barr virus infection and the p53 tumour suppressor pathway in gastric stump cancer compared with carcinoma in the non-operated stomach

AIM: To evaluate similarities and differences between gastric stump cancer and conventional carcinoma in the non-operated stomach. METHODS: 26 stump carcinomas were compared with 24 conventional stomach cancers. Stage, histological type, and demographics were comparable in the two groups. Expression of p53 and p21-Waf1/Cip1 was evaluated by immunohistochemical staining. Helicobacter pylori infection was evaluated by examining haematoxylin-eosin stained slides and immunohistochemistry. Epstein-Barr virus infection was evaluated by RNA in situ hybridisation. RESULTS: Expression of p53 and p21-Waf1/Cip1 was similar in both groups and positive in more than half of the patients. H pylori infection was observed in six stump carcinomas and 17 conventional carcinomas in the intact stomach (p < 0.01). RNA in situ hybridisation (EBER1-ISH) for Epstein-Barr virus was positive in nine stump carcinomas and two carcinomas in the non-operated stomach (p < 0.05). CONCLUSIONS: There appear to be aetiological differences between stump carcinoma and cancer in the intact stomach. Further study of these differences may improve our understanding of gastric carcinogenesis in general.

Helicobacter pylori-related and -non-related gastric cancers do not differ with respect to chromosomal aberrations.

Gastric carcinogenesis is strongly associated with Helicobacter pylori infection, but the underlying genetic mechanisms are largely unknown. The aim of this study was to correlate chromosomal aberrations in gastric cancer to H. pylori status and its different strains, as well as to histological type and other clinico‐pathological variables. DNA from 46 gastric cancers (male/female 35/11, age 27–85 years) was extracted from formaldehyde‐fixed, paraffin‐embedded material and tested for chromosomal gains and losses by comparative genomic hybridization (CGH). Chromosomal aberrations with frequencies of 20% or higher were considered to be non‐random changes associated with gastric cancer. The mean number of chromosomal events per tumour was 9.7 (range 0–27), with a mean of 3.2 gains (range 0–16) and 6.5 losses (range 0–15). Gains were most frequently found at chromosomes 8q and 13q (24% and 26%, respectively). Losses were predominantly found on chromosome arms 2q, 9p, 12q, 14q, 15q, 16p, 16q, 17p, 17q, 19p, 19q, and 22q (22%, 30%, 43%, 22%, 33%, 50%, 28%, 50%, 39%, 33%, 39%, and 37%, respectively). Common regions of overlap narrowed down to 2q11–14, 8q23, 9p21, 12q24, 13q21–22, 14q24 and 15q11–15. The mean number of gains was higher in tumours with metastases than in localized tumours (4.1 vs. 1.9, p=0.04). Tumours with a loss at 17p showed a higher number of losses than tumours without a 17p loss (9.5 vs. 4.7 on average, p<0.001). Neither H. pylori status (+, n=25; −, n=21) nor H. pylori strain was correlated to the total number of events or to any specific chromosomal aberration, nor were there differences between intestinal (n=30) and diffuse (n=15) cancers or any other clinico‐pathological variable tested. In conclusion, a complex of chromosomal aberrations is involved in gastric cancer, but their pattern does not depend on H. pylori status or strain, nor on the histological type of the tumour. The exact biological meaning of these aberrations in carcinogenesis needs further clarification. Copyright

Inhibition of angiogenesis and HGF-cMET-elicited malignant processes in human hepatocellular carcinoma cells using adenoviral vector-mediated NK4 gene therapy

NK4 is an hepatocyte growth factor (HGF)-antagonist and a broad angiogenesis inhibitor. NK4 gene therapy has confirmed antitumor efficacy on cancers with intact HGF-cMET signaling pathway. However, the feasibility to treat tumors in which the effect of the HGF-cMET signaling pathway is less unambiguous or may even be inhibitory on carcinogenesis, such as hepatocellular carcinoma (HCC) with NK4 needs further assessment. Therefore, we evaluated the effects of adenoviral vector-mediated expression of NK4 on the biological behavior of a series of HCC cell lines in vitro and on HepG2 xenografts in vivo. In vitro, transduction of HCC cell lines with the replication-deficient recombinant adenoviral vector AdCMV.NK4 resulted in significant inhibition of proliferation over and above the antimitogenic effects of HGF. In addition, HGF-induced scattering and invasion through matrigel were inhibited effectively. Moreover, transduced HCC cells produced sufficient amounts of NK4 protein to achieve bystander effects involving reduced migration of nontransduced tumor cells and reduced proliferation of endothelial cells. Finally, treatment of established HepG2 xenografts with AdCMV.NK4 resulted in significant tumor growth delay and significant reduction of intratumoral microvessel density. In conclusion, NK4 gene therapy is a promising strategy to treat HCC based on the pleiotropic functions of NK4 interfering with tumor growth, invasion, metastasis and angiogenesis.

Absence of tpr-met and expression of c-met in human gastric mucosa and carcinoma

The c‐met proto‐oncogene, encoding the hepatocyte growth factor receptor, can be activated by various mechanisms. These include, among others, gene amplification with concomitant overexpression and the tpr–met oncogenic rearrangement. In the case of gastric cancer, contradictory results on the presence of the tpr–met oncogenic rearrangement have been published. The current study aimed therefore to assess the prevalence of tpr–met expression in Caucasian gastric adenocarcinomas, to evaluate the importance of this oncogene in their carcinogenesis. In addition, the level of c‐met expression was determined, to evaluate the role of this alternative mode of activation of the proto‐oncogene. A series of Caucasian gastric adenocarcinomas (n=43) and normal gastric mucosal samples (n=14) was analysed for tpr–met and c‐met expression. Expression of tpr–met mRNA in the samples was performed by two reverse transcriptase polymerase chain reaction (RT‐PCR) assays, with excellent correlation. The specificity of both methods was confirmed by direct sequencing of the PCR products of the MNNG‐HOS cell line, which is known to contain the rearrangement. The level of c‐met expression was assessed using semi‐quantitative RT‐PCR assays and immunohistochemistry (IHC). None of the normal gastric mucosal or gastric adenocarcinoma samples expressed tpr–met mRNA, as determined by both RT‐PCR assays. Seventy per cent of the adenocarcinomas showed overexpression of c‐met, according to elevated c‐met mRNA levels, compared with the expression level of normal gastric mucosa. A significant correlation was found between the level of c‐met mRNA and protein expression. In conclusion, these results strongly suggest that tpr–met activation does not play a role in Caucasian gastric carcinogenesis, while overexpression of the c‐met gene occurs in the majority of Caucasian gastric adenocarcinomas. Copyright

Selective gene transfer into primary human gastric tumors using epithelial cell adhesion molecule-targeted adenoviral vectors with ablated native tropism.

Currently, application of adenoviral vectors (AdV) in gastric cancer gene therapy would be improved by increases in the specificity of transduction. Previously, we found that epithelial cell adhesion molecule (EpCAM) was expressed on gastric tumors but not on gastric epithelium. In this study, we evaluated doubly-ablated AdV lacking native binding ability together with bispecific single-chain antibodies targeted toward EpCAM for gene therapy of gastric cancer. Specific binding to EpCAM augmented the gene transfer efficiency of doubly-ablated AdV on gastric cancer cell lines up to 144-fold, reaching levels similar to or exceeding those achieved with native AdV. In contrast, EpCAM-targeted doubly-ablated AdV-mediated gene transfer into an EpCAM-negative cell line was reduced 38-fold compared with transduction by native AdV. Most importantly, EpCAM-targeted doubly-ablated AdV showed selectivity for primary human gastric tumors versus the surrounding nonneoplastic gastric mucosa of the same patients and normal liver tissue samples. Targeting these doubly-ablated AdV toward EpCAM resulted in similar transduction efficiency as obtained with native AdV for EpCAM-expressing primary human gastric tumors, whereas transduction of gastric epithelium and liver tissue was reduced at least 10-fold. This study thus indicates that application of EpCAM-targeted doubly-ablated AdV for gastric cancer gene therapy results in a favorable tumor-over-normal tissue transduction ratio.

p21Waf1/Cip1 expression and the p53/MDM2 feedback loop in gastric carcinogenesis

Data are non‐existent regarding coincidental alterations in the expression of p53 and its downstream target genes MDM2 and p21Waf1/Cip1 in gastric carcinogenesis. An immunohistochemical study was therefore performed to examine the interrelationships of p53, MDM2, and p21Waf1/Cip1 expression in a series of Caucasian early gastric carcinomas and precursor lesions. In normal gastric mucosa, chronic gastritis, and intestinal metaplasia, the surface cells expressed p21Waf1/Cip1 in the absence of detectable nuclear p53 and MDM2 protein. Nuclear p53 protein accumulation was found in 60 per cent of the carcinomas, with significant differences in staining characteristics between the Lauren types in the absence of detectable MDM2 protein ( p< 0·005). Nearly 80 per cent of the carcinomas expressed p21Waf1/Cip1, irrespective of Lauren type. Stratification of the carcinomas according to histological grade and growth pattern did not result in significant differences in p53 and p21Waf1/Cip1 expression. Finally, no significant correlation was found between overall p53 and p21Waf1/Cip1 expression in early gastric carcinomas. It is concluded that p21Waf1/Cip1 expression in the non‐neoplastic mucosa most likely relates to cell senescence and/or terminal differentiation, perhaps even in a p53‐independent manner. In view of p53/MDM2 homeostasis, the differences in p53 staining characteristics between intestinal and diffuse‐type carcinomas probably result, at least in part, from a difference in the prevalence of p53 gene mutations. Moreover, p53‐independent induction of p21Waf1/Cip1 expression apparently occurs in a considerable proportion of early carcinomas. Finally, in contrast to other carcinomas, p21Waf1/Cip1 expression is not significantly correlated with histological grade in gastric carcinomas, suggesting possible defects downstream of p21Waf1/Cip1 as an underlying cause for this apparent discrepancy. Copyright

A Rapid and Reliable Enzyme Immunoassay PCR-Based Screening Method to Identify EBV-Carrying Gastric Carcinomas

Epstein-Barr virus (EBV) is associated with a substantial number of gastric adenocarcinomas worldwide, as confirmed by EBER1/2-RNA in situ hybridization (RISH). In the present study, we developed a rapid and sensitive PCR-based prescreening method for the detection of EBV in gastric carcinomas to reduce the amount of laborious EBER1/2-RISH assays to be performed. The method was evaluated by testing gastric adenocarcinomas (n = 242) using both BamHI W PCR-enzyme immunoassay (EIA) and EBER1/2-RISH, in combination with appropriate DNA and RNA quality controls. Seventy-four percent of the paraffin-embedded gastric adenocarcinomas had good DNA quality as shown by β-globin polymerase chain reaction (PCR) after proteinase K and boiling pretreatment, whereas after DNA purification this was increased to 90%. Thirty-two percent of all cases were EBV-DNA positive after PCR-EIA, whereas 10% of these gastric cancers contained EBV transcripts in the neoplastic cells as confirmed by EBER1/2-RISH. Interestingly, only samples with high optical density (OD) 405/630 values in PCR-EIA, equivalent to the maximum reading of the assay as determined by the positive control, contained EBV-positive tumor cells in the EBER1/2-RISH. In contrast, the weak positive samples, as determined by low OD readings in the PCR-EIA were EBER1/2-RISH negative. In conclusion, high OD values in EBV PCR-EIA are very valuable to prescreen EBV-carrying gastric carcinomas as confirmed by EBER1/2-RISH. Only these samples and those with poor DNA quality will require testing in the EBER1/2-RISH, thereby reducing the amount of laborious RISH assays with 85%.