In oesophageal squamous cell carcinoma vascular endothelial growth factor is associated with p53 mutation, advanced stage and poor prognosis.

Vascular endothelial growth factor (VEGF) affects malignant tumours by promoting angiogenesis. The tumour-suppressor gene p53 has been thought to regulate VEGF. We investigated the effect of VEGF on oesophageal carcinoma and the connection between VEGF and p53. One hundred and nine resected oesophageal squamous cell carcinomas were examined. VEGF expression was analysed by immunohistochemical staining. Sixty-five tumours (59.6%, 65 out of 109) were classified as VEGF positive. A significant correlation was found between the VEGF expression and both the depth of invasion (P = 0.0001) and lymph node metastasis (P < 0.0001). With regard to p53, we compared the expression of VEGF with the mutation of p53, examined using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing in tumour samples obtained from 36 patients who we have reported previously. The VEGF expression was significantly correlated to p53 mutation (P = 0.0291). To evaluate the angiogenesis, microvascular density (MVD) was counted, and endothelial cells were stained immunohistochemically using anti-CD34 monoclonal antibody against 29 cases with invasion limited to the submucosal layer. The average MVD had a tendency to correlate to VEGF expression (P = 0.1626). The prognoses of patients with VEGF-positive primary tumours were significantly worse than for those with VEGF-negative primary tumours (P = 0.0077). We have assumed that VEGF contributes to aggressive characteristics in oesophageal carcinomas and that VEGF expression might be affected by p53 status.

Genetic alterations in patients with esophageal cancer with short- and long-term survival rates after curative esophagectomy.

OBJECTIVE: The objective of this study was to ascertain the exact relation between specific oncogenes and long- and short-term survival rates in patients with esophageal cancer. SUMMARY BACKGROUND DATA: Recent developments in molecular biology have shown that several oncogenes and suppressor genes are involved in the development of esophageal cancer. However, the role of these genes still is unknown. METHODS: The clinical outcome of 84 cases of R0-resected esophageal carcinomas (from 1986-1993) and the molecular and biologic characteristics of these tumors were studied. The patients studied were divided into three groups, which were designated as follows: shortest term survivors (up to 6 months), short-term survivors (7-12 months), and long-term survivors (>5 years). These groups included 23, 17, and 44 subjects, respectively. For the genomic analysis, CyclinD1, int-2, murine double minute 2 (MDM2), retinoblastoma, p53, adenomatous polyposis coli (APC), deleted in colorectal carcinoma (DCC), and human papillomavirus were studied in these patients. The regrowth capability of primary cultures and the clinicopathologic characteristics of these patients also were analyzed. RESULTS: The CyclinD1 and int-2 genes, which are located in the 11q13 chromosome, and the MDM2 gene were related to short survival. However, the p53 mutation and human papillomavirus infection were not related to short-term survival. The average ratio of genomic abnormalities to genes examined was higher in the shortest and short-term survival groups than in the long-term survival group. Regrowth capability in primary cultures also was related to short-term survival. Among the long-term survival patients, 7 (16%) of 44 cases suffered further cancer after esophagectomy. CONCLUSIONS: These results suggest that the 11q13 amplicon and MDM2 may play an important role in the progression of esophageal cancer, and an accumulation of genomic abnormalities may result in poor prognosis. Careful follow-up testing for double cancer is needed in long-term survivors of esophageal cancer.

Multiple types of aberrations in the p16 (INK4a) and the p15(INK4b) genes in 30 esophageal squamous-cell-carcinoma cell lines.

To determine the role and mode of inactivation of the p16 and p15 genes in human esophageal tumors, we examined alterations and expression of the alpha and beta forms of the p16 gene, 5' CpG island methylation of p16 exon 1 alpha, and alterations of the p15 gene in 30 esophageal squamous-cell-carcinoma cell lines. Of 30 such cell lines examined, 28 (93%) showed aberrations of the alpha form of the p16 gene: 18 homozygous deletions, 6 point mutations and 4 hypermethylation. Methylation was exclusively observed in cell lines with the wild-type alpha form. Of the 6 point mutations, one was observed in exon 1 alpha, one in the splice acceptor site of intron 1 and the remaining 4 were in exon 2. In the beta form, 18 homozygous deletions and 3 point mutations in exon 2 were detected, but no point mutation was found in exon 1 beta. All mutations in exon 2 gave rise to premature termination codons in the reading frame of the alpha transcript, while no non-sense mutations were observed in the reading frame of the beta transcript. Among 12 cell lines without homozygous deletions of the alpha and beta forms of the p16 gene, the expected wild-type beta transcript was observed in 8 cell lines, whereas only one cell line expressed the expected wild-type alpha transcript. Homozygous deletions of the p15 gene were observed in 16 cell lines (53%), and no point mutations were detected. Twelve cell lines had alterations only in the alpha form of the p16 gene, while none showed aberrations exclusively in the p15 gene. Taken together, these results indicate that inactivation of the beta form of the p16 gene and the p15 gene are not so frequent as that of the alpha form of the p16 gene in ESC cell lines, suggesting that aberration of the alpha form of p16 gene is the primary target of 9p loss in ESC.

Prognostic value of monolayer culture patterning in primary cell culture of oesophageal cancer.

The growth of primary cell cultures of oesophageal cancer was compared with the clinical outcome of patients from whom the cancers were taken. Ninety-three patients underwent curative resection, with no operative deaths, and were divided into three groups according to the monolayer culture pattern of the primary cell culture: culture from 43 patients (46 per cent) grew no malignant cells (group 1), 21 (23 per cent) produced monolayer epithelial growth (group 2) and the rest (from 29 patients) established cell lines (group 3). The 5-year survival rate of patients in group 2 (29 per cent) and group 3 (23 per cent) was significantly lower (P < 0.005) than that of those in group 1 (51 per cent). Monolayer epithelial growth potential is a significant prognostic factor in patients with oesophageal cancer.

Characterization of p53 gene mutations in esophageal squamous cell carcinoma cell lines: increased frequency and different spectrum of mutations from primary tumors.

We screened 29 human esophageal squamous cell carcinoma (ESC) cell lines for mutations of the p53 gene through all coding exons and exon-intron junctions. Mutations were found in 22 cell lines (76%), consisting of 20 single-base substitutions, 2 small deletions and 1 single-base insertion. Out of 20 single-base substitution, 5 were located at the exon-intron junctions and mRNAs with abnormal splicing were detected by RT-PCR in 4 of them. A G:C to T:A transversion, which occurred rather frequently in resected tumors of ESC, was observed in only 1 cell line, and, instead, frequent transitions at CpG sites were detected. We also examined 65 fresh tumor materials, from all of which we tried to establish cell lines, and detected mutations in 26 samples (40%). Compared with the results in these fresh tumor materials, the mutation incidence in cell lines was significantly high and the mutation spectrum was also different. From these 65 tumors, 10 cell lines were established, including 3 cell lines from 26 tumors with p53 mutations and 7 cell lines from 39 without mutations, which indicates that there was no significant correlation between the status of the p53 gene in each fresh tumor and its establishment as a cell line. In 7 cell lines established from mutation-free tumors, newly acquired mutations were detected in 5, which suggests that mutations might occur during the process of establishing cell lines.

p53 mutation, murine double minute 2 amplification, and human papillomavirus infection are frequently involved but not associated with each other in esophageal squamous cell carcinoma.

We examined the relationship between p53 mutation, murine double minute 2 (MDM2) gene amplification, and human papillomavirus (HPV) infection in 72 esophageal squamous cell carcinomas. We identified p53 mutations in 29 tumors (40.3%) by PCR-single-strand conformation polymorphism analysis and direct sequencing. Amplification of the MDM2 gene was detected by Southern blot hybridization in 13 (18.1%) of 72 tumor tissues and in 4 (33.3%) of 12 cultured esophageal squamous cell lines. All four cell lines with MDM2 amplifications showed overexpression of the MDM2 mRNA in Northern blotting. We observed HPV infection in 15 (20.8%) of 72 tumor tissues by specific PCR amplification and Southern blot hybridization. In most tumors, amplification of the MDM2 gene or infection of HPV was not associated with p53 mutations, except in four cases with p53 mutation and MDM2 amplification, and three cases with p53 mutation and HPV infection. Since p53 mutations, MDM2 overexpression, and HPV infection are all considered to abrogate the normal function of p53 protein, each of these genetic changes may be equally important in tumorigenesis. In addition, we found that patients with MDM2 amplification exhibited a significantly shorter survival period (P = 0.0053).

Amplification and overexpression of the cyclin D1 gene in aggressive human hepatocellular carcinoma.

We analyzed the genetic alterations of the cyclin D1 and INT-2 genes in hepatocellular carcinomas (HCCs) from 45 patients. Among these, expression of the cyclin D1 mRNA was also analyzed in 18 of them by Northern blotting. The cyclin D1 gene was amplified 3-16 fold in five HCCs (11%); among these, the INT-2 gene was also amplified 2-10 fold in four HCCs. We analyzed the mRNA of cyclin D1 in four HCCs with gene amplifications, and 6-10 fold overexpressions were detected in all of them. Because the cyclin D1 gene was amplified in patients at an advanced stage of HCC with rapid tumor growth, it appeared to be associated with the aggressive behavior of tumors. Studies on loss of heterozygosity on chromosome 13q, where the retinoblastoma (RB) gene is located, indicated that all HCCs with an amplified cyclin D1 gene retained heterozygosity on chromosome 13q. These results suggest that amplification and overexpression of the cyclin D1 gene result in the rapid growth of a subset of HCC, even though the function of the RB gene is retained.

Allelotype analysis of esophageal squamous cell carcinoma.

In previous studies, we have shown that allelic loss on chromosome 17p, on which the p53 gene is located, is very frequent, and loss-of-function mutations of the p53 gene are closely associated with the tumorigenesis of esophageal cancer. In this study, we performed allelotype analysis to investigate whether other tumor suppressor genes are also involved in esophageal cancer. Using 55 polymorphic DNA markers covering every autosomal arm except 13p, 21p, and 22p, restriction fragment length polymorphism analysis was performed on 36 esophageal squamous cell carcinomas (ESCs) and their adjacent normal tissue samples. Frequent loss of heterozygosity (LOH) of > 30% of the informative cases was observed on chromosomes 3p (41.1%), 5q (52.6%), 6p (30.4%), 8p (33.3%), 9p (35.7%), 9q (30.8%), 11p (32.4%), 13q (52.7%), 17p (55.2%), 17q (33.3%), 18q (45.7%), and 19q (30.4%). Among these, LOH on 5q, 13q, 17p, and 18q was previously reported in ESC and is considered to involve the APC, RB, p53, and DCC genes, respectively. However, our deletion analysis of chromosome 18q revealed that the region commonly lost did not include the DCC locus, suggesting that a possible tumor suppressor gene on 18q other than the DCC gene is involved in ESC. We screened 60 primary ESC tumors and 20 cultured ESC cell lines for the mutation of the APC gene within a mutation cluster region in exon 15, where the "hot spot" of somatic mutation for colorectal and pancreatic cancers is thought to be. We could not find any mutation despite the high frequency of LOH on chromosome 5q. We also analyzed the relationship between the clinicopathological data and the allelic loss and found that LOH on chromosomes 6p and 13q was associated with poor prognosis.

Loss of 17p, mutation of the p53 gene, and overexpression of p53 protein in esophageal squamous cell carcinomas.

We analyzed mutations of the p53 gene by single-strand conformation polymorphism analysis of polymerase chain reaction products and direct sequencing through all coding exons and exon-intron junctions in 32 cases with esophageal squamous cell carcinoma. Mutations were detected in 15 of 32 (47%) tumor samples, in which G:C to T:A transversions were rather frequent (33%). Previously, we reported deletion of chromosome 17p where the p53 gene is located in 45% of Japanese esophageal squamous cell carcinoma, and here the relationship between mutation of the p53 gene and loss of 17p was analyzed. Mutations were observed in 12 of 16 patients with loss of 17p, whereas only 2 of 11 without loss were positive for mutations, suggesting that mutations of the p53 gene were closely associated with a 17p deletion. Furthermore, we immunohistochemically analyzed the expression of p53 protein in esophageal squamous cell carcinoma tumor tissues using a monoclonal antibody. Five of 6 tumors with missense mutations of the p53 gene were positively stained, while in tumors with nonsense mutations or without mutations of the p53 gene staining was very weak or negative. These results suggest a good correlation between mutations and abnormal expression of the p53 gene.