AIM: To verify the effectiveness of denaturing highperformance liquid chromatography (DHPLC) in detecting somatic mutation of p53 gene in gastric carcinoma tissues. The superiority of this method has been proved in the detection of germline mutations, but it was not very affirmative with respect to somatic mutations in tumor specimens. ST7 gene, a candidate tumor suppressor gene identified recently at human chromosome 7q31.1, was also detected because LOH at this site has also been widely reported in stomach cancer. METHODS: DNA was extracted from 39 cases of surgical gastric carcinoma specimen and their correspondent normal mucosa. Seven fragments spanning the 11 exons were used to detect the mutation of p53 gene and the four exons reported to have mutations in ST7 gene were amplified by PCR and directly analyzed by DHPLC without mixing with wild-type allele. RESULTS: In the analysis of p53 gene mutation, 9 aberrant DHPLC chromatographies were found in tumor tissues, while their normal-adjacent counterparts running in parallel showed a normal shape. Subsequent sequencing revealed nine sequence variations, 1 polymorphism and 8 mutations including 3 mutations not reported before. The mutation rate of p53 gene (21%) was consistent with that previously reported. Furthermore, no additional aberrant chromatography was found when wild-type DNA was added into the DNA of other 30 tumor samples that showed normal shapes previously. The positivity of p53 mutations was significantly higher in intestinal-type carcinomas (40%) than that in diffuse-type (8.33%) carcinomas of the stomach. No mutation of ST7 gene was found. CONCLUSION: DHPLC is a very convenient method for the detection of somatic mutations in gastric carcinoma. The amount of wild type alleles supplied by the non-tumorouscells in gastric tumor specimens is enough to form heteroduplex with mutant alleles for DHPLC detection. ST7 gene may not be the target gene of inactivation at 7q31 site in gastric carcinoma.

PTEN/MMAC1/TEP1, encoding a dual-specificity phosphatase, is a tumor suppressor gene which has recently been cloned and mapped to chromosome 10q23.3. We have shown that germline mutations of PTEN are present in individuals with two hamartoma syndromes: Cowden Syndrome, associated with a predisposition to breast and thyroid cancers, and Bannayan-Zonana syndrome. Somatic mutations of PTEN have been reported in a variety of human cancer cell lines, suggesting a potential role for this gene in the pathogenesis of human malignancies. We report the identification of a highly conserved PTEN processed pseudogene, cPTEN, which shares over 98% homology with the coding region of functional PTEN, and its localisation to chromosome 9p21. The high sequence homology of cPTEN with the PTEN transcript may potentially lead to misinterpretation when performing mutation analyses based on cDNA templates. Caution should be exerted when using such screening approaches.

We examined loss of heterozygosity (LOH) at the retinoblastoma susceptibility gene (RB1) locus on chromosome 13q14 in 20 non-small-cell lung cancers (NSCLCs) using polymorphic markers. The expression of RB protein was examined by immunohistochemical analysis of paraffinembedded specimens of the same tumors. The results revealed that 10 of 16 informative cases showed an LOH at the RB1 locus, whereas only 2 of the 10 tumors lost expression of the RB protein. These 2 tumors had mutations in the remaining RB1 allele. Thus, inactivation of the RB1 gene appears to be involved in a small subset of NSCLCs only. To elucidate the presence of tumor-suppressor genes other than RB1 on 13q, heterozygosity at 15 different loci was investigated. Of 20 tumors analyzed, 15 showed an LOH at least at one locus, and the regions 13q12.1-qter, 13q12.2-14.2 and 13q14.1-q14.3, including the RB1 locus, were deleted in significant numbers of the tumors. Our results suggest that, in addition to the RB1 gene, abnormalities of other tumorsuppressor genes on chromosome 13q are involved in the development of human NSCLCs. Int. J. Cancer 74: 45-49.

AIM: Survivin, a recently identified member of the inhibitor of apoptosis protein family, is expressed during development and in various human cancers. However, its expression in normal tissues and clinical relevance in cancers are still debated. In the present study, we analyzed the expression of the survivin gene in human primary and metastatic gastric cancer cells as well as in paired epithelial cells from normal gastric mucosa by means of a novel laser capture microdissection (LCM) technique coupled with reverse transcription-polymerase chain reaction (RT-PCR). METHODS: Thirty patients who had undergone gastrectomy with lymph node dissection for gastric cancer without preoperative treatments were included. Neoplastic tissue, metastatic lymph nodes, and apparently uninvolved normal tissue were collected from each patient. LCM-captured "pure" cell groups were espectively subjected to RT-PCR analysis with primers specific for the survivin gene. RESULTS: Of the paired samples from 30 gastric cancer patients studied, 24 (80%) primary gastric cancer cell groups and 7 (23%) adjacent morphologically "normal" gastric epithelial cell groups were shown to have a detectable survivin expression. There was a statistically significant difference in suvivin expression between these two groups (P<0.01). Meanwhile, 95% (19/20) of the metastatic gastric cancer cell groups from lymph nodes had a clear expression of the survivin gene. However, no significant correlation between survivin expression and clinicopathological features of gastric cancer was bserved in the present study.CONCLUSION: Survivin expression is present in the majority of gastric cancer cell groups obtained by LCM techniques. The high expression rate in metastatic lesions suggests a possible role of survivin in cancer invasiveness and metastasis. It may contribute to the detection of gastriccancer micrometastasis as a potential molecular marker. In addition, the high expression percentage renders survivin a potential target in the therapy for gastric cancer.

Background Peritoneal dissemination is the most common pattern of metastasis in advanced gastric carcinoma with serosal invasion In the present study, we reported the clinical relevance of a new diagnostic method involving RT-PCR, using su rvivin as the target gene, for the detection of free cancer cells in peritoneal washes Methods Intraoperative peritoneal washes were obtained from 48 patients who underwent su rgery for gastric cancer RT-PCR analysis with primers specific for survivin and conventional cytological examinations were both performed Results Su rvivin mRNA was not detected in any peritoneal wash samples from patients with benign disease, but was detected in 28 of 48 samples taken from patients with gastric cancer and in all metastastic nodules Survivin expression in the peritoneal cavity significantly correlated with depth of cancer invasion, lymph node etastasis, and TNM stage There were 92% of clinically evident peritoneal metastasis cases showed detectable su rvivin expression The combination of survivin RT-PCR and cytological examination yielded positive results in 66 7% (32/48) of patients with gastric cancer, much higher than the results produced by cytological method alone Conclusions Survivin mRNA detected in peritoneal lavage fluid might indicate the presence of free cancer cells in the peritoneal cavity. The high sensitivity of the RT-PCR-based survivin assay suggests that su rvivin serves as a molecular marker for detecting peritoneal micrometastasis Its ubiquitous expression in peritoneal cancer cells and metastatic nodules also suggests a promising future therapeutic strategy based on su rvivin inhibition for cases of gastric cancer involving peritoneal metastasis