Alterations in exon 4 of the p53 gene in gastric carcinoma.

BACKGROUND & AIMS: Our long-term goal was to evaluate the role of p53 in the prognosis of gastric cancer. We previously showed a discrepancy between p53 expression and the presence of mutations when only exons 5-9 were examined. We then evaluated exon 4. METHODS: DNA was sequenced from 217 gastric cancers to detect exon 4 alterations. Codon 72 was examined by restriction enzyme digestion. RESULTS: Mutations were present in 3.2% of tumors. In addition, 2 polymorphic sites were found at codons 36 and 72. Polymorphisms at codon 36 were only found in 2 patients. In contrast, the codon 72 polymorphism was very frequent. The genotype frequency was arg/arg (54%), arg/pro (33%), and pro/pro (14%). The genotype of the polymorphic site varied with race (P = 0.001): 64% of whites had the arg/arg genotype, compared with 24% of blacks. The difference in genotype by site, sex, or histological tumor type was not statistically significant (P = 0.067). CONCLUSIONS: There are several exon 4 alterations in gastric cancers. These include the rare mutations and the very rare codon 36 polymorphism. The most common change is the codon 72 polymorphism, the genotype of which differs significantly with race. The more common arg/arg genotype in whites may explain why whites are more prone to develop cardiac cancer, whereas the more common proline allele in blacks may explain why they are more prone to develop antral cancers. Further studies are required to determine whether the codon 72 polymorphism affects patient predisposition to gastric cancer.

p53 immunoreactivity and single-strand conformational polymorphism analysis often fail to predict p53 mutational status.

The intent of this study was to investigate the ability of p53 expression and single-strand conformational polymorphism analysis (SSCP) to predict p53 mutational status in archival, paraffin-embedded tissues of gastric cancer. We evaluated paraffin-embedded tissues from 78 patients with advanced gastric cancer. The mutational status of the p53 gene (exons 5-9) was examined by SSCP analysis and by direct sequencing. These results were compared with p53 expression as assessed by immunohistochemical analysis (IHC). We graded p53 expression on a scale from 0 to 8 on the basis of both the intensity and the number of cells staining. Overall, we detected p53 immunoreactivity in 75.6% of the gastric cases; 19 (32.2%) of these cases scored from 1 to 4, and 40 (67.8%) cases scored from 5 to 8. p53 gene mutations were detected in 18 cases (23.1%) by SSCP and in 28 cases (36%) by direct sequencing. Thus, SSCP failed to detect 38% of the mutations found by sequencing. The majority of missed mutations involved exons 7 and 8. The concordance between IHC and SSCP was 37%, and the concordance between IHC and direct sequencing was 50%. Forty-five percent of cases positive by IHC failed to show mutations in exons 5 through 9. Five percent of cases negative by IHC (4 cases) contained mutations. One had a 1-base pair insertion; one had a mutation that resulted in a stop codon; the third had a mutation in exon 8; and the fourth had a mutation in both exons 5 and 8. Our findings indicate that p53 immunoreactivity correlates with the presence or absence of gene mutations in 50% of advanced gastric cancers when exons 5 through 9 are examined and that IHC cannot be reproducibly used as a marker of mutation in the most commonly mutated exons of the p53 gene. Furthermore, the sensitivity of SSCP for detecting mutations is only 62%. Thus, SSCP analysis cannot be used reliably to screen for p53 mutations.

Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors.

Carcinoid tumors are potentially malignant neoplasms that arise in various body sites, including the lung and gastrointestinal tract. Those that appear cytologically atypical are more likely to behave aggressively than more typical carcinoid tumors. However, in the absence of cytological atypia or large tumor size, it is difficult to predict the biology of an individual tumor, because some lesions metastasize, whereas others do not. This study had four aims: (1) To study the expression pattern of p53, Ki-67, NCAM, and S-100 in carcinoid tumors and to relate these expression patterns to classical histopathologic features and to tumor location. (2) To identify nonhistological markers that might more accurately predict the early behavior of carcinoid tumors. (3) To determine whether sustentacular cells are present in carcinoid tumors arising in tissues derived from different embryological derivatives. (4) To determine the synaptophysin and chromogranin immunoreactivity in neuroendocrine tumors arising in various locations. The immunostaining reactions were quantitatively scored by three observers. Only 3 of the 39 tumors (all histologically atypical) were strongly positive for Ki-67; two of these were also strongly p53 immunoreactive. NCAM immunostaining differed according to the site of origin: 76.5% of foregut lesions, 58% of the midgut lesions, and 20% of hindgut lesions were positive. S-100 immunostaining ranged from 41% in foregut lesions to 50% in both the hindgut- and midgut-derived tumors. S-100-positive sustentacular cells were present in 20.5% of carcinoid tumors. All tumors stained with antibodies against synaptophysin. In contrast, 100% of midgut, 60% of hindgut, and 88% of foregut tumors were chromogranin positive. Carcinoid tumors tend to have low proliferative rates. p53 immunostaining tends to be strongly positive in tumors that are histologically atypical, but it is negative in typical carcinoid tumors arising in the gastrointestinal tract and lungs. Immunostaining reactions with antibodies to NCAM, S-100, and chromogranin differ depending on the site of origin. Synaptophysin stains 100% of carcinoid tumors regardless of their site of origin. In contrast, antibodies to chromogranin fail to stain 40% of hindgut tumors and 12% of foregut carcinoid tumors. S-100-positive sustentacular cells are present in foregut and midgut tumors but not in hindgut tumors.

Staurosporine-induced versus spontaneous squamous metaplasia in pre- and postmenopausal breast tissue.

Breast cancers from pre- vs. postmenopausal women display unique characteristics that may be related to differences in epithelial differentiation between these two populations. In addition to lobular development, lactational changes, and involution, breast epithelium can undergo metaplastic alterations, often in association with carcinoma. Because protein kinase C (PKC) regulates differentiation and proliferation in many cell types, we asked whether modulation of PKC activity could define biochemical differences in breast epithelium from pre- vs. postmenopausal women. Organ cultures of normal human breast were treated with PKC agonists and antagonists. Epithelial differentiation was evaluated based on morphologic criteria and the expression of cell-type specific proteins. Staurosporine, a nonspecific but extremely potent inhibitor of PKC, induced squamous metaplasia in eight of eight cases within 2 weeks of treatment. Other inhibitors of PKC, such as calphostin C and tamoxifen, had no effect on epithelial differentiation. Long-term treatment with phorbol esters also did not induce squamous metaplasia. However, stimulation of cAMP levels by forskolin and isobutyl-methyl-xanthene (IMX) rapidly induced squamous metaplasia, as has been previously reported. Surprisingly, squamous metaplasia occurred in 10 of 12 cultures derived from postmenopausal women in the absence of exogenous agents. Untreated cultures derived from premenopausal women never developed this type of epithelium (0 of 11). Therefore, breast epithelium from pre- and postmenopausal women responded differently to in vitro culture. Forskolin/IMX or staurosporine can reproduce these conditions, acting independent of menopausal status. Because staurosporine's action was unique among PKC inhibitors, staurosporine may induce squamous metaplasia of breast epithelium by a PKC-independent mechanism.

Hemorrhagic infarct conversion in experimental stroke.

STUDY OBJECTIVE: This study investigated the relations between hemorrhagic infarction and occlusion, release, levels of glycemia, brain energy state, and lactate content after cerebrovascular occlusion. DESIGN: Prospective, controlled laboratory investigation. TYPE OF PARTICIPANTS: One hundred six pentobarbital-anesthetized cats. INTERVENTIONS: The middle cerebral artery was occluded with a Yasargil clip transorbitally either temporarily (0.5, four, and eight hours) or permanently. Normoglycemic and hyperglycemic animals were closely monitored for eight hours. Brain pathology was assessed after two weeks' survival or at the time of spontaneous animal death. Topographic brain metabolite studies were carried out after four hours of middle cerebral artery occlusion. MEASUREMENTS AND MAIN RESULTS: Morphometric quantitation of cerebral hemorrhage and infarction and fluorometric determinations of blood and brain tissue, glucose, glycogen, lactate, adenosine triphosphate, and phosphocreatine from 16 topographic brain sites were carried out. Twenty-one of 82 (25.6%) animals evaluated neuropathologically showed hemorrhagic infarcts. Occluding the artery in hyperglycemic animals caused fivefold more frequent and 25-fold more extensive hemorrhage into infarcts than in normoglycemic animals. Temporary occlusion with clip release after four hours in hyperglycemic animals caused the most extensive hemorrhage into infarcts. Most hemorrhages into infarcts (81%) took place in animals that died within a few hours after they experienced ischemia and that showed infarction and marked edema of the entire middle cerebral artery territory. Linear regression analyses demonstrated a close relation between hemorrhage into infarcts and near-total energy depletion (adenosine triphosphate, less than 0.3 microM/g; phosphocreatine, less than 0.5 microM/g) in brain sites that showed extremely high tissue lactate concentrations (more than 30 microM/g). The biochemical changes that correlated with hemorrhage into infarcts were more marked than those with infarcts without hemorrhage. CONCLUSION: Hyperglycemia and restoration of blood flow to ischemic territories were strong risk factors for hemorrhagic infarct conversion. Concomitant tissue metabolic changes suggest that marked tissue energy depletion accompanied by acidosis damages brain vessels and renders them penetrable for edema fluid and, ultimately, red blood cell extravasation.