Accumulation of p53 protein as an indicator for p53 gene mutation in breast cancer. Occurrence of false-positives and false-negatives.

Accumulation of p53 protein resulting in levels detectable by immunohistochemistry (IHC) has been proposed as an indicator of mutation of the p53 gene. We have investigated a panel of 23 fresh-frozen breast cancers by IHC (PAb 1801), Southern and Northern blot analysis, and direct sequencing of the mutation hot spot regions (exons 5-8) of the p53 gene. Three tumors (13%) showed an intense nuclear staining in the majority of malignant cells, but only one of these showed a mutation of the p53 gene (codon 237, Arg to His). Furthermore, a mutation (5-bp deletion) was identified in a tumor that showed no p53 immunoreactivity. Our results indicate that accumulation of p53 protein, as detectable by IHC, is not a reliable indicator for p53 gene mutation in human breast cancer.

Expression of stromelysin 3 in the stromal elements of human basal cell carcinoma.

In basal cell carcinoma, release of proteolytic activity is implicated in extracellular matrix degradation and tumor infiltration. The stromelysin metalloproteinase family is a major candidate for the matrix proteolytic activity in infiltrative tumors. However, in murine models of basal cell carcinoma, neither stromelysin 1 nor 2 appears to play a role in tumor infiltration. We have analyzed the expression of the newly described stromelysin 3 in human basal cell carcinoma using Northern blot analysis and in situ hybridization. In 12 of 14 cases, levels of stromelysin 3 expression were more than tenfold above those observed in normal skin. In one of five cases of squamous cell carcinoma, stromelysin 3 expression was tenfold above levels seen in normal skin. Stromelysin 3 expression was either undetectable or extremely weak in all five cases of infiltrative malignant melanoma. In basal cell carcinoma, stromelysin 3 transcripts were localized by in situ hybridization to the stromal tissue immediately adjacent to basal cell carcinoma, the tumor cells themselves being negative. Therefore, expression of stromelysin 3 in stromal cells may be expected to play a significant role in destruction of the basal membrane zone and extracellular matrix in basal cell carcinoma invasion.

Oncogene expression in endocrine pancreatic tumors.

The mRNA expression of the (proto)oncogenes Ha-ras, Ki-ras, fos, c-myc, N-myc, and sis was studied in five pancreatic endocrine tumors and two non-neoplastic pancreatic tissues. Compared with non-tumorous pancreatic tissue, Ha-ras and Ki-ras mRNA was overexpressed up to 42-fold in all the tumors; metastasizing tumors showed 2-6 times higher Ha-ras mRNA levels than benign neoplasias. In contrast, c-myc mRNA levels were higher in normal tissue than n tumors and fos mRNA levels did not differ significantly between tumors and normal tissue. The activities of Ki-ras, fos and c-myc mRNA expression did not correlate with any of the histological or biological properties of the tumors, nor with the clinical course of disease. Our results, although based on a limited number of cases, suggest tha Ha-ras and Ki-ras mRNA overexpression is associated with the development of pancreatic endocrine tumors. The measurement of Ha-ras mRNA levels may contribute to the assessment of tumor prognosis.

Oncogene expression in a medullary thyroid carcinoma.

We report the expression of Ha-ras, fos, c-myc and N-myc mRNA in a human medullary carcinoma of the thyroid gland, both in primary tumor and lymph node metastasis, as demonstrated by in situ hybridization and Northern blot analysis. A significant difference in the oncogene expression in the primary tumor and the metastasis was not observed. Tumor tissue revealed a significant overexpression of Ha-ras, c-myc and N-myc mRNA as compared to the normal thyroid gland. The amount of fos mRNA expression in non tumorous thyroid gland did not significantly differ from tumor tissue, sis, fms and abl mRNA expression was not detectable in tumor tissue and non tumorous thyroid gland. We conclude, that the (over)expression of the oncogenes Ha-ras, c-myc and N-myc may be associated with initiation and progression of medullary thyroid carcinoma. Similar studies on additional cases of human medullary thyroid carcinoma will be necessary to reveal further information.

Simultaneous localization of calcitonin mRNA and peptide in a medullary thyroid carcinoma.

We report the visualization of calcitonin gene expression products at the mRNA and peptide levels on the same section of a medullary thyroid carcinoma by combined in situ hybridization and immunohistochemistry. mRNA detection was accomplished by hybridization with radioactively labeled antisense RNA probes followed by autoradiography and immunohistochemically using the avidin-biotin complex method. Best results were obtained when in situ hybridization preceded immunohistochemistry, as determined by quantitative analysis of the autoradiographs. When immunohistochemistry was performed prior to in situ hybridization, the RNase inhibitor heparin had to be added to the antibodies to retain hybridizable mRNA. The intensity of the two reactions varied in individual cells, indicating a functional heterogeneity of tumor cells with regard to calcitonin mRNA content and storage of the related immunoreactive peptide. These results, in combination with elevated serum calcitonin levels, suggest significant differences in the rate of secretion of individual tumor cells. Simultaneous localization of mRNA and its peptide within the same cell may, therefore, provide further insight into gene expression and secretory activity at the single cell level.

Expression of c-myc and c-fos mRNA in colorectal carcinoma in man.

Thirty colorectal carcinomas, 1 adenoma of the colon, 1 case of Crohn's disease and 13 specimens of non-neoplastic colorectal mucosa were examined for qualitative and quantitative expression of the c-myc and c-fos protooncogenes. These genes encode nuclear proteins, which are both believed to regulate gene transcription. Oncogene expression was evaluated at the mRNA level by in situ hybridization and Northern blot analysis. Densitometric analysis of the specific bands on Northern blots revealed a highly significant overexpression of c-myc mRNA in colorectal carcinomas compared with non-neoplastic tissue (p less than 0.001). Furthermore, increased expression of c-myc mRNA was found in moderately and poorly differentiated carcinomas compared with well differentiated ones. In contrast to c-myc, c-fos mRNA expression was significantly lower in carcinomas than in non neoplastic tissue (p less than 0.02). Neither, c-myc nor c-fos mRNA levels showed a clear-cut correlation with tumor stage. We conclude that c-myc mRNA overexpression plays an important role in the progression of colorectal carcinomas. In contrast enhanced c-fos mRNA expression may be related to cell differentiation, both in tumors and non-neoplastic tissue.

c-myc mRNA expression in non-Hodgkin's lymphomas.

Steady state c-myc mRNA levels determined by Northern blot analysis were examined in non-Hodgkin's lymphomas (NHL) of both high (n = 29) and low malignancy (n = 18), and in non-specific chronic lymphadenitis (n = 6). High grade NHL, classified according to the updated Kiel classification, revealed significantly larger amounts of c-myc mRNA compared with low grade NHL and lymphadenitis. mRNA levels in non-specific lymphadenitis were lower than in low grade NHL, but the differences were not statistically significant. No correlation between c-myc mRNA levels and the immunologic phenotype was discernible. Growth fractions of the NHL were determined by immunostaining with the monoclonal antibody Ki-67. Significant correlations between the percentages of Ki-67-positive cells, as well as the amounts of c-myc mRNA, and classification into high or low grade NHL were found. However, the percentage of Ki-67 positive cells and c-myc mRNA levels in individual cases and in the various histologic entities of NHL did not correlate. Our results indicate the overexpression of the c-myc gene in NHL, and a highly significant correlation of steady state c-myc mRNA levels with the prognosis-related histomorphologic Kiel classification of NHL into different subgroups of low and high grade malignancy.