Expression of the 21,000 molecular weight ras protein in a spectrum of benign and malignant human mammary tissues

Monoclonal antibodies RAP-5 and Y13-259, directed against the ras gene product [a protein with a molecular weight of 21,000 (p21)] have been used to evaluate ras p21 expression in malignant and benign mammary tissues as well as in the lesions of intermediate stature such as atypical hyperplasia using immunohistochemical assays. Invasive carcinoma demonstrated enhanced expression of ras p21, with generally decreasing expression in carcinoma in situ, atypical hyperplasia, and nonatypical hyperplasia, respectively. Heterogeneous expression of ras p21 was observed among primary as well as metastatic mammary carcinomas. Carcinomas from postmenopausal patients generally demonstrated higher levels of ras p21 than those from premenopausal patients, but no significant difference in ras p21 expression in carcinomas between estrogen-receptor rich and estrogen-receptor poor patients was found. Normal mammary epithelium in terminal duct lobular units from patients with hyperplasia generally demonstrated higher levels of ras p21 expression than did epithelium in large ducts. This demonstration of enhanced ras p21 expression by the epithelium of peripheral lobular portion of the breast is consistent with the previous hypothesis that these areas preferentially undergo malignant transformation. Analyses of the limited number of specimens available from patients with 15-yr follow-up revealed a generally higher level of ras p21 in hyperplasia from patients who subsequently developed carcinoma, as compared to those from patients without carcinoma development. However, no conclusions regarding the potential for malignant transformation could be drawn for any individual patient on the basis of ras p21 expression. Concomitant analyses of ras p21 expression in mammary carcinomas and benign lesions using liquid competition radioimmunoassay and immunohistochemical assay demonstrated the complementary nature of these alternative approaches. These results suggest that enhanced ras p21 expression may be involved in the early stages of mammary carcinogenesis but is probably not involved in the maintenance of the transformed phenotype.     

Histologic demonstration of antigens reactive with anti-p21 ras monoclonal antibody (RAP-5) in human stomach cancers

The immunohistochemical reactivity of RAP-5, a monoclonal antibody (MoAb) raised against a synthetic peptide corresponding to positions 10-17 of the ras gene product from T24 bladder carcinoma, was studied in 96 surgically resected stomach cancers of humans. The cytoplasm of cancer cells in 65 cases (68%) was positively stained with MoAb RAP-5, although the staining was heterogeneous among cancer cells. There was no definite correlation between depth of tumor invasion and reactivity to MoAb RAP-5. Cancer cells of poorly differentiated tumors showed a tendency to react less frequently and less intensely to MoAb RAP-5. In nontumorous gastric mucosa, parietal cells and some portions of intestinal metaplasia were stained with MoAb RAP-5. These findings suggest an increased expression of the ras gene product (p21) in about two-thirds of gastric adenocarcinomas and in some nonneoplastic gastric epithelial cells.     

Immunohistochemical demonstration of ras p21 oncogene product in normal, benign, and malignant human thyroid tissues

The p21 protein product of the cellular oncogene ras, designated ras p21, has been detected immunohistochemically in normal, benign and malignant human thyroid tissues. With the monoclonal antibody RAP-5 generated against a synthetic peptide corresponding to amino acid positions 10 to 17 of the ras p21 protein and an avidin-biotin-peroxidase complex (ABC), the expression of the ras p21 was evaluated in paraffin-embedded sections. Western blot analysis using fresh thyroid carcinoma tissue revealed double protein bands, one band was at molecular weight 21,000 and the other was a more rapidly migrating band at the molecular weight 17,500. Immunohistochemically, papillary adenocarcinomas of the thyroid showed moderate to intense stainings for ras p21 in most cases. Cytoplasmic and apical surface stainings were the most common patterns of immunoreactivity. Adenomas showed variable ras p21 positivity in cytoplasm and apical surface stainings of adenomas were negative to borderline in most cases. The cytoplasm of tissues of Hashimoto's thyroiditis, Graves' disease, and normal thyroid tissues was uniformly ras p21 positive, but the apical cell surface was nonreactive for ras p21 in all tissues. Judging from the findings obtained on this large series of normal, benign, and malignant thyroid tissues, the elevation of ras p21 may be a common event in thyroid neoplasm, and especially elevated ras expression in the apical cell surface may be characteristic to papillary carcinomas of the thyroid. This suggests that apical surface expression of ras p21 may be important in the development of thyroid carcinomas and be useful in differentiation of papillary adenocarcinoma.     

An immunohistochemical analysis of ras oncogene expression in epithelial neoplasms of the colon

Colonic epithelial tumors (101) including villoglandular adenomas, carcinomas in situ, adenocarcinomas, and neuroendocrine (NE) carcinomas were studied immunohistochemically with monoclonal antibodies (MoAb) RAP-5 and RAS-10 recognizing altered and unaltered ras oncogene products. In addition, 20 samples from multiple polyposis including adenomas with and without dysplasia, carcinomas in situ, and invasive carcinomas were studied. Using immunostaining techniques, normal mucosa was weakly stained, whereas the mucosa in the vicinity of tumors or inflammation showed enhanced staining. More tumors stained intensely with MoAb RAP-5 than with MoAb RAS-10. With MoAb RAP-5, most benign and malignant tumors showed enhanced staining. No significant differences in staining were noted in relation to superficial versus deeply invasive carcinomas or clinical staging. Immunostaining was also noted in some metastases. No significant differences in enhanced staining were found in carcinomas. Interestingly, the most extensive and enhanced immunostaining was noted in the villoglandular adenomas, dysplastic adenomas, and carcinomas in situ. The authors conclude that (1) ras protein expression is detectable in most benign, borderline, and malignant epithelial tumors of the colon as determined with MoAb RAP-5 and RAS-10, whereas enhanced expression is more often detected with RAP-5; (2) enhanced ras product expression in colon carcinomas does not seem to correlate with advanced tumor stages or with exocrine, NE, or phenotypically mixed tumors; and (3) the finding of the most intensely enhanced ras products expression in villoglandular polyps and carcinomas in situ suggests a possibly significant role for the oncogene in the early phases of transformation.     

Monoclonal antibody highly sensitive for the detection of ras p21 in immunoblotting analysis

Four murine monoclonal antibodies (NCC-RAS-001, -004, -005, -017) reactive with ras p21 were produced by using recombinant c-Ha-ras p21 as an immunogen. Among these antibodies, NCC-RAS-004 was extremely sensitive when used in immunoblotting analysis, facilitating semiquantitative detection of c-Ha-, c-Ki- and N-ras p21 in cell and tissue lysates. In cells carrying a point-mutationally activated ras, p21 with abnormal mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis was clearly detected.     

Quantitation of Harvey ras p21 enhanced expression in human breast and colon carcinomas

Molecular studies have demonstrated increased expression of the Harvey (Ha) ras oncogene in human breast and colon carcinomas. With the use of a direct-binding liquid competition radioimmunoassay (RIA), capable of providing truly quantitative analysis of the 21,000-dalton (p21) ras oncogene and protooncogene products, absolute levels of Ha-ras p21 have been determined in human breast and colon carcinomas, benign lesions, and/or their respective normal tissues. Enhanced Ha-ras expression was documented in 66% of breast and 100% of colon carcinomas as compared with their normal counterparts, with levels in breast carcinomas ranging from 10.1 to 50.4 pg ras p21/micrograms protein and those in colon carcinomas ranging from 18.4 to 51.7 pg ras p21/micrograms protein. Some dysplastic lesions of the breast and colon also contained elevated Ha-ras p21. Relative levels of Ha-ras p21 expression, detected by competition RIA, correlated with percent Ha-ras p21-positive cells as determined by immunohistochemical assays. By use of liquid competition RIA and immunohistochemical assays, it has been shown that levels of ras p21 expression did not always correlate between primary and metastatic colon lesions of the same patient. The use of the quantitative RIA and semiquantitative immunohistochemical assays, in concert with cDNA probes for identification of specific ras point-mutated oncogenes or protooncogenes, may now provide the means for definitive quantitative analyses of ras p21 in human carcinomas and benign lesions.     

Tumor-associated glycoprotein (TAG-72) detected in adenocarcinomas and benign lesions of the stomach

Murine monoclonal antibody (MAb) B72.3, prepared against a membrane-enriched extract of metastatic carcinoma and reactive with a high-molecular-weight determinant, designated tumor-associated glycoprotein (TAG)-72, was shown to be reactive immunohistochemically with 97% of a variety of primary adenocarcinomas of the stomach (n = 40). All "early" gastric carcinomas were reactive with MAb B72.3, although the average percentage cellular reactivity was lower than in "advanced" carcinomas. TAG-72 antigen was detected in benign lesions (i.e. adenomatous polyps and hyperplastic polyps) with intestinal metaplasia. Dysplastic lesions characterized by cellular atypia, abnormal differentiation, and disorganized mucosal architecture demonstrated higher TAG-72 expression than non-dysplastic epithelia. In contrast, normal gastric mucosa was generally non-reactive with MAb B72.3. Assays using serial sections of normal, benign and malignant gastric tissues with two MAbs (B1.1 and COL-6) directed against distinct epitopes of carcinoembryonic antigen (CEA) demonstrated differential reactivity, confirming that TAG-72 and CEA are distinct, non-coordinately expressed antigens. Our results suggest that TAG-72 antigen may be expressed in malignant and dysplastic epithelial cells, as well as in intestinalized epithelium of the stomach which has been closely related to subsequent carcinoma development. Hence, MAb B72.3 may be a useful immunohistochemical adjunct for detecting early foci of adenocarcinomas and premalignant lesions of the stomach.     

Comparison of EGFR, c-erbB-2 product and ras p21 immunohistochemistry as prognostic markers in primary breast cancer.

In a prospective study of 75 breast carcinomas, monoclonal antibodies EGFR1, Anti-serum 21N (As21N) and RAP-5 were used to assess immunohistochemically expression of Epidermal Growth Factor Receptor (EGFR), c-erbB-2 oncoprotein and ras protein p21. A careful comparison was made of their relative prognostic significance. Positive staining was seen with EGFR1 in 12/71 cancers (17%) and with As21N in 16/75 cancers (21%). Positive staining with RAP-5 occurred in all cancers and benign breast tissue, but varied in intensity. EGFR expression correlated with the number of involved lymph nodes, histological grade, estrogen and progesterone receptor (ER, PR) levels and the Melbourne Prognostic Index. C-erbB-2 and ras expression both correlated with ER levels and EGFR, but not with the Prognostic Index. Based on an immunohistochemical technique, EGFR expression emerges as the parameter with strongest prognostic associations.     

Expression of ras oncogene p21 protein in early gastric carcinoma and adjacent gastric epithelia

The expression of the ras gene product p21 in normal gastric mucosa, early gastric carcinoma of diffuse (gastric) and intestinal types, and in adjacent mucosal abnormalities is reported. The analysis was performed on paraffin sections by an immunohistochemical assay using the mouse monoclonal antibody RAP-5 and the rat monoclonal antibody Y13-259. Expression of ras p21 was assessed by staining intensity and percentage of positively stained cells. In comparison to normal gastric mucosa of non-cancer patients, p21 was overexpressed in nearly all early carcinomas of both types and in the dysplastic and/or metaplastic mucosal alterations accompanying intestinal type of gastric cancer. Increased p21 expression was also observed in the normal-appearing mucosa adjacent to early carcinomas of diffuse type, but not in the morphologically normal gastric epithelium adjacent to the intestinal type. The results of this investigation suggest that ras p21 overexpression may be related to early events of human gastric carcinogenesis. The study supports the notion of different pathways in the development of diffuse (gastric) and intestinal types of gastric carcinomas.     

ras oncogene p21 as a tumor marker in the cytodiagnosis of gastric and colonic carcinomas

This study was undertaken to determine whether the expression of ras oncogene product p21 can be used as a tumor cell marker of gastric and colonic carcinoma in brush smears. To detect p21 an immunocytochemical assay with RAP-5 monoclonal antibody was used. Benign epithelial gastric cells obtained from normal gastric mucosa or benign gastric lesions reacted negatively in 12 out of 13 cases. Similarly, benign epithelial colonic cells from normal colon or benign colonic lesions were negative for p21 in nine out of ten cases. Weakly positive reaction, confined to a few cell clusters only, was observed in one smear of a benign gastric ulcer and one smear of chronic ulcerative colitis. All 20 smears from colonic carcinoma and all 20 smears of gastric carcinoma contained cells that stained positively for p21, and the degree of tumor differentiation had no impact on the staining pattern. The results recorded in this study show that the immunocytochemical assay for the ras oncogene product may prove to represent a new tool for the cytodiagnosis of gastric and colonic carcinomas.     

Immunohistochemical study of the ras oncogene expression in human breast lesions

An immunohistochemical study of ras oncogene expression in human breast lesions was carried out using a monoclonal antibody, Y13 259, to the ras encoded p21 protein. A total of 75 cases of breast disease examined included: 33 simple and complex cystic disease; 22 simple and hyperplastic fibroadenomas; 18 ductal, lobular and mixed carcinomas and 2 in situ carcinomas. Most of the complex cystic disease, hyperplastic fibroadenomas and all types of carcinomas showed high p21 expression as indicated by staining intensity. These results suggest that elevated ras expression may play an important role in the development of some premalignant and malignant breast lesions.     

Evaluation of a monoclonal antibody to ras peptide, RAP-5, claimed to bind preferentially to cells of infiltrating carcinomas

RAP-5, a monoclonal antibody raised against a p21ras peptide, has been claimed to show immunohistochemical localisation of cells with infiltrative properties in human tumours. We confirmed that this antibody reveals pronounced cellular heterogeneity in human colonic neoplasms but could find no obvious relationship to infiltrative activity. RAP-5 bound to many different cell types, neoplastic and normal. In order to clarify the specificities of RAP-5 we applied it to two cell lines: nontumorigenic hamster fibroblasts in which ras expression is barely detectable, and a vigorously tumorigenic line derived from these fibroblasts by insertion of the human mutated Ha-ras oncogene in a high expression vector. Another antibody to p21ras, Y13-259, clearly distinguished between these cell lines both on immunoblots and immunocytochemically, but RAP-5 did not. Rather, it bound to proteins of a variety of molecular weights in both cell lines. The results show that RAP-5 is unlikely to be a useful reagent for detection of ras associated proteins in human tissues.     

Point Mutation of c-Ki-ras Oncogene in Gastric Adenoma and Adenocarcinoma with Tubular Differentiation

The presence of point mutation at codons 12,13 and 61 of the c-Ki- ras oncogene was investigated in 7 cases of gastric adenoma and 35 cases of gastric adenocarcinoma using DNA samples from formalin-fixed and paraffin-embedded tissues. Oligonucleotides encompassing the three codons were amplified by using the polymerase chain reaction (PCR), and then examined for point mutation by the selective oligonucleotide hybridization technique. Point mutation was detected in three of the 7 adenomas (43%) and three of the 35 carcinomas (9%). All the gastric adenomas showed the histology of tubular adenoma, being very similar to that of colonic adenoma. The 35 cases of gastric adenocarcinoma were classified into 17 cases of differentiated type and 17 cases of undifferentiated type including signet-ring cell carcinoma. The point mutation of c-Ki- ras oncogene was detected only in the differentiated type (3/17, 18%), and there was no case with point mutation in the undifferentiated type. These results suggest that the genetic mechanism of carcinogenesis differs between the differentiated type and the undifferentiated type of gastric adenocarcinoma, and also that c-Ki- ros activation is possibly involved in a relatively early step of the "adenoma-carcinoma sequence," which leads to the development of a portion of differentiated adenocarcinomas in the stomach.     

Immunohistochemical detection of the ras oncogene p21 product in an experimental tumour and in human colorectal neoplasms

The monoclonal antibody Y13 259 to the ras oncogene protein product p21 was used in an immunohistochemical study of ras expression in human colorectal neoplasms. The ability of the antibody to detect enhanced levels of ras expression was confirmed by its use with an experimental neoplasm known to express ras at high levels. Human colonic adenocarcinomas in general showed a similar staining intensity to that seen in normal mucosa. Adenomas however showed consistently high p21 expression as indicated by staining intensity. This suggests that elevated ras expression may be important in the development of adenomas, but that high levels need not be sustained in the conversion to invasive carcinoma.     

Differential expression of p21ras gene products among histological subtypes of fresh primary human lung tumors

Activation of the ras oncogene has been associated with a number of human tumors. In this study, expression of p21ras in different histological types of fresh primary bronchogenic carcinomas was examined. p21ras products were detected in all lung tissues that were analyzed. Only 1 of 23 tumors demonstrated aberrant migration of p21ras. In contrast, 10 tumors had substantially elevated levels of p21ras products with respect to the adjacent normal lung tissues and with respect to the other lung tumors. There was no correlation between increased ras protein expression and tobacco exposure of the patient or extent of disease at the time of diagnosis. However, 9 of 11 tumors with a squamous component as opposed to only 1 of 12 tumors belonging to other histological classifications demonstrated increased p21ras. These data suggest that, in bronchogenic carcinomas, mutations associated with structural abnormalities and aberrant migration of p21ras occur infrequently as compared to quantitative changes in p21ras. Furthermore, differential expression of c-ras products in primary human lung tumors correlates with pathological cell type, and may be a common event in squamous cell carcinomas, but not adenocarcinomas or small cell carcinomas of the lung.     

Ha-ras oncogene product in human gastric carcinoma: correlation with invasiveness, metastasis or prognosis

c-Ha-ras oncogene product in human gastric carcinomas was examined by Western blotting and immunohistochemistry using anti-Ha-ras p21 antibody. In Western blotting, high levels of c-Ha-ras p21s were found in gastric carcinomas. Immunohistochemically, c-Ha-ras p21 was detected in 3 (11.1%) of 27 early carcinomas and in 63 (43.8%) of 144 advanced carcinomas. In advanced carcinomas, c-Ha-ras p21-immunoreactivity was correlated with the depth of tumor invasion and was stronger in metastatic tumors than in primary tumors. Patients with c-Ha-ras p21-positive carcinomas had a significantly worse prognosis than those with p21-negative carcinomas.     

Activation of H-ras oncogene in rat bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine

Ras-oncogene activation was investigated in the bladder tumors of F344 male rats given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water. DNA from one of the nine transitional cell carcinomas contained an H-ras oncogene detectable by the NIH/3T3 transfection assay. Analysis of p21 ras proteins suggested that the activating mutation resided within codon 61 of the H-ras gene and that such activating mutations were not present in other tumors. In contrast to mutational activation of ras genes, enhanced expression of p21 was observed in all tumors examined by immunohistochemical techniques with the use of Formalin-fixed paraffin-embedded tissue sections and an anti-ras p21 antibody, RAP-5. Further histochemical analysis of bladder tissues at various stages of the BBN-induced carcinogenic process indicated that the enhanced expression of p21 appeared early; the reactivity with RAP-5 was observed in diffuse hyperplastic epithelia after 5 weeks of exposure to BBN. The frequency of ras oncogenes, activated either by point mutations or overexpression of p21, in BBN-induced rat bladder carcinomas has thus been shown to be similar to that observed in human bladder carcinomas.     

Site-specific Mutation of the Human c-Ha-ras Transgene Induced by Dimethylbenzanthracene Causes Tissue-specific Tumors in Mice

Forestoraach squamous cell carcinomas, lung adenocarcinomas and spleen angiosarcomas were induced by dimethylbenzanthracene (DMBA) in the rasH2 transgenic mouse line carrying human c-Ha-ras genes with their own promoter, encoding the prototype p21 gene product. Fifteen out of 21 mice (71%) developed forestomach squamous cell carcinomas, while 15 out of 21 (71%) had lung adenocarcinomas and 3 out of 21 (14%) showed spleen angiosarcomas within 8 weeks after a single administration of 50 mg/kg DMBA intraperitoneally. Somatic mutation at the 61st codon of the transgenes, from CAG(Gln) to CTG(Len), was detected in all these newly developed tumors. However, non-transgenic littermates demonstrated no tumors at all. These findings provide strong evidence that the somatic mutational activation of human c-Ha- ras genes is a critical event in tumorigenesis and a close relationship is therefore strongly suggested between the tissue-specific development of tumors and the somatic mutation of human c-Ha-ras genes in these rasH2 transgenic mice.     

Immunohistochemical study of oncogene product ras p21, c-myc and growth factor EGF in breast carcinomas.

The expression of the oncogene products ras p21, c-myc and the growth factor EGF (epidermal growth factor) was studied immunohistochemically in the tissue of 119 benign and malignant human breasts. In most cases, histologically normal breast tissues and benign lesions were found to be negative or poorly-expressive for reactivity with each antibody. Similar findings were observed in carcinoma in situ. Invading breast carcinomas demonstrated a significantly higher percentage of stained cells than that observed in benign lesions or carcinoma in situ; forty-two of 66 invasive breast carcinomas (63.6%) were highly-expressive for ras p21, thirty-eight (57.6%) for c-myc and twenty (30.3%) for EGF, but overall correlations between each oncogene expression and the clinical stage, tumor size or degree of differentiation were not found. The overall 5-year survival rate was studied in 58 patients with Stage II and III in association with each oncogene or EGF expression. Their survival rate was significantly effected by the EGF expression (0.05 less than p less than 0.1) but not by ras p21 or c-myc expression. Analysis of 36 specimens available with ER (estrogen-receptor) level revealed a significant correlation between the ER status and c-myc or E2 (estradiol) and a significant inverse correlation between ER status and ras p21 or EGF expression (P less than 0.05). The expression of ras p21, EGF and c-myc was not associated with metastatic tumor progression.     

Expression of ras oncogene p21 protein in relation to regional spread of human breast carcinomas

The oncogenes most frequently detected in human tumors belong to the ras gene family (Ha-ras, Ki-ras, and N-ras). These genes encode a group of closely related 21,000 dalton proteins termed p21. An immunohistochemical study of ras p21 expression was carried out on paraffin sections of 54 human breast carcinomas using monoclonal antibodies to p21. The control group consisted of ten cases of benign fibrocystic disease. The p21 expression was significantly higher in cancer cells than in epithelial cells of control specimens. No correlations, however, were observed between oncogene product expression and tumor size, histologic type, or grade. As a group, tumors with axillary lymph node metastases expressed higher levels of ras p21 than nonmetastasizing tumors. However, because of the significant overlap in individual p21 values, it is unlikely that the immunohistochemical assay for p21 could be used to predict the behavior of mammary carcinomas.