Evidence for a second tumor suppressor gene on 17p linked to high S-phase index in primary human breast carcinomas.

The short area of chromosome 17 is a frequent target for deletions in human tumors, including breast cancer. We have investigated by restriction fragment polymorphism analysis the pattern of loss of heterozygosity (LOH) at four loci on 17p13.1-17pter in a panel of 110 primary human breast carcinomas. A copy of the p53 gene was lost in 23% of the informative cases. Point mutations in the p53 gene were statistically associated with LOH at the same locus (p = 0.003) but not at other loci on 17p13.3-17pter. A second region bordered by the loci D17S5/D17S28 (17p13.3) and D17S34 (17pter) is also affected by LOH, independent of point mutations in the p53 gene. We propose the presence of a second tumor suppressor gene within this region. In support of this hypothesis is the significant association (p = 0.005) between LOH at the D17S5/D17S28, but not at the TP53 or D17S34 loci, and tumors having a high S-phase index.

Genetic and molecular heterogeneity of breast cancer cells.

We have undertaken a systematic study of primary human breast tumor DNAs to identify and characterize frequently occurring somatic mutations. Loss of heterozygosity (LOH) was found on chromosomes 1p (37%), 1q (20%), 3p (30%), 7 (41%), 13q (30%), 17p (49%), 17q (29%) and 18q (34%) in our tumor DNA panel. Specific subsets of tumors could be defined based on the particular collection of mutations they contained. One goal of these studies has been to determine whether there is a significant association between specific mutations and clinical parameters of the disease. We have found that LOH on chromosome 17p in tumor DNAs is associated with breast tumors having a high proliferative index and that LOH on chromosome 7 is associated with patients having a poor prognosis. Our analysis of chromosome 17 suggests that there may be as many as four tumor suppressor genes affected in primary human breast tumors.

In primary human breast carcinomas mutations in exons 5 and 6 of the p53 gene are associated with a high S-phase index.

A series of 121 human breast tumors was screened for point mutations in exons 5 through 8 of the p53 gene, by SSCP analysis. On the same tumor samples, the S-phase index (SPI) was determined by the incorporation of BUdR in fresh tissue. p53 mutations were observed in 29% of the cases. The frequency of point mutations for the individual exons was: exon 5, 10.0%; exon 6, 9.9%; exon 7, 7.1% and exon 8, 5.5%. Two mutations detected by SSCP were confirmed by sequencing the p53 cDNA. The presence of a p53 mutation, irrespective of its location, correlates (p = 0.003) with a high SPI. This association appears to primarily reflect mutations in exon 5 (p = 0.0002) and exon 6 (p = 0.05), since mutations in exons 7 and 8 failed to show any association. These results indicate that mutations in the p53 gene identify highly proliferating tumors, and that the position of the p53 mutation may have different effects upon the proliferative activity of tumor cells in vivo.

Survival of breast cancer patients from Piedmont, Italy.

A population-based series of 4,764 women from Piedmont, Italy, who were diagnosed with breast cancer during 1979-81 and for whom information on social and demographic factors was available, was followed-up for mortality until 1986 or 1987. Relative survival rates at one, three, and five years were 94.6, 81.6, and 71.1 percent, respectively, and were similar to those of other European series. Women aged 40-49 years at diagnosis experienced a better survival than women in other age groups. The mortality was highest between one and four years after diagnosis, and lowest between five and seven years. Survival rates were lowest for women above the age of 80, and single women had a worse prognosis than married women. Women with less than seven years of education had nonsignificantly lower survival rates than more educated women. No difference in survival was found according to occupation, size of town of residence, place of birth, or type of hospital.

Cell proliferation of breast cancer evaluated by anti-BrdU and anti-Ki-67 antibodies: its prognostic value on short-term recurrences.

The prognostic value of breast cancer proliferative activity was evaluated in 385 women operated for primary, non-metastasised mammary carcinoma. Cell kinetics was measured using two immunohistochemical techniques. Cells in S-phase of cell cycle were labelled in vitro by incubation of fresh tissue fragments with 5-bromo 2-deoxyuridine (BrdU), a thymidine analogue. Nuclei of cells in active DNA synthesis were stained by an anti-BrdU monoclonal antibody (Mab). Cells in interphase and mitosis were detected with Ki-67, a Mab that is known to react with a nuclear antigen present in G1/S/G2/M phases of cell cycle, but not in resting cells. This reagent provides a means of evaluating the growth fraction of neoplastic cells. BrdU was incorporated in a proportion of tumour cells ranging from 0.1 to 65.5% (median 6.8%). In the panel of tumours presented in this report the median percentage of Ki-67 positive cells (Ki-67 score) was 9.0% (range 0.1-77%). The relationship between disease-free survival (DFS), BrdU labelling index, Ki-67 score and 13 different clinico-pathological variables was investigated by multivariate analysis, using the Cox proportional hazards model. Axillary node status (P = 0.009) and Ki-67 score (P = 0.038) emerged as independent prognostic factors. Nodal status and tumour growth fraction allowed division of patients into groups at different risk of relapse: tumours with a proliferative index below the median value showed a lower recurrence rate than tumours with a high proliferative activity (P < 0.001). In particular, no relapse occurred in pN0 patients bearing carcinomas with a Ki-67 labelling < 9.0% 4 years after surgery. These findings suggest that the evaluation of proliferative activity in breast cancer enhances the probability of correctly predicting outcome after surgery and could be of assistance in the planning of adjuvant therapies.

Somatic mutations and human breast cancer. A status report.

A systematic study of primary human breast tumor DNA demonstrated that three proto-oncogenes or regions of the genome (c-myc, int-2, and c-erbB2) are frequently amplified and that there is loss of heterozygosity (LOH) on chromosomes 1p(37%), 1q(20%), 3p(30%), 7(41%), 11p(20%), 13q(30%), 17p(49%), 17q(29%), and 18q(34%). Specific subsets of tumors can be defined based on the particular collection of mutations they contain. For instance, LOH on chromosomes 11p, 17p, and 18q frequently occurs in the same tumor. A search for putative tumor suppressor genes within the regions of the genome affected by LOH has been started. In a comprehensive molecular analysis of the p53 gene on chromosome 17p, 46% of the tumors contained a point mutation in the p53 gene.

Loss of heterozygosity on chromosome 17p13 in breast carcinomas identifies tumors with high proliferation index.

The capacity of breast tumor cells to proliferate is considered a potential prognostic factor together with other histopathologic parameters. The authors determined the proliferation index on a large panel of human primary breast tumors by measuring the levels of incorporation of bromodeoxyuridine (BrdU) by fresh tumor specimens in culture. Previous analysis showed that the percentage of cells entering the S-phase of the cell cycle strongly correlates with tumor grade, tumor size, and estrogen and progesterone receptor status. The capacity of tumor cells to proliferate might be associated with specific genetic mutations in primary tumors. To test this hypothesis, a panel of 96 human breast carcinomas, for which the BrdU labeling index (LI) was known, were tested for loss of heterozygosity (LOH) or increased copy number (ICN) at chromosomes 1q, 3p, 13q, 17p, and 18q. On chromosome 17p, LOH and ICN were observed in 27% and 12%, respectively, of the informative breast tumors. The LOH on chromosome 17p was significantly associated with tumors having an elevated BrdU proliferation index (P = 0.022). No association (P = 0.45) was observed between BrdU LI and tumor size (T2 + T3 compared with T1), tumor grade, and lymph node status. Increased copy number on chromosome 17p, LOH or ICN on 1q, and LOH on 13q14, 18q, and 3p also showed no significant correlation with cell kinetic parameters. These data are consistent with the presence of a gene or genes on chromosome 17p13 near the YNZ22.1 locus whose normal functioning is necessary for controlling breast tumor cells proliferation in vivo.

Mutations in the p53 gene in primary human breast cancers.

Twenty-six primary breast tumors were examined for mutations in the p53 tumor suppressor gene by an RNase protection assay and nucleotide sequence analysis of PCR-amplified p53 complementary DNAs. Each method detected p53 mutations in the same three tumors (12%). One tumor contained two mutations in the same allele. Single strand conformation polymorphism analysis of genomic DNA and complementary DNA proved more sensitive in the detection of mutations. Combining this technique with the other two a total of 12 mutations in the p53 gene were demonstrated in 11 tumors (46%), and a polymorphism at codon 213 was detected in another tumor. Loss of heterozygosity on chromosome 17p was detected by Southern blot analysis in 30% of the tumor DNAs. Not all of the tumors containing a point mutation in p53 also had loss of heterozygosity of the remaining allele, suggesting that loss of heterozygosity may represent a later event.

[Tyrosine-phosphorylated proteins in human breast carcinoma]. / Proteine fosforilate in tirosina nel carcinoma mammario umano.

Proteins phosphorylated on tyrosine are detectable by antibodies against phosphotyrosine (P-Tyr antibodies) in cells transformed by oncogene-encoded tyrosine kinases. We used P-Tyr antibodies to investigate the existence of abnormal levels of phosphoproteins in human breast cancer. Three human breast cancer cell lines (SK-BR-3, MCF-7 and CG-5) and 37 human breast cancer specimens were examined by Western blot analysis and "in vitro" kinase assay. In the SK-BR-3 cell line three major phosphoproteins of the approximate Mr of 185,000 (p185), 135,000 (p135) and 110,000 (p110) were detected. The former was identified as the HER-2 gene product by specific antibodies against HER-2 encoded protein. In the other cell lines, a product of the approximate Mr of 170,000 (p170), together with a p135 and a p110, were phosphorylated on tyrosine. P185 and p170 were shown to have an associated tyrosine kinase activity. Two proteins, comigrating with p135 and p110, were found to be highly phosphorylated on tyrosine in 50% of the breast cancer samples, but not in samples harvested from 12 human tumors of the gastro-intestinal tract. These data show that 50% of human breast cancer samples display an abnormal level of tyrosine phosphorylated proteins.

Late cytokinetic abnormalities in irradiated rectal mucosa.

Late cytokinetic changes of the colonic crypt epithelium after radiation therapy were investigated. A monoclonal antibody to bromodeoxyuridine (anti-BrdU MAb) was used in tissue specimens previously incubated with BrdU to show S-phase cells by immunohistochemical technique. Endoscopic rectal biopsies were taken from 30 patients previously treated with radiotherapy for gynaecological cancer and from 50 patients with comparable but untreated neoplasms, as controls. Number and height distribution of S-phase cells were evaluated by dividing each crypt column into 5 equal longitudinal compartments. No statistically significant differences were found in total Labelling Index (LI) between controls and irradiated mucosa, whereas LI per crypt compartment, percentage of labelled compartments and percentage of BrdU-positive cells in the middle and superficial portions of the crypt were significantly higher in patients submitted to radiation therapy. This kinetic abnormality corresponds to a progressive shift of the major zone of DNA synthesis to the upper third of the crypt as a late reaction to radiation and represents an early step in the histogenesis of colorectal cancer. These results lend support to the view that there is a higher risk of colorectal carcinoma after pelvic irradiation.

Risk of breast cancer, diet and internal migrations in northern Italy.

The protective effect of birth in southern Italy as opposed to other regions was evaluated in a population-based case-control study of diet and breast cancer among residents in the province of Vercelli. Cases were 250 women with breast cancer diagnosed during 1983-1984 and controls 499 women randomly selected from the general population. The crude relative risk of breast cancer for women born in the south was 0.76 (95% confidence interval, 0.43-1.3). After adjustment for dietary and other potential confounders in multivariate analyses, the protective effect of place of birth disappeared (RR 1.0; 95% CI, 0.57-1.9). The study indicates that north-south differences in the incidence of breast cancer in Italy may in large part be attributed to different dietary habits.

Breast cancer and alcohol consumption: a case-control study in northern Italy.

From 1983 to 1986, a population-based case-control study of alcohol and breast cancer (250 cases and 499 controls) was conducted in a grape-farming area of northern Italy, where wine consumption is widespread. In the study population, 30% of women were abstainers and 15% reported alcohol intakes of 30 g/day or more. After adjustment for potential confounders, no appreciable association was evident for alcohol consumptions as high as 40 g/day. Women reporting intakes of more than 40 g/day showed approximately a 2-fold increase in the risk of breast cancer (relative risk, 1.9; 95% confidence interval, 1.1-3.3). A 2-fold increase in risk was observed for consumptions of more than 40 g/day of alcohol from wine, the most common alcoholic beverage in this population. These findings suggest that an association between alcohol intake and breast cancer may exist. However, the moderate risk observed seems to be limited to the relatively small group of women consuming daily amounts of alcohol in excess of 40 g, the equivalent of about half a bottle of wine or more.

Calorie-providing nutrients and risk of breast cancer.

A case-control study was conducted in Italy to investigate the role of diet in breast cancer. Cases were 250 women with breast cancer, and controls were a stratified random sample of 499 women from the general population. A dietary history questionnaire was used to measure the intake of total fat, saturated fat, animal proteins, and other macronutrients. In multivariate analyses, the relative risks of breast cancer for women in the highest quintile of consumption of saturated fat and animal proteins were 3.0 (95% confidence interval, 1.9-4.7) and 2.9 (1.8-4.6), respectively. A reduced risk was found for women who derived less than 28% of calories from fat versus greater than 36%. A similarly reduced risk was found for women who derived less than 9.6% of calories from saturated fat or less than 5.9% from animal proteins. These data suggest that during adult life, a reduction in dietary intake of fat and proteins of animal origin may contribute to a substantial reduction in the incidence of breast cancer in population subgroups with high intake of animal products.

Generation and selection of monoclonal antibodies identifying surface molecules discriminating between fetal and adult fibroblasts.

The present report describes the reactivity of two murine monoclonal antibodies, i.e., CB11 and CB43, the first obtained by using embryonic material as immunizer and the second elicited against a human cell line. These reagents recognize two distinct molecules marking a peculiar differentiation stage on fetal/embryonic fibroblasts. The first structure is a single chain of 14.5 kd, the second one a heterodimer of 40-80 kd. Even if these reagents are not strictly fibroblast restricted, translineage expression does not diminish the medical relevance of the reagents; in fact, they may have application in prenatal diagnostics and amniocentesis testing, in which it is necessary to discriminate between fetal and maternal cells.

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins.

A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture. Unlike what is commonly observed on glass or plastic, where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state.

Expression of the monoclonal antibody-defined CAR-3 epitope on neoplastic and preneoplastic lesions of the colon mucosa.

The AR-3 monoclonal antibody, which defines the tumor-associated antigen CAR-3, was previously found to be able to discriminate between neoplastic cells in gastric, pancreatic, colonic, ovarian and endometrial carcinomas and their normal counterparts. In fact, it strongly reacts with carcinomatous cells at the level of both the glycocalix and the cytoplasm, while its reactivity with normal tissues is restricted to the glycocalix of few mucin-producing epithelial cells. We have now investigated the reactivity of this antibody with immunohistochemical techniques on a series of formalin-fixed paraffin-embedded specimens, from precancerous and cancerous lesions of the large bowel which were classified as adenomas with mild, moderate or severe dysplasia, adenomas with cancer and adenocarcinomas, respectively. It was found that the intensity and extent of the staining correlated with the degree of dysplasia and that the highest expression of the CAR-3 epitope was detectable in adenocarcinomas. Also the localization of the staining in the lesions displayed an increasingly complex pattern, going from linear in adenomas with mild dysplasia to a very strong intracytoplasmic and/or intraluminal expression in adenomas with severe dysplasia or adenocarcinomas.