Reduced breast cancer risk with increasing serum folate in a case-control study of the C677T genotype of the methylenetetrahydrofolate reductase gene.

Breast cancer risk may be associated with folate status or the C677T genotype of the methylenetetrahydrofolate reductase (MTHFR) gene. We compared serum folate concentrations and C677T genotype in 141 breast cancer patients and 109 age-matched controls. Serum folate was significantly lower in cases compared to controls (geometric means, 5.7 versus 6.6 microg/l; P=0.005). Breast cancer risk was not associated with C677T genotype. After adjusting for age of menarche, parity, alcohol intake and total fat intake we observed reductions in odds ratios for breast cancer risk comparing the highest with the lowest quartiles of serum folate concentrations of 0.23 (95% confidence interval (CI) 0.09, 0.54) for the entire group, 0.27 (CI 0.09, 0.80) for the wild-type and 0.08 (CI 0.01, 0.52) for the heterozygous C677T genotype. We conclude that for the whole group, and the wild-type and heterozygous C677T genotypes, increased serum concentrations of folate were associated with reduced risks of breast cancer.

Hypermethylation of HpaII recognition sequences within the 5' coding region of the estrogen receptor gene is not associated with estrogen receptor negativity in primary breast tumours.

Estrogen receptor negative tumours are unable to respond to antiestrogen therapy. The underlying molecular mechanisms of estrogen receptor negativity are poorly understood. Cytosine mechylation is one of the mechanisms of gene control and previous studies, particularly on breast tumour derived cell lines have suggested that hypermethylation of HpaII recognition sequences within the 5' coding region of estrogen receptor may be responsible for gene inactivity. This study has examined the methylation status of HpaII recognition sequences in estrogen receptor positive and negative breast tumours taking into account a polymorphic HpaII site in the 5' coding region of the estrogen receptor gene. It is concluded that hypermethylation of one or more of the 4 or 5 HpaII recognition sequences in the 5' coding region of estrogen receptor is not associated with ER negativity in primary breast tumours.

Detection of p53 gene mutation by rapid PCR-SSCP and its association with poor survival in breast cancer.

We examined the association between mutation of the p53 gene and survival in a large cohort of breast cancer patients. Using a rapid, non-isotopic single-strand conformation polymorphism (SSCP) method we screened for mutations in exons 4-10 of the p53 gene in 375 primary breast cancers from patients with a median follow-up of 57 months. Mutations were found in 19% of tumours. Statistically significant associations were found between p53 mutation and histological grade, hormone receptor status, ploidy and S-phase fraction. No association was found between p53 mutation and axillary lymph node involvement, histological type, tumour size, vascular invasion or patient age. In univariate survival analysis, p53 mutation was strongly associated with poor prognosis. This was maintained in the lymph node-negative and hormone receptor-positive patient subgroups. In multivariate analysis, p53 mutation was associated with poor survival independent of lymph node status, estrogen receptor status and S-phase fraction. Our results demonstrate the feasibility of using a rapid and simple polymerase chain reaction-SSCP screening procedure to detect p53 gene mutation in breast cancer for the provision of prognostic information.

Expression of the PIP/GCDFP-15 gene and survival in breast cancer.

Expression of the PIP/GCDFP-15 gene was determined by measuring PIP/GCDFP-15-mRNA in breast carcinomas of 91 patients. The patients were followed-up for an average of 47 months after initial diagnosis and treatment of the disease. There were no deaths in the group of 14 patients with tumours of high PIP/GCDFP-15-mRNA levels, while 16 of 77 patients of the group with low PIP/GCDFP-15-mRNA tumour levels died. A similar advantage for high PIP/GCDFP-mRNA expression was observed with regard to disease free survival.

Hypermethylation of the myogenic gene Myf-3 in human breast carcinomas.

The methylation status of Myf-3 was studied in 34 human primary breast carcinomas and 9 normal breast tissues. One third of the carcinomas contained hypermethylated Myf-3. All normal tissues contained unmethylated Myf-3. Myf-3 hypermethylation was more common in poorly differentiated grade 3 tumours than in better differentiated grade 1 and 2 tumours.

Prospective study of fatty acid synthetase (FAS) expression in breast cancer and progression of disease.

Fatty acid synthetase (FAS) mRNA was determined in breast carcinomas of 91 patients. The patients were followed for an average of 47 months. Disease-free survival and overall survival were compared with the expression of FAS-mRNA in the tumour. Sixty-five patients remained disease-free during the follow-up period; twenty-six had recurrent disease, and of those 18 died of the disease. Seventy-five percent of the tumours had low FAS-mRNA levels and there were fewer deaths in this group of patients (15%) than in that with high FAS-mRNA levels (35%). The proportion of recurrences was also smaller in the group with low FAS-mRNA. The probability of survival for five years was over 80% for the low FAS-mRNA group, and about 50% for the high FAS-mRNA group.

The interaction between protein-kinase-C (pkc) and estrogens (review).

The interactions between protein kinase C (PKC) and the steroid hormone estradiol or its receptor (ER) are reviewed. Estradiol upregulates PKC both in vitro and in vivo in the ovary, the anterior pituitary and in mammary tissue of several mammalian species. The antiestrogen tamoxifen inhibits PKC. Activation of PKC leads to a marked decrease of ER protein and ERmRNA in human breast cancer cells and some other cell lines. Inhibition or down-regulation of PKC enhances ER binding. These results indicate that there are links between the PKC signal transduction pathway and the steroid receptor family. Further studies are needed to clarify the role of PKC isoforms in normal and cancerous tissues which are known to be influenced by estradiol.

Prospective-study of stromelysin-3 expression in breast-cancer biopsies and disease-free survival.

Stromelysin-3 was estimated by Northern blot analysis in primary breast carcinomas. The patients were followed for an average of 47 months after initial surgery and the dates of recurrent carcinoma and of death, if applicable, were recorded. Stromelysin-3 expression in the primary tumour was found equally in patients with subsequent recurrent disease and in patients who remained disease-free during the study period. There was no correlation between stromelysin-3 levels and disease-free survival.

Evaluation of the expression levels of nm23-H1 mRNA in primary breast cancer, benign breast disease, axillary lymph nodes and normal breast tissue.

Expression levels of nm23-H1 were evaluated in a variety of normal benign and malignant breast tissues by Northern and slot blot. Tissues from 153 patients presenting with palpable breast lesions were studied: 132 primary infiltrating breast cancers, 9 pure duct carcinoma in situ lesions, a phyllodes tumor, 9 benign lesions and 2 local recurrences of carcinoma. In addition to lesional tissue, 49 samples of macroscopically normal breast tissue, 37 axillary lymph nodes and 9 samples from patients undergoing cosmetic reduction mammoplasty were studied. Sets of normal breast tissue, primary tumor and lymph node tissue from individual patients were available for comparison in 37 cases. A wide range of gene expression was detected in the various tissue types. The highest levels of expression were detected in malignant samples with in situ carcinomas being associated with the highest levels of gene expression. The expression levels of nm23-H1 in normal breast tissue were lower than the corresponding tumors from the same patients (p < 0.0005). Benign breast lesions (including 6 fibroadenomas) had levels of gene expression approximating those of the normal tissue samples. Normal axillary lymph nodes had significantly lower levels of nm23-H1 expression than nodes with metastatic deposits (p < 0.03). No significant association was observed between nm23-H1 expression levels and axillary node status in patients with infiltrating carcinoma, although there was a slight trend toward lower nm23-H1 mRNA levels in the node negative group.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of hormone replacement therapy on prognostic indices in women with breast cancer.

OBJECTIVE: To examine the effect of hormone replacement therapy (HRT) on breast cancer biology. PATIENTS: Four hundred and sixty Western Australian women with breast cancer who were 40 years or over at the time of their breast cancer surgery. DESIGN AND SETTING: A questionnaire was sent to women seeking information on the use of HRT before breast cancer surgery. To qualify as HRT users, HRT had to have been used continuously for six months or more up to within two weeks of surgery. MAIN OUTCOME MEASURES: Biochemical indices of oestrogen receptor, progesterone receptor, cathepsin D and protein levels, and pathological indices of tumour size, tumour differentiation and lymph node involvement for users and nonusers of HRT. RESULTS: Eighty-seven per cent of questionnaires were recovered and 39 HRT users and 258 non-users were analysed. Twenty-five HRT users used a combination of oestrogen and progestogen and 14 used oestrogen only. Twenty-six had used HRT for more than two years and 13 for two years or less at the time of breast cancer diagnosis. There were no significant differences in the tumour indices between these two groups. The mean level of oestrogen receptors appeared to be lower in oestrogen-only users than in combination HRT users and non-users. The mean cathepsin D level was significantly higher in oestrogen-only users than in non-users. The percentage of all HRT users with involved lymph nodes (23%) was significantly lower than the percentage of non-users (44%). CONCLUSION: Oestrogen-only HRT may have a detrimental effect on tumour biology. The use of a progestogen in combination with oestrogen may offer some protection. On the other hand, HRT users had less lymph node involvement with tumour. This may reflect early detection with increased surveillance in women using HRT.

Expression of stromelysin-3 and nm23 in breast carcinoma and related tissues.

Biological functions proposed for the ST3 and nm23 genes in tumour development and progression seem to be directly opposed. Stromelysin-3 (ST3) is a putative member of the matrix metalloproteinase family. ST3 has been implicated in the progression of epithelial malignancies, specifically with regard to an invasive (and therefore potentially metastasizing) phenotype. The nm23 gene, on the other hand, encodes a nucleoside diphosphate kinase which allegedly has a metastasis-suppressor-type function. It was therefore of interest to compare the expression of ST3 and nm23 in various surgically excised normal and neoplastic breast tissues. RNA was isolated from over 200 surgical specimens and studied by Northern blots. Normal breast tissues did not express ST3, and ST3 expression was detected in only 1 of 20 normal axillary lymph nodes. None of 7 fibroadenomas expressed ST3. In contrast, 60% of primary and metastatic breast carcinomas contained ST3-mRNA. The expression of ST3 was mainly confined to invasive carcinomas and was observed less frequently in pure ductal carcinoma in situ (DCIS) lesions. Our results support the suggestion that ST3 expression is related to the malignant process in breast cancer. The role of nm23 is far less clear-cut.

Hormone-regulated genes (pS2, PIP, FAS) in breast cancer and nontumoral mammary tissue.

Expression of the hormone-regulated genes, pS2, prolactin-inducible protein (PIP) and fatty acid synthetase (FAS), was investigated by Northern blotting in primary breast carcinoma, metastatic breast cancer in axillary lymph nodes, in uninvolved breast tissue from mastectomies and in normal lymph nodes. There were considerable differences in expression of the genes between the tissues. The proportion of tissues containing PIP-mRNA decreased from uninvolved breast tissue to primary breast carcinoma to metastatic carcinoma. The reverse applied to FAS-mRNA which was found more often in metastatic cancer than in primary cancer, and least frequently in uninvolved breast tissue. Yet another pattern was observed for pS2 expression. The highest proportion of tissues demonstrating gene expression was found in primary breast cancer with both metastatic tumor and uninvolved breast tissue expressing the gene less frequently. pS2-mRNA and PIP-mRNA could only rarely be detected in trace amounts in normal lymph nodes. In contrast, FAS-mRNA was present in about one third of normal lymph nodes. Only pS2-mRNA showed an association with estrogen and progesterone receptor status.

The relative prognostic significance of total cathepsin D and HER-2/neu oncogene amplification in breast cancer. The South Australian Breast Cancer Study Group.

Total tumor cathepsin D (TCD) levels were determined prospectively by a radioimmunometric assay in tumor cytosol of 858 primary breast cancer patients diagnosed between 1989-1991. In 581 of these patients, tumor HER-2/neu oncogene amplification was simultaneously determined. In a "training-set" of 313 patients, "high" TCD was associated with significantly shorter disease-free survival (DFS). For the whole group, there was no correlation between TCD and pathologic stage, number of axillary nodes with tumor deposits, tumor size, histologic type and grade, or hormone receptor levels. In the node-positive group, high TCD level was associated with HER-2/neu amplification. After a median follow-up duration of 31 months, univariate analysis indicated that high TCD level was significantly associated with shorter DFS only in node-positive patients. The shorter DFS in association with high TCD levels was observed in both estrogen-receptor-positive and -negative patients. Cox multivariate analysis of DFS confirmed that high TCD level was predictive of shorter DFS in node-positive patients only. Because of the short duration of follow-up, the significance of TCD in overall survival was not determined. We conclude that high tumor TCD in node-positive patients is predictive of shorter DFS, and is often associated with HER-2/neu amplification. The possibility exists that high tumor TCD may act in combination with HER-2/neu amplification to promote dissemination of metastases.

Expression of the pS2 gene in normal breast tissue.

pS2 expression in normal breast tissue removed for cosmetic reasons was significantly lower than in uninvolved breast tissues from mastectomies for breast carcinomas. It is speculated that the presence of the carcinoma, or factors related to its development, could be the reason for this difference.

Cathepsin-D in human breast cancer: correlation with vascular invasion and other clinical and histopathological characteristics.

The relationship between the Cathepsin-D concentration in breast cancer cytosols and clinical and histopathological characteristics of the tumours was investigated, including vascular invasion, histological type, histological grade, lymph node involvement and tumour size. The median cathepsin-D concentration of a series of 738 primary breast carcinomas was used to define "low" and "high" cathepsin-D. High cathepsin-D concentration was associated with peritumoral vascular invasion, with high grade infiltrating duct carcinomas, with tumours of > or = 2 diameter, and with metastases in the axillary lymph nodes. Low cathepsin-D concentration was associated with in-situ carcinomas.

Stromelysin-3 expression in breast cancer biopsies: clinico-pathological correlations.

Stromelysin-3 expression was studied by Northern blotting in 222 tissue samples including primary and metastatic breast carcinoma and normal breast tissue. Uninvolved breast tissue from mastectomy specimens, normal breast tissue from reduction mammoplasties and normal lymph nodes did not contain stromelysin-3 mRNA. About 62% of primary and metastatic breast carcinomas, but only 1 of 10 in situ ductal carcinomas, expressed stromelysin-3. Stromelysin-3 mRNA was found more often in estrogen-receptor-positive carcinomas and in histological grade-1 carcinomas. There was no significant correlation between stromelysin-3 expression and other prognostic factors, including tumor size, lymph-node involvement, age of patient, vascular invasion and cathepsin-D.

Expression of the pS2 gene in breast cancer--a comparison of pS2 protein radioimmunoassay and immunohistochemistry.

pS2 expression was studied in a series of 82 primary breast carcinomas using and comparing a radioimmunoassay (RIA) technique and immunohistochemistry (IPOX). There was close correlation of the results obtained with each technique. Accurate and reliable determination of pS2 status in breast cancer can be made on the basis of immunohistochemistry using formalin fixed paraffin embedded sections. Immunohistochemical determination of pS2 status may be used in situations where the RIA technique cannot be applied, i.e. instances when fresh tumor tissue is not available.

Detection of estradiol-induced messenger RNA (pS2) in uninvolved breast tissue from mastectomies for breast cancer.

pS2 mRNA was estimated in uninvolved breast tissue and breast carcinoma from the same patients. pS2 mRNA was clearly detected in 14 of 59 uninvolved breast tissues and in 30 of 58 breast carcinomas. pS2 mRNA was found more frequently in uninvolved breast tissue of premenopausal women than in that of postmenopausal women.

Expression of the pS2 gene in breast tissues assessed by pS2-mRNA analysis and pS2-protein radioimmunoassay.

The expression of the pS2 gene in breast tissues was assessed by measuring pS2-protein using a radioimmunoassay, and by determining pS2-mRNA using Northern blotting. There was a good correlation between the two measurements, indicating that expression of the pS2 gene in breast tissues may be assessed by either method. Since radioimmunoassay is technically easier and more efficient than Northern blotting, radioimmunoassay will be the method of choice in routine applications.