Methylation profile of triple-negative breast carcinomas.

Breast cancer is a group of clinically, histopathologically and molecularly heterogeneous diseases, with different outcomes and responses to treatment. Triple-negative (TN) breast cancers are defined as tumors that lack the expression of estrogen receptor, progesterone receptor and epidermal growth factor receptor 2. This subgroup accounts for 15% of all types of breast cancer and its prevalence is higher among young African, African-American and Latino women. The hypermethylation of CpG islands (CpGI) is a common epigenetic alteration for suppressing gene expression in breast cancer and has been shown to be a key factor in breast carcinogenesis. In this study we analyzed the hypermethylation of 110 CpGI within 69 cancer-related genes in TN tumors. For the methylation analysis, we used the methyl-specific multiplex-ligation probe amplification assay. We found that the number of methylated CpGI is similar between TN and non-TN tumors, but the methylated genes between the groups are different. The methylation profile of TN tumors is defined by the methylation of five genes (that is, CDKN2B, CD44, MGMT, RB and p73) plus the non-methylation of 11 genes (that is, GSTP1, PMS2, MSH2, MLH1, MSH3, MSH6, DLC1, CACNA1A, CACNA1G, TWIST1 and ID4). We conclude that TN tumors have a specific methylation profile. Our findings give new information for better understanding tumor etiology and encourage future studies on potential drug targets for triple-negative breast tumors, which now lack a specific treatment.

Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer.

Using the clinically relevant 4T1-derived syngeneic murine model of spontaneous mammary metastasis to bone, we have identified the cysteine cathepsin inhibitor Stefin A as a gene differentially expressed in primary and metastatic mammary tumours. In primary tumours, Stefin A expression correlated inversely with metastatic potential in 4T1-derived lines and was not detected in tumour cells in culture, indicating induction only within the tumour microenvironment. Enforced expression of Stefin A in the highly metastatic 4T1.2 cell line significantly reduced spontaneous bone metastasis following orthotopic injection into the mammary gland. Consistent with the mouse data, Stefin A expression correlated with disease-free survival (absence of distant metastasis) in a cohort of 142 primary tumours from breast cancer patients. This was most significant for patients with invasive ductal carcinoma expressing Stefin A, who were less likely to develop distant metastases (log rank test, p = 0.0075). In a multivariate disease-free survival analysis (Cox proportional hazards model), Stefin A expression remained a significant independent prognostic factor in patients with invasive ductal carcinoma (p = 0.0014), along with grade and progesterone receptor (PR) status. In human lung and bone metastases, we detected irregular Stefin A staining patterns, with expression often localizing to micrometastases (<0.2 mm) in direct contact with the stroma. We propose that Stefin A, as a cysteine cathepsin inhibitor, may be a marker of increased cathepsin activity in metastases. Using immunohistology, the cathepsin inhibitor was detected co-expressed with cathepsin B in lung and bone metastases in both the murine model and human tissues. We conclude that Stefin A expression reduces distant metastasis in breast cancer and propose that this may be due to the inhibition of cysteine cathepsins, such as cathepsin B.

c-erbB-2 (HER-2/neu) protein and drug resistance in breast cancer patients treated with induction chemotherapy.

Expression of c-erbB-2 protein has been associated with poor prognosis and poor response to chemotherapy in breast cancer patients. In the present prospective study, we have analyzed whether c-erbB-2, p53 and P170 proteins may be determinants of tumor resistance in locally advanced breast cancer patients treated with induction chemotherapy. Biopsies (n = 60) were examined by immuno-histochemistry; in 62% of cases core or incisional biopsies were taken before drug administration, allowing comparison in paired biopsies of the cytological and molecular changes induced by treatment Sixty percent of the patients received relatively high doses of FAC or FEC (5-fluorouracil, doxorubicin or epirubicin and cyclophosphamide), and 40% received relatively high doses of doxorubicin or epirubicin alone. No significant changes were observed in the molecular markers studied following chemotherapy; in the few biopsies where changes appeared, the changes did not exhibit any significant or similar trend. For 30 of the patients who received FAC/FEC treatment, follow-up reached a median of 34 months. In these cases, neither the clinical (reduction in tumor size) nor the histological (evaluated after neoadjuvant chemotherapy) responses showed statistically significant differences between the patients who developed distant metastases and the disease-free patients. c-erbB-2 was over-expressed in 50% of patients who developed distant metastases vs. 7% of the disease-free patients. Disease free survival (DFS) curves between c-erbB-2-positive and c-erbB-2-negative patients were statistically significant. No correlation between p53 or P170 expression with DFS was found. Our results suggest that c-erbB-2 protein expression is associated with development of distant metastases in breast cancer patients treated with relatively high doses of anthracyclines in induction chemotherapy.

Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy.

Heat shock proteins (Hsps) are induced in vitro by several cytotoxic drugs; in human breast cancer cells these proteins appear to be involved in anti-cancer drug resistance. The present report was designed to analyze whether chemotherapy affects in vivo the expression of Hsp27, Hsp70, Hsc70 and Hsp90 in breast cancer patients treated with induction chemotherapy and whether these proteins may be determinants of tumor resistance to drug administration. We have analyzed 35 biopsies from breast cancer patients treated with induction chemotherapy. Expression of the Hsps in the tumors was compared with (i) histological and clinical responses to chemotherapy, (ii) tumor cell proliferation measured by proliferating cell nuclear antigen (PCNA) immunostaining and nucleolar organizer regions (AgNORs) staining and (iii) the expression of estrogen and progesterone receptors. We also compared disease-free survival (DFS) and overall survival (OS) with the expression of the Hsps studied. After chemotherapy, nuclear Hsp27 and Hsp70 expression was increased and Hsp70 and Hsc70 cytoplasmic expression was decreased. A high nuclear proportion of Hsp70 in tumor cells (>10%) correlated significantly with drug resistance. We also observed that patients whose tumors expressed nuclear or a high cytoplasmic proportion (>66%) of Hsp27 had shorter DFS. The combination of Hsp27 and Hsp70 levels showed a strong correlation with DFS. Neither the cellular proliferation nor the levels of steroid receptors showed any significant difference before or after drug administration or during follow-up of patients. Our results suggest that Hsp27 and Hsp70 are involved in drug resistance in breast cancer patients treated with combination chemotherapies.

Integration of estrogen and progesterone receptors with pathological and molecular prognostic factors in breast cancer patients.

In this study we have examined biopsies from women with localized primary breast cancer to investigate the prognostic performance of estrogen receptors (ER) and progesterone receptors (PR) for estimating the metastatic probability of the patients, and to explore whether discrimination gets better by combining clinicopathological and other molecular parameters into a score. This prospective study involved 205 patients with a median follow-up of 5 y. Among the evaluated clinicopathological data were: patient's age; tumor size; axillary lymph node involvement; and tumor grade. The most representative tumor samples were derived to a single laboratory for immunohistochemical evaluation of the following molecular markers: ER, PR, proliferating cell nuclear antigen (PCNA), p53 protein product, erbB-2 (HER-2/neu) oncoprotein, and P170 glycoprotein (mdrl gen product). Distant metastases (study endpoint) appeared in 19.5% (40/205) of the patients, most of these patients presented a mixture of poor, regular and good prognostic factors. Disease-free survival analysis procedures (Kaplan-Meier method) identified tumor size, axillary lymph node involvement, tumor grade, receptor status, PCNA, p53, erbB-2 and P170 as useful prognostic factors. Proportional hazard regression analysis (Cox) identified in order of importance erbB-2, tumor size, receptors status, tumor grade and PCNA as useful prognostic factors. To facilitate the evaluation of the prognostic factors, a practical and simple score system was derived. A high pathological score identified 65% of the patients that developed distant metastases, while a high molecular score was obtained in 57% of patients with metastatic disease. There was a significant improvement in the diagnosis of probability of being with distant metastases when the pathological score was combined with the molecular score, 82% of the patients with distant metastases showed an elevated combined score. Validation of this scoring system will need further larger studies (validation set as opposed to the training set used in the present study). Due to the complexity of events in cancer, the evaluation of a combination of prognostic factors should be of value to clinicians to make a more objective estimate of the prognosis of individual breast cancer patients.

Heat shock proteins and cell proliferation in human breast cancer biopsy samples.

Human breast cancers may overexpress certain heat shock protein (hsp) family members, proteins which are involved with cell proliferation and differentiation as well as with disease prognosis and drug resistance. Here, we have studied the relationship between the expression of two hsps (hsp27 and hsp70) and the proliferative activity of tumor cells in 40 biopsies from breast cancer patients. Twenty of these tumors were selected for a detailed colocalization study. Immunocytochemistry was done using specific antibodies against hsp27 and hsp70. Cell proliferation was studied analyzing the expression of proliferating cell nuclear antigen (PCNA) (late G1, S, and G2 phases of the cell cycle) and the number of silver-staining nucleolar organizer regions (AgNORs) (G1 phase). The colocalization study revealed a statistically significant inverse correlation between hsp27 expression and cell proliferation in 16/19 (84%) of the cases evaluated by PCNA immunostaining, and in 11/16 (69%) of the cases evaluated by AgNORs. In contrast, a statistically significant positive correlation between hsp70 expression and elevated cell proliferation was seen in almost 85% of the cases evaluated by PCNA staining, and in almost 50% of the cases evaluated by AgNORs. Moreover, in 22% (9/40) of the breast cancer samples examined, hsp70 was clearly associated with the mitotic spindle. A Western blot analysis revealed that hsp70 was coprecipitated with taxol-polymerized tubulin. The association of hsp70 with the mitotic spindle was not clearly noted in lung carcinoma samples (N = 20) or in normal cells displaying elevated mitotic activity. These studies thus demonstrate that in a significant percentage of clinical breast cancers hsp27 overexpression is inversely correlated with cell proliferation, while hsp70 is clearly associated with the mitotic spindle and cell proliferation. These results add evidence to the concept that in human breast cancers hsp27 may be involved in cell growth arrest and increased differentiation while, in contrast, hsp70 may be involved in cell proliferation; further studies will be necessary to elucidate these possible cause-and-effect relationships.

Estrogen receptors, progesterone receptors, and cell proliferation in human breast cancer.

The breast is a target organ for estrogens and progesterone. These hormones control several functions of the normal and abnormal mammary epithelium including cell proliferation. Most of the actions of estrogens and progesterone are mediated via specific steroid receptors, and one would expect that proliferating cells should contain estrogen receptors (ER) and/or progesterone receptors (PR). However, the correlation between receptor expression and cell proliferation is still controversial. In the present study we have examined 29 human breast cancer samples; in 17 of them we evaluated the simultaneous ER and PR localization with that of proliferating cell nuclear antigen (PCNA) and silver-stained nucleolar organizer regions (AgNORs) in a cell-by-cell study. We found that in almost 50% of the tumor biopsies examined, the cells expressing ER were significantly associated with elevated cell proliferation. In another group (38%) there were not significant differences between ER expression and cell proliferation. In only one of the samples (6%) the cells expressing ER showed lower cell proliferation. The study also revealed that in 44% of the tumors the PR expressing cells were associated with elevated cell proliferation. In a second group the PR expression was not significantly associated with cell proliferation (33% of the cases). Finally, in 22% of the samples the cells carrying PR showed lower cell proliferation. We also detected lower ER immunoreactivity in 30% of the breast cancer biopsies with one of the monoclonal antibodies against ER (antibody 1D5 directed against the A/B domain). This group of tumors was PR-negative (or very weakly positive) and had high proliferation. The presence of tumors with 'abnormal' ER proteins and displaying ER/PR significantly associated with elevated cell proliferation could have implications in human breast cancer treatment.

Corticotropin-releasing hormone, luteinizing hormone-releasing hormone, growth hormone-releasing hormone, and somatostatin-like immunoreactivities in biopsies from breast cancer patients.

The presence of immunoreactive adrenocorticotropin-releasing hormone (CRH), luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), and somatostatin has been investigated by immunohistochemistry in forty biopsies from breast cancer patients. All of these hypothalamic hormones were found in about 30% of the samples, seen in the cytoplasm or in the nuclei of the tumor cells. Positive immunostaining for the hypothalamic hormones was present in colloid, lobular, and infiltrating ductal carcinomas. There was not a clear relationship between occurrence of staining for the hypothalamic hormones and the histologic grade of tumors or the clinical stage of the disease. Immunoreactive LHRH was more frequently found in breast tumors with estrogen and progesterone receptors. On the other hand, preneoplastic breast lesions expressed mainly somatostatin, while immunoreactivity was absent in normal mammary tissue.