ERα-negative and triple negative breast cancer: Molecular features and potential therapeutic approaches

Triple negative breast cancer (TNBC) is a type of aggressive breast cancer lacking the expression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor-2 (HER-2). TNBC patients account for approximately 15% of total breast cancer patients and are more prevalent among young African, African-American and Latino women patients. The currently available ER-targeted and Her-2-based therapies are not effective for treating TNBC. Recent studies have revealed a number of novel features of TNBC. In the present work, we comprehensively addressed these features and discussed potential therapeutic approaches based on these features for TNBC, with particular focus on: 1) the pathological features of TNBC/basal-like breast cancer; 2) E₂/ERβ-mediated signaling pathways; 3) G-protein coupling receptor-30/epithelial growth factor receptor (GPCR-30/EGFR) signaling pathway; 4) interactions of ERβ with breast cancer 1/2 (BRCA1/2); 5) chemokine CXCL8 and related chemokines; 6) altered microRNA signatures and suppression of ERα expression/ERα-signaling by micro-RNAs; 7) altered expression of several pro-oncongenic and tumor suppressor proteins; and 8) genotoxic effects caused by oxidative estrogen metabolites. Gaining better insights into these molecular pathways in TNBC may lead to identification of novel biomarkers and targets for development of diagnostic and therapeutic approaches for prevention and treatment of TNBC.     

Prognostic evaluation of breast cancer in cytologic specimens

OBJECTIVE: To evaluate prognostic factors in breast cancer using cytologic samples and to determine the correlation between those factors and ploidy. STUDY DESIGN: Two hundred sixteen fine needle aspirates from patients with primary breast cancer were analyzed for expression of estrogen receptors (ERs), progesterone receptors (PRs), Ki-67 antigen, expression of p53 tumor suppressor gene and overexpression of c-erbB-2 using a standard immunochemical method. Not all subjects had all biomarker information because of the study design (c-erbB2 added later). The specimens were analyzed also for ploidy. We used the SAMBA 4000 image analysis system for quantification of the percent of cells stained positively by the different immunocytochemical stains andfor ploidy. RESULTS: A significant correlation wasfound between ER and PR and between Ki-67 and positive p53. Steroid receptor content was not significantly related to p53, Ki-67 or c-erbB2. No correlation was found between c-erbB2 and the other biomarkers. Ploidy had a significant correlation with all the biomarkers used. CONCLUSION: A reliable and rapid evaluation of markers for breast cancer can be achieved by measuring cells stained positively by immunocytochemical stains, as well as ploidy, by means of an image analysis system. ER, PR Ki-67, p53 and c-erbB2 had a significant correlation with ploidy and overall prognostic value in breast cancer.     

Human breast cancer associated fibroblasts exhibit subtype specific gene expression profiles

ABSTRACT: BACKGROUND: Breast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2) of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs) within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes. METHODS: To address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC) and six Her2+. RESULTS: We observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER + cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs likely contribute to the enhanced migration of breast cancer cells in transwell assays and may contribute to the unfavorable prognosis of Her2+ breast cancer. CONCLUSIONS: These data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer.     

Expression of hormone receptors predicts survival and platinum sensitivity of high-grade serous ovarian cancer

High-grade serous ovarian cancer (HGSOC) has abundant expression of hormone receptors, including androgen receptor (AR), estrogen receptor α (ER), and progesterone receptor (PR). The effects of hormone receptors on prognosis of HGSOC were first evaluated in online databases. Their prognostic values were then explored and validated in our inhouse TJ-cohort (92 HGSOC patients) and in a validation cohort (33 HGSOC patients), wherein hormone receptors were detected immunohistochemically. High expression of hormone receptors denoted longer progression-free survival (PFS), overall survival (OS), and platinum-free interval (PFI). Platinum-sensitive patients had higher expression of hormone receptors than their counterparts. Correlation analysis revealed significant positive correlations between hormone receptors expression and survival. AR, ER, and PR had predictive and prognostic values, alone and in combination. By receiver operating characteristic curve (ROC) analysis, co-expression of AR, ER, and PR had an improved predictive performance with an area under the curve (AUC) value of 0.945. Expression of hormone receptors predicts survival and platinum sensitivity of HGSOC. AR, ER, and PR might be feasible prognostic biomarkers for HGSOC by immunohistochemical analysis.     

Correlation between sex hormone receptors in normal and cancerous target tissues

According to the classic model of regulation of sex hormone receptors biosynthesis in target tissues, oestrogen stimulates and progesterone inhibits biosynthesis in both receptors. One of the consequences of this model is a direct correlation between oestrogen (ER) and progesterone receptors (PR) in target tissues. Here we investigate a correlation between ER and PR in calf endometrium and breast cancer (BC) tissues of women. A direct correlation was found between receptors in the calf endometrium (r = +0.70; p < 0.01). There were three variants of BC tissues: without correlation, with positive correlation (r = +0.49; p < 0.01), and with non-linear negative correlation between ER and PR. The last variant of samples were detected exclusively in patients operated in spring and fall. The non-linear negative correlation between ER and PR in BC tissues in spring and fall may indicate disregulation of sex hormone receptors biosynthesis under the influence of external factors.     

Epidermal growth factor receptors in human breast and endometrial carcinomas

The incidence and levels of epidermal growth factor receptor (EGFR) were studied in 67 breast tumors and 22 endometrial carcinomas. Estrogen receptors (ER) were also measured in all samples and progesterone receptors (PR) were analyzed in 57 breast samples and 21 endometrial tumors. A high level of EGFR expression is found in both breast and endometrial carcinomas, although the incidence of EGFR content is greater in breast carcinomas. 36% of breast tumors had EGFR at levels 3-49.5 fmol/mg membrane protein, whereas this percentage of positivity was 27% for endometrial tumors. In 51% of breast carcinoma and 73% of endometrial tumors, there was a positive ER content, whereas 53% of breast tumors and 62% of endometrial carcinomas were positive. A clear inverse relationship between EGFR content and ER and PR status has been observed in breast tumors. Our data confirm the previously described inverse correlation between expression of EGFR and estrogen receptors in human breast cancer. We also show here that there is a similar inverse relationship between EGFR content and ER levels in endometrial tumors.     

Expression of oestrogen receptor-beta in invasive breast tumours

Steroid hormone receptors are used routinely to predict endocrine responsiveness in patients with breast cancer. Two oestrogen receptors (ERs): ER alpha and ER beta have been identified. Although ER alpha and ER beta genes share a large degree of homology, it is generally thought that their distribution and function are substantially different in many tissues. Both of them may be expressed in normal and neoplastic tissues of the breast. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. Recent development of reliable antibodies to ER beta has provided opportunity to test immunohistochemical reactions detecting ER beta in archival breast tumours. The aim of our study was to learn more about the cellular mechanisms underlying the relationship of ER beta and progesterone receptor (PR) in breast cancer tissues, discriminating between hormone-dependent and hormone-independent tumours. ER alpha and PR content of tumour tissues of 154 patients with breast cancer were tested by in situ indirect immunohistochemical method parallel with ligand binding biochemical assay. ER beta was detected in 8 ER alpha-/PR+ breast carcinomas by immunohistochemical method too. Steroid hormone receptor content was analysed comparing to the histologic type and grading of the tumours. CONCLUSIONS: A considerable part of breast carcinomas belongs to the ER+/PR+ and ER-/PR- groups. About 1-2% of the tumours is expected to be ER alpha-/ER beta+/PR+ type. In such cases ER alpha negative reaction together with PR positivity can signal the necessity of the immunohistochemical detection of ER beta in routine histopathological practice, presenting the precise steroid hormone receptor status for the most effective endocrine therapy of the patients.     

Identification of oestrogen,progesterone receptor and human epidermal growth factor receptor 2 expression in mediastinal metastases of breast cancer obtained by endobronchial ultrasound‐guided transbronchial needle aspiration

Background

In breast cancer patients, the expression statuses of oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are crucial in the choice of treatment. Receptor expression in metastatic lesions can differ from the primary tumour. The aim of our study was to analyse the utility of endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) to obtain samples allowing the identification of ER, PR and HER2 expression in patients with mediastinal metastases of breast cancer.

Patients and methods

The clinical files of all patients with a final diagnosis of breast cancer mediastinal metastases diagnosed by EBUS‐TBNA in our institution were retrospectively analysed. The ability of EBUS‐TBNA to obtain samples that allowed hormone receptor and HER2 expression analysis was calculated.

Results

Twenty‐four patients were included. ER, PR and HER2 assessments could be performed in 22, 20 and 22 patients, respectively. In 20 of the 24 patients it was possible to investigate all three types of receptor expression. In the remaining four cases, where ER, PR or HER2 expression tests could not be performed, it was due to a lack of tissue. In cases with adequate results for EBUS‐TBNA and the primary tumour agreement was greater for ER (16/19) and HER2 (12/14) than PR (8/17). Based on receptor status, there was a change in the choice of treatment for five patients.

Conclusion

In patients with breast cancer mediastinal metastases, ER, PR and HER2 expression can be assessed in samples obtained by EBUS‐TBNA whenever a sufficient tissue sample is collected.     

Agreement of self-reported hormone receptor status with cancer registry data in young breast cancer patients

BackgroundThough breast cancer subtype is a key determinant of treatment choice and prognosis, few studies have assessed breast cancer patients’ knowledge of estrogen and progesterone receptor (ER/PR) status.MethodsWomen diagnosed with invasive breast cancer at age 18–64 years in 2007 were recruited from the Pennsylvania Cancer Registry, and mailed a questionnaire that asked respondents to identify their ER/PR status. There were 2191 respondents included in the analysis. Agreement between self-report and cancer registry ER/PR status was assessed using kappa statistic. Logistic regression was used to assess the association of demographic, socioeconomic, and tumor factors with inaccurate self-report of ER/PR status.ResultsFifty-nine percent of respondents reported ER/PR positive status, 15% reported ER/PR negative status, 17% responded ‘don’t know’, and 9% did not respond. Overall, there was 69% agreement between self-report and cancer registry data, and fair agreement as measured by kappa (0.36). After excluding women who did not know or did not report their ER/PR status, there was 93% agreement, and substantial agreement as measured by kappa (0.76). Women who were older, non-white, less educated, lower income, and had ER/PR negative disease were significantly more likely to inaccurately report their ER/PR status.ConclusionsThough a significant proportion of women do not know their hormone receptor status, women who reported their ER/PR status were accurate. Our results suggest room for improvement in patient knowledge of tumor subtypes, but also that self-reported ER/PR status may be a useful surrogate when medical record or cancer registry data is unavailable.     

Serum Thyrotropin Concentrations are not Predictive of Aggressive Breast Cancer Biology in Euthyroid Individuals

Objective: The potential influence of hypothyroidism on breast cancer remains incompletely understood. The objective of this study was to investigate the relationship between serum thyrotropin (thyroid-stimulating hormone, TSH) concentration and markers of aggressive breast cancer biology as defined by receptor expression profile, tumor grade, and American Joint Committee on Cancer (AJCC) stage characteristics.Methods: This was a retrospective cohort study of patients from 2002 to 2014. All breast cancer patients who had complete receptor (estrogen receptor, ER; progesterone receptor, PR; and human epidermal growth factor receptor 2, Her2/neu) and prediagnosis serum TSH data (n = 437) were included. All patients had 1 of 6 receptor profiles: ER+ PR+ Her2/neu-, ER+ PR- Her2/neu-, ER+ PR+ Her2/neu+, ER+ PR- Her2/neu+, ER- PR- Her2/neu+, or ER- PR- Her2/neu-. Log-transformed serum TSH concentrations were analyzed using multinomial and logistic regressions to identify potential relationships with markers of breast cancer aggressiveness.Results: Increasing serum TSH concentration was associated with a lower probability of having the receptor expression profile ER+ PR+ Her2/neu+ compared to patients with the ER+ PR+ Her2/neu- profile (odds ratio [OR] = 0.52, P = .0045). No significant associations between other receptor expression profiles and serum TSH concentration were found. All time-weighted and unweighted median serum TSH concentrations were within normal limits. No significant associations between serum TSH concentration and tumor grade, overall AJCC stage, tumor size (T), lymph node positivity (N), or presence of metastasis (M) were observed.Conclusions: Serum TSH was not associated with markers of breast cancer aggressiveness in our cohort.Abbreviations: AJCC = American Joint Committee on Cancer ER = estrogen receptor Her2/neu = human epidermal growth factor receptor 2 M = metastasis N = lymph node positivity OR = odds ratio PR = progesterone receptor T = tumor size TSH = thyroid stimulating hormone UCLA = University of California Los Angeles     

Immunocytochemistry on scrape cytology in breast cancer: will it unearth the weaker positives?

OBJECTIVE: To standardize the technique of immunocytochemical (ICC) assessment of estrogen (ER) and progesterone receptor (PR) status in breast cancer by scrape cytology and to compare the results with immunohistochemistry on paraffin blocks. STUDY DESIGN: ICC assessment for ER and PR was done on scrape smears from tissue samples in 200 cases of primary breast cancer. The results were compared to those obtained from immunohistochemical (IHC) evaluation of formalin-fixed paraffin same tissue samples. RESULTS: ER/PR positivity rates as well as staining scores were compared between the scrape smears and tissue sections. The concordance between cytology and histology was 84% for ER and 90% for PR. Both the positivity rates and the staining intensity scores were higher for cytochemistry than for histochemistry. CONCLUSION: The ICC method on scrape smears is a simple test with rapid turnaround time. The sample required is small, and antigen loss due to fixation and processing is minimal. This new method gives a higher yield of hormone receptor positivity and, when used in conjunction with the IHC method, may improve the pickup rate of ER-positive cases, thereby playing an important role in risk stratification and therapeutic decision making in patients with breast cancer.     

Comparative expression analysis of four breast cancer subtypes versus matched normal tissue from the same patients

Gene expression studies have been widely used in an effort to identify signatures that can predict clinical progression of cancer. In this study we focused instead on identifying gene expression differences between breast tumors and adjacent normal tissue, and between different subtypes of tumor classified by clinical marker status. We have collected a set of 20 breast cancer tissues, matched with the adjacent pathologically normal tissue from the same patient. The cancer samples representing each subtype of breast cancer identified by estrogen receptor ER(+/-) and Her2(+/-) status and divided into four subgroups (ER+/Her2+, ER+/Her2-, ER-/Her2+, and ER-/Her2-) were hybridized on Affymetrix HG-133 Plus 2.0 microarrays. By comparing cancer samples with their matched normal controls we have identified 3537 overall differentially expressed genes using data analysis methods from Bioconductor. When we looked at the genes in common of the four subgroups, we found 151 regulated genes, some of them encoding known targets for breast cancer treatment. Unique genes in the four subgroups instead suggested gene regulation dependent on the ER/Her2 markers selection. In conclusion, the results indicate that microarray studies using robust analysis of matched tumor and normal samples from the same patients can be used to identify genes differentially expressed in breast cancer tumor subtypes even when small numbers of samples are considered and can further elucidate molecular features of breast cancer.     

Lack of relationship between the expression of hsp27 heat shock estrogen receptor-associated protein and estrogen receptor or progesterone receptor status in male breast carcinoma

Estrogen, through estrogen receptors (ERs), may regulate the synthesis of progesterone receptors (PRs) and of a heat shock estrogen receptor-associated protein (hsp27). In female breast carcinoma (FBC) both proteins serve as surrogate indicators for the presence of functional ERs. In addition, the expression of these proteins was related to other prognostic indicators of value in female breast tumours. Endocrine disorders, hormone therapy and altered estrogen metabolism have been associated with the development of male breast cancer (MBC), suggesting that evaluation of the expression of ER, PR and hsp27 might improve our understanding of the biology of this tumour. ER and PR status and hsp27 expression were evaluated by immunohistochemistry in 16 primary MBC patients. The interrelationships between these parameters were established and compared with the clinicopathological data on the tumours. Ten (56%) MBC patients were ER-positive, 69% were PR-positive and all samples were hsp27-positive. Our series of MBC patients showed a positive correlation between ERs and PRs, however there was a lack of correlation between hsp27 and ERs or PRs. MBCs did not exhibit any correlation between the biomarkers studied and known prognostic indicators for females (e.g. Scarff-Bloom-Richardson (SBR) or modified SBR (MSBR) grade, T stage, lymph node status). This is the first published series reporting the incidence of hsp27 in MBC. The lack of association between the expression of ERs and hsp27 found in MBC differs from the results reported for FBC, moreover the expression of ERs, PRs or hsp27 did not correlate with the clinicopathological parameters that have prognostic value in females. Although the data were obtained from a relatively small sample population, our findings suggest that MBC and FBC are biologically different tumours with respect to the expression of the studied proteins.     

Assessment of hormone receptors in breast carcinoma by immunocytochemistry and image analysis. II. Estrogen receptors

Frozen sections of 30 invasive breast carcinomas were stained for estrogen receptors (ERs) and the tumor cell proliferative rate by an immunoalkaline phosphatase technique. The stained sections were evaluated for ER by the microTICAS image analysis system. Seventeen tumors were ER positive and 13 were ER negative by image analysis. There was 93% concordance between the ER results obtained by image analysis and those obtained by biochemical methods. One case that was ER negative by image analysis was weakly positive by biochemical assay; a second case was ER positive by image analysis but ER negative by biochemical assay. Twelve of the 17 ER-positive tumors were diffusely positive while 5 displayed considerable intratumoral heterogeneity, with tumor cells exhibiting a broad range of intensity of receptor expression. In most cases, the image analysis ER status coincided with the progesterone receptor (PR) status, but in a large minority of cases (41%) the ER status and the PR status differed. Tumors with a high growth fraction (greater than 30%), as measured by Ki-67 immunostaining, were uniformly ER negative. The results of this investigation suggest that immunohistochemical staining of frozen sections for ER aided by automated image analysis (1) reliably detects the receptor in breast carcinoma, (2) allows for the assessment of heterogeneity within tumors and (3) may be used as part of a panel of antibodies to markers of potential prognostic importance in a single small tissue sample.     

Estrogen and progesterone receptor status in primary breast cancer--a study of 11,273 patients from the year 1990 to 2002

The aim of this study was to gain insight of the breast cancer hormone receptor status of our patients, its stratification according to age as well as its changes during the period of 13 years. 11,273 patients with primary breast cancer from several towns in Croatia were included in this study. Patients' tumour specimens were collected from 1990 to 2002 and were analysed on estrogen (ER) and progesterone (PR) receptors in the Laboratory of the Department of Medical Oncology, University Hospital Centre Zagreb. More than half of our breast cancer patients had ER positive tumours (54.3%). We observed ER + tumours increased with age continuously, with highest percentage in the age group of 70 to 79 years (68.1%). Similarly, proportion of PR + tumours was higher in the older age groups, being the highest between 40 and 49 years (55.9%). During 13 years of the study, the increase in frequency and proportion of ER + tumours was observed (from 52% in 1990 to 62% in 2002) and decrease of PR + tumours (56% to 53%). We confirm previous findings that the risk of hormone dependent breast cancer increases with aging. Risk of ER + breast cancer increased for 10% from 1990 to 2002 and PR + tumours decreased for 3.5% in the same period.     

Comprehensive analysis of novel three-long noncoding RNA signatures as a diagnostic and prognostic biomarkers of human triple-negative breast cancer

Currently, traditional predictors of prognosis (tumor size, nodal status, progesterone receptor [PR], estrogen receptor [ER], or human epidermal growth factor receptor-2 [HER2]) are insufficient for precise survival prediction for triple-negative breast cancer (TNBC). Long noncoding RNAs (lncRNAs) have been observed to exert critical functions in cancer, including in TNBC. Nevertheless, systematically tracking expression-based lncRNA biomarkers based on the sequence data for the prediction of prognosis in TNBC has not yet been investigated. To ascertain whether biomarkers exist that can distinguish TNBC from adjacent normal tissue or nTNBC, we implemented a comprehensive analysis of lncRNA expression profiles and clinical data of 1097 BC samples from The Cancer Genome Atlas database. A total of 1510 differentially expressed lncRNAs in normal and TNBC samples were extracted. Similarly, 672 differentially expressed lncRNAs between nTNBC and TNBC samples were detected. The receiver operating characteristic curve analysis indicated that three upregulated lncRNAs (AC091043.1, AP000924.1, and FOXCUT) may be of strong diagnostic value for predicting the existence of TNBC in the training and validation sets (area under the curve (AUC > 0.85). Kaplan-Meier analysis demonstrated that the other three lncRNAs (AC010343.3, AL354793.1, and FGF10-AS1) were associated with the prognosis of TNBC patients (P < 0.05). We used the three overall survival (OS)-related lncRNAs to establish a three-lncRNA signature. Multivariate Cox regression analysis suggested that the three-lncRNA signature was a prognostic factor independent of other clinical variables ( P < 0.01) for predicting OS in TNBC patients that could be utilized to classify patients into high- or low-risk subgroups. Our results might provide efficient signatures for clinical diagnosis and prognostic evaluation of TNBC.     

Promoter hypermethylation of BRCA1 correlates with absence of expression in hereditary breast cancer tumors

Germline mutations in BRCA1 account for a low proportion of hereditary cases in diverse populations. Several efforts have been made to find new genes involved in the inheritance of breast cancer with no success until today. The participation of BRCA1 in the development of breast cancer has been proposed in several studies where hypermethylation of its promoter and a decrease in expression has been reported for sporadic cases and one study on familial cases. To explore the participation of BRCA1 in hereditary carcinogenesis through a different mechanism than the inheritance of germline mutations, we studied the methylation status of its promoter in breast tumors, from patients previously screened for BRCA1/BRCA2 germline mutations. We also determined the presence of the BRCA1 protein in these tumors and correlated both events with tumor grade, hormone receptors and ERBB2 presence. Promoter hypermethylation of the BRCA1 gene was detected in 51% of our biopsies, among which 67% did not express the respective protein. This result leads us to suggest that hypermethylation could be considered as an inactivating mechanism for BRCA1 expression, either as a first or second hit. Moreover, a number of biopsies with absence of expression on BRCA1 showed negative detection of estrogen and progesterone receptors, a similar phenotype to BRCA1 mutated breast tumors.