Andrew R. Green

Prognostic markers in triple-negative breast cancer.

Triple‐negative breast cancer (estrogen receptor‐negative, progesterone receptor‐negative, and HER2‐negative) is a high risk breast cancer that lacks the benefit of specific therapy that targets these proteins.

MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype

BackgroundMicroRNAs (miRNAs), a class of short non-coding RNAs found in many plants and animals, often act post-transcriptionally to inhibit gene expression.ResultsHere we report the analysis of miRNA expression in 93 primary human breast tumors, using a bead-based flow cytometric miRNA expression profiling method. Of 309 human miRNAs assayed, we identify 133 miRNAs expressed in human breast and breast tumors. We used mRNA expression profiling to classify the breast tumors as luminal A, luminal B, basal-like, HER2+ and normal-like. A number of miRNAs are differentially expressed between these molecular tumor subtypes and individual miRNAs are associated with clinicopathological factors. Furthermore, we find that miRNAs could classify basal versus luminal tumor subtypes in an independent data set. In some cases, changes in miRNA expression correlate with genomic loss or gain; in others, changes in miRNA expression are likely due to changes in primary transcription and or miRNA biogenesis. Finally, the expression of DICER1 and AGO2 is correlated with tumor subtype and may explain some of the changes in miRNA expression observed.ConclusionThis study represents the first integrated analysis of miRNA expression, mRNA expression and genomic changes in human breast cancer and may serve as a basis for functional studies of the role of miRNAs in the etiology of breast cancer. Furthermore, we demonstrate that bead-based flow cytometric miRNA expression profiling might be a suitable platform to classify breast cancer into prognostic molecular subtypes.

Subtyping of breast cancer by immunohistochemistry to investigate a relationship between subtype and short and long term survival: a collaborative analysis of data for 10,159 cases from 12 studies

Paul Pharoah and colleagues evaluate the prognostic significance of immunohistochemical subtype classification in more than 10,000 breast cancer cases with early disease, and examine the influence of a patients survival time on the prediction of future survival.

Prognostic Value of a Combined Estrogen Receptor, Progesterone Receptor, Ki-67, and Human Epidermal Growth Factor Receptor 2 Immunohistochemical Score and Comparison With the Genomic Health Recurrence Score in Early Breast Cancer

PURPOSE We recently reported that the mRNA-based, 21-gene Genomic Health recurrence score (GHI-RS) provided additional prognostic information regarding distant recurrence beyond that obtained from classical clinicopathologic factors (age, nodal status, tumor size, grade, endocrine treatment) in women with early breast cancer, confirming earlier reports. The aim of this article is to determine how much of this information is contained in standard immunohistochemical (IHC) markers. PATIENTS AND METHODS The primary cohort comprised 1,125 estrogen receptor-positive (ER-positive) patients from the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial who did not receive adjuvant chemotherapy, had the GHI-RS computed, and had adequate tissue for the four IHC measurements: ER, progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), and Ki-67. Distant recurrence was the primary end point, and proportional hazards models were used with sample splitting to control for overfitting. A prognostic model that used classical variables and the four IHC markers (IHC4 score) was created and assessed in a separate cohort of 786 patients. RESULTS All four IHC markers provided independent prognostic information in the presence of classical variables. In sample-splitting analyses, the information in the IHC4 score was found to be similar to that in the GHI-RS, and little additional prognostic value was seen in the combined use of both scores. The prognostic value of the IHC4 score was further validated in the second separate cohort. CONCLUSION This study suggests that the amount of prognostic information contained in four widely performed IHC assays is similar to that in the GHI-RS. Additional studies are needed to determine the general applicability of the IHC4 score.

Triple-Negative Breast Cancer: Distinguishing between Basal and Nonbasal Subtypes

Purpose: Triple-negative (TN; estrogen receptor, progesterone receptor, and HER-2 negative) cancer and basal-like breast cancer (BLBC) are associated with poor outcome and lack the benefit of targeted therapy. It is widely perceived that BLBC and TN tumors are synonymous and BLBC can be defined using a TN definition without the need for the expression of basal markers. Experimental Design: We have used two well-defined cohorts of breast cancers with a large panel of biomarkers, BRCA1 mutation status, and follow-up data to compare the clinicopathologic and immunohistochemical features of TN tumors expressing one or more of the specific basal markers (CK5/6, CK17, CK14, and epidermal growth factor receptor; BLBC) with those TN tumors that express none of these markers (TN3BKE−). Results: Here, we show that although the morphologic features of BLBC are not significantly different from that of TN3BKE- tumors, BLBC showed distinct clinical and immunophenotypic differences. BLBC showed a statistically significant association with the expression of the hypoxia-associated factor (CA9), neuroendocrine markers, and other markers of poor prognosis such as p53. A difference in the expression of cell cycle-associated proteins and biomarkers involved in the immunologic portrait of tumors was seen. Compared with TN3BKE- tumors, BLBC was positively associated with BRCA1 mutation status and showed a unique pattern of distant metastasis, better response to chemotherapy, and shorter survival. Conclusion: TN breast cancers encompass a remarkably heterogeneous group of tumors. Expression of basal markers identifies a biologically and clinically distinct subgroup of TN tumors, justifying the use of basal markers (in TN tumors) to define BLBC.

Morphological and immunophenotypic analysis of breast carcinomas with basal and myoepithelial differentiation

The aim of this study was to assess the morphological characteristics and immunohistochemical profile of breast carcinomas with basal and myoepithelial phenotypes to obtain a better understanding of their biological behaviour and nature. One thousand nine hundred and forty‐four invasive breast carcinomas were examined, using tissue microarray (TMA) technology and immunohistochemistry, to identify those tumours that showed basal and myoepithelial phenotypes, and their immunophenotype profile was characterized using a variety of markers. In addition, haematoxylin and eosin‐stained sections of these tumours were studied for several morphological parameters. The findings were correlated with patient and tumour characteristics and outcome data. Tumours were classified into two groups: (1) tumours with basal phenotype [expressing one or both basal markers (CK5/6 and/or CK14)] and (2) tumours with myoepithelial phenotype (expressing SMA and/or p63). Group 1 was further subdivided into two subgroups: (A) dominant basal pattern (more than 50% of cells positive) and (B) basal characteristics (10–50% of cells positive). Group 1 tumours constituted 18.6% (8.6% and 10% for groups 1A and 1B, respectively) and group 2 constituted 13.7% of the cases. In both groups, the most common histological types were ductal/no specific type, tubular mixed and medullary‐like carcinomas; the majority of these tumours were grade 3. There were positive associations with adenoid cystic growth pattern, loss of tubule formation, marked cellular pleomorphism, poorer Nottingham prognostic index, and development of distant metastasis. In addition, associations were found with loss of expression of steroid hormone receptors and FHIT proteins and positive expression of p53 and EGFR. The most common characteristics in group 1 were larger size, high‐grade comedo‐type necrosis, development of tumour recurrence, and absence of lymph node disease. Group 2 tumours were more common in younger patients and were associated with central acellular zones, basaloid change, and positive E‐cadherin protein expression. Group 1 characteristics were associated with both reduced overall survival (OS) [log rank (LR) = 22.5, p < 0.001] and reduced disease‐free interval (DFI) (LR = 30.1, p < 0.001), while group 2 characteristics showed an association with OS (LR = 5, p = 0.02) but not with DFI. Multivariate analysis showed that basal, but not myoepithelial, phenotype has an independent value in predicting outcome. Breast cancers with basal and myoepithelial phenotypes are distinct groups of tumours that share some common morphological features and an association with poor prognosis. The basal rather than the myoepithelial phenotype has the strongest relationship with patient outcome. Copyright

Global Histone Modifications in Breast Cancer Correlate with Tumor Phenotypes, Prognostic Factors, and Patient Outcome

Post-translational histone modifications are known to be altered in cancer cells, and loss of selected histone acetylation and methylation marks has recently been shown to predict patient outcome in human carcinoma. Immunohistochemistry was used to detect a series of histone lysine acetylation (H3K9ac, H3K18ac, H4K12ac, and H4K16ac), lysine methylation (H3K4me2 and H4K20me3), and arginine methylation (H4R3me2) marks in a well-characterized series of human breast carcinomas (n = 880). Tissue staining intensities were assessed using blinded semiquantitative scoring. Validation studies were done using immunofluorescence staining and Western blotting. Our analyses revealed low or absent H4K16ac in the majority of breast cancer cases (78.9%), suggesting that this alteration may represent an early sign of breast cancer. There was a highly significant correlation between histone modifications status, tumor biomarker phenotype, and clinical outcome, where high relative levels of global histone acetylation and methylation were associated with a favorable prognosis and detected almost exclusively in luminal-like breast tumors (93%). Moderate to low levels of lysine acetylation (H3K9ac, H3K18ac, and H4K12ac), lysine (H3K4me2 and H4K20me3), and arginine methylation (H4R3me2) were observed in carcinomas of poorer prognostic subtypes, including basal carcinomas and HER-2-positive tumors. Clustering analysis identified three groups of histone displaying distinct pattern in breast cancer, which have distinct relationships to known prognostic factors and clinical outcome. This study identifies the presence of variations in global levels of histone marks in different grades, morphologic types, and phenotype classes of invasive breast cancer and shows that these differences have clinical significance.

Beta-Blocker Drug Therapy Reduces Secondary Cancer Formation in Breast Cancer and Improves Cancer Specific Survival

Laboratory models show that the beta-blocker, propranolol, can inhibit norepinephrine-induced breast cancer cell migration. We hypothesised that breast cancer patients receiving beta-blockers for hypertension would show reduced metastasis and improved clinical outcome. Three patient subgroups were identified from the medical records of 466 consecutive female patients (median age 57, range 28-71) with operable breast cancer and follow-up (>10 years). Two subgroups comprised 43 and 49 hypertensive patients treated with beta-blockers or other antihypertensives respectively, prior to cancer diagnosis. 374 patients formed a non-hypertensive control group. Metastasis development, disease free interval, tumour recurrence and hazards risk were statistically compared between groups. Kaplan-Meier plots were used to model survival and DM. Beta-blocker treated patients showed a significant reduction in metastasis development (p=0.026), tumour recurrence (p=0.001), and longer disease free interval (p=0.01). In addition, there was a 57% reduced risk of metastasis (Hazards ratio=0.430; 95% CI=0.200-0.926, p=0.031), and a 71% reduction in breast cancer mortality after 10 years (Hazards ratio=0.291; 95% CI=0.119-0.715, p=0.007). This proof-of-principle study showed beta-blocker therapy significantly reduces distant metastases, cancer recurrence, and cancer-specific mortality in breast cancer patients suggesting a novel role for beta-blocker therapy. A larger epidemiological study leading to randomised clinical trials is needed for breast and other cancer types including colon, prostate and ovary.

The shaping and functional consequences of the microRNA landscape in breast cancer.

MicroRNAs (miRNAs) show differential expression across breast cancer subtypes, and have both oncogenic and tumour-suppressive roles. Here we report the miRNA expression profiles of 1,302 breast tumours with matching detailed clinical annotation, long-term follow-up and genomic and messenger RNA expression data. This provides a comprehensive overview of the quantity, distribution and variation of the miRNA population and provides information on the extent to which genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture, suggesting an important role for post-transcriptional regulation. The key clinical parameters and cellular pathways related to the miRNA landscape are characterized, revealing context-dependent interactions, for example with regards to cell adhesion and Wnt signalling. Notably, only prognostic miRNA signatures derived from breast tumours devoid of somatic copy-number aberrations (CNA-devoid) are consistently prognostic across several other subtypes and can be validated in external cohorts. We then use a data-driven approach to seek the effects of miRNAs associated with differential co-expression of mRNAs, and find that miRNAs act as modulators of mRNA–mRNA interactions rather than as on–off molecular switches. We demonstrate such an important modulatory role for miRNAs in the biology of CNA-devoid breast cancers, a common subtype in which the immune response is prominent. These findings represent a new framework for studying the biology of miRNAs in human breast cancer.

Expression of mucins (MUC1, MUC2, MUC3, MUC4, MUC5AC and MUC6) and their prognostic significance in human breast cancer

Mucins are a large family of glycoproteins expressed by many epithelial cells and their malignant counterparts. Much interest has been focused on expression of its members in breast cancer because of their potential role as prognostic indicators and their involvement in cancer therapy. We have examined 1447 cases of invasive breast carcinoma with a long-term follow-up, using tissue microarray (TMA) technology and immunohistochemistry to evaluate the expression profiles of several mucins (MUC1, MUC2, MUC3, MUC4, MUC5AC and MUC6) and to assess their prognostic value. We detected MUC1 expression in 91% of tumours. MUC1 overexpression was associated with a lower grade, smaller tumour size, a higher oestrogen receptor (ER)-positive phenotype and absence of both regional recurrence and distance metastasis. The subcellular localization but not the level of expression had a prognostic value in predicting outcome. The aberrant cytoplasmic and membranous localization of MUC1 was associated with poor outcome compared with apical localization, which is the normal physiological site of expression. MUC2 expression was noticed in only 8.3% of all cases and was restricted to the cytoplasm of the tumour cells. An inverse trend was identified between MUC2 expression and lymph node stage and vascular invasion status. On excluding cases of mucinous carcinoma from the analysis, the inverse association with vascular invasion was still defined and in addition an inverse association with ER status emerged. MUC3 expression was detected in 91% of cases and its expression was associated with increased local recurrence, and lymph node stage. The membranous expression of MUC3 was found to be a potentially poor prognostic feature, with higher grade and poorer Nottingham Prognostic Index (NPI), and negative ER expression. MUC4, MUC5AC and MUC6 were expressed in 95, 37 and 20% of cases, respectively. Apart from an association between MUC4 expression and tumour grade and between MUC6 and ER-negative tumours, no other associations with any clinicopathological variables were found. Apart from the higher expression of MUC2 and MUC6 in mucinous carcinomas, no association was found between the expression of different mucins and tumour type. No association between the level of expression of any of the studied mucins and patient outcomes has been identified. In conclusion, most breast carcinomas express MUC1, MUC3 and MUC4. Among the various mucins expressed in breast cancer, MUC1 and MUC3 are potential prognostic indicators, MUC1 having the strongest relationship with patient outcome.