Expression In Breast Carcinoma Research Articles

Stromal Caveolin-1 Expression in Breast Carcinoma. Correlation with Early Tumor Recurrence and Clinical Outcome

Caveolin- (cav-1) has been linked to tumor progression and clinical outcome in breast cancer, but its role as a prognostic marker is still unclear. We evaluated stromal and tumor caveolin-1 expression in 91 breast carcinomas, and assessed the association between their expression and clinicopathologic variables as well as patient outcome and early tumor recurrence. Absence of stromal caveolin-1 expression was detected in 18.7% of cases, while 25.3% of cases revealed tumor epithelial caveolin-1 expression. Combined stromal and tumor caveolin-1 immunopositivity was seen in 24.2% of cases. Absence of stromal cav-1 associated with larger tumor size, higher grade, higher nodal stage, higher number of positive nodes, higher TNM stage, positive HER2 status, higher recurrence rate, and shorter mean progression free survival (PFS). Stromal cav-1 status was a significant predictor of PFS in ER+, PR +, and HER2 + tumors. In tamoxifen-treated patients, absence of stromal Cav-1 was a significant predictor of poor clinical outcome, suggestive of tamoxifen resistance. Conversely, tumor epithelial and combined caveolin-1 expression, didnot associate with patient outcome. In multivariate analysis, only TNM stage independently associated with survival. Loss of stromal caveolin-1 is a novel breast cancer biomarker that can predict early tumor recurrence, short PFS, and tamoxifen- resistance. Thus, its use as a predictive biomarker, especially in lower grade, lower stage, ER+, PR+, HER2+, and tamoxifen treated patients may allow for early interventions with more aggressive therapies. Thus, stromal marker expression and epithelial-stromal cross talk may be critical for tumor progression and metastasis.

Apoptosis gene signature of Survivin and its splice variant expression in breast carcinoma

Survivin, an anti-apoptotic protein, was described as strongly expressed in human cancers including breast cancer. However, little is known about the association between Survivin variants (Survivin-2B, Survivin-ΔEx3, Survivin-3B, and Survivin-2α) and the other apoptotic-related genes. In this study, we analyzed the apoptosis gene signature of Survivin and its variant expression in breast cancer. Human Apoptosis Gene Arrays were used to screen genes that could be associated with Survivin variants. Expression of the five transcripts was measured by RT-PCR in 135 breast carcinomas and Cox survival analysis was analyzed according to the patient outcome. Significant associations between Survivin transcripts and apoptotic genes were found. Interestingly, Survivin-3B variant showed major inverse correlations with pro-apoptotic genes. In addition, in vitro results indicated that overexpression of Survivin-3B strongly inhibits 5-fluorouracil/epirubicin/cyclophosphamide-induced apoptosis in breast tumor cell lines. In breast carcinomas, uni- and multivariate analysis showed patients with high level of Survivin-3B expression had a shorter overall (P=0.030 and P=0.042 respectively), and disease-free (P=0.024 and P=0.009) survival. Our data suggest that Survivin-3B contributes to cell survival through the anti-apoptotic pathway and that its expression level could be an important factor in determining therapeutic strategies for breast carcinoma.

PHLDA1 is a crucial negative regulator and effector of Aurora A kinase in breast cancer

Aurora A kinase is overexpressed in the majority of breast carcinomas. A chemical genetic approach was used to identify the malignant targets of Aurora A, which revealed pleckstrin-homology-like domain protein PHLDA1 as an Aurora A substrate. PHLDA1 downregulation is a powerful prognostic predictor for breast carcinoma, which was confirmed in our study. We further show that downregulation of PHLDA1 is associated with estrogen receptor (ER) expression in breast carcinoma. Aurora A directly phosphorylates PHLDA1 leading to its degradation. PHLDA1 also negatively regulates Aurora A, thereby triggering a feedback loop. We demonstrate the underlying mechanisms by which PHLDA1 upregulation strongly antagonizes Aurora-A-mediated oncogenic pathways, thereby revealing PHLDA1 degradation as a key mechanism by which Aurora A promotes breast malignancy. Thus, not surprisingly, PHLDA1 upregulation acts synergistically with Aurora A inhibition in promoting cell death. PHLDA1 overexpression might therefore be an alternative method to modulate Aurora A deregulation in breast carcinoma. Finally, this study led to the discovery of a mutation in the Aurora A active site that renders it amenable to the chemical genetic approach. Similar mutations are required for Aurora B, suggesting that this modified approach can be extended to other kinases that have hitherto not been amenable to this methodology.

The significance of Survivin and Nectin-4 expression in the prognosis of breast carcinoma

To investigate the prognostic significance of Survivin and Nectin-4 expression in breast carcinomas. Imprint smears were obtained from 140 breast carcinoma specimens and studied immunocytochemically for the expression of Survivin and Nectin-4. The results were correlated with several clinicopathological parameters, including five-year survival. Increased Survivin staining pattern correlated with increased grade (p < 0.0001), increased lymph node invasion (p < 0.0001), increased tumor size and reduced survival (p < 0.0001). Elevated Nectin-4 expression also correlated significantly with increased grade (p < 0.0001), increased tumor size (p < 0.0001) and reduced survival (p < 0.0001). In addition, Survivin and Nectin-4 staining patterns correlated strongly with one another (p < 0.0001). However, on multivariate analysis, neither Survivin nor Nectin-4 expression seemed to have an independent impact on survival in our study cases. The findings of our study suggest that increased expression of Survivin and Nectin-4 may indicate a worse prognosis in breast cancer patients. The exact implications of the expression of these markers in breast cancer prognosis and treatment remain to be clarified.

Abstract 1035: WAS/WASL interacting protein family, member 2 (WIPF2) expression in breast carcinomas

Abstract ERBB2 over-expression in breast carcinomas is associated with poor prognosis and early metastatic dissemination. The gene WIPF2 is located on the position q21.2 of the chromosome 17 between ERBB2 and TOP2A (17q21-q22) genes and encodes a WASP interacting protein (WIRE protein). It has been reported that this protein has a role in the WASP-mediated organization of the actin cytoskeleton. This protein is a potential link between the activated platelet-derived growth factor receptor and the actin polymerization machinery and its expression has never been evaluated in cancer tissues. The objectives of this investigation were the characterization of the levels of expression of WIPF2 gene in breast carcinomas and its correlation with EGFR 1 stimuli. Material and Methods: We studied the expression of WIRE protein in 87 ductal invasive carcinomas and WIPF2 gene in three breast cancer cell lines (Hb4a-C5.2, MCF7 and SKBR3). Breast cancer samples were mounted in a TMA block and the immunohistochemistry was performed to evaluate the expression of WIRE protein, estrogen receptor, progesterone receptor, HER-2 and Ki-67. The HER2, WIRE and KI-67 expression was further studied by using ImageScope software after digital image capture (Aperio). Tumors were classified according ER, PgR, HER2 and Ki-67 expression in luminal A or B, HER2 positive and basal-like subtypes. The WIPF2 and ERBB2 genes expression (real time RT-PCR) and WIRE protein (immunofluorescence) was analyzed in all cell lines before and after EGF treatment (40ng/µl for 12 and 72 hours). Results: We observed that WIRE protein is ubiquitously expressed in breast carcinomas. According to digital analysis, HER2 positive tumors have a higher expression of WIRE protein (p = 0.02). SKBR3 cell line (HER2 over expressed) has a significant 11 fold increase in WIPF2 gene expression comparing to MCF7. After EGF (EGFR 1 ligand) treatment, we observed a significant decrease in WIPF2 gene expression in SKBR3. Additionally, we observed a subcellular modification of WIRE protein, specially in SKBR3 cell line. The protein is usually dispersed into the cytoplasm. After EGF treatment, there is a reduction in its expression and the protein concentrate in endoplasmic reticulum (peri-nuclear). The decrease in WIPF2 gene expression and the subcellular protein modification is time dependent. They are more expressive after 72 hours of EGF exposure. Conclusion: WIRE protein is over-expressed in HER2 ductal breast carcinomas. EGF stimuli (EGFR1) changes its sub-cellular localization and reduces the WIPF2 gene expression in SKBR3 (HER2 positive) cell line. It is possible to infer that the expression of WIPF2 could be associated with ERFR signaling and the actin cytoskeleton motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1035. doi:10.1158/1538-7445.AM2011-1035

Abstract 3207: Automated quantitative analysis of p53, cyclin D1 and pErk expression in breast carcinoma does not differ from expert pathologist scoring and correlates well with clinico-pathological characteristics

Abstract Prognosis and risk assessment of breast cancer patients are currently driven by TNM stage, ER/PR/HER2 expression, tumor grade and lymphovascular invasion (LVI). However there is critical need for improved biomarker assessment platforms to better predict systemic treatment response. One roadblock is the lack of semi-quantitative methods to reliably measure expression, activity and localization of biomarkers in formalin-fixed tumor specimens. The present study assesses reliability of automated IHC scoring compared to manual scoring of routine and non-routine biomarkers (HER2, cyclin D1, p53 and phospho(p)-ERK) on a human breast cancer tissue microarray according to REMARK guidelines, and correlates these markers with clinical-pathological data. Using a triplicate core TMA of formalin-fixed paraffin embedded tissues, we investigated 63 primary invasive breast cancers, for which ER/PR/HER2 status, LVI, grade and recurrence status were recorded. IHC was performed on the TMA for the above biomarkers (pH 6 citrate buffer conditions). Histologic (H) scores (% positive tumor area × staining intensity 0-3) were determined manually by two independent evaluators with resolution of discordant cases by a senior pathologist. Excellent replicability was observed between H scores for each marker compared on replicate slides, as determined by Spearman correlations (0.79-0.82). Each TMA slide was then scanned into the Ariol Imaging System, algorithms were trained for each marker, and H scores were calculated. Pearson correlation coefficients (with data left as continuous) and Kappa statistics (with dichotomized data) were used for inter-method comparisons. Associations between biomarker positivity and clinical data were assessed by Fisher's exact test. Excellent concordance between manual and automated Ariol scores was observed for all four markers based on Kappa statistics (0.667-0.813) and Pearson correlation coefficients (0.790-0.885). Distinct proportions of tumor cases showed any positive staining for membranous HER2 (19/63), nuclear p53 (16/56), cyclin D1 (26/57) and pERK (32/59). A statistically significant association of pERK positivity with absence of LVI (p=0.0025) and lymph node negativity (p=0.0006) was observed. In contrast, pERK positivity was associated with high-grade tumors (p=0.0040), consistent with a role of pERK in poorly differentiated high-grade primary tumors. p53 over-expression, characteristic of dysfunctional p53 in breast cancer, was also associated with high tumor grade (p=0.0074). Thus automated quantitation of immunostaining yields objective results that do not differ from pathologists’ scoring, and provide meaningful associations with clinico-pathological data. (Supported by CIHR, PSI, and Queen's Dept. Pathol. &amp; Mol. Med.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3207. doi:10.1158/1538-7445.AM2011-3207

Identification of PUMA as an estrogen target gene that mediates the apoptotic response to tamoxifen in human breast cancer cells and predicts patient outcome and tamoxifen responsiveness in breast cancer

Recognition of the pivotal role of estrogen in the aetiology of breast cancer has led to the development of antiestrogens (AE), such as tamoxifen (TAM) as effective therapies for the treatment and prevention of this disease. However, despite their widespread clinical efficacy, response to AEs is often short-lived, and acquired or innate therapeutic resistance remains a major obstacle in the successful treatment of breast cancer. Thus, delineating the intracellular pathways that mediate the cellular response to estrogen could potentially lead to new, more effective approaches to the treatment of breast cancer, particularly endocrine-resistant disease. Here, we have identified the BCL-2 homology 3 (BH3)-only, pro-apoptotic regulator, PUMA (p53 upregulated modulator of apoptosis) as an estrogen target gene that is acutely downregulated in response to estrogen in breast cancer cell lines, independently of their p53 status. PUMA is transcriptionally upregulated following treatment with TAM, and knock down of PUMA expression in these cells attenuates the apoptotic response to TAM. Furthermore, low PUMA expression in breast carcinomas is significantly associated with breast cancer-specific death (P=0.0014 and P=0.0115, for mRNA and protein, respectively), and worse outcome in TAM-treated patients (mRNA, P=1.49e-05). These findings suggest that the dysregulation of apoptotic signaling pathways such as those executed through PUMA, can significantly impact on both the progression and therapeutic responsiveness of breast cancer. Moreover, they provide a convincing rationale for exploring new therapeutic approaches involving endocrine and non-endocrine therapies that target apoptotic pathways as an effective strategy for tackling endocrine refractory disease.

Enhancer of Zeste Homologue 2 Expression in Breast Carcinoma Smears in Relationship with p53, Ki-67 and Other Prognostic Parameters

Background: The polycomb group protein enhancer of zeste homologue 2 (EZH 2) has been reported as a marker of aggressive breast cancer. The aim of this study was to investigate the correlation between the expression of EZH 2 with p53 and Ki-67 expression and other clinicopathological parameters in primary breast carcinomas in order to determine the role of the above marker as a prognosticator of tumor aggressiveness and patient outcome. Patients and Methods: One hundred primary operable breast cancer patients were investigated in order to identify the expression of EZH 2, Ki-67 and p53 in imprint smears immunocytochemically. The prevalence of expression of these markers was then correlated with clinicopathological parameters. Follow-up was available for all patients. Results: EZH 2 was expressed in 64% of the cases and correlated with higher levels of p53 (relative risk = 3.00, p < 0.0001) and Ki-67 (relative risk = 3.25, p < 0.0001). Malignant cells showed immunoreactivity for all markers in the nucleus. Univariate analysis revealed a strong association between EZH 2 protein expression and tumor grade and size, lymph node metastasis, and HER-2 and estrogen and progesterone receptor status. Multivariable statistical analysis revealed that lymph node metastasis was the main predictor for EZH 2 expression. Decreased patient survival was also significantly associated with EZH 2 expression (p < 0.0001). Conclusions: EZH 2 expression may be a marker of poor prognosis in breast carcinoma patients and has been suggested as a candidate for targeted therapy.

Stromal Expression of CD10 in Invasive Breast Carcinoma and Its Correlation with ER, PR, HER2-neu, and Ki67

CD10 is a cell surface zinc-dependent endopeptidase, which degrades many bioactive peptides. CD10 expression in tumour stroma is associated with biological aggressiveness of many epithelial malignancies. To date, only one study has correlated with expression of CD10 with well-known prognostic markers of breast, that is, ER, PR, HER2-neu, and tumour grade; however, its correlation with ki67 is still not studied. The aim of this study is to evaluate stromal CD10 expression in breast carcinoma and to examine its correlation with ER, PR, HER2-neu, and Ki67. Methods and Results. CD10 expression in fifty patients was assessed by immunohistochemistry and scored as negative, weak and strong. CD10 was found to be positive in stroma of 40/50 (80%) cases. Stromal CD10 showed positive correlation with tumour grade, HER2-neu (P = .000), and ki67 (P = .027), negative correlation with ER and PR. Conclusions. Hence CD10 expression correlated strongly with well-established negative prognostic markers, that is, HER2-neu and ki67 positivity, ER/PR negativity, and higher tumour grade, thus indicating that CD10 can be used as independent marker indicating poor prognosis and can be used as target for the development of novel therapies.

Protein kinase CK2α subunit over-expression correlates with metastatic risk in breast carcinomas: Quantitative immunohistochemistry in tissue microarrays

Background CK2α is a signalling molecule that participates in major events in solid tumour progression. The aim of this study was to evaluate the prognostic significance of the immunohistochemical expression of CK2α in breast carcinomas. Methods Quantitative measurements of immunohistochemical expression of 33 biomarkers using high-throughput densitometry, assessed on digitised microscopic tissue micro-array images were correlated with clinical outcome in 1000 breast carcinomas using univariate and multivariate analyses. Results In univariate analysis, CK2α was a significant prognostic indicator ( p < 0.001). Moreover, a multivariable model allowed the selection of the best combination of the 33 biomarkers to predict patients’ outcome through logistic regression. A nine-marker signature highly predictive of metastatic risk, associating SHARP-2, STAT1, eIF4E, pmapKAPk-2, pAKT, caveolin, VEGF, FGF-1 and CK2α permitted to classify well 82.32% of patients (specificity 81.59%, sensitivity 92.55%, area under ROC curve 0.939). Importantly, in a node negative subset of patients an even more (86%) clinically relevant association of eleven markers was found predictive of poor outcome. Conclusion A strong quantitative CK2α immunohistochemical expression in breast carcinomas is individually a significant indicator of poor prognosis. Moreover, an immunohistochemical signature of 11 markers including CK2α accurately (86%) well classifies node negative patients in good and poor outcome subsets. Our results suggest that CK2α evaluation together with key downstream CK2 targets might be a useful tool to identify patients at high risk of distant metastases and that CK2 can be considered as a relevant target for potential specific therapy.

Abstract P3-04-05: Hypermethylation and Downregulation of Glutathione Peroxidase-3 in Inflammatory Breast Carcinogenesis

Abstract Background: The breast tumor microenvironment is characterized by the release of endogenous reactive oxygen species (ROS), and has been suggested as being associated with disease aggressiveness. Normal cells have an antioxidant system that controls the balance between production and removal of oxygen radicals, thereby protecting against oxidative damage, such as the Glutathione peroxidases (GPXs) enzymes. Epigenetics and DNA methylation play important roles in several inflammatory disorders. We therefore, analyzed the promoter of GPX3 and found a dense CpG island closest to the transcription start-site. The aim of the present study is to 1) investigate GPX3 expression in breast carcinoma and normal breast tissues, 2) identify whether GPX3 is epigenetically regulated in breast carcinoma tissues versus normal tissue, and 3) compare the levels of expression of GPX3 in Inflammatory Breast Cancer (IBC) versus non-IBC tissues. Material and Methods: We enrolled 40 breast cancer patients with tumor mass range from 1.8-9cm (with median size 4.77 ± 3.9). Patients were sub-grouped as IBC (n = 20) or non-IBC (n = 20), healthy breast tissues from the same patients were used as control. Using real-time PCR and immunohistochemistry, we assessed the level of expression of GPX3 at mRNA and protein levels in breast cancer tissues versus control. To identify whether the CpG island of the GPX3 gene was epigenetically regulated, we analyzed the methylation profile of GPX3 in breast carcinoma versus normal tissues using DNA bisulfate treatment and methylation-specific PCR (MSP). Results: GPX3-mRNA expression was down regulated in breast cancer samples compared to control tissues. There was a significant decrease (P = 0.01) in the mRNA expression level of IBC compared to non-IBC carcinoma tissues. MSP showed that the GPX3 gene was hypermethylated in breast carcinoma tissues compared to control. Moreover, confirmatory semiquantitative immunohistochemical scoring revealed weak or negative immunostain of carcinoma tissues compared to healthy breast tissues. Conclusion: These preliminary data suggest that epigenetic inactivation of GPX3 is a frequent finding in inflammatory breast cancer. Silencing of GPX3 in IBC versus non-IBC carcinoma cells suggested that GPX3 may be critical in the development and progression of IBC. To our knowledge, this is first study to test the role of GPX3 in breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-05.

Abstract P4-06-05: Silencing of Cyclin D1 Increases Breast Cancer Cell Migration through Increased Id1 Expression and Induction of Epithelial-Mesenchymal Transition

Abstract Backround: The oncogene cyclin D1 is overexpressed in many breast cancers and despite proliferation activating properties, has been linked to a less malignant phenotype. We have previously shown that down-regulation of cyclin D1 in actively cycling breast cancer cells significantly increased their migratory capacity whilst also decreasing proliferation. Gene expression analysis and animal modeling have highlighted an increase in the inhibitor of differentiation-1 (Id1) gene following cyclin D1 gene (CCND1) silencing. Notably, increased Id1 is associated not only with enhanced migratory and invasive features of breast cancer cells but also with prevention of cell differentiation and induction of an epithelial-mesenchymal-like (EMT) phenotype. Here we sought to determine if the increase in cell motility following cyclin D1 silencing was through increased Id1 expression and induction of an EMT phenotype, and confirm our findings in a large patient cohort. Materials and Methods: Expression of cyclin D1, Id1 and SNAI2 was determined in MDA-MB-231 and ZR75 cells by qPCR and western blot, whilst cell migration was monitored by transwell assay. Gene expression of CCND1, ID1 and EMT markers was analysed from a pooled cohort of batch-mean centered publically available data sets, with data analysis performed in SPSS. Results: Silencing of cyclin D1 increased cell migration 1.38 and 1.72 fold in MDA-MB-231 (P&amp;lt;0.001), and ZR75 (P&amp;lt;0.001) cells respectively, an effect abolished by silencing of Id1. qPCR analysis confirmed an increase in Id1 (1.73) and SNAI2 (1.50) expression following siRNA treatment of cyclin D1, an effect reversed following siRNA treatment of Id1. CCND1 gene exression was negatively, while Id1 was positively, associated with the EMT markers Vimentin (P&amp;lt;0.001), SNAI2 (P&amp;lt;0.001) and TWIST1 (P&amp;lt;0.001) in 1,100 breast tumors. In ER+ patients CCND1 was negatively correlated to Id1 expression (0.050) and in CCND1 low patients increasing Id1 expression was predictive of worse cumulative survival (0.027). Conclusion: Decreased CCND1 expression enhances MDA-MB-231 and ZR75 cell migration through increased Id1 expression and induction of EMT. Moreover, both genes are correlated to EMT markers in breast cancer patients and in a subset of the cohort, confer a worse survival. These results may explain the conflicting data regarding cyclin D1 expression in breast carcinomas and solidifies the rationale of the protein as a clinical target. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-05.

The effect of raloxifene on Bax protein expression in breast carcinomas of postmenopausal women

The actual role of Bax protein as a prognostic biomarker that predicts response to therapy remains unclear. The objective of this study was to evaluate the effect of raloxifene on Bax protein expression in breast carcinomas of postmenopausal women. Twenty postmenopausal patients with operable stage II, estrogen receptor-positive, infiltrating ductal breast carcinoma were treated with oral raloxifene at a dose of 60 mg/day for a period of 28 days before definitive surgery. Tumor samples were obtained by incisional biopsy at the time of diagnosis and again at the time of definitive surgical treatment. Immunohistochemical evaluation of Bax expression was assessed semiquantitatively based on the fraction of stained tumor cells and intensity of staining. McNemar's test was used to analyze data (P < 0.05). Eleven of the 20 (55%) patients were classified as positive for Bax expression before raloxifene treatment, whereas 9(45%) were classified as positive after raloxifene treatment (P = 0.479). In conclusion, raloxifene did not alter Bax expression significantly in estrogen receptor-positive breast carcinomas of postmenopausal women.

Expression of midkine and endoglin in breast carcinomas with different immunohistochemical profiles

The aim of this study was to investigate the midkine and endoglin expression in breast carcinomas with five different immunohistochemical profiles and their relevance to histopathologic and clinicopathologic features. We analyzed 161 archival tissues immunohistologically. The level of midkine expression in breast cancer significantly correlated with lymph node metastasis (p = 0.001) and TNM staging (p = 0.003). High microvessel density (MVD) was associated with higher midkine reactivity group (p = 0.036). Although the basal-like subtype had higher midkine expression level and MVD, no significant difference with the other breast cancer subtypes was found. In conclusion, midkine was a promising target for tumor prognosis in clinical diagnosis and treatment. This study found no significant differences in tumor angiogenesis in different molecular subtypes of breast cancer.

Thyroid Transcription Factor-1 Expression in Breast Carcinomas

Immunostaining for thyroid transcription factor-1 (TTF-1) is frequently used to help assess the site of origin of metastatic carcinomas. TTF-1 expression is most frequently seen in carcinomas of thyroid and lung origin. Furthermore, it has been assumed that the expression of TTF-1 in a carcinoma excludes the possibility of a breast origin. We have recently encountered in our consultation practice 4 cases of invasive breast carcinoma (confirmed by clinical findings and other immunophenotypic features) that showed unequivocal tumor cell expression of TTF-1. However, the frequency with which TTF-1 expression is observed in breast carcinomas is unknown. To address this, we carried out immunostaining for TTF-1 on 546 primary breast carcinomas submitted for routine estrogen receptor, progesterone receptor, and/or HER2 testing. Cases were considered TTF-1 positive if they showed any nuclear staining for this marker. TTF-1 expression was identified in 13 cases (2.4%). Expression varied from focal and weak to diffuse and strong and was seen in both invasive and in situ components. We conclude that a small proportion of breast carcinomas show TTF-1 expression. Therefore, the presence of TTF-1 immunoreactivity in a carcinoma cannot by itself be used to exclude the possibility of a breast origin.

Expression of DBC1 and SIRT1 is associated with poor prognosis for breast carcinoma

Recently, it has been reported that SIRT1 and DBC1 may be involved in the development of tumors and predict poor survival in some cancers. However, their exact role is not clear. Therefore, we investigated the expression status and clinical significance of DBC1 and SIRT1 expression in breast carcinomas. We evaluated the immunohistochemical expression of DBC1, SIRT1, and p53 using a 3-mm core from 122 patients with breast cancer for tissue microarray. Positive expression of DBC1 and SIRT1 were seen in 71% and 67% of patients, respectively. In the patients with breast cancer, overall, expression of DBC1 and SIRT1 was significantly associated with distant metastatic relapse and shorter relapse-free survival and overall survival by univariate analysis. Tumor stage and DBC1 and SIRT1 expression were also independent prognostic factors by multivariate analysis. Among the patients who had received chemotherapy, DBC1 and SIRT1 expression was significantly associated with distant metastatic relapse and shorter survival by univariate analysis. DBC1 expression was also associated with distant metastatic relapse and shorter survival in patients who had received endocrine therapy, according to univariate and multivariate analysis. In conclusion, this study shows that expression of DBC1 and SIRT1 is a significant prognostic indicator for breast carcinoma patients.

Intratumoral Heterogeneity of Immunohistochemical Marker Expression in Breast Carcinoma

Core needle biopsies of breast carcinomas provide diagnostic, prognostic, and predictive information before neoadjuvant therapy. Possible intratumoral heterogeneity of biomarker expression questions the validity of core needle biopsy interpretation in small biopsy specimens. Using tissue microarray (TMA) technology, we studied intratumoral heterogeneity of 7 immunomarkers. Five TMAs were constructed from 44 breast carcinomas and 5 normal breast tissues, each represented by 1-mm cores in triplicate from each of 3 foci. TMAs were immunostained for monoclonal estrogen receptor (ER), monoclonal progesterone receptor (PR), polyclonal human epidermal growth factor receptor 2 (HER2), monoclonal E-cadherin (E-cad), monoclonal epidermal growth factor receptor (EGFR), monoclonal p53, and monoclonal MIB-1. Expression was quantified visually by light microscopy and by image cytometry as intensity, percentage of cells positive, and score. Using intraclass correlation coefficient, heterogeneity in the expression of the immunomarkers within subjects was compared with the overall variance. Intratumoral heterogeneity was seen with 5 immunomarkers: ER, PR, HER2, p53, and MIB-1. E-cad and EGFR failed to show intratumoral heterogeneity. Intratumoral heterogeneity in ER, PR, HER2, p53, and MIB-1 indicates their problematic interpretation in small biopsy specimens as indicative of the status of the entire tumor. A negative result does not exclude the expression of these markers in the remainder of the tumor. E-cad (positive in ductal carcinomas) and EGFR lacked heterogeneity.

Prognostic relevance of light chain 3 (LC3A) autophagy patterns in colorectal adenocarcinomas

AimsThe microtubule-associated protein 1 light chain 3 (LC3A) is an essential component of the autophagic vacuoles, forming a reliable marker of autophagic activity. In a previous study, the authors showed...

Reactivation of epigenetically silenced HER4/ERBB4 results in apoptosis of breast tumor cells

Experimental and clinical data support a growth inhibitory role for HER4 in breast cancer. Clinically HER4 expression is extinguished during breast tumorigenesis supporting a tumor suppressor function for HER4, however, a molecular mechanism to explain the selective loss of HER4 expression has remained elusive. Epigenetic mechanisms, for example, aberrant gene promoter hypermethylation, have been shown to ablate tumor suppressor gene expression in breast carcinomas. We identified a CpG island within the HER4 promoter and show by pyrosequencing of bisulfite-treated DNA an inverse correlation between HER4 expression and the extent of promoter methylation. Treatment of the HER4-negative BT20 cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine (DAC)-enhanced HER4 expression, confirming a role for DNA methylation in suppressed HER4 expression. DAC treatment to reactive HER4 expression in combination with the HER4 ligand heregulin-β1 (HRG) resulted in apoptosis of BT20 cells providing a novel therapeutic strategy for triple-negative tumors. The BT20 cells were rescued from apoptosis when preincubated with HER4 small interfering RNA, thereby confirming a role for HER4 in DAC/HRG-induced apoptosis. We verified HER4 promoter methylation in primary breast carcinomas and detected a significant increase in HER4 promoter methylation in HER4-negative breast tumors (P<0.001). Furthermore, increased levels of HER4 promoter methylation were significantly associated with worse patient prognosis (P=0.0234). Taken together, our data support a tumor suppressor function for HER4, which is epigenetically suppressed in breast tumors through promoter hypermethylation.

821 Dissecting the genetic components of gene expression in breast carcinoma

821 Dissecting the genetic components of gene expression in breast carcinoma