Phenotype Of Breast Carcinoma Research Articles

Cytologic features of triple‐negative breast carcinoma

Triple-negative breast cancer (TNBC) is distinct from other breast cancers, because the tumor cells lack estrogen and progesterone receptors (hormone receptors) and also are negative for human epidermal growth factor receptor 2 (HER2). They comprise a heterogeneous group of tumors with various histologic features and clinical behaviors. High-grade, invasive ductal carcinoma not otherwise specified is the most frequent type, and a substantial fraction of TNBCs belongs to the basal-like tumor type. The purpose of this study was to determine whether some cytologic features could predict the triple phenotype of breast carcinoma. Fine-needle aspiration cytology samples of 62 TNBCs were compared with samples of 82 hormone receptor-positive, high-grade, invasive carcinomas (HRBC) and with samples of 33 hormone receptor-negative, HER2 positive, invasive carcinomas (HER2BC) for the following cytomorphologic features: cellularity, necrosis, lymphocytes, syncytial clusters, tubular/ductal-like clusters, large bare nuclei, streaming within the clusters, and calcifications. Moreover, single cell features, such as cellular borders, cytoplasm, cytoplasmic vacuoles, nuclear pleomorphism, nucleoli, and type of chromatin pattern, were evaluated. Descriptive analyses and 2 multivariate regression models were performed to compare TNBC, HRBC, and HER2BC and to identify the cytologic factors that were associated with tumor type. TNBCs were more likely to have an abundant necrotic background, many lymphocytes, many syncytial clusters, and ill defined cell borders than non-TNBCs. A tubular/ductal pattern was observed only rarely in TNBCs. Multivariate logistic analysis indicated a 90.8% probability of identifying TNBC versus HRBC by the following cytologic variables: lymphocytes, ill defined cell borders and syncytial clusters, tubular/ductal clusters, cytoplasmic vacuoles, and cellular pleomorphism; whereas there was a 77.5% probability of identifying TNBC rather than HER2BC by the following variables: cellularity, ill defined cellular borders and syncytial clusters, and tubular/ductal clusters. Although TNBCs embrace a heterogeneous group of tumors, in this study, they exhibited some common cytologic features that can help to distinguish them from other high-grade breast carcinomas in daily practice.

CD44+/CD24−/low cancer stem/progenitor cells are more abundant in triple-negative invasive breast carcinoma phenotype and are associated with poor outcome

Women classified as having triple-negative tumors have a poor prognosis. The importance of CD44(+)/CD24(-/low) (stem/progenitor cell-phenotype) in breast cancer patients has also been appreciated. However, correlation between triple negativity and CD44(+)/CD24(-/low) with tumor recurrence remains elusive. In the present study, we evaluated tumor specimens of 50 breast cancer patients with known hormone receptor status for whom we had follow-up information and outcome data available, and performed immunohistochemistry analysis to determine CD44 and CD24 expression. Gene expression arrays were also independently performed on 52 breast cancer specimens with banked frozen tissue. Lastly, we used FVBN202 transgenic mouse model of breast carcinoma and determined the hormone receptor status, the proportion of CD44(+)/CD24(-/low) breast cancer stem-like cells, and the behavior of the tumor. We determined that patients with triple-negative tumors had significantly higher incidence of recurrence or distant metastasis associated with increased frequency of breast cancer stem cell phenotypes compared with those with non-triple-negative tumors. Preclinical studies in FVBN202 transgenic mice confirmed these findings by showing that relapsed tumors were triple negative and had significantly higher frequency of breast cancer stem cells compared with their related primary tumors. Unlike non-triple-negative primary tumors, relapsed triple-negative tumors were tumorigenic at low doses when inoculated into FVBN202 transgenic mice. These findings suggest that CD44(+)/CD24(-/low) breast cancer stem-like cells play an important role in the clinical behavior of triple-negative breast cancer and that development of therapeutic targets directed to breast cancer stem-like cells may lead to reduction in the aggressiveness of triple-negative breast cancers.

Suppression of Heregulin‐β1/HER2‐Modulated Invasive and Aggressive Phenotype of Breast Carcinoma by Pterostilbene via Inhibition of Matrix Metalloproteinase‐9, p38 Kinase Cascade and Akt Activation

Invasive breast cancer is the major cause of death among females and its incidence is closely linked to HER2 (human epidermal growth factor receptor 2) overexpression. Pterostilbene, a natural analog of resveratrol, exerts its cancer chemopreventive activity similar to resveratrol by inhibiting cancer cell proliferation and inducing apoptosis. However, the anti-invasive effect of pterostilbene on HER2-bearing breast cancer has not been evaluated. Here, we used heregulin-β1 (HRG-β1), a ligand for HER3, to transactivate HER2 signaling. We found that pterostilbene was able to suppress HRG-β1-mediated cell invasion, motility and cell transformation of MCF-7 human breast carcinoma through down-regulation of matrix metalloproteinase-9 (MMP-9) activity and growth inhibition. In parallel, pterostilbene also inhibited protein and mRNA expression of MMP-9 driven by HRG-β1, suggesting that pterostilbene decreased HRG-β1-mediated MMP-9 induction via transcriptional regulation. Examining the signaling pathways responsible for HRG-β1-associated MMP-9 induction and growth inhibition, we observed that pterostilbene, as well as SB203580 (p38 kinase inhibitor), can abolish the phosphorylation of p38 mitogen-activated protein kinase (p38 kinase), a downstream HRG-β1-responsive kinase responsible for MMP-9 induction. In addition, HRG-β1-driven Akt phosphorylation required for cell proliferation was also suppressed by pterostilbene. Taken together, our present results suggest that pterostilbene may serve as a chemopreventive agent to inhibit HRG-β1/HER2-mediated aggressive and invasive phenotype of breast carcinoma through down-regulation of MMP-9, p38 kinase and Akt activation.

P-cadherin, vimentin and CK14 for identification of basal-like phenotype in breast carcinomas: an immunohistochemical study.

The most suitable immunohistochemical criterion to identify basal-like breast carcinomas (BLBC), a molecular subgroup of breast cancer associated with poor prognosis, is the triple negative phenotype along with CK5 and/or EGFR immunoreactivity. However, several putative basal markers have been suggested as alternatives to identify BLBC with more accuracy. The expression of CK5, EGFR, P-cadherin, CK14, Vimentin and p63 were evaluated in 462 invasive breast carcinomas to determine their sensitivity and specificity for BLBC identification. P-cadherin and CK5 showed higher sensitivity values, while EGFR, Vimentin and CK14 were the most specific markers. The combination of CK5 with P-cadherin, Vimentin or CK14 proved to be a reliable option for distinguishing the basal phenotype, compared to the "gold standard" pair CK5/EGFR. Furthermore, P-cadherin was still able to recognize a large number of putative BLBC among the "unclassified" group (ER-/PR-/HER2-/CK5-/EGFR-). P-cadherin, Vimentin and CK14 can recognize BLBC already identified in triple negative/ CK5 and/or EGFR+ tumors, and due to P-cadherin sensitivity for BLBC identification this marker can reliably recruit a large number of breast carcinomas with basal phenotype among immunohistochemistry triple negative/ CK5 and/or EGFR - pool of tumors. Although they need GEP validation, our results can introduce the idea of these markers as additional options in the daily workup of breast pathology laboratories to identify BLBC.

Pathway-focused proteomic signatures in HER2-overexpressing breast cancer with a basal-like phenotype: New insights into de novo resistance to trastuzumab (Herceptin)

Pioneering clinical studies in de novo refractoriness to the anti-HER2 monoclonal antibody trastuzumab have suggested that HER2 gene-amplification can take place also in a basal-like molecular background to generate basal/HER2+ tumors intrinsically resistant to trastuzumab. Here, we first investigated the unique histogenesis of the basal/HER2+ phenotype in breast carcinomas. The presence of basal CK5/CK6 cytokeratin expression in HER2+ tumors revealed a significant overlap in the histological features of HER2+/CK5/6+ and basal-like breast carcinomas. Basal/HER2+ tumors were typically poorly differentiated, high-grade invasive ductal carcinomas with large geographic necrosis, pushing margins of invasion, syncytial arrangement of tumor cells, ribbon- or festoon-like architecture, squamous metaplasia, stromal lymphocytic infiltrates, high mitotic index and strong p53 positivity. Secondly, we performed low-scale proteomic approaches in JIMT-1 cells, a unique model of HER2-gene amplified trastuzumab-resistant breast carcinoma with a basal-like phenotype, to develop biomarker signatures that may differentiate trastuzumab-responsive from non-responsive tumors. When applying antibody-based array technology to the extracellular milieu of trastuzumab-refractory JIMT-1 and trastuzumab-sensitive SKBR3 cell cultures, JIMT-1 cells were found to secrete higher amounts of several growth factors including amphiregulin, EGF, IGFBP-6, PDGF-AA, neurotrophins, TGFbeta and VEGF. Semi-quantitative signaling node multi-target sandwich ELISAs revealed that JIMT-1 cells drastically overactivate RelA, the prosurvival subunit of NF-kappaB as compared to trastuzumab-sensitive luminal/HER2+ SKBR3 cells. When simultaneously assessing the activation status of 42 receptor tyrosine kinases (RTK) using a human phospho-RTK array, JIMT-1 cells were found to constitutively display hyperactivation of the insulin-like growth factor-I receptor (IGF-1R). High-content immunofluorescence imaging revealed that activated IGF-1R mainly localized at focal adhesion-like structures in JIMT-1 cells. In vitro wound healing assays suggested that this functional reorganization of the JIMT-1 cytoskeletal reorganization may account for an exacerbated trastuzumab-refractory 'migratogenic' phenotype. Forthcoming studies should validate the notion that identification of basal-like immunophenotypes and/or basal-like molecular signatures within HER2+ breast carcinomas may provide rapid means to define subgroups of breast cancer patients likely to display resistance to trastuzumab ab initio.

YB-1 alters MT1-MMP trafficking and stimulates MCF-7 breast tumor invasion and metastasis

YB-1 is a member of the cold shock domain family, with complex roles in DNA structure, gene transcription and translation. YB-1 promotes chromosomal instability, and mammary gland transgenic expression induces tumors with 100% penetrance. YB-1 is linked to poor prognosis in breast carcinoma and is a strong predictor of relapse and disease-specific survival. Survival is directly tied to the extent of local invasion and distal metastasis, processes dependent upon the activity of the membrane type I-matrix metalloproteinase, MT1-MMP. Non-invasive MCF-7 breast adenocarcinoma cells were transfected with YB-1/EGFP. YB-1 protein was detected in the invadopodia of cells with a migratory phenotype. There was increased expression of MT1-MMP protein concentrated at the leading edges of motile cells, which were highly invasive in collagen three-dimensional culture. The rates of MT1-MMP protein endocytosis and recycling to the cell surface were elevated in clones expressing higher levels of YB-1 protein. Control MCF-7 cells formed nonfatal, non-invasive, differentiated adenocarcinomas in vivo. MCF-7 cells expressing a twofold increase in YB-1 formed highly anaplastic tumors with local invasion, pulmonary metastases and high lethality. We conclude that YB-1 contributes to the development of an invasive, metastatic breast carcinoma phenotype by enhanced presentation of MT1-MMP at the sites of cellular invasion.

Combined effects of IL-8 and CXCR2gene polymorphisms on breast cancer susceptibility and aggressiveness

BackgroundInterleukin-8 (IL-8/CXCL-8) is a prototype of the ELR+CXC chemokines that play an important role in the promotion and progression of many human cancers including breast cancer. We have recently showed the implication of polymorphism (-251) T/A of IL-8 gene in the susceptibility and prognosis of breast carcinoma. IL-8 acts through its CXCR1 and CXCR2 receptors. CXCR2, expressed on the endothelial cells, is the receptor involved in mediating the angiogenic effects of ELR+CXC chemokines and in particular IL-8.In the current study, we investigated the susceptibility and prognostic implications of the genetic variation in CXCR2 in breast carcinoma. We also confirmed the implication of IL-8 (-251) T/A polymorphism in a larger cohort. Finally, we combined the IL-8 and CXCR2 variant alleles and analyzed their effects in breast cancer risk and prognosis.MethodsWe used the allele-specific polymerase chain reaction to characterize the variation of IL-8 and CXCR2 for 409 unrelated Tunisian patients with breast carcinoma and 301 healthy control subjects. To estimate the relative risks, Odds ratios and 95% confidence intervals were calculated using unconditional logistic regression after adjusting for the known risk factors for breast cancer. Associations of the genetic marker with the rates of breast carcinoma-specific overall survival and disease-free survival were assessed using univariate and multivariate analyses.ResultsA highly significant association was found between the homozygous CXCR2 (+ 1208) TT genotype (adjusted OR = 2.89; P = 0.008) and breast carcinoma. A significantly increased risk of breast carcinoma was associated with IL-8 (-251) A allele (adjusted OR = 1.86; P = 0.001). The presence of two higher risk genotypes (the TA and TT in IL-8, and the TT in CXCR2) significantly increased the risk of developing breast carcinoma (adjusted OR = 4.15; P = 0.0004).The CXCR2 (+ 1208) T allele manifested a significant association with an aggressive phenotype of breast carcinoma as defined by a large tumor size, a high histological grade, and auxiliary's lymph node metastasis. A significant association between the IL-8 (-251) A allele and the aggressive form of breast carcinoma was also found.Moreover, the presence of the IL-8 (-251) A and/or the CXCR2 (+ 1208) T allele showed a significant association with a decreased overall survival and disease-free survival in breast carcinoma patients.ConclusionOur results indicated that the polymorphisms in IL-8 and CXCR2 genes are associated with increased breast cancer risk, as well as disease progress, supporting our hypothesis for IL-8 and ELR+CXC chemokine receptor (CXCR2) involvement in breast cancer pathogenesis.

Tight junction protein, claudin-6, downregulates the malignant phenotype of breast carcinoma

Claudin-6 is a protein component of tight junctions and its expression has been found to be undetectable or at low levels in some human and rat breast cancer cells. Here we investigated the effect of claudin-6 upregulation on the malignant phenotype of human MCF-7 breast cancer cells. MCF-7 sublines with stable claudin-6 expression were established by transfection with a pcDNA3.1-claudin-6 expressing vector. The expression of claudin-6 on mRNA and protein levels was confirmed by reverse transcription-PCR, western blot and immunofluorescent assays. Then the effects of claudin-6 on cell proliferation and cell cycle were examined by 5-diphenyl terazolium-bromide assay and flow cytometry, respectively. Colony-forming assays were used to examine two-dimensional and three-dimensional colony forming ability. Invasive and migratory traits of claudin-6 expressing cells were determined by matrigel-based Boyden chamber invasion assay and monolayer wound-healing assay. The structure and function of tight junctions in both parental and claudin-6 expression MCF-7 cells were evaluated by measuring transepithelial electrical resistance. Immunofluorescent assays showed that transfected cells expressed claudin-6 on their membranes. Cells with high level expression of claudin-6 grew slowly and had a higher rate of death than control cells. Anchorage-independent growth, invasive and migratory traits were also substantially decreased in cells with claudin-6 expression; whereas the transepithelial electrical resistance was increased in the claudin-6 transfected cells. In conclusion, these results suggest that claudin-6 may function as a cancer suppressor; its downregulation may contribute to the malignant progression of certain types of breast cancers.

The alternative lengthening of telomeres phenotype in breast carcinoma is associated with HER-2 overexpression

Approximately 10–15% of human cancers do not show evidence of telomerase activity, and a subset of these maintain telomere lengths by a recombination-based mechanism termed alternative lengthening of telomeres (ALT). The ALT phenotype, relatively common in certain sarcomas and germ cell tumors, is very rare in carcinomas. In this study we describe evidence for the ALT phenotype in molecular subclasses of breast carcinoma, specifically a subset of cancers with HER-2 overexpression. Tissue microarrays were created from 71 invasive ductal carcinomas of the breast categorized into subclasses, and telomere lengths were directly assessed using fluorescence in situ hybridization with combined promyelocytic leukemia (PML) protein immunofluorescence. The ALT phenotype was identified in 3 of 21 HER-2-positive cases, but in none of the other 50 cases (P=0.023). This is the first direct observation of this mechanism of telomere maintenance in breast carcinoma unrelated to Li–Fraumeni syndrome. The correlation of the ALT phenotype with HER-2 positivity, both of which involve abnormal DNA amplification, suggests a possible common underlying mechanism. This telomere phenotype confers a poor prognosis in some cancers; two of the three cases in our study showed rapid tumor progression, possibly suggesting that it may adversely affect outcome in breast carcinoma as well. As cancers using the ALT pathway are predicted to be resistant to therapies based on telomerase inhibition, these results may have therapeutic consequences.

Expression and prognostic significance of metalloproteases and their inhibitors in luminal A and basal-like phenotypes of breast carcinoma

To analyze the expression and prognostic value of matrix metalloproteases and their tissue inhibitors in luminal A and basal-like breast carcinomas, an immunohistochemical study was performed on cancer specimens from 93 randomly selected patients with invasive primary ductal tumors of the breast (46 with and 47 without distant metastasis) and with luminal A (n = 48) (ER+, HER2-) or basal-like (HER2-, ER-, PgR-) (n = 45) lesions. Luminal B cases were too few to analyze. Specimens were also studied using tissue microarrays and specific antibodies against matrix metalloproteases 1, 2, 7, 9, 11, 13, and 14 and tissue inhibitors 1, 2, and 3. There were no significant differences in matrix metalloprotease or tissue inhibitor expression in the 2 phenotypes of tumors. In basal-like carcinomas, high scores for matrix metalloproteases 9 and 11 were significantly associated with a high distant metastasis rate. Likewise, data showed associations between matrix metalloprotease/tissue inhibitor expression by either stromal fibroblasts or mononuclear inflammatory cells and distant relapse-free survival in both tumor phenotypes. In addition, in infiltrating luminal A and basal-like tumors, we identified a prometastatic phenotype of mononuclear inflammatory cells, showing a high matrix metalloprotease/tissue inhibitor molecular profile. Expression of matrix metalloproteases and tissue inhibitors is related to the characteristics of breast tumor cells. As prognostic factors in breast carcinomas of both luminal A and basal-like phenotypes, our results point to the importance of the expression of matrix metalloproteases and tissue inhibitors by the stromal cells.

Cytological criteria to predict basal phenotype of breast carcinomas

Breast carcinoma is a heterogeneous disease. It can be classified into phenotypes based on the expression of certain proteins, with distinct differences in prognosis. The basal phenotype is associated with worse prognosis and it still remains without specific treatment. However, there is currently no international consensus on the cytological criteria that could predict this phenotype. The purpose of the study was to evaluate the cytological criteria in fine-needle aspiration biopsy and to identify their association with the basal phenotype of breast carcinoma. Fine-needle aspiration biopsy specimens and tissue sections (mastectomy specimen) from 74 cases of high-grade invasive ductal breast carcinomas were consecutively retrieved from the files of three institutions. Breast carcinomas were studied using the tissue microarray technique, being classified into phenotypes: luminal A, luminal B, HER2 overexpression, and basal. The cytological criteria for all cases were reviewed blindly by two pathologists according to five cytological criteria: cellularity, cell pattern, presence of necrosis, nucleoli, and nuclear atypia. Exact Fisher test was used to test the association between cytological criteria and the phenotypes of breast carcinoma. Necrosis was present in 64.7% of basal breast carcinomas, and 31.1% of nonbasal breast carcinomas, and that result was statistically significant, showing an odds ratio (OR) of 3.80. The basal phenotype, compared with the luminal A, showed more necrosis (OR = 6.97), present/prominent nucleoli (OR = 8.18), and cellularity more frequently (OR = 18.03). Necrosis, as well as present/prominent nucleoli and abundant cellularity are criteria more frequently associated to the basal phenotype of breast carcinoma.

CK5 Is More Sensitive Than CK5/6 in Identifying the “Basal-like” Phenotype of Breast Carcinoma

Multiple immunohistochemical stains, including cytokeratin (CK)5/6, are used in a panel format to identify "basal-like" carcinomas. We set out to determine the sensitivity and specificity of the CK5 antibody (clone XM26) and compared its expression with that of CK5/6 (clone D5/16B4) in a variety of breast carcinoma cases. The study was performed on 3 breast carcinoma tissue microarrays (TMAs). TMA-1 consisted of 59 consecutive breast carcinoma cases. TMA-2 (n = 16) and TMA-3 (n = 11) consisted of basal-like breast carcinomas previously characterized morphologically and immunohistochemically at our institution. Of the 86 total cases, 20 were positive for CK5 and CK5/6, 14 were positive for CK5 only, and 52 were negative for both. The sensitivity of CK5 for identifying basal-like tumors was 97% compared with 59% for CK5/6. Both antibodies had comparable specificity of more than 95%. For positive cases, the percentage and intensity of staining was much higher with CK5 than with CK5/6 (P = .0001).

Basaler und myoepithelialer Phänotyp des invasiven Mammakarzinoms

AimsThe aim of the present study was to evaluate the prognostic impact of basal and myoepithelial phenotype in breast carcinomas (BBC and MBC) in the palliative situation.MethodsParaffin-embedded material from 244 primary breast carcinomas of patients with subsequent metastatic disease was stained immunohistochemically for CK 5/6, CK14, smooth-muscle actin, p63, estrogen receptor and progesterone receptor. BBC was defined as positive for CK5/6 and/or CK14 and MBC as positive for SMA and/or p63. Clinical and pathological data were available for all patients and follow-up data for 96.3% (range 5 days-151 months).ResultsUntil the end of the follow-up period 90.2% of patients died and 6.1% are still alive. Of the tumours 28.7% could be classified as BBC and 8.2% as MBC. Kaplan Meier analysis revealed a trend for reduced survival after first diagnosis of metastasis (OASM) for BBC and MBC. Differences in survival were significant for BBC (log-rank =5.0; p=0.025), but not for MBC. CK5/6+ and CK14+ double positive tumours (n=18; 7.4%) were identified as a subgroup of BBC associated with reduced OASM (log-rank=8,6; p=0.003). This subgroup, but not BBC or MBC was an independent negative prognostic factor in a multivariate analysis including age, typing, tumour size, grading, axillary nodes, HR, Her2/neu, site of first metastasis and disease-free interval.ConclusionThe association of BBC and MBC with reduced OASM in metastatic breast carcinomas is not independent from established prognostic factors. CK5/6+ CK14+ double positive tumours may be a subgroup of BBC with particularly unfavourable outcome.

Expression of EGFR and c‐kit is associated with the basal‐like phenotype in breast carcinomas of African women

Epidermal growth factor receptor (EGFR) and c-kit are tyrosine kinase growth factor receptors which are frequently expressed in basal-like breast carcinomas, and tyrosine kinase inhibition is now a promising strategy in treatment of breast cancer. The aim of this study was to evaluate the expression of EGFR and c-kit in breast cancer with special focus on the basal-like phenotype (BLP) and other prognostic factors in an African population. We analyzed 65 archival tissues immunohistologically. EGFR and/or c-kit were expressed in 55% of basal-like tumors. Expression of EGFR and/or c-kit was strongly associated with high histologic grade (P=0.001), high nuclear grade (P=0.017), high mitotic counts (P=0.002), ER negativity (P=0.003), PR negativity (P=0.007), and HER2 negativity (P=0.014). EGFR and/or c-kit positive tumors were more likely to express the BLP (OR 9.1, CI 2.6-32.0, P<0.0005) than the negative tumors. In conclusion, there is a high expression of EGFR and/or c-kit in basal-like breast carcinoma in this series from Uganda and their expression is associated with features of poor prognosis. More studies are required to assess the clinical significance of EGFR and c-kit in breast cancer patients in Uganda.

WW domain containing E3 ubiquitin protein ligase 1 (WWP1) expression in breast cancer and its correlation with estrogen receptor (ER) and insulin-like growth factor receptor 1 (IGF-1R) status

22076 Background: WWP1, a HECT type E3 ubiquitin ligase, is frequently amplified and overexpressed in breast cancer and may associated with positive ER status. IGF-1R is a tyrosine kinase growth factor receptor that has been linked to prognosis in a variety of malignancies. Activation of IGF-1R by autocrine, paracrine and endocrine stimulation via exposure to its activating ligand, insulin, leads to cell growth. Methods: Formalin-fixed, paraffin-embedded tissue sections from 122 cases of invasive mammary carcinoma (90 ductal carcinomas (IDC) and 32 lobular carcinomas (ILC) were immunostained by automated methods (Ventana Medical Systems Inc., Tucson, AZ) using mouse anti-human WWP1 (Novus Biologicals, Littleton, CO) and mouse anti-human IGF-1R antibody (sc-462; Santa Cruz Biotechnology, Santa Cruz, CA). Cytoplasmic immunoreactivity was semiquantitatively scored based on staining intensity and distribution for each protein and the results were correlated with morphologic and prognostic variables. The correlations between WWP1 and ER/IGF-1R were also analyzed in a panel of breast cancer cell lines by Western blot. WWP1 knockdown studies were used to test WWP1 regulation of ER and IGF-1R in ER positive breast cancer cell lines. Results: The adjacent benign epithelium was essentially negative for both proteins. Cytoplasmic WWP-1 immunoreactivity was observed in 53/122 (43%) tumors and showed a positive correlation with ER status (p=0.038). Cytoplasmic IGF-1R positivity was observed in 66/122 (54%) tumors and correlated with tumor subtype (61% IDC vs. 34% ILC, p=0.011) and ER status within the IDC subgroup (p=0.036). There was a significant co-expression of both proteins (p=0.001). The positive correlations between WWP-1 and ER/IGF-1R were also observed in a panel of breast cancer cell lines assessed by Western blot. WWP1 knockdown decreased the expression levels of both ER and IGF-1R in MCF7 and T47D cell lines. Conclusions: WWP-1 and IGF-1R proteins are overexpressed and are associated with each other and the ER+ phenotype in breast carcinoma. Further development of WWP-1 and IGF-1R as biomarkers for breast cancer management appears warranted. No significant financial relationships to disclose.

The basal‐like breast carcinoma phenotype is regulated by SLUG gene expression

Basal-like breast carcinoma is an aggressive form of breast cancer, characterized by the absence of oestrogen receptor and HER2 expression, the presence of cytokeratin 5 and epidermal growth factor receptor expression, and by the up-regulation of stem cell regulatory genes. We show here that tumour tissues expressing high levels of SLUG mRNA show a basal-like breast carcinoma phenotype and that such tumours also express high levels of stem cell-regulatory genes, ie CD133, Bmi1. Further, we show that stem/progenitor cells, isolated from ductal breast carcinoma and from normal mammary gland as mammospheres, express SLUG, CD133, and Bmi1 mRNA and show a phenotype similar to that of basal-like breast carcinoma. We also report that SLUG expression in tumour tissues correlates with that of the hypoxia survival gene carbonic anhydrase IX. In this regard, we report that the exposure of SLUG-negative/luminal-like MCF-7 cells to a hypoxic environment promotes the onset of the basal-like breast carcinoma phenotype, together with up-regulation of the SLUG gene, which in turn blunts oestrogen receptor-alpha and boosts carbonic anhydrase IX gene expression. Finally, we show that SLUG expression promotes the invasiveness of MCF-7 cells exposed to hypoxia and sustains the in vivo aggressiveness of hypoxia-selected, MCF-7-derived cells in xenografts. These data indicate that SLUG gene expression is part of a hypoxia-induced genetic programme which sets up a basal/stem cell-like, aggressive phenotype in breast cancer cells.

Epigenetic Reversion of Breast Carcinoma Phenotype Is Accompanied by Changes in DNA Sequestration as Measured by AluI Restriction Enzyme

The importance of microenvironment and context in regulation of tissue-specific genes is well established. DNA exposure to or the sequestration from nucleases detects differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we used an established three-dimensional assay in which normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like versus tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion in which the malignant cells resist digestion relative to nonmalignant cells. Treatment of T4-2 breast cancer cells in three-dimensional culture with cAMP analogs or a phosphatidylinositol 3-kinase inhibitor not only reverted their phenotype from nonpolar to polar acinar-like structures but also enhanced chromatin sensitivity to AluI. By using different cAMP analogs, we show that cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin produced the same effect. These experiments underscore the concept that modifying the tumor microenvironment can alter the organization of tumor cells and demonstrate that architecture and global chromatin organization are coupled and highly plastic.

Advanced stages and poorly differentiated grade are associated with an increased risk of HER2/neu positive breast carcinoma only in White women: findings from a prospective cohort study of African-American and White-American women.

The primary objective of this study was to evaluate the race-specific risk associated with HER2/neu positive breast carcinoma in a prospective cohort design. Our secondary objectives were to assess prevalence of different breast cancer phenotypes between African-American and White women and to determine if race was associated with the risk of basal-like breast carcinoma phenotype in this cohort. Demographic, clinical and pathologic data were collected from existing databases. The status of HER2/neu and hormone receptors was dichotomized as either positive or negative. Immunohistochemistry taxonomy was used to assess prevalence of different breast carcinoma phenotypes. Risk estimates were calculated using the multivariable logistic regression statistics. The risk of HER2/neu positive breast carcinoma differs between African-American and White women. For White women only, this risk was statistically significant and increased almost linearly within each TNM stage with grade dedifferentiation. The statistically significantly higher prevalence of "ER(-)/PR(-), HER2(- )" phenotype in African American women potentially is the attributing factor to observed lack of an association between the risk of HER2/neu positive breast carcinoma with advanced stages and poorly differentiated grade. Among women diagnosed with "ER(-)/PR(-), HER2(-)" phenotype the odds ratios of being African-American and pre-menopausal was 1.72 (95% CI 1.17-2.54, P = 0.006) and 1.94 (95% CI 1.27-2.96, P = 0.002), respectively. The histologic features of basal-like and ER(-)/HER2(+ )carcinomas overlaps. Differences in the biology of breast carcinoma between African American and White women are partially attributed to the disparity in more adverse pathologic prognostic indicators at the initial clinical presentation of this disease.

Effect of the different phosphorylated Smad2 protein localizations on the invasive breast carcinoma phenotype

Smad2 participates in the TGF-beta signaling pathway, where it cooperates with transcription factors to regulate expression of defined genes. The purpose of this study was to investigate the expression pattern of phosphorylated Smad2 (pSmad2) in association with clinicopathological parameters and biological markers of proliferation and invasion. Immunohistochemistry was applied on paraffin-embedded sections from 164 patients with invasive breast carcinomas to detect the expression of the proteins pSmad2, ER, PR, Ki67, topoisomerase IIa, ERK2, catenin-p120, MMP-14 and TIMP-2. pSmad2 protein was detected in the nuclei of the malignant cells (68.1%) and in the tumor fibroblasts (55.2%). Nuclear pSmad2 was inversely correlated with histological grade and LN (p=0.047 and p=0.05) as well as with Ki67 and topoIIa (p=0.003 and p=0.021, respectively). There was also an inverse relation between nuclear pSmad2 and normal immunoexpression of the adhesion molecule catenin-p120 (p=0.028). Both nuclear and stromal pSmad2 were positively correlated with ERK2 of tumor fibroblasts (p=0.008 and p=0.0001, respectively), while stromal pSmad2 was furthermore related to stromal MMP-14 and tumor TIMP-2 (p=0.006 and p=0.022, respectively). Patients with high expression of cancerous pSmad2 tended to have a better prognosis, although statistic significance was never reached. pSmad2 was found to play a dual role, according to its distribution. Nuclear localization was thus found to be related to a less aggressive tumor phenotype, whereas stromal location was associated with an invasive phenotype.

Basal phenotype of ductal carcinoma in situ: recognition and immunohistologic profile

The basal phenotype of breast carcinoma was demonstrated from a study of gene expression profiles, which demonstrated five carcinoma phenotypes with differing immunohistologic profiles and outcomes. The basal phenotype, so-named because of an immunohistologic profile that is similar to myoepithelial cells of the breast, has poor outcomes. While the invasive basal phenotype has been described, there is a paucity of literature regarding the existence or recognition of a precursor lesion. We searched our CoPath database for breast carcinomas in the age group of 37 years or less, and this yielded 98 cases from the years 2001 to April 2006. Pathology reports were screened for those cases that were negative for estrogen and progesterone receptors and HER-2/neu (triple negative). A total of 16 cases (16/98, 16%) fulfilled these criteria. Histology was reviewed and immunostains were performed for Cytokeratins 14, 17, and 5/6, vimentin, EGFR, c-kit, smooth muscle actin and p63. All 16 cases had a high-grade invasive ductal carcinoma, Nottingham score 9/9, with geographic necrosis, good circumscription and lymphoid infiltrates. Of the 16 cases, 13 exhibited at least one area of ductal carcinoma in situ (DCIS). The DCIS types were solid, flat or micropapillary, high nuclear grade, with comedonecrosis and invariably associated with intense lymphoid inflammatory cell infiltration. Of 16 invasive cases, 14 (88%) were positive for CK14, CK17, CK5/6 and EGFR; 94% were vimentin positive, while half or less of cases were positive for smooth muscle actin, c-kit or p63. All of the DCIS components demonstrated the same immunohistologic profile as the invasive component. A DCIS component of solid, flat or micropapillary type exists in the basal phenotype of breast carcinoma, and it demonstrates the same immunophenotype as the invasive carcinoma, typically positive for CK5/6, CK14, CK17, vimentin and EGFR, but negative for ER/PR and HER-2/neu.