Basal Phenotype In Breast Cancer Research Articles

Overexpression of HIF1α and CAXI predicts poor outcome in early-stage triple negative breast cancer.

Dysregulated energy metabolism is one of the main mechanisms for uncontrolled growth in solid tumors. Hypoxia-inducible factor 1-alpha (HIF1α) is a transcription factor implicated in regulating several genes that are responsible for cell metabolism, including carbonic anhydrase IX (CAIX). The aim of this study is to determine the clinical significance of immunohistochemical metabolic alteration in early-stage triple negative breast cancer (TNBC) patients who received cyclophosphamide-based chemotherapy or radiotherapy and those with basal phenotype. Immunohistochemical staining for HIF1α and CAIX was performed to determine the correlation with clinicopathologic variables and survival outcome on tissue microarrays from 270 early-stage TNBC patients. In vitro experiments with multiple human TNBC cell lines, suppression of HIF1α by small interfering RNA (siRNA) significantly reduced CAIX protein expression in all cell lines. In multivariate analyses for different therapeutic modalities and basal phenotype, combined HIF1α and CAIX protein overexpression was significantly associated with disease-free survival in the total cohort (OR=2.583, P=0.002), stratified cohorts expressing basal phenotype (OR=2.234, P=0.021), and in those patients who received adjuvant chemotherapy (OR=3.078, P=0.023) and adjuvant radiotherapy (OR=2.111, P=0.050), respectively. In early TNBC, combined HIF1α and CAIX protein expression may serve as an unfavorable prognostic indicator particularly in patients treated with cyclophosphamide-based chemotherapy or radiotherapy as well as those with basal phenotype of breast cancer.

Caveolin-1 in Tumour Cells is Predominately Expressed in the Basal Subtype of Triple-Negative Breast Carcinoma

Abstract Triple-negative breast cancer (TNBC) is a subtype of breast cancer which is clinically negative for expression of steroid hormones and HER2 protein. According to DNA microarray profiling, breast cancer was divided into several molecular subgroups, including Luminal A and Luminal B; HER2 overexpressing; normal breast- like and basal-like subtype. TNBC comprises tumours that express heterogeneous molecular and immunohistochemical phenotype, thus determining different prognosis. The majority of TNBC carry the “basal-like” molecular profile on gene expression arrays. Basal-like carcinomas of the breast associated with poor prognosis often correlate with expression of Caveolin-1. The study aimed to investigate the expression of caveolin-1 in the tumour cells and in the stromal component of the tumour among the group of TNBC and compare it to the expression of caveolin-1 in a control group with non-TN breast cancer. Whole tissue sections were used. Formalin fixed, paraffin embedded tissue materials from 101 patients diagnosed with invasive breast cancer during the period 2004-2007 were investigated in a retrospective study. A multistep approach was used to separate the different subtypes of breast cancer. During the first step the breast tumours were separated according to their ER, PgR, HER 2 and proliferative activity, using the Ki-67 index. The triple- negative tumours were additionally tested for EGFR and CK5/6. The basal breast cancer group was finally subdivided into basal and baso-luminal according to the type of expression of basal cytokeratins. caveolin-1 expression was examined in the tumour parenchyma and stroma. According to our results, caveolin-1 expression in breast cancer was significantly associated with basal biomarkers expression (basal and baso-luminal type of BC) χ2= 33.4; p<0.0001. Caveolin-1 can be used as a potential marker to examine the presence of basal phenotype in breast cancer.

Abstract 750: Identification of tumor antigens in transgenic mouse model as vaccine targets for the prevention of human breast cancer

Abstract The development of a vaccine to prevent breast cancer would be facilitated if tumor antigens were identified which were associated with the initiation of the malignant transformation. Transgenic mouse models have been proven to demonstrate a similar antigenic repertoire as that seen in breast cancer patients, therefore, we propose to use these models to identify tumor antigens as vaccine targets for breast cancer prevention. We have chosen two mouse models: TgMMTV-neu and TgC3(1)-Tag for study. TgMMTV-neu mouse has a genotype similar to luminal breast cancer and the TgC3(1)-Tag model reflects a basal phenotype of breast cancer. We identified potential vaccine candidate antigens in one of two ways; (1) determining which proteins expressed by the cancer are immunogenic very early in the malignant process by screening pre-diagnostic sera for immunogenicity against tumor cDNA libraries, and (2) injecting parental animals with a syngeneic tumor cell line resulting in “tumor rejection” which we have shown previously is mediated by T cells. To date, we have identified nine pre-diagnostic tumor antigens in TgMMTV-neu mouse; Rpl5, TNFaip3/A20, Pdhx, Otud6b, Stk39, Zfp238, Dnajc10, Lgals8 and Vps35. Moreover, we have identified four rejection antigens in TgC3(1)-Tag mouse; Ddx21, Sfrs11, Edh1 and Tdg. We have initiated vaccination experiments to examine the anti-tumor effect of vaccination with plasmids encoding these antigens. We found that vaccination targeting Ddx21, Dnajc10, and Pdhx mediated tumor growth inhibition in implant models compared with a control group (p<0.01). Our preliminary studies also demonstrate that several of the proteins are immunogenic in humans and we have identified putative promiscuous class II epitopes suitable for screening human PBMC for T cells specific to these proteins. 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 750. doi:10.1158/1538-7445.AM2011-750

Utilization of genomic signatures to identify phenotype-specific drugs.

Genetic and genomic studies highlight the substantial complexity and heterogeneity of human cancers and emphasize the general lack of therapeutics that can match this complexity. With the goal of expanding opportunities for drug discovery, we describe an approach that makes use of a phenotype-based screen combined with the use of multiple cancer cell lines. In particular, we have used the NCI-60 cancer cell line panel that includes drug sensitivity measures for over 40,000 compounds assayed on 59 independent cells lines. Targets are cancer-relevant phenotypes represented as gene expression signatures that are used to identify cells within the NCI-60 panel reflecting the signature phenotype and then connect to compounds that are selectively active against those cells. As a proof-of-concept, we show that this strategy effectively identifies compounds with selectivity to the RAS or PI3K pathways. We have then extended this strategy to identify compounds that have activity towards cells exhibiting the basal phenotype of breast cancer, a clinically-important breast cancer characterized as ER-, PR-, and Her2- that lacks viable therapeutic options. One of these compounds, Simvastatin, has previously been shown to inhibit breast cancer cell growth in vitro and importantly, has been associated with a reduction in ER-, PR- breast cancer in a clinical study. We suggest that this approach provides a novel strategy towards identification of therapeutic agents based on clinically relevant phenotypes that can augment the conventional strategies of target-based screens.

The proteins FABP7 and OATP2 are associated with the basal phenotype and patient outcome in human breast cancer

The basal-like or basal phenotype (BP) class of breast cancers have recently attracted attention as a poor prognostic form of breast cancer. However, BP appears to encompass biologically and clinically heterogeneous tumours, resulting in a lack of consensus definition of BP. We analysed 48,000 gene transcripts in 132 invasive breast carcinomas to identify two novel genes (OATP2 and FABP7) significantly associated with BP [defined by cytokeratin (CK)5/6 and/or CK14 positivity]. Using a series of invasive breast carcinoma cases (n = 899), prepared as tissue microarrays, we assessed OATP2 and FABP7 protein expression using immunohistochemistry to investigate associations with clinicopathological variables, patients' outcome and ability to refine BP classification. A total of 7.9 and 15.6% cases were OATP2 and FABP7 positive, respectively. OATP2 was associated with tumours of high histological grade (p < 0.01), ER and PgR negativity (p < 0.01) and shorter breast cancer-specific survival (p = 0.04). FABP7 expression was associated with lower lymph node stage (p < 0.01), ER and PgR negativity (p < 0.01). BP tumours which were FABP7 positive had a significantly longer BCSS (p = 0.05) and disease-free survival (p = 0.01) compared with FABP7 negative basal tumours (p < 0.01). OATP2 positive tumours were associated with adverse survival and increased risk of early recurrence. This study confirms the biological and clinical heterogeneity of the BP in breast cancer. We have identified a novel subgroup of basal tumours showing FABP7 expression that have significantly better clinical outcome. Further studies analysing the role of FABP7 are therefore warranted.

Antibody-based immunotherapy: targeting CSPG4 on human basal breast cancer stem cells.

Abstract Abstract #5046 Introduction: Basal-like breast cancer is associated with high grade, poor prognosis, and younger patient age. Because tumors are estrogen receptor (ER) and HER2 negative, effective targeted treatment for basal breast cancer remains elusive. The lack of efficacy of currently used chemotherapies may reflect their inability to eradicate cancer stem cells (CSC) within a basal breast tumor.&amp;#x2028; Chondroitin sulfate proteoglycan 4 (CSPG4), a molecule initially identified in melanoma cells, is a cell surface antigen involved in migration and invasion of tumor cells. The rat homolog of the human CSPG4, named NG2, is expressed by progenitor cells in several types of tissues. Furthermore, NG2 expression is typically maximal when progenitor cells are mitotic and motile. As one important characteristic of cancer stem cells is their ability to migrate and metastasize, and both CSPG4 and NG2 play a role in tumor cell migration, we tested whether CSPG4 is expressed on cells with the CSC phenotype in ER negative/HER2 negative (basal-like) breast cancer, and can thus serve as a target for immunotherapy of breast CSC.&amp;#x2028; Methods and Results: The mRNA levels of CSPG4 in ER- and Her2- breast cancer were compared to ER-/Her2+, ER+/Her2-, and ER+/Her2+ breast cancer using a publicly available, clinically annotated breast cancer data set. The gene expression level of CSPG4 was elevated in the ER- and Her2- subgroup when compared to ER-/Her2+ (Mann Whitney, p=0.03), ER+/Her2- (Mann Whitney, p=0.01), and ER+/Her2+ (Mann Whitney, p=0.06). These results suggest that CSPG4 expression is best correlated with the basal phenotype of breast cancer. Flow cytometric analysis showed that CSPG4 is expressed on CD44+, CD24-/lo cells in the human basal breast cancer cell lines, i.e., MDA-MD-231, MDA-MB-435, HS578T and SUM149, but is not detectable on the luminal breast cancer cell lines, i.e., MCF-7, SK-BR-3 and T-47D. Furthermore CSPG4 is expressed on the cell surface of putative breast CSC identified as lineage-negative (CD2,3,10,16,18,31,45,64,140b), CD44+, CD24-/lo cells in pleural effusions of 12 patients with breast cancer. Further, inoculation of flow cytometric sorted CD44+CD24-/low CSPG4+ cells from the MDA-MB-231-luciferease expressing cell line, to the mammary fat pads of immunodeficient mice, led to the development of tumors detected by whole-body bioluminescent reporter imaging 3 weeks after the injection. In contrast, inoculation of CD44+CD24-/low CSPG4- cells, sorted from the same cell line, did not generate tumors. More importantly, CSPG4-specific mAb significantly inhibited post-surgery tumor recurrence and lung metastases in SCID mice transplanted with the human basal breast cancer cell line MDA-MB-435, which has the putative breast CSC phenotype (CD44+, CD24-/lo) in more than 99% of cells.&amp;#x2028; Conclusion: These results suggest that the CSPG4 is a potential new immunotherapeutic target for basal-like breast cancer and breast CSC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5046.

Fragile histidine triad protein, WW domain‐containing oxidoreductase protein Wwox, and activator protein 2γ expression levels correlate with basal phenotype in breast cancer

The expression of fragile histidine triad protein (Fhit) and WW domain-containing oxidoreductase protein (Wwox), tumor suppressors that are encoded by fragile (FRA) loci FRA3B and FRA16D, are lost concordantly in breast cancers. In the current study, the authors examined correlations among Fhit, Wwox, the activator protein 2 transcription factors AP2alpha and AP2gamma, cytokeratins 5 and 6 (CK5/6), epidermal growth factor receptor (EGFR), estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) and their associations with breast cancer phenotypes. Tissue microarrays constructed from 837 breast cancer blocks were immunostained. Expression in >10% of tumor cells was considered positive for cytoplasmic CK5/6, membranous EGFR, and nuclear AP2alpha and AP2gamma. Cytoplasmic Fhit and Wwox staining was scored according to staining intensity. ER, PR, and HER-2 status of tumors was derived from records. Correlations among immunohistochemical markers and tumor subtypes were assessed by univariate and multivariate statistical methods. Triple-negative tumors had more frequent expression of EGFR, CK5/6 (P < .001), and AP2gamma (P = .003) and more frequent loss of Fhit and Wwox (P < .001), and an inverse correlation was observed between Fhit, Wwox expression and EGFR, ER, and PR expression (P < .001). Reduced Fhit expression was more common in HER-2-positive and AP2gamma-positive cases (P < .001 and P = .002, respectively). There was a direct correlation noted between Fhit and Wwox (P < .001) and a borderline positive relation between AP2alpha and AP2gamma (P = .054). The results from this investigation suggested that reduced expression levels of Fhit, Wwox, and nuclear AP2gamma have roles in the pathogenesis of basal-like differentiation in breast cancer. Alteration in the expression of fragile site genes occurs in most of these cancers and may contribute to defects in DNA repair, as observed in breast cancer 1 (BRCA1)-deficient cancers. Thus, DNA damage response checkpoint proteins may be targets for treatment.

The combination of high cyclin E and Skp2 expression in breast cancer is associated with a poor prognosis and the basal phenotype

Gene expression studies have identified a basal phenotype of breast cancer; these are hormone receptor and HER2-negative cancers with poor prognosis. High levels of cyclin E and Skp2, and low levels of p27 have previously been individually associated with both basal-like breast cancer and a poor outcome after diagnosis. The goal of this study was to first confirm the prognostic value of these biomolecular markers using a breast cancer tissue microarray. Second, we also test the hypothesis that the combined phenotype of high cyclin E, low p27, and high Skp2 would be a strong predictor of outcome and would be closely associated with the basal phenotype of breast cancer. Our cohort consisted of 438 cases of breast cancer and the median follow-up was 15.4 years. The tissue microarray was constructed from archival tumor blocks and we used commercially available antibodies for biomarker immunostaining. Cyclin E was positive in 46% of cases, p27 was negative in 62%, and Skp2 was positive in 35%. We found cyclin E and Skp2 to be prognostic for breast cancer-specific survival in univariate analyses, but p27 was not prognostic. The strongest predictor of outcome was the combination of cyclin E positive and Skp2 positive (difference in survival of 19% at 10 years, P = .0009). This combination was present in 78 (27%) of 288 cases for which data on both biomarkers were available. This combination was also highly associated with young age at diagnosis, grade 3 tumors, ER-negative status, HER2-negative status, and the basal biomarkers epidermal growth factor receptor and cytokeratin 5/6. However, in a multivariate model including standard clinicopathologic variables, this combination was not found to have independent prognostic significance. In conclusion, the combination of high cyclin E and Skp2 expression predicts for poor prognosis in breast cancer in univariate analysis only, it is associated with high risk features, and it is associated with the basal phenotype.

O-29 Effect of dietary energy restriction on gene expression in normal breast and subcutaneous adipose tissues of overweight women at increased breast cancer risk

Breast cancer is recognised to be a heterogeneous disease with a range of morphological appearances and behaviours. The recently recognised basal phenotype (BP) is associated with poor survival, but the clinical implications of this class of breast cancers remain to be adequately defined.We have examined a well-characterised series of 1872 invasive breast carcinomas with a long term follow-up to assess the clinical significance of BP.A pragmatic definition of the BP as immunophenotypic evidence of basal cytokeratins CK5/6 and/or CK14 expression was used. These tumours were associated with shorter overall survival and disease-free interval in our series as a whole and in both the lymph node (LN) negative and LN positive subgroups. When stratified by histological grade, BP was of highly significant prognostic value in grade 3 but not in grades 1 or 2 tumours. Similarly, it was associated with poor survival in the moderate group of the Nottingham prognostic Index but not in the other groups. In a subgroup comprising LN negative grade 3 tumours, BP was the most powerful prognostic marker followed only by tumour size, while the other variables were non-significant. Patients with BP were more likely to respond to chemotherapy than those with non-basal tumours.Our results provide robust evidence that BP is an important class of breast cancers with a particularly aggressive behaviour in patients with LN negative grade 3 disease. We recommend routine identification of BP in breast cancer and the development of effective adjuvant treatment strategies. These are important observations as these tumours typically lack hormone receptor and HER-2 overexpression limiting the range of relevant adjuvant therapies.

262 POSTER S100A7 and the basal phenotype in breast cancer

262 POSTER S100A7 and the basal phenotype in breast cancer

Basal phenotype identifies a poor prognostic subgroup of breast cancer of clinical importance

Background Breast cancer is recognised to be a heterogeneous disease with a range of morphological appearances and behaviours. The recently recognised basal phenotype (BP) is associated with poor survival, but the clinical implications of this class of breast cancers remain to be adequately defined. Methods We have examined a well-characterised series of 1872 invasive breast carcinomas with a long term follow-up to assess the clinical significance of BP. Results A pragmatic definition of the BP as immunophenotypic evidence of basal cytokeratins CK5/6 and/or CK14 expression was used. These tumours were associated with shorter overall survival and disease-free interval in our series as a whole and in both the lymph node (LN) negative and LN positive subgroups. When stratified by histological grade, BP was of highly significant prognostic value in grade 3 but not in grades 1 or 2 tumours. Similarly, it was associated with poor survival in the moderate group of the Nottingham prognostic Index but not in the other groups. In a subgroup comprising LN negative grade 3 tumours, BP was the most powerful prognostic marker followed only by tumour size, while the other variables were non-significant. Patients with BP were more likely to respond to chemotherapy than those with non-basal tumours. Conclusions Our results provide robust evidence that BP is an important class of breast cancers with a particularly aggressive behaviour in patients with LN negative grade 3 disease. We recommend routine identification of BP in breast cancer and the development of effective adjuvant treatment strategies. These are important observations as these tumours typically lack hormone receptor and HER-2 overexpression limiting the range of relevant adjuvant therapies.

P63 correlates with both BRCA1 and cytokeratin 5 in invasive breast carcinomas: further evidence for the pathogenesis of the basal phenotype of breast cancer

To study the expression of p63, cytokeratin (CK) 5 and CK8/18 in invasive ductal carcinomas and their relationship with BRCA1 and other pathological and immunohistochemical features of clinical significance. Immunohistochemistry with the antibodies p63, CK5, CK8/18, BRCA1, oestrogen receptor, progesterone receptor, p53, c-erbB-2 and Ki67 was performed in 102 formalin-fixed paraffin-embedded samples of invasive ductal carcinomas. The CK5+ cases were submitted to a double-immunolabelling study with p63. There was a strong relationship between CK5 and p63 expression and both markers were associated with hormonal receptor-negative high-grade carcinomas with high proliferative rate. Furthermore, there was coexpression of CK5 and p63 in neoplastic cells, indicating that p63, like CK5, is a marker of the basal phenotype of breast cancer. There was a strong relationship between reduced expression of BRCA1 with both p63 and CK5 expression as well as an inverse correlation between p63 and CK8/18 expression, suggesting that loss of p63 expression is required for the transition between a basal to a luminal phenotype of breast carcinoma. Since p63 is thought to be a marker of stem cells and may act as an oncogene, our data support the idea that BRCA1 acts as stem cell regulator.