Tumor ER Research Articles

A microRNA Expression Profile Consisting of 12 microRNAs Is Associated with Inflammatory Breast Cancer.

Abstract Introduction: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with a dismal clinical outcome. In the past we showed that IBC is characterized by a specific gene expression pattern. In this study, we investigate if microRNAs (miRNAs) are responsible for the specific IBC gene signature.Materials and methods: 377 miRNAs were profiled in 20 IBC tumor samples and 50 non-stage matched nIBC tumour samples using the human MicroRNA A Array Set version 2.0 (Applied Biosystems). The IBC and nIBC study populations are matched for tumor grade and ER status. All miRNAs with Ct-values less than 35 in 25% of the samples were included for analysis. Data normalization was performed relative to the median miRNA expression level per sample and expression values were log2-transformed. Principal component analysis (PCA) was performed to identify metagenes of miRNAs, associated with the IBC phenotype. Relevant metagenes were compared with prognostic/predictive gene signature-classifications (wound healing response, invasiveness gene signature, 70-gene prognostic signature, genomic grade index, recurrence score, HOXB13/IL17RB-expression ratio and the molecular breast cancer subtypes). Data were validated by analysing relevant miRNA target gene sets on an IBC/nIBC gene expression data set.Results: Using PCA, we identified a metagene associated with the IBC phenotype (Hazard Ratio: 4.500; P=0.011). This metagene was also significantly associated with the Luminal B phenotype, the IBC signature and with an elevated HOXB13/IL17RB-expresion ratio (range Rs = 0.302 – 0.366; P<0.05). MiRNAs significantly (FDR<0.1) associated with this metagene were: miR29A, miR544, miR574-3p, miR548C-3p, miR451, miR548B-5p, miR24, miR28-5p, miR137, miR302B, miR148A and miR302A (range Rs: -0.367 – 0.307). Cluster analysis using this miRNA signature identified two sample clusters, one containing 70% of the IBC samples and a second one containing 64% of the nIBC samples (Pearson X2; P=0.021). Using gene set enrichment analysis for miR24-, miR28-, miR29A-, miR137-, miR148A-, miR302A- and miR302B target gene sets, we confirmed the differential expression for the above mentioned miRNAs (P<0.01) except for miR28. Using Oncomine analyses we were able to associate the identified miRNAs with NFkB activation (miR29, miR148), steroid receptor signalling (miR302A, miR302B, and miR137), cell adhesion (miR29), stem cell signalling (miR137, miR28) and regulation of the extracellular matrix composition (miR29, miR148).Discussion: Through PCA we identified a miRNA signature associated with IBC. The miRNA signature was partly validated on the gene expression level. Interestingly, most identified miRNAs regulate biological processes previously associated with IBC through gene expression analysis, confirming our results also at the biological level. The association of the IBC-specific miRNA-metagene with the Luminal B phenotype and elevated HOXB13/IL17RB-expression ratios confirms our data that ER+ IBC samples, which constitute the majority of the present IBC study population, generally belong to the Luminal B subtype and are associated with resistance to endocrine therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6119.

Biomarkers of Oxidative Stress in Breast Cancer.

Abstract INTRODUCTIONDespite significant improvements in cancer diagnosis and treatment, approximately one-quarter of breast cancer patients will die of their disease. Oxidative stress has been implicated as a contributory factor in the development of breast cancer. Reactive oxygen species can initiate lipid peroxidation, resulting in generation of reactive secondary products which can induce oxidative DNA damage and mutations in the p53 tumour suppressor gene, which is one of the most frequent mutations associated with breast cancer development. Oxidative stress has also been associated with activation of transcription factors and tamoxifen resistance. The aim of the present study was to identify the levels of lipid peroxdation products; malondialdehyde and 8-iso-Prostaglandin F2α (8-iso-PGF2α), in patients with primary operable breast cancer, and to correlate these levels with pathological factors of disease including tumour grade, ER and HER2 status and also Nottingham prognostic index (NPI) status.METHODS:89 patients with primary operable breast cancer and 10 healthy subjects were studied. Plasma levels of MDA (total and free) and 8-iso-PGF2α were measured.RESULTS:The majority of patients were over 50 years (87%), had grade II/III tumours (68%), were axillary lymph node positive (41%) and had ER-positive tumours (82%). Compared to the control, patients had higher levels of total MDA (p<0.001), free MDA (p<0.001), and 8-iso-PGF2α (p<0.001). Both free (p<0.05) and total MDA (p=0.001) were significantly correlated with increasing tumour grade. Furthermore, total MDA levels were also associated with increased tumour size (p<0.05) and ER-negative tumours (p<0.05). Taken together, both total (p<0.001) and free (p <0.05) MDA were significantly associated with higher NPI categories. 8-iso-PGF2α showed only a correlation with increasing age (p<0.05)CONCLUSION:The present study shows that patients with breast cancer have evidence of increased levels of oxidative stress. Furthermore, MDA concentration was significantly associated with conventional markers of poor prognosis. This data suggests oxidative stress may be involved in tumour progression to a more aggressive phenotype. Thus MDA maybe a useful marker of tumour associated oxidative damage and may provide a promising marker for risk prediction and targets for preventative measures in breast cancer patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2141.

B7-H4gene polymorphisms are associated with sporadic breast cancer in a Chinese Han population

BackgroundB7-H4, a co-inhibitory molecule of the B7 family, can restrain T cell proliferation, cytokine secretion and the development of cytotoxicity. B7-H4 is expressed in tumor tissues at a higher level than in normal tissues, and has a potential effect to protect tumors from anti-tumor immune responses. This case-control study was carried out to determine the potential influences of B7-H4 gene polymorphisms on the susceptibility and progression of breast cancer in Han women of Northeast China.MethodsWe genotyped three B7-H4 variants (rs10754339, rs10801935 and rs3738414) and tagged all common haplotypes (frequency greater than or equal to 1%) in a Chinese population consisting of 500 breast cancer cases and 504 control individuals matched for age. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to determine the genotypes.ResultsOur data indicated that, compared with the common genotype and allele of each SNP, the rs10754339 AG genotype and G allele showed a significantly increased risk of breast cancer (OR = 1.455, 95% CI 1.119-1.892; OR = 1.325, 95% CI 1.073-1.637, respectively). The rs10801935 CC genotype, the rs3738414 AA genotype and the rs3738414 A allele were associated with a significantly decreased risk of breast cancer (OR = 0.328, 95% CI 0.145-0.739; OR = 0.412, 95% CI 0.203-0.835; OR = 0.698, 95% CI 0.564-0.864, respectively). Additionally, the rs10754339 GG genotype was significantly associated with lymph node metastasis and PR status, and the G allele and the AG genotype were respectively associated with lymph node metastasis and ER status. In haplotype analysis, we observed that compared with the AAG haplotype, the AAA haplotype showed a significantly decreased risk of breast cancer (OR = 0.689, 95% CI 0.539-0.881), but the GAG haplotype was associated with a significantly increased risk of breast cancer (OR = 1.511, 95% CI 1.125-2.031). And the AAA and the GCG haplotypes also respectively have significant influences on tumor size and ER status.ConclusionThese results suggest that B7-H4 gene polymorphism may contribute to the sporadic breast cancer risk and prognosis in Chinese Han women.

Tissue estradiol is selectively elevated in receptor positive breast cancers while tumour estrone is reduced independent of receptor status

Previous studies have suggested elevated estrogen production in tumour-bearing breast quadrants as well as in breast cancers versus benign tissue. Using highly sensitive assays, we determined breast cancer tissue estrogen concentrations together with plasma and benign tissue estrogen concentrations in each quadrant obtained from mastectomy specimens (34 postmenopausal and 13 premenopausal women). We detected similar concentrations of each of the three major estrogens estradiol (E 2), estrone (E 1) and E 1S in tumour-bearing versus non-tumour-bearing quadrants. Considering malignant tumours, intratumour E 1 levels were reduced in cancer tissue obtained from pre- as well as postmenopausal women independent of tumour ER status (average ratio E 1 cancer: benign tissue of 0.2 and 0.3, respectively; p < 0.001 for both groups), suggesting intratumour aromatization to be of minor importance. The most striking finding was a significant (4.1–8.6-fold) increased E 2 concentration in ER positive tumours versus normal tissue ( p < 0.05 and <0.001 for pre- and postmenopausal patients, respectively), contrasting low E 2 concentrations in ER− tumours ( p < 0.01 and <0.001 comparing E 2 levels between ER+ and ER− tumours in pre- and postmenopausals, respectively). A possible explanation to our finding is increased ligand receptor binding capacity for E 2 in receptor positive tumours but alternative factors influencing intratumour estrogen disposition cannot be excluded.

Angiogenesis and expression of estrogen and progesterone receptors as predictive factors of recurrence in meningiomas: A long-term prospective study

e13013 Background: Meningioma is a bening tumor, with a high rate of recurrence after surgery (80%); the most important relapse predictive factor is the extent of surgical resection; other potentially predictive factors have been studied with poor results. Angiogenesis has an important role in growth and spread of neoplasic cells; previous studies have shown a high incidence of cyclin E (CE), estrogen, and progesterone receptors (ER, PR) in meningiomas. The aim of this prospective study was to evaluate the prognostic significance of clinical-pathological factors (CPF), vascular density index (VDI), cell proliferation index (CPI), CE, ER, and PR tissue expression in meningioma recurrence of patients submitted to surgical resection. Methods: From January 1995 to December 2000, we enrolled 42 patients with histopathological diagnosis of meningioma and treated only with surgical resection. Tumor VDI, CPI, CE, ER, and PR tissue expression were evaluated by immunohistochemistry in patients with or without recurrence. CPF, VDI, CPI, and expression of CE, ER, and PR were associated with recurrence. Results: Complete surgical resection was achieved in 32.5% of patients. Minimal follow-up was 6 years. Recurrence of meningioma was found in 17 patients (40%). Mean time of recurrence was 32 + 8 months. Tissue expression was positive for CE, ER, and PR in 90.9%, 27.6%, and in 64% of patients, respectively. VDI was &gt;8 (40X in 10 fields) in 52.6% of patients and CPI was &gt;600 (40X in 10 fields) in 54.2% of patients. Significant recurrence-associated factors were extent of resection (RR = 2.4, 95% CI 1.7–2.9, p = 0.03) and VDI &gt;8 (RR = 1.7, 95% CI 1.003–2.9, p = 0.001). CPI, CE, ER, and PR expressions were not statistically significant (p = 0.83, p = 0.16, p = 0.46, and p = 0.74, respectively). Conclusions: Patients with partially resected meningioma and high VDI have an increased recurrence risk and could benefit of additional therapeutic measures. No significant financial relationships to disclose.

Detection of methylation in the CpG islands of the RASSF1A and RAR2b gene promoters in breast cancer (BC)

e14632 Background: Aberrant methylation of CpG islands of cancer-related genes has emerged as an important epigenetic mechanism during carcinogenesis. We examined the methylation of RASSF1A (Ras-association domain family 1, isoform A) and RAR2b (Retinoic Acid Receptor 2b) genes in BC. The knowledge of their role in BC is limited and confused to date. Methods: Promoter methylation was measured in 37 pts, 26 with BC and 11 with benign lesions. The specimens were obtained from archive formalin-fixed paraffin-embedded tumors. After DNA extraction, the methylation was determined by chemical modification of DNA with sodium bisulfite and subsequent double “hot start” Methylation-Specific PCR (MSP), followed by detection on agarose gel. Results: Methylation of at least one of the genes was observed in 18/26 pts (p&lt;0.05). Methylation of RASSF1A gene was observed in 15/26 and RAR2b in 11/26 pts. Both genes were methylated in 8/26 pts. Correlation between methylation and clinicopathological features revealed an association of the RASSF1A gene with T2 tumors (12/15 pts, p&lt;0.05) and ER (+) status (12/15 pts, p&lt;0.05). T2 tumors and ER (+) status presented together in 9/15 pts. The methylation of RAR2b gene was not associated with any known clinicopathological features. In benign lesions methylation of at least one of the genes was observed in 8/11 pts (p&lt;0.05). Methylation of RASSF1A gene was observed in 5/11 pts and RAR2b in 7/11 pts. Both genes were methylated in 4/11 pts. Conclusions: Both genes are frequently methylated in both benign and malignant breast lesions. The association of methylation of RASSF1A gene with tumor size and ER status is noteworthy. The small number of pts does not allow us to confirm the exact role of gene methylation in BC yet. Larger studies are required in order to assess if these epigenetic alterations point out new BC markers, which could be helpful for BC control. No significant financial relationships to disclose.

Triple-negative breast cancers are increased in black women regardless of age or body mass index

IntroductionWe investigated clinical and pathologic features of breast cancers (BC) in an unselected series of patients diagnosed in a tertiary care hospital serving a diverse population. We focused on triple-negative (Tneg) tumours (oestrogen receptor (ER), progesterone receptor (PR) and HER2 negative), which are associated with poor prognosis.MethodsWe identified female patients with invasive BC diagnosed between 1998 and 2006, with data available on tumor grade, stage, ER, PR and HER2 status, and patient age, body mass index (BMI) and self-identified racial/ethnic group. We determined associations between patient and tumour characteristics using contingency tables and multivariate logistic regression.Results415 cases were identified. Patients were racially and ethnically diverse (born in 44 countries, 36% white, 43% black, 10% Hispanic and 11% other). 47% were obese (BMI > 30 kg/m2). 72% of tumours were ER+ and/or PR+, 20% were Tneg and 13% were HER2+. The odds of having a Tneg tumour were 3-fold higher (95% CI 1.6, 5.5; p = 0.0001) in black compared with white women. Tneg tumours were equally common in black women diagnosed before and after age 50 (31% vs 29%; p = NS), and who were obese and non-obese (29% vs 31%; p = NS). Considering all patients, as BMI increased, the proportion of Tneg tumours decreased (p = 0.08).ConclusionsBlack women of diverse background have 3-fold more Tneg tumours than non-black women, regardless of age and BMI. Other factors must determine tumour subtype. The higher prevalence of Tneg tumours in black women in all age and weight categories likely contributes to black women's unfavorable breast cancer prognosis.

RET finger protein expression in invasive breast carcinoma: Relationship between RFP and ErbB2 expression

RET finger protein (RFP), which belongs to the large B-box RING finger protein family, has been reported to be expressed in breast carcinoma cell lines. We conducted this study to evaluate the expression level of RFP in breast carcinomas. Tissue microarrays were constructed from 133 cases of invasive breast carcinoma. Sections obtained from tissue arrays and whole sections from 10 non-neoplastic breast tissues were stained for ER, PR, ErbB2, and RFP using immunohistochemistry, and ErbB2 gene status was evaluated by FISH. The correlation between various immunohistochemical markers was determined. We found nuclear RFP expression in 41.4% of invasive carcinomas and in none of the non-neoplastic breast tissues. We also stained whole sections of 29 cases of invasive breast carcinoma, which included the TMA study, and 10 cases of ductal carcinoma in situ (DCIS) by RFP. We observed that four (40%) of the DCIS cases were positive with RFP. In statistical analysis, there was a significant, positive association between RFP and ErbB2 status at the protein level ( p=0.002) and the gene level ( p=0.049). There was no statistically significant association between the expression of RFP and tumor size, LN status, ER, PR, and grade ( p>0.05). However, we found a significant association between age and RFP expression. RFP expression was stronger in patients 50 years or older ( p=0.048). In our study, the expression of RFP correlated strongly with ErbB2 status. Our study is the first in the literature to show expression of RFP in patients with breast carcinoma. However, the possible role of RFP in breast carcinogenesis needs to be investigated in more detailed clinical and experimental studies.

Is the prognostic value of Ki-67 dependent on ER status and histological grade?.

Abstract Abstract #1076 Purpose: Histological grade, tumor size, age, hormone receptor, and HER2 are established prognostic indicators for distant recurrences in node negative breast cancer. However, the role of the proliferation marker Ki-67 as a prognostic factor is still under debate. The aim of this study is to evaluate the additional prognostic value of Ki-67 in lymph node negative breast cancer.&amp;#x2028; Patients and methods: In 200 consecutive premenopausal women with lymph node negative breast cancer, Ki-67 was determined using the monoclonal MIB-1 antibody on tissue microarray. High Ki-67 proliferation index was defined as &amp;gt;20% positive tumor cells. Cox proportional hazards regression was used to model the impact of the prognostic factors on distant disease-free survival (DDFS). Due to non-proportional hazards, the analysis was restricted to the first 5 years after diagnosis, a time period during which 38 patients developed distant recurrences.&amp;#x2028; Results: Strong positive correlations were observed between Ki-67, histological grade, S-phase, and HER-2. In univariate analysis, Ki-67 was a prognostic factor for DDFS (HR 2.9 95%CI: 1.5-5.7, p=0.002), as well as HER2, histological grade, age and ER. Tumor size was not a significant factor. In multivariate analysis, Ki-67 was an independent prognostic factor for DDFS (HR 2.3 95% CI: 1.0-5.0, p=0.044) together with HER2 and age, whereas tumor size and ER were not significant factors. Models including both Ki-67 and histological grade were not fitted due to colinearity. When stratifying for ER status Ki-67 was a significant prognostic factor in the ER positive group only (HR 5.1 95% CI: 2.0-13, p=0.001, univariate analysis) and significant interaction between the two markers was found (p=0.02). When stratifying for histological grade, Ki-67 could divide the patients with histological grade 2 (n=69; 10 distant recurrences) into two separate groups with significant difference in DDFS (HR 15 95% CI: 3.7-57, p=0.0001). This remained significant after adjustment for age and HER2 status. In histological grade 1 and 3, Ki-67 provided no additional prognostic information.&amp;#x2028; Conclusion: This study demonstrates that Ki-67 is an independent prognostic factor for premenopausal patients with lymph node negative breast cancer. It is noteworthy that Ki-67 adds prognostic information in ER positive patients, but not in ER negative, and in patients with histological grade 2, but not in patients with histological grade 1 and 3. Taken together, Ki-67 may be clinically helpful for prognostic considerations and for selection of adjuvant treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1076.

Prognostic Factors Influence on the Systemic Recurrence in Axillary Lymph Node Negative Breast Cancer

Purpose: Axillary lymph node metastasis is one of the most important prognostic factors in breast cancer. Previous reports show differences that clinicopathologic factors influence the systemic recurrence and survival in axillary lymph node negative breast cancer. Thus, we have attempted to determine the prognostic factors influence on the systemic recurrence and survival in axillary lymph node negative breast cancer. Methods: We retrospectively reviewed the data of 1,351 node negative breast cancer patients who underwent curative surgery to determine the prognostic factors such as age, sex, body mass index (BMI), family history, bilateral breast cancer, operation method, tumor size, stage, histologic grade, number of resected lymph nodes, hormone receptor status, overexpression of p53 and c-erbB2, and adjuvant therapy that influence the systemic recurrence and 10-year-distant relapse-free survival. Results: Systemic recurrence occurred in 58 patients (4.3%) during 53.3 months median follow up period. The tumor size (P=0.001), stage (P=0.005), histologic grade (P=0.049). ER (P=0.028), PR (P=0.002), overexpression of p53 (P=0.001) and bilateral breast cancer (P=0.043) were statistically significant factors that influenced the systemic recurrence. In multivariate analysis, only tumor size was associated with the systemic recurrence (P=0.003). Tumor size (P=0.004), histologic grade (P=0.035), ER (P=0.046), PR (P=0.001) and bilateral cancer (P=0.003) were statistically significant factors that influenced 10-year-distant relapse-free survival. Conclusion: The larger tumor size was determined to be an independent prognostic value in axillary lymph node negative breast cancer.

Outcome Prediction for Estrogen Receptor-Positive Breast Cancer Based on Postneoadjuvant Endocrine Therapy Tumor Characteristics

BackgroundUnderstanding how tumor response is related to relapse risk would help clinicians make decisions about additional treatment options for patients who have received neoadjuvant endocrine treatment for estrogen receptor–positive (ER+) breast cancer.MethodsTumors from 228 postmenopausal women with confirmed ER+ stage 2 and 3 breast cancers in the P024 neoadjuvant endocrine therapy trial, which compared letrozole and tamoxifen for 4 months before surgery, were analyzed for posttreatment ER status, Ki67 proliferation index, histological grade, pathological tumor size, node status, and treatment response. Cox proportional hazards were used to identify factors associated with relapse-free survival (RFS) and breast cancer–specific survival (BCSS) in 158 women. A preoperative endocrine prognostic index (PEPI) for RFS was developed from these data and validated in an independent study of 203 postmenopausal women in the IMPACT trial, which compared treatment with anastrozole, tamoxifen, or the combination 3 months before surgery. Statistical tests were two-sided.ResultsMedian follow-up in P024 was 61.2 months. Patients with confirmed baseline ER+ clinical stage 2 and 3 tumors that were downstaged to stage 1 or 0 at surgery had 100% RFS (compared with higher stages, P < .001). Multivariable testing of posttreatment tumor characteristics revealed that pathological tumor size, node status, Ki67 level, and ER status were independently associated with both RFS and BCSS. The PEPI model based on these factors predicted RFS in the IMPACT trial (P = .002).ConclusionsBreast cancer patients with pathological stage 1 or 0 disease after neoadjuvant endocrine therapy and a low-risk biomarker profile in the surgical specimen (PEPI score 0) have an extremely low risk of relapse and are therefore unlikely to benefit from adjuvant chemotherapy.

Inflammatory breast carcinoma signature and relapse-free survival in patients with noninflammatory breast cancer

11048 Background: Inflammatory Breast Carcinoma (IBC) is an aggressive form of breast cancer associated with poor patient survival. We hypothesize that a gene expression profile characteristic for IBC might be related to tumour aggressiveness in nIBC. Methods: RNA from 19 IBC samples and 40 nIBC samples was hybridized onto Affymetrix chips. A gene signature predictive of IBC was identified and applied onto 7 publicly available gene expression data sets (1157 nIBC samples) with survival data of 881 nIBC samples (4 data sets). Samples were classified as “IBC-like” or “nIBC-like”. Relapse Free Survival (RFS) was compared between these groups by the Kaplan-Meyer method. We classified the 1157 nIBC breast cancer samples according to other prognostically relevant gene signatures and compared these classifications with the IBC signature classification. Cox regression analysis was performed to identify the most predictive signature with respect to RFS. Results: Patients with an “IBC-like” phenotype demonstrate a shorter RFS interval in all 4 data sets (p=0.049, p=0.032, p<0.0001, p=0.0005). Classification according to the IBC signature is significantly (p<0.0001) associated with the cell-of-origin subtypes-, the Wound Healing Response (WHR)-, the Invasive Gene Signature-, the Genomic Grade Index (GGI)- and the Fibroblastic Neoplasm Signature. Significant associations (p<0.0001) were found between the IBC signature and tumour grade and ER status. Cox regression analysis on the entire data set of 881 nIBC samples identified the IBC signature as an independent predictor of RFS (RR=1.532, C.I.=1.100–2.133, p=0.012), together with the WHR and GGI. Conclusions: We demonstrate that nIBC breast tumours having an “IBC-like” phenotype have a reduced RFS interval. This suggests that IBC and nIBC tumours demonstrate the same phenotypic traits with respect to aggressive tumour cell behaviour. Gene signatures related to tumour stroma and tumour grade add information regarding patient survival. Hence the IBC signature represents a different aspect of aggressive tumour behaviour. No significant financial relationships to disclose.

Pathologic node involvement as a prognostic factor in HER2-amplified primary breast cancer (BC) patients (pts) treated without trastuzumab

11537 Background: Current management of BC pts with HER2 overexpression includes chemotherapy and trastuzumab treatment. In order to assess the potential individual benefit of this treatment we analyzed outcome data and prognostic factors in a prospectively characterized cohort of HER2 positive BC pts treated before the trastuzumab era. Methods: Medical records from 103 HER2 positive BC pts treated between 1996 and 2005 according to our standard institutional protocols without adjuvant trastuzumab were analyzed. Clinicopathologic variables such as age, menopausal status, clinical stage, pathologic tumor size (pTS), histologic grade (HG), ER and PR status, lymphovascular invasion (LVI) and axillary lymph node (ALN) status were extracted from the institutional database that has been prospectively collected since 1996. Variables with a 0.15 significance level in univariate analysis were entered in a multivariate Cox model. A backward stepwise procedure discriminated non-influential variables at 0.05 level. R...

Expression of TRPC6 channels in human epithelial breast cancer cells

BackgroundTRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues.MethodsMolecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration).ResultsA non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPM7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours.ConclusionOur results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.

CDKN1C/p57kip2is a candidate tumor suppressor gene in human breast cancer

BackgroundCDKN1C (also known as p57KIP2) is a cyclin-dependent kinase inhibitor previously implicated in several types of human cancer. Its family members (CDKN1A/p21CIP1 and B/p27KIP1) have been implicated in breast cancer, but information about CDKN1C's role is limited. We hypothesized that decreased CDKN1C may be involved in human breast carcinogenesis in vivo.MethodsWe determined rates of allele imbalance or loss of heterozygosity (AI/LOH) in CDKN1C, using an intronic polymorphism, and in the surrounding 11p15.5 region in 82 breast cancers. We examined the CDKN1C mRNA level in 10 cancers using quantitative real-time PCR (qPCR), and the CDKN1C protein level in 20 cancers using immunohistochemistry (IHC). All samples were obtained using laser microdissection. Data were analyzed using standard statistical tests.ResultsAI/LOH at 11p15.5 occurred in 28/73 (38%) informative cancers, but CDKN1C itself underwent AI/LOH in only 3/16 (19%) cancers (p = ns). In contrast, CDKN1C mRNA levels were reduced in 9/10 (90%) cancers (p < 0.0001), ranging from 2–60% of paired normal epithelium. Similarly, CDKN1C protein staining was seen in 19/20 (95%) cases' normal epithelium but in only 7/14 (50%) cases' CIS (p < 0.004) and 5/18 (28%) cases' IC (p < 0.00003). The reduction appears primarily due to loss of CDKN1C expression from myoepithelial layer cells, which stained intensely in 17/20 (85%) normal lobules, but in 0/14 (0%) CIS (p < 0.00001). In contrast, luminal cells displayed less intense, focal staining fairly consistently across histologies. Decreased CDKN1C was not clearly associated with tumor grade, histology, ER, PR or HER2 status.ConclusionCDKN1C is expressed in normal epithelium of most breast cancer cases, mainly in the myothepithelial layer. This expression decreases, at both the mRNA and protein level, in the large majority of breast cancers, and does not appear to be mediated by AI/LOH at the gene. Thus, CDKN1C may be a breast cancer tumor suppressor.

QS111. Correlation of COX-2 Expression With Triple Receptor Negative Status in Operable Human Breast Cancer

Objective: COX-2 expression has been associated with large tumor size, high histological grade, negative hormone receptor status, high proliferation rate, axillary node metastases and ductal type of histology. We hypothesized that increased COX-2 expression is associated with triple receptor-negative status (negative for the activation of ER and PgR, and for the expression/amplification of HER2) in primary breast tumors as these tumors are aggressive and have a worse prognosis. Methods: Clinical and pathological data were collected from 109 patients with operable breast cancer enrolled in a prospective clinical trial comparing primary tumor characteristics from tumor tissue collected at definitive surgery for primary breast cancer (Stage I-III) with micrometastatic disease in bilateral bone marrow aspirates and peripheral blood. Primary tumors were immunostained with a monoclonal antibody directed against COX-2. Positive status was determined as 5% or more of tumor cells staining positive. The CellSearch system (Veridex Corporation, Warren NJ) was used to detect CTCs in whole peripheral blood. CTCs were defined as nucleated cells lacking CD45 but expressing cytokeratins 8, 18, or 19. The presence of 1 or more epithelial cells per 10 mL of blood was considered a positive result. Bone marrow aspirates were obtained from bilateral iliac crests. Anti-cytokeratin antibody was used to assess for the presence of DTCs. One or more cytokeratin positive cells were considered a positive result for DTCs. Primary tumor characteristics studied: tumor size, ER, PgR, HER2, tumor grade, histological type, lympho-vascular invasion (LVI), Ki-67 and lymph node metastasis. Statistical analysis utilized Chi squared test. Results: Currently, we have assessed 83 primary tumor specimens for COX-2 expression. Triple-negative status was present in 29% (32/109) patients and COX-2 expression was present in 29% (24/93). There was no statistically significant correlation between triple-negative receptor status and tumor size, histological type, lymph node metastasis, CTC status, and DTC status. Patients with triple receptor-negative status tended to express COX-2 more frequently than patients with any one of the receptors being positive (p =0.001, OR =5.79). Statistically significant correlations were also obtained between triple receptor-negative status and grade of tumor (p <0.001, OR =6.53) and Ki-67 (p=0.01, OR=6.43). Conclusions: Triple receptor-negative primary breast tumors express high levels of COX-2 as compared to tumors expressing any one of the three primary tumor markers. Identifying this subset of patients expressing increased COX-2 production could open a potential avenue of treating them with COX-2 inhibitors.

Androgen Receptor As A Target For the Treatment of Hormone Receptor-Negative Breast Cancer: An Unchartered Territory

Estrogen receptor-negative (ER-) and progesterone receptor-negative (PR-) breast cancers represent approximately 30% of all breast cancers and, in general, have a more aggressive clinical course. They are unresponsive to antiestrogens, more likely to be poorly differentiated, of higher histological grade and are associated with a higher recurrence rate and decreased overall survival. Androgen receptor (AR) expression has been reported in over 70% of breast cancer and in 45-50% of patients with ER-negative breast cancer. There is emerging evidence that the androgen signaling pathway plays a critical role in breast carcinogenesis, independent of ER. Preclinical data have suggested the inhibitory role of adrenal steroids, such as dehydroepiandosterone (DHEA) and its sulfate on the growth of human ER-negative breast cancer cell lines, when these demonstrate a strong expression of AR. This potentially results in decreased AR gene expression. However, DHEA has been shown to stimulate growth in breast cancer cells when an ER is expressed in ER-positive breast cancer cells. Therefore, the effect of adrenal steroids may differ based on the tumor hormone receptor status and ER-/PR- breast tumors may not be truly hormone 'insensitive'. Exploration of new androgen-based hormonal therapy is warranted in this patient population. This article reviews the role of the AR in breast cancer and discusses potential avenues for the treatment of ER-/PR-/AR+ tumors with 'hormonal therapy'.

Prognostic factors in women with breast cancer: inequalities by ethnicity and socioeconomic position in New Zealand.

To investigate differences in breast cancer prognostic factors between ethnic and socioeconomic groups in New Zealand. We analyzed all 21,586 breast cancer cases on the New Zealand Cancer Registry (July 1994-June 2004). Māori, Pacific, and non-Māori/non-Pacific women were categorized according to ethnicity on the Registry. Deprivation was analyzed as quintiles of the New Zealand Deprivation Index 2001, an area-based measure of socioeconomic position. Logistic regression was used to estimate age-adjusted odds ratios (OR) (95% confidence intervals (CI)). Māori and Pacific women were more likely to have non-local stage, less well differentiated cancer, larger tumors and positive human epidermal growth factor receptor-type 2 (HER-2) status than non-Māori/non-Pacific women. Māori were less likely and Pacific women more likely than non-Māori/non-Pacific women to have negative oestrogen (ER) and progesterone receptor (PR) status. Adjusting for deprivation did not materially alter the results. Women living in more deprived areas had a higher risk of non-local stage and larger tumors. These associations were only partially explained by ethnicity. There was no relationship between tumor grade, ER, PR or HER-2 status and deprivation. Our results confirm that Māori, Pacific and low socioeconomic women present with poor prognosis breast tumors.

Forkhead box A1 expression in breast cancer is associated with luminal subtype and good prognosis

Aims:Forkhead box A1 (FOXA1) is a forkhead family transcription factor expressed in breast cancer cells. It is essential for optimal expression of ∼50% of oestrogen receptor (ER)-related genes. This study...

JAG1 expression is associated with a basal phenotype and recurrence in lymph node-negative breast cancer

Expression of the JAG1 Notch ligand has previously been shown to correlate with poor overall survival in women with advanced breast cancer. We undertook to test whether expression of JAG1 is associated with reduced disease free survival (DFS) in 887 samples from a prospectively accrued LNN cohort with a median follow-up greater than 8 years. Moderate to high JAG1 mRNA expression was associated with reduced DFS in univariate analysis (hazard ratio of 1.58; 95% confidence interval, 1.03-2.40; P = 0.034) and correlated with large tumor size, ER and PgR negativity, high tumor grade, and p53 antibody reactivity. Although elevated risk of reduced DFS in patients with high JAG1 mRNA did not persist with adjustment for other prognostic factors, it did in combination with HER2. JAG1 mRNA was positively associated with expression of basal breast cancer markers, however, in contrast to the finding that basal gene expression is most strongly associated with reduced DFS in the first 36 months of follow-up, JAG1 mRNA expression was associated with reduced DFS through the full follow-up period. Also, tumors expressing high levels of both mRNA and protein showed reduced DFS as compared to all other groups in univariate analysis (hazard ratio of 1.73; 95% confidence interval, 1.09-2.74; P = 0.020). Thus, JAG1 expression is associated with poor DFS in LNN breast cancer. As JAG1 is a target of several oncogenic signaling pathways, and is a ligand for Notch, these data provide novel insights into signaling that may contribute to progression of early stage breast cancer.