Specific Breast Cancer Subtypes Research Articles

Abstract IA39: Variations in breast cancer risk by race/ethnicity in the US

Abstract The American Cancer Society recommends average-risk women begin annual screening at age 45, with an option for women to begin screening earlier at age 40. This guideline is for all women; however, breast cancer risk varies substantially by race/ethnicity in the US. In this presentation, I will provide an overview of the most current information on breast cancer rates in the US, focusing on differences in risk by race/ethnicity and age, as well as for specific breast cancer subtypes. Citation Format: Carol DeSantis. Variations in breast cancer risk by race/ethnicity in the US. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr IA39.

Diabetes and Breast Cancer Subtypes.

BackgroundWomen with diabetes have a worse survival after breast cancer diagnosis compared to women without diabetes. This may be due to a different etiological profile, leading to the development of more aggressive breast cancer subtypes. Our aim was to investigate whether insulin and non-insulin treated women with diabetes develop specific clinicopathological breast cancer subtypes compared to women without diabetes.Methods and FindingsThis cross-sectional study included randomly selected patients with invasive breast cancer diagnosed in 2000–2010. Stratified by age at breast cancer diagnosis (≤50 and >50 years), women with diabetes were 2:1 frequency-matched on year of birth and age at breast cancer diagnosis (both in 10-year categories) to women without diabetes, to select ~300 patients with tumor tissue available. Tumor MicroArrays were stained by immunohistochemistry for estrogen and progesterone receptor (ER, PR), HER2, Ki67, CK5/6, CK14, and p63. A pathologist scored all stains and revised morphology and grade. Associations between diabetes/insulin treatment and clinicopathological subtypes were analyzed using multivariable logistic regression. Morphology and grade were not significantly different between women with diabetes (n = 211) and women without diabetes (n = 101), irrespective of menopausal status. Premenopausal women with diabetes tended to have more often PR-negative (OR = 2.44(95%CI:1.07–5.55)), HER2-negative (OR = 2.84(95%CI:1.11–7.22)), and basal-like (OR = 3.14(95%CI:1.03–9.60) tumors than the women without diabetes, with non-significantly increased frequencies of ER-negative (OR = 2.48(95%CI:0.95–6.45)) and triple negative (OR = 2.60(95%CI:0.88–7.67) tumors. After adjustment for age and BMI, the associations remained similar in size but less significant. We observed no evidence for associations of clinicopathological subtypes with diabetes in postmenopausal women, or with insulin treatment in general.ConclusionsWe found no compelling evidence that women with diabetes, treated with or without insulin, develop different breast cancer subtypes than women without diabetes. However, premenopausal women with diabetes tended to develop breast tumors that do not express hormonal receptors, which are typically associated with poor prognosis.

What Health Care Providers Know and How They Communicate Breast Cancer Risk to Patients

Racial disparities in breast cancer mortality continue, partly due to higher prevalence of an aggressive breast cancer subtype called basal-like breast cancer (BBC) in African-Americans. Health care providers (HCPs) are uniquely positioned to discuss cancer risk and prevention with patients. We investigated breast cancer knowledge and risk communication among HCPs to identify factors that influenced communication with patients. Interviews were conducted with 34 HCPs in North Carolina. We found limited evidence of breast cancer risk education, and specific subtypes of breast cancer were not discussed. Barriers to communication about prevention include limited time, perceived patient receptivity and education level, and scientific misinformation. Factors that prompted discussions included patient characteristics (age, race, and socioeconomic status). To broaden the conversation, HCPs must receive and communicate more accurate information on breast cancer risk. Given these barriers to breast cancer education, additional opportunities to intervene with high-risk populations must be identified.

THE RESULTS OF A STUDY OF CHEMOTHERAPY WITH BEVACIZUMAB, OXALIPLATIN AND PACLITAXEL IN PATIENTS WITH METASTATIC TRIPLE NEGATIVE BREAST CANCER

Breast cancer (BC) is the most frequently diagnosed cancer among women. Triple negative breast cancer (TNBC) is a special subtype of BC, representing 15% of all breast cancers, is characterized by the absence of receptors to estrogen, progesterone and expression of HER-2 growth factor. This subtype carries an aggressive course, more frequently affects younger patients, a poor prognosis and a high risk of early recurrence with development of metastases. The heterogeneity of the disease and the absence of well-known molecular targets (hormone receptors and amplification of HER-2/neu) explains the complicity in choosing the optimal therapeutics regimens. Less than 30% of women with metastatic TNBC survive 5 years. There is a major need to develop new effective treatment options for patients with this aggressive subtype of breast cancer, that would lead to improve patient outcome.

Microenvironmental networks promote tumor heterogeneity and enrich for metastatic cancer stem-like cells in Luminal-A breast tumor cells.

The roles of the tumor microenvironment (TME) in generating intra-tumoral diversity within each specific breast cancer subtype are far from being fully elucidated. In this study, we exposed Luminal-A breast cancer cells in culture to combined “TME Stimulation”, representing three typical arms of the breast TME: hormonal (estrogen), inflammatory (tumor necrosis factor α) and growth-promoting (epidermal growth factor). In addition to enriching the tumor cell population with CD44+/β1+ cells (as we previously published), TME Stimulation selected for CD44+/CD24low/− stem-like cells, that were further enriched by doxorubicin treatment and demonstrated high plasticity in vitro and in vivo. Knock-down experiments revealed that CD44 and Zeb1 regulated CD24 and β1 expression and controlled differently cell spreading and formation of cellular protrusions. TME-enriched CD44+/CD24low/− stem-like cells promoted dissemination to bones and lymph nodes, whereas CD44+/β1+ cells had a low metastatic potential. Mixed co-injections of TME-enriched CD44+/CD24low/− and CD44+/β1+ sub-populations generated metastases populated mostly by CD44+/CD24low/−-derived cells. Thus, combined activities of several TME factors select for CD44+/CD24low/− stem-like cells that dictate the metastatic phenotype of Luminal-A breast tumor cells, suggesting that therapeutic modalities targeting the TME could be introduced as a potential strategy of inhibiting the detrimental stem-like sub-population in this disease subtype.

Dysregulation of long non-coding RNA in breast cancer: an overview of mechanism and clinical implication.

Long non-coding RNAs (lncRNAs), which occupy nearly 98% of genome, have crucial roles in cancer development, including breast cancer. Breast cancer is a disease with high incidence. Despite of recent progress in understanding the molecular mechanisms and combined therapy strategies, the functions and mechanisms of lncRNAs in breast cancer remains unclear. This review presents the currently basic knowledge and research approaches of lncRNAs. We also highlight the latest advances of seven classic lncRNAs and three novel lncRNAs in breast cancer, elucidating their mechanisms and possible therapeutic targets. Additionally, association between lncRNA and specific molecular subtype of breast cancer is reported. Lastly, we briefly delineate the potential roles of lncRNAs in clinical applications as biomarkers and treatment targets.

LIMT is a novel metastasis inhibiting lncRNA suppressed by EGF and downregulated in aggressive breast cancer.

Long noncoding RNAs (lncRNAs) are emerging as regulators of gene expression in pathogenesis, including cancer. Recently, lncRNAs have been implicated in progression of specific subtypes of breast cancer. One aggressive, basal‐like subtype associates with increased EGFR signaling, while another, the HER2‐enriched subtype, engages a kin of EGFR. Based on the premise that EGFR‐regulated lncRNAs might control the aggressiveness of basal‐like tumors, we identified multiple EGFR‐inducible lncRNAs in basal‐like normal cells and overlaid them with the transcriptomes of over 3,000 breast cancer patients. This led to the identification of 11 prognostic lncRNAs. Functional analyses of this group uncovered LINC01089 (here renamed LncRNA Inhibiting Metastasis; LIMT), a highly conserved lncRNA, which is depleted in basal‐like and in HER2‐positive tumors, and the low expression of which predicts poor patient prognosis. Interestingly, EGF rapidly downregulates LIMT expression by enhancing histone deacetylation at the respective promoter. We also find that LIMT inhibits extracellular matrix invasion of mammary cells in vitro and tumor metastasis in vivo. In conclusion, lncRNAs dynamically regulated by growth factors might act as novel drivers of cancer progression and serve as prognostic biomarkers.

Abstract 1779: Risk factors for early-onset ER- and ER+ breast cancer in African American women

Abstract Introduction: Relative to white women, African American (AA) women have a disproportionately high incidence of early-onset breast cancer, particularly higher incidence of aggressive breast cancer subtypes such as estrogen receptor (ER) negative tumors. Elucidation of risk factors for early-onset of specific subtypes of breast cancer is needed. Methods: We evaluated associations of reproductive and lifestyle factors with early-onset invasive breast cancer, defined as diagnosis before age 50, in 47,212 AA women ages 21-49 in the Black Women's Health Study (BWHS). The BWHS is an ongoing prospective cohort study in the U.S. that began in 1995. Risk factor information is updated every two years via mailed questionnaires. Incident cases were self-reported on biennial questionnaires or identified through death records and confirmed by medical records and cancer registry data. Through 2013, we identified 192 ER- and 322 ER+ invasive breast cancers diagnosed before age 50. We used multivariable Cox proportional hazards regression, stratified by age, to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations of family history of breast cancer, age at menarche, parity, age at first birth, years since last birth, lactation, body mass index (recent and at age 18), waist-to-hip ratio, oral contraceptive use, alcohol consumption, smoking history, and physical activity, with risk of ER- and ER+ early-onset breast cancer, separately. Results: The strongest associations were observed for family history of breast cancer, early age at menarche, and lower body mass index at age 18, and these factors were associated with increased incidence of both ER- and ER+ breast cancer. Oral contraceptive use within the past 10 years was also associated with increased risk of both subtypes. High waist-to-hip ratio (HR for top tertile vs. bottom: 1.46, 95% CI: 0.98, 2.19) and high parity (HR for 3+ births vs. 1: 1.43, 95% CI: 0.88, 2.32) were associated with increased risk of ER- breast cancer, while breastfeeding was associated with a reduced risk (HR for ever vs. never: 0.68, 95% CI: 0.47, 0.97). In contrast, higher parity was associated with a reduced risk of ER+ cancer (HR for 3+ births vs. 1: 0.68, 95% CI: 0.45, 1.01). Recent pregnancy (HR for <10 years vs. 10 or more years: 1.33, 95% CI: 0.94, 1.88) and greater alcohol consumption (HR for 7+ drinks/week vs. <1/week: 1.33, 95% CI: 0.81, 2.17) were associated with an increased risk of ER+ tumors. Conclusions: Differential associations of risk factors for ER- vs. ER+ breast cancers in young AA women suggest etiological heterogeneity by tumor subtypes. These differences could explain, in part, disparities in breast cancer incidence and mortality between black and white women. Some identified risk factors are potentially modifiable, suggesting possible opportunities for prevention. Citation Format: Kimberly A. Bertrand, Lynn Rosenberg, Julie R. Palmer. Risk factors for early-onset ER- and ER+ breast cancer in African American women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1779.

Abstract 1813: Insulin-like growth factor receptor and sphingosine kinase co-expression has prognostic and therapeutic potential in breast cancer

Abstract The search for reliable prognostic markers of breast cancer disease outcome is ongoing. The insulin-like growth factor receptor (IGF1R) and sphingosine kinase (SphK1) signaling pathways are known to contribute to breast oncogenesis and are therapeutic targets [1, 2]. Since IGF-I signaling activates SphK1 [3] we hypothesized that high IGF1R and SphK1 co-expression may be clinically relevant, and potential dual therapeutic targets in breast cancer. Kaplan-Meier analysis was performed to determine the prognostic significance of IGF1R and SphK1 high co-expression on disease free survival (DFS) and overall survival (OS) probability in breast cancer (http://glados.ucd.ie/BreastMark/). Cell viability studies and clonogenic assays were undertaken using the luminal, estrogen receptor (ER)-positive MCF7 and T47D and the basal-like, ER-negative HCC-1806 and HCC70 breast cancer cells to test the effectiveness of the dual IGF1R and insulin receptor inhibitor (OSI-906; 0.1 - 10 μM) and the SphK1 inhibitor (SKI-II; 1 - 20 μM) as single and combined agents. Statistical analysis was completed using a one-way ANOVA (p<0.05), repeated measures (p<0.05) and calculation of drug synergism by a combination index (CI) <1 (CommSyn). Immunoblot analysis was used to show effective drug target inhibition of IGF1R and SphK1. IGF1R and SphK1 high co-expression was associated with decreased DFS (HR; 2.184 (1.007 - 4.735, p = 0.042; total 270 patients) and OS (HR; 2.433 (1.136 - 5.214, p = 0.018; total 170 patients) in ER-positive, luminal A, lymph-node positive breast cancers compared to no significance using single-gene analysis. A reduction in OS in basal, ER-negative, lymph node negative breast cancers was associated with high IGF1R and SphK1 co-expression (HR; 5.687 (1.534 - 21.084, p = 0.003; total 55 patients), compared to high IGF1R expression alone (HR; 4.103 (1.34 - 12.57, p = 0.007; total 55 patients). In all four cell-lines 4 μM SKI-II acted synergistically with OSI over the range 0.1 - 6.4 μM (CI<1), and significantly increased sensitivity towards OSI (p<0.05). p-IGF1R and SphK1 protein levels were both reduced by SKI-II and OSI-906 in the HCC-1806 and HCC70 cell-lines, revealing a reciprocal regulation of signaling and effective drug treatment. Based on the limited clinical success of the IGF1R mono-therapies better predictive markers of IGF1R activity are now required for the development of more effective IGF1R-directed combination therapies. Collectively, these studies have identified: i) IGF1R and SphK1 expression are potentially co-dependent and have prognostic significance and ii) a novel IGF1R targeted combination therapy that may be of clinical benefit to specific molecular subtypes of breast cancer. 1. Martin, J.L., et al., Mol Cancer Ther, 2014. 13(2): p. 316-28. 2. Beckwith, H. and D. Yee., Endocr Pract, 2014. 20(11): p. 1214-21. 3. Granata, R., et al., J Thromb Haemost, 2007. 5(4): p. 835-45. Citation Format: Aleksandra M. Ochnik, Robert C. Baxter. Insulin-like growth factor receptor and sphingosine kinase co-expression has prognostic and therapeutic potential in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1813.

Abstract CT086: Omega-3 fatty acids and ERPR(-) and HER-2/neu(+) breast cancer prevention

Abstract Background: Breast cancer is a family of diseases, each with distinctive biologic behaviors and clinical outcomes. Refining therapy for specific breast cancer subtypes improves survival and limits toxicity. The application of these concepts to prevention has been limited and is a novel aspect of this clinical trial. Our pre-clinical research demonstrated that diets enriched in fish oil decreased the incidence and multiplicity of estrogen receptor negative, progesterone receptor negative (ERPR-), HER-2/neu positive (HER2+) mammary tumors in HER-2/neu transgenic mice, with decreased mammary atypia, Ki67 and COX-2 expression (Yee et al, 2005, 2012). Of interest is whether human breast cancers with poor prognostic features of negative hormone receptor status ± HER-2/neu overexpression might be uniquely responsive to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the long chain ω-3 fatty acids (FAs) found in fish oil. This study aims to determine biological changes associated with a low vs high dose of ω-3 FAs over 12 months in women at high risk for recurrent breast cancer. Doses were based on our dose finding study of ω-3 FAs in high risk women that showed superiority of ? 2.52 g EPA+DHA/day vs 0.84 g/day to increase serum and breast adipose tissue EPA+DHA content over 6 months (Yee et al, 2010). The objectives of the trial are to develop novel mammary epithelial, adipose tissue markers of exposure and response to ω-3 FAs that can be carried forward into definitive intervention trials of ω-3 FAs for breast cancer prevention. Methods: This is a double blinded randomized trial of high dose (∼5.4 g EPA+DHA) or low dose (∼0.9 g EPA+DHA in fatty acid mix of the typical American diet) of ω-3 FAs provided in 5 capsules/day for 12 months. Cellular samples of breast epithelial and adipose tissue are obtained by fine needle aspiration (FNA) to determine the effects of ω-3 FAs on mammary tissue biomarkers of ω-3 exposure, DNA methylation patterns, and intermediate anti-carcinogenic endpoints related to inflammation. Sample size of 40 participants per arm was justified to provide at least 80% power to detect a difference of interest (e.g. 1.5 fold) for each primary endpoint (six in total) based on two-sided t test with Bonferroni adjustment for multiplicity (α = 0.05/6 = 0.008, CV = 50%). This study focuses on women survivors of high risk breast cancer subtypes, namely triple negative or ERPR(-),HER2(+) disease, who are currently without long term adjuvant options. Eligibility criteria include prior diagnosis of ERPR(-) Stage 0 to III breast cancer, ?5 years from completion of standard therapy. Ineligibility criteria include current malignancy, inability to undergo breast FNA, chronic use of ω-3 FAs/fish oil, NSAIDs or full dose aspirin. The trial opened in September 2014 with first enrollment 9/16/14. 43% of planned participants have enrolled as of January 2016. The trial is continuing as planned. This study is registered at ClinicalTrials.gov (NCT02295059) and supported by the National Cancer Institute (1R01CA164019-01A1). Citation Format: Lisa D. Yee, Joanne L. Lester, Shana R. Straka, Sarah Woelke, Jia-Yu Ke, Rulong Shen, Pearlly Yan, Jianying Zhang, Martha A. Belury, Steven K. Clinton. Omega-3 fatty acids and ERPR(-) and HER-2/neu(+) breast cancer prevention. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT086.

Expression of Cytokeratin - 18 in Serum as a Biomarker of Apoptosis & Necrosis in Carcinoma Breast

Background: Cytokeratin expression is helpful in differentiating between specific breast cancer subtypes and as prognostic indicator. Objectives: To compare the cytokeratin-18 levels between breast cancer and healthy control. To compare cytokeratin-18 levels before and after neo-adjuvant chemotherapy (NAC) and to correlate cytokeratin-18 levels of pre and post chemotherapy with the percentage response to chemotherapy as evaluated by the RECIST criteria. Methods: Thirty patients of histologically confirmed invasive breast carcinoma and ten healthy individuals were taken as controls. The estimation of cytokeratin-18 was done at presentation and after two cycles of NAC. The change in level of serum cytokeratin-18 levels following two cycles of chemotherapy has been correlated with the RECIST criteria of response to chemotherapy. Results: Mean age of the patients was 48.73 years (range 28-80 years). The mean cytokeratin-18 level in carcinoma breast patient pre-treatment was 156.27 IU/L and in control group was 279.30 IU/L. The mean Cytokeratin 18 levels in patients before and after chemotherapy was 156.27 IU/L and 181.77 IU/L respectively (p<0.05). The average diameter of tumor before and after chemotherapy was 5.33 and 3.73 cm respectively. On correlation of percentage change of Cytokeratin 18 levels before and after chemotherapy and response to chemotherapy as evaluated by the RECIST criteria the following result was obtained: complete response (n=2): 76% rise of CK-18, partial response (n=21): 24% rise of CK-18, stable disease (n=5): 22% decline of CK-18, progressive disease (n=2), 21% decline of CK-18. Conclusion: The expression of cytokeratin-18 is a useful biomarker in breast cancer patient. Serum levels of CK-18 can be used to judge the efficacy of neo-adjuvant chemotherapy. Depending on post chemotherapy changes of CK-18 levels, the most appropriate chemotherapy regimen can be selected for individual patients.

Three-Dimensional Breast Cancer Models Mimic Hallmarks of Size-Induced Tumor Progression.

Tumor size is strongly correlated with breast cancer metastasis and patient survival. Increased tumor size contributes to hypoxic and metabolic gradients in the solid tumor and to an aggressive tumor phenotype. Thus, it is important to develop three-dimensional (3D) breast tumor models that recapitulate size-induced microenvironmental changes and, consequently, natural tumor progression in real time without the use of artificial culture conditions or gene manipulations. Here, we developed size-controlled multicellular aggregates ("microtumors") of subtype-specific breast cancer cells by using non-adhesive polyethylene glycol dimethacrylate hydrogel microwells of defined sizes (150-600 μm). These 3D microtumor models faithfully represent size-induced microenvironmental changes, such as hypoxic gradients, cellular heterogeneity, and spatial distribution of necrotic/proliferating cells. These microtumors acquire hallmarks of tumor progression in the same cell lines within 6 days. Of note, large microtumors of hormone receptor-positive cells exhibited an aggressive phenotype characterized by collective cell migration and upregulation of mesenchymal markers at mRNA and protein level, which was not observed in small microtumors. Interestingly, triple-negative breast cancer (TNBC) cell lines did not show size-dependent upregulation of mesenchymal markers. In conclusion, size-controlled microtumor models successfully recapitulated clinically observed positive association between tumor size and aggressive phenotype in hormone receptor-positive breast cancer while maintaining clinically proven poor correlation of tumor size with aggressive phenotype in TNBC. Such clinically relevant 3D models generated under controlled experimental conditions can serve as precise preclinical models to study mechanisms involved in breast tumor progression as well as antitumor drug effects as a function of tumor progression. Cancer Res; 76(13); 3732-43. ©2016 AACR.

An exome-wide analysis of low frequency and rare variants in relation to risk of breast cancer in African American Women: the AMBER Consortium.

A large percentage of breast cancer heritability remains unaccounted for, and most of the known susceptibility loci have been established in European and Asian populations. Rare variants may contribute to the unexplained heritability of this disease, including in women of African ancestry (AA). We conducted an exome-wide analysis of rare variants in relation to risk of overall and subtype-specific breast cancer in the African American Breast Cancer Epidemiology and Risk (AMBER) Consortium, which includes data from four large studies of AA women. Genotyping on the Illumina Human Exome Beadchip yielded data for 170 812 SNPs and 8287 subjects: 3629 cases (1093 estrogen receptor negative (ER-), 1968 ER+, 568 ER unknown) and 4658 controls, the largest exome chip study to date for AA breast cancer. Pooled gene-based association analyses were performed using the unified optimal sequence kernel association test (SKAT-O) for variants with minor allele frequency (MAF) ≤ 5%. In addition, each variant with MAF >0.5% was tested for association using logistic regression. There were no significant associations with overall breast cancer. However, a novel gene, FBXL22 (P = 8.2×10(-6)), and a gene previously identified in GWAS of European ancestry populations, PDE4D (P = 1.2×10(-6)), were significantly associated with ER- breast cancer after correction for multiple testing. Cases with the associated rare variants were also negative for progesterone and human epidermal growth factor receptors-thus, triple-negative cancer. Replication is required to confirm these gene-level associations, which are based on very small counts at extremely rare SNPs.

Chemotherapy principles of managing stage IV breast cancer in the United States.

The therapeutic landscape for metastatic breast cancer (MBC) has expanded greatly over the last three decades with an increasing availability of targeted therapies for specific breast cancer subtypes. However, cytotoxic chemotherapy remains an essential component for the management of endocrine refractory or triple negative MBC. Multiple chemotherapy agents have demonstrated activity in MBC as single agents and in combination. While taxanes are frequently recommended as the initial treatment of metastatic disease, capecitabine is a convenient oral therapy with well received toxicity profile. Eribulin is the only agent that demonstrated overall survival (OS) benefit in a phase III clinical trial when compared to treatment of physician choice in heavily pre-treated patients. Ixabepilone, gemcitabine, vinorelbine and platinum agents have demonstrated activity and, therefore, constitute additional therapeutic options. In this review, we will discuss the data supporting the use of different cytotoxic agents and the general principles in guiding the use of chemotherapy.

The voltage gated Ca(2+)-channel Cav3.2 and therapeutic responses in breast cancer.

BackgroundUnderstanding the cause of therapeutic resistance and identifying new biomarkers in breast cancer to predict therapeutic responses will help optimise patient care. Calcium (Ca2+)-signalling is important in a variety of processes associated with tumour progression, including breast cancer cell migration and proliferation. Ca2+-signalling is also linked to the acquisition of multidrug resistance. This study aimed to assess the expression level of proteins involved in Ca2+-signalling in an in vitro model of trastuzumab-resistance and to assess the ability of identified targets to reverse resistance and/or act as potential biomarkers for prognosis or therapy outcome.MethodsExpression levels of a panel of Ca2+-pumps, channels and channel regulators were assessed using RT-qPCR in resistant and sensitive age-matched SKBR3 breast cancer cells, established through continuous culture in the absence or presence of trastuzumab. The role of Cav3.2 in the acquisition of trastuzumab-resistance was assessed through pharmacological inhibition and induced overexpression. Levels of Cav3.2 were assessed in a panel of non-malignant and malignant breast cell lines using RT-qPCR and in patient samples representing different molecular subtypes (PAM50 cohort). Patient survival was also assessed in samples stratified by Cav3.2 expression (METABRIC and KM-Plotter cohort).ResultsIncreased mRNA of Cav3.2 was a feature of both acquired and intrinsic trastuzumab-resistant SKBR3 cells. However, pharmacological inhibition of Cav3.2 did not restore trastuzumab-sensitivity nor did Cav3.2 overexpression induce the expression of markers associated with resistance, suggesting that Cav3.2 is not a driver of trastuzumab-resistance. Cav3.2 levels were significantly higher in luminal A, luminal B and HER2-enriched subtypes compared to the basal subtype. High levels of Cav3.2 were associated with poor outcome in patients with oestrogen receptor positive (ER+) breast cancers, whereas Cav3.2 levels were correlated positively with patient survival after chemotherapy in patients with HER2-positive breast cancers.ConclusionOur study identified elevated levels of Cav3.2 in trastuzumab-resistant SKBR3 cell lines. Although not a regulator of trastuzumab-resistance in HER2-positive breast cancer cells, Cav3.2 may be a potential differential biomarker for survival and treatment response in specific breast cancer subtypes. These studies add to the complex and diverse role of Ca2+-signalling in breast cancer progression and treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0299-0) contains supplementary material, which is available to authorized users.

New insights on PI3K/AKT pathway alterations and clinical outcomes in breast cancer.

PI3K/AKT signaling pathway plays an important role in tumorigenesis and regulates critical cellular functions including survival, proliferation and metabolism. PIK3CA mutations and AKT activation by phosphorylation (pAKT) are often detected in many cancers and especially at high frequencies in breast cancer. Mounting data suggest that PIK3CA mutations or pAKT are mostly associated with better or insignificant outcomes in estrogen receptor-positive (ER+) early stage breast cancer and tend to be with worse prognosis in ER- disease. pAKT expression has been identified to predict paclitaxel chemotherapy benefit in node-positive breast cancer. Preclinical and neoadjuvant trial data suggest that PIK3CA alterations confer resistance to HER2-targeted therapy and are associated with lower pathological complete response (pCR) rate in HER2-positive breast cancer. However, recent results from randomized clinical trials of adjuvant and metastatic settings show that patients with mutant and wildtype PIK3CA tumors derived similar benefit from anti-HER2 therapy. This article, with our new insights, aims to decipher the mixed data and discusses the influence of the potential confounding factors in the assessments. We also share our views for validation of PI3K/AKT alterations in relation to clinical outcome in the context of specific breast cancer subtypes and treatment modalities towards further advance of the precision medicine for breast cancer treatment.

Abstract IA22: Mammographic density: A risk factor for all breast cancers or only specific subtypes?

Abstract Gene expression profiling has identified molecular subtypes that classify invasive breast cancers into distinct categories that vary in their clinical behavior and response to treatment. These subtypes highlight the many possible biologically and clinically distinct types of breast cancer. Immunohistochemical (IHC) staining of tumor sections using antibody panels has been used as a surrogate for the molecular subtypes identified through gene expression profiling, resulting in a set of intrinsic molecular subtypes. Given the heterogeneity within breast cancer, one might expect that different risk factors influence specific subtypes of breast cancer through various etiologic pathways. We evaluated whether percent mammographic density (MD), the proportion of fibroglandular or white tissue on a mammogram and one of the strongest and most consistent risk factors for breast cancer, is associated equally with all intrinsic molecular subtypes. Data were pooled from six cohort or case-control studies including 3389 women with invasive breast cancer and 9860 without, who underwent screening mammography. Percent MD was assessed from digitized film-screen mammograms (for cases, at a median 4 years prior to diagnosis) using a computer-assisted threshold technique, and categorized as 0-10%, 11-25%, 26-50% and 51%+. Receptor status was abstracted from clinical pathology records and supplemented by IHC staining of tumor sections or microarrays. With estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2) information, we classified tumors into the intrinsic molecular subtypes: Luminal A ( ER+ and/or PR+ and HER2- and grade 1 or 2), Luminal B (ER+ and/or PR+ and HER2+ or Luminal A and grade 3), HER2 expressing (HER2+/ER-/PR-) and triple negative (ER-/PR-/HER2-). For cancers found to be triple negative (TN), we also stained for epidermal growth factor receptor (EGFR) and cytokeratins (CK) 5/6 to differentiate basal-like tumors (positive for EGFR and/or CK 5/6) from the unclassifieds (negative on both EGFR and CK 5/6). We used polytomous logistic regression to calculate the odds (OR) of each of the intrinsic subtypes of breast cancer by categories of percent MD, adjusting for age, BMI and study. Contrasts were constructed and tested within the polytomous model framework to investigate differences in the strength of association of percent MD across subtypes. On average, cases were slightly older (56.4 years [SD=11.3]) than controls (55.8 [10.9]). Of the 3389 invasive breast cancer cases, 2146 (63%) were classified as Luminal A, 709 (21%) as Luminal B, 169 (5%) as HER-2 expressing, and 365(11%) as triple negative (TN). Of the TN tumors, 203 had tumor tissue available and were evaluated for CK 5/6 and EGFR, with 167 (82%) classified as basal-like and 36 (18%) as unclassified. Percent (%) MD was associated with breast cancer risk across all intrinsic subtypes. For Luminal A, compared to women with 11-25% MD (reference), women with 0-10% MD had a reduced risk of breast cancer (OR=0.64 [95% confidence interval (CI): 0.55, 0.74]) while women with 26-50% MD had an OR=1.5 (1.3,1.7) and women with 51%+ MD had the highest risk, OR=2.4 (2.0, 2.8). Similar associations were seen for Luminal B, HER2 expressing, and TN across percent MD categories. The small differences among the percent MD-breast cancer associations observed for the different subtypes were not statistically significant, either when comparing the four (Luminal A, Luminal B, HER-2 expressing and TN; p=0.61) or five categories of subtypes (Luminal A, Luminal B, HER-2 expressing, basal-like, unclassified; p=0.66). Our results suggest percent MD is a risk factor for all intrinsic molecular subtypes. Additional study is needed to examine whether these results hold for younger and older aged women. Understanding the importance of mammographic density measures for subtypes of breast cancer has significance for development of subtype-specific risk models. Citation Format: Celine Vachon, Rulla Tamimi, Yunn-Yi Chen, Christopher Scott, Kimberly Bertrand, Matthew Jensen, Daniel Visscher, Aaron Norman, Fergus Couch, John Shepherd, Bo Fan, Fang-Fang Wu, Lin Ma, Andrew Beck, Steve Cummings, V. Shane Pankratz, Karla Kerlikowske. Mammographic density: A risk factor for all breast cancers or only specific subtypes? [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr IA22.

Abstract P1-07-03: Reproductive factors and subtype specific breast cancer risk

Abstract Introduction: Reproductive history and breast cancer risk reportedly differ by the estrogen receptor (ER±) and by the joint expression of ER and the human epidermal growth factor-2 receptor (ER±/HER2±). However, large sample sizes are needed to identify risk factor associations for the relatively less common ER- subtypes. Material and Methods: We, therefore, linked two large-scale and population-based Danish registries to assess the associations for parity, number of live births, and age at first live birth (AFLB) with receptor-specific breast cancer risk. Relative risks (RRs) and 95% confidence intervals (CIs) for associations were estimated with Poisson regression models. Results: With nearly 31 million women-years of follow-up, there were 45786 Danish women between the ages 20-84 years who developed an invasive breast cancer during the study period 1992-2011. Parity significantly reduced risk for ER+ and ER+/HER2- subtypes (RR for ER+/HER2- = 0.92; 0.87, 0.98) and suggestively increased risk for ER- and ER-/HER2- subtypes (RR for ER-/HER2- = 1.16; 0.99, 1.36). RRs increased with advancing AFLB for ER+ cancers, especially among premenopausal women; and were elevated for ER- cancers among age groups 12-19 years and 30-34 years compared to the reference age group 20-24 years. Conclusion: Associations of breast cancer risk and reproductive history varied among Danish women by ER± and by ER±/HER2±, consistent with receptor-specific etiological heterogeneity. Risk estimates for ER+ and ER+/HER2- cancers were similar to the well-established associations for breast cancer overall, whereas relative risks for ER- and ER/HER2- cancers tended to be null or the inverse of ER+ associations. Citation Format: Anderson WF, Pfeiffer RM, Wohlfahrt J, Ejlertsen B, Jensen M-B, Kroman NT. Reproductive factors and subtype specific breast cancer risk. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-07-03.

Identification of differentially expressed miRNAs in individual breast cancer patient and application in personalized medicine.

MicroRNAs (miRNAs) have key roles in breast cancer progression, and their expression levels are heterogeneous across individual breast cancer patients. Traditional methods aim to identify differentially expressed miRNAs in populations rather than in individuals and are affected by the expression intensities of miRNAs in different experimental batches or platforms. Thus it is urgent to conduct miRNA differential expression analysis at an individual level for further personalized medicine research. We proposed a straightforward method to determine the differential expression of each miRNA in an individual patient by utilizing the reversal expression order of miRNA pairs between two conditions (cancer and normal tissue). We applied our method to breast cancer miRNA expression profiles from The Cancer Genome Atlas and two other independent data sets. In total, 292 miRNAs were differentially expressed in individual breast cancer patients. Using the differential expression profile of miRNAs in individual patients, we found that the deregulations of miRNA tend to occur in specific breast cancer subtypes. We investigated the coordination effect between the miRNA and its target, based on the hypothesis that one gene function can be changed by copy number alterations of the corresponding gene or deregulation of the miRNA. We revealed that patients exhibiting an upregulation of hsa-miR-92b and patients with deletions of PTEN did not tend to overlap, and hsa-miR-92b and PTEN coordinately regulated the pathway of ‘cell cycle' and so on. Moreover, we discovered a new prognostic signature, hsa-miR-29c, whose downregulation was associated with poor survival of breast cancer patients.

Family History of Cancer in Relation to Breast Cancer Subtypes in African American Women.

The evidence on the relation of family history of cancers other than breast cancer to breast cancer risk is conflicting, and most studies have not assessed specific breast cancer subtypes. We assessed the relation of first-degree family history of breast, prostate, lung, colorectal, ovarian, and cervical cancer and lymphoma or leukemia, to the risk of estrogen receptor-positive (ER(+)), ER(-), and triple-negative breast cancer in data from the African American Breast Cancer Epidemiology and Risk Consortium. Multivariable logistic regression models were used to calculate ORs and 95% confidence intervals (CI). There were 3,023 ER(+) and 1,497 ER(-) breast cancer cases (including 696 triple-negative cases) and 17,420 controls. First-degree family history of breast cancer was associated with increased risk of each subtype: OR = 1.76 (95% CI, 1.57-1.97) for ER(+), 1.67 (1.42-1.95) for ER(-), and 1.72 (1.38-2.13) for triple-negative breast cancer. Family history of cervical cancer was associated with increased risk of ER(-) (OR = 2.39; 95% CI, 1.36-4.20), but not ER(+) cancer. Family history of both breast and prostate cancer was associated with increased risk of ER(+) (3.40; 2.42-4.79) and ER(-) (2.09; 1.21-3.63) cancer, but family history of both breast and lung cancer was associated only with ER(-) cancer (2.11; 1.29-3.46). A family history of cancers other than breast may influence the risk of breast cancer, and associations may differ by subtype. Greater surveillance and counseling for additional screening may be warranted for women with a family history of cancer.