HER2-positive Breast Cancer Subtypes Research Articles

Tools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies.

BackgroundLarge population-based translational epigenetic studies are emerging due to recent technological advances that have made molecular analyses possible. For example, the Infinium HumanMethylation450 Beadchip (HM450K) has enabled studies of genome-wide methylation on a scale not previously possible. However, application of the HM450K to DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour material has been more challenging than application to high quality DNA extracted from blood. To facilitate the application of this assay consistently across a large number of FFPE tumour-enriched DNA samples we have devised a modification to the HM450K protocol for FFPE that includes an additional quality control (QC) checkpoint.ResultsQC checkpoint 3 was designed to assess the presence of DNA after bisulfite conversion and restoration, just prior to application of the HM450K assay. DNA was extracted from 474 archival FFPE breast tumour material. Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3. Genome-wide methylation was measured for the remaining 428 tumour-enriched DNA. Of these, only 4 samples failed our stringent HM450K data criteria thus representing a 99 % success rate. Using prior knowledge about methylation marks associated with breast cancer we further explored the quality of the data. Twenty probes in the BRCA1 promoter region showed increased methylation in triple-negative breast cancers compared to Luminal A, Luminal B and HER2-positive breast cancer subtypes. Validation of this observation in published data from The Cancer Genome Atlas (TCGA) Network (obtained from DNA extracted from fresh frozen tumour samples) confirms the quality of the data obtained from the improved protocol.ConclusionsThe modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples. This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1487-z) contains supplementary material, which is available to authorized users.

Neoadjuvant chemotherapy: what does it take to tAnGo?

Use of additional chemotherapy agents is a time-proven method of achieving an incremental survival gain in early breast cancer. A decade has passed since the publication of trials showing the benefit of adding taxanes to anthracycline-based adjuvant chemotherapy. Neo-tAnGo and the companion tAnGo trial set out to test whether additional benefit could be achieved by adding gemcitabine in the neoadjuvant and adjuvant settings, respectively. Neo-tAnGo also investigated the effect of sequencing paclitaxel either before or after epirubicin and cyclophosphamide (EC). The addition of gemcitabine was not associated with an improvement in pathological complete response (pCR) or survival, consistent with other trials that incorporated gemcitabine into neoadjuvant or adjuvant chemotherapy. Administration of paclitaxel prior to EC resulted in a 5% higher pCR rate compared with the reverse sequence (P=0.03). The pCR difference did not translate into a survival advantage. Neoadjuvant therapy is an important clinical trial platform to assess the effectiveness of new agents in early and locally advanced breast cancer. Early endpoints such as pCR, serial biopsies and imaging can be used to evaluate the activity of new therapies. The link between improvements in pCR and survival in the neoadjuvant setting, and translation of benefit into the adjuvant setting is tantalisingly close - particularly in the triple negative breast cancer and HER2 positive breast cancer subtypes - but the association remains inconsistent. This editorial will contextualise the results of the Neo-tAnGo in the current and future neoadjuvant trial landscape.

Novel Insights into Breast Cancer Genetic Variance through RNA Sequencing

Using RNA sequencing of triple-negative breast cancer (TNBC), non-TBNC and HER2-positive breast cancer sub-types, here we report novel expressed variants, allelic prevalence and abundance, and coexpression with other variation, and splicing signatures. To reveal the most prevalent variant alleles, we overlaid our findings with cancer- and population-based datasets and validated a subset of novel variants of cancer-related genes: ESRP2, GBP1, TPP1, MAD2L1BP, GLUD2 and SLC30A8. As a proof-of-principle, we demonstrated that a rare substitution in the splicing coordinator ESRP2 (R353Q) impairs its ability to bind to its substrate FGFR2 pre-mRNA. In addition, we describe novel SNPs and INDELs in cancer relevant genes with no prior reported association of point mutations with cancer, such as MTAP and MAGED1. For the first time, this study illustrates the power of RNA-sequencing in revealing the variation landscape of breast transcriptome and exemplifies analytical strategies to search regulatory interactions among cancer relevant molecules.

Triple Negative but not HER2 Positive Breast Cancer Subtype is Associated with Higher Locoregional Recurrence after Breast-conserving Therapy and Modern Systemic Therapy

Triple Negative but not HER2 Positive Breast Cancer Subtype is Associated with Higher Locoregional Recurrence after Breast-conserving Therapy and Modern Systemic Therapy

Comparison of locoregional recurrence in triple-negative and HER2-positive breast cancer subtype after breast-conserving therapy and modern systemic therapy.

112 Background: Triple-negative (basal) and HER2+ subtypes have been associated with increased risk of locoregional recurrence (LRR) in earlier studies of breast-conserving therapy (BCT), in which fewer patients received systemic therapy compared with recent standards, including HER2-targeted therapy. We analyzed whether basal and HER2+ subtypes are independent prognostic factors for LRR in BCT patients treated with modern systemic therapy. Methods: We retrospectively studied 415 patients with invasive breast cancer who received BCT from 1992 to 2009. Axillary surgery was done in 404 patients (97%). Forty eight percent of patients received chemotherapy (anthracycline/taxane 34%, trastuzumab-containing 4%, other 10%). Hormone therapy was given in 67%. Progression-free survival (PFS) and OS were estimated by the Kaplan-Meier method. Rate of LRR was estimated by the method of cumulative incidence allowing for competing risks. The effect of prognostic factors was examined by Fine and Gray’s test. Results: Median follow-up was 60 months. Median age was 54 (26–86). Stage: T1 70%, T2 26.3%, T3 8%; N0 75%, N1 23.5%, N2 1.7%. Receptor status was available in 301 patients to approximate subtypes: 66% Luminal A, 9% Luminal B, and 5% HER2+, and 20% Basal. Overall, there were 11 LRR, 15 distant metastasis, and 20 deaths. The 10-year PFS and OS were 85.4% and 90.5%, respectively. The 5 and 10-year cumulative incidence of LRR were 2.8% (95%CI: 1.4–5.2) and 4.5% (95%CI: 2.2–8.2) respectively. On univariate analysis, Basal vs. Luminal A (HR: 9.12, p<0.001) and tumor size >2cm vs. ≤2cm (HR: 4.00, p=0.022) were significant prognostic factors for LRR as first failure. On multivariate analysis, only basal subtype (HR: 6.29, p=0.011) retained significance; others effects, including luminal B/HER2 combined subtypes, were not significant. Conclusions: Long-term clinical outcomes were excellent in this cohort of breast cancer patients treated with BCT and modern systemic therapy, with 10-year LRR 4.5% and OS 90.5%. Basal subtype was an independent predictor for LRR. Conversely, HER2+ subtype was not associated with increase in LRR, in this cohort treated with BCT and trastuzumab.

Prognostic Implications of MicroRNA-21 Overexpression in Invasive Ductal Carcinomas of the Breast

PurposeAmong more than 500 microRNAs, microRNA-21 (miR-21) is known to act as an oncogene. The aim of this study was to investigate the significance of miR-21 expression level in relation with clinicopathological factors and prognosis in breast cancer.MethodsMicroRNA was extracted from cancer and normal breast tissue of 109 breast cancer patients who underwent surgery from 2002 to 2004 using the Taqman® MicroRNA Assay. The correlation between miR-21 expression and clinicopathologic features was analyzed and the significance of miR-21 as a prognostic factor and its relationship with survival was determined.ResultsMiR-21 expression was higher in cancer tissues than in normal tissues (p<0.0001). High miR-21 expression was associated with mastectomy, larger tumor size, higher stage, higher grade, estrogen receptor (ER) negative, human epidermal growth factor receptor 2 (HER2) positive, HER2 positive breast cancer subtype, high Ki-67 expression, and death. On multivariate analysis, prognostic factors for overall survival were ER and miR-21. High miR-21 expression was significantly related to lower overall survival (p=0.031).ConclusionThis study supports the role of miR-21 as an oncogene and a biomarker for breast cancer with its high expression in cancer tissues and its relationship with other prognostic factors and survival.

COX-2 activation is associated with Akt phosphorylation and poor survival in ER-negative, HER2-positive breast cancer

BackgroundInducible cyclooxgenase-2 (COX-2) is commonly overexpressed in breast tumors and is a target for cancer therapy. Here, we studied the association of COX-2 with breast cancer survival and how this association is influenced by tumor estrogen and HER2 receptor status and Akt pathway activation.MethodsTumor COX-2, HER2 and estrogen receptor α (ER) expression and phosphorylation of Akt, BAD, and caspase-9 were analyzed immunohistochemically in 248 cases of breast cancer. Spearman's correlation and multivariable logistic regression analyses were used to examine the relationship between COX-2 and tumor characteristics. Kaplan-Meier survival and multivariable Cox proportional hazards regression analyses were used to examine the relationship between COX-2 and disease-specific survival.ResultsCOX-2 was significantly associated with breast cancer outcome in ER-negative [Hazard ratio (HR) = 2.72; 95% confidence interval (CI), 1.36-5.41; comparing high versus low COX-2] and HER2 overexpressing breast cancer (HR = 2.84; 95% CI, 1.07-7.52). However, the hazard of poor survival associated with increased COX-2 was highest among patients who were both ER-negative and HER2-positive (HR = 5.95; 95% CI, 1.01-34.9). Notably, COX-2 expression in the ER-negative and HER2-positive tumors correlated significantly with increased phosphorylation of Akt and of the two Akt targets, BAD at Ser136 and caspase-9 at Ser196.ConclusionsUp-regulation of COX-2 in ER-negative and HER2-positive breast tumors is associated with Akt pathway activation and is a marker of poor outcome. The findings suggest that COX-2-specific inhibitors and inhibitors of the Akt pathway may act synergistically as anticancer drugs in the ER-negative and HER2-positive breast cancer subtype.

Asian ethnicity and breast cancer subtypes: a study from the California Cancer Registry

The distribution of breast cancer molecular subtypes has been shown to vary by race/ethnicity, highlighting the importance of host factors in breast tumor biology. We undertook the current analysis to determine population-based distributions of breast cancer subtypes among six ethnic Asian groups in California. We defined immunohistochemical (IHC) surrogates for each breast cancer subtype among Chinese, Japanese, Filipina, Korean, Vietnamese, and South Asian patients diagnosed with incident, primary, invasive breast cancer between 2002 and 2007 in the California Cancer Registry as: hormone receptor-positive (HR+)/HER2- [estrogen receptor-positive (ER+) and/or progesterone receptor-positive (PR+), human epidermal growth factor receptor 2-negative (HER2-)], triple-negative (ER-, PR-, and HER2-), and HER2-positive (ER±, PR±, and HER2+). We calculated frequencies of breast cancer subtypes among Asian ethnic groups and evaluated their associations with clinical and demographic factors. Complete IHC data were available for 8,140 Asian women. Compared to non-Hispanic White women, Korean [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.5-2.2], Filipina (OR = 1.3, 95% CI = 1.2-1.5), Vietnamese (OR = 1.3, 95% CI = 1.1-1.6), and Chinese (OR = 1.1, 95% CI = 1.0-1.3) women had a significantly increased risk of being diagnosed with HER2-positive breast cancer subtypes after adjusting for age, stage, grade, socioeconomic status, histology, diagnosis year, nativity, and hospital ownership status. We report a significant ethnic disparity in HER2-positive breast cancer in a large population-based cohort enriched for Asian-Americans. Given the poor prognosis and high treatment costs of HER2-positive breast cancer, our results have implications for healthcare resource utilization, cancer biology, and clinical care.

Role of HER2 in the activation of c-Abl in breast cancer cells.

e21096 Background: HER2/neu, a member of the epidermal growth factor receptor (EGFR) family, is over expressed in approximately 25-30% of breast cancer tumors. HER2-positive breast cancer subtype has been associated with a more aggressive phenotype, but the precise molecular mechanisms that link HER2 over-expression to breast carcinogenesis remain poorly understood. Increase activity of c-Abl, a cytoplasmic kinase of tyrosine residues linked to motility, has been associated with a more aggressive breast cancer phenotype. Methods: In this study we evaluated the possible participation of c-Abl in the HER2/neu signaling pathway. Results: Our results showed that the total amount of c-Abl differs among the three HER2-positive breast cancer cell lines analyzed (T47D, ZR-75-30 and MBCD5). This different level of expression of c-Abl might be related with a more aggressive tumor phenotype. We demonstrated for the first time that in vitro addition of trastuzumab to breast cancer cell lines reduces the level of phosphorylation of the tyrosine-245 residue of c-Abl in all three cancer cell lines. This result strongly indicates that the phosphorylation of c-Abl on Tyr245, an event linked to c-Abl activation, depends on the activity of HER2. The combined use of imatinib (an inhibitor of c-Abl) with trastuzumab had no synergistic effect on the generalized reduction in tyrosine residue phosphorylation, under these conditions only the effect of imatinib dominated. In addition, treatment with trastuzumab decreased cell proliferation in a magnitude that correlated with the level of HER2-overexpression. Conclusions: Taken together, these results strongly support the idea that c-Abl is downstream of the HER2-signaling pathway. These results can contribute to a better understanding of the process of breast carcinogenesis and aid in the design of new therapeutic strategies against this tumor. No significant financial relationships to disclose.

Triple Negative Breast Cancer Patients with Brain Metastasis Are Associated with More Concurrent Lung Metastases.

Abstract Background: Breast cancer is a heterogeneous disease with different molecular subtypes. It is known that triple negative breast cancer and the HER-2 positive breast cancer subtypes are associated with more brain metastases. We further explored different subtypes of breast cancer patients with brain metastasis and analyzed their clinical behaviour with particular reference to the Hong Kong Chinese population. Methodology: We reviewed all breast cancer patients with brain metastasis presented to the Department of Clinical Oncology in the Queen Mary Hospital, the University of Hong Kong which is a teaching hospital during January 2004 to June 2008. Sociodemographic factors, date of primary breast cancer diagnosis and the tumour features, clinical behaviour at presentation, treatment received such as surgery, chemotherapy, hormonal therapy and targeted therapy, time of local recurrence, metastasis including brain metastasis, the recursive partitioning analysis (RPA) classes and treatment for the brain metastasis were all recorded. We classified the patients into 3 main subtypes, hormone positive, triple-negative (TNC) and HER-2 positive subtypes and further studied the clinical features and brain metastasis behaviour. Statistical analysis using SPSS 16.0 version was applied. Results: Altogether, there were 60 breast cancer patients with brain metastasis occurred during January 2004 to June 2008 requiring further management in our Department. Hormone positive patients constituted 46.6% while TNC accounted for 15.5% and HER-2 positive patients accounted for 37.9% of the cohort. The majority presented with symptoms at the time of the brain metastasis (93.1%) with headache being the most common symptom (43.1%), and 54 patients received whole brain radiotherapy (WBRT) and 8 patients received surgical resection of the brain metastasis. There were no significant statistical significance between the breast cancer subtypes and the metastatic behaviour except that the TNC patients were associated with concurrent lung metastasis when they first presented with brain metastasis (p = 0.03) when compared to the hormone positive and HER-2 positive patients. The ANOVA analysis showed there was a statistically significant difference between the RPA Classes of the patients and their time of survival after the brain metastasis (p = 0.002). Further ANOVA Post Hoc showed the RPA Class 3 patients were associated with shorter time of survival after brain metastasis when compared to the Class 1 patients (2.4 months vs 11.8 months, p = 0.001). However, patients with brain metastasis and different breast cancer subtypes did not have statistically significant different time of survival after their brain metastases. Conclusion: Hong Kong Chinese triple negative breast cancer patients with brain metastasis were associated with more concurrent lung metastasis. The RPA Classification still serves as a prognostic indicator with reference to the survival after brain metastasis in the breast cancer patients when compared to the breast cancer subtypes. Further large-scale multi-centre epidemiology study is warranted to confirm the above findings. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3072.

The antidiabetic drug metformin suppresses HER2 (erbB-2) oncoprotein overexpression via inhibition of the mTOR effector p70S6K1 in human breast carcinoma cells

Population studies have revealed that treatment with the anti-diabetic drug metformin significantly associates with reduced breast cancer risk. Animal studies have shown that metformin suppresses the development of mammary carcinomas in transgenic female mice carrying a HER2 oncogene, but not that of spontaneous tumors. We herein demonstrate that HER2 oncoprotein itself may represent a key cellular target involved in the anti-breast cancer actions of metformin. First, ectopical overexpression of HER2 oncogene significantly enhances metformin-induced breast cancer cell growth inhibition. Second, metformin treatment drastically down-regulates HER2 protein levels (up to 85% reduction) in a dose- and time-dependent manner. Metformin-induced inhibition of HER2 take places regardless the molecular mechanism contributing to HER2 overexpression (i.e., human HER2 cDNA exogenously driven by a viral promoter and naturally occurring endogenous HER2 gene amplification). Mechanistically, metformin-induced suppression of HER2 overexpression appears to occur via direct (AMPK-independent) inhibition of p70S6K1 activity. Compound C- and small interference RNA (siRNA)-induced blockade of AMPK activity/expression fail to prevent the anti-HER2 effect of metformin while AMPK hyperactivation following exposure to the AMP analog AICAR is not sufficient to down-regulate HER2 expression. HER2-positive breast cancer cells transfected with p70S6K1 siRNA become completely refractory to metformin-induced HER2 suppression. Of note, co-incubation with agents that block reactive oxygen species (ROS) production (e.g. N-acetylcisteine) dramatically enhanced the ability of metformin to decrease HER2 expression. From the perspective of chemoprevention, these findings altogether suggest that metformin might exert a protective mostly confined to the HER2-positive breast cancer subtype. From the perspective of intervention, the presence/absence of molecular hallmarks such as HER2 overexpression and/or p70S6K1 hyperactivation might dictate alternative responses in metformin-based treatment of early breast cancer. The importance of mTOR/p70S6K1-sensed ROS status at mediating the anti-oncogenic effects of metformin might represent a previously unrecognized linkage molecularly connecting its anti-aging and anti-cancer actions.

Giacomo Castelvetro’s salads. Anti-HER2 oncogene nutraceuticals since the 17th century?

We are accumulating evidence to suggest that 17(th) century Renaissance foodways -largely based on the old "Mediterranean dietary traditions"- may provide new nutraceutical management strategies against HER2-positive breast cancer disease in the 21st century. Epidemiological and experimental studies begin to support the notion that "The Sacred Law of Salads" (i.e., "raw vegetables... plenty of generous (olive) oil") -originally proposed in 1614 by Giacomo Castelvetro in its book The Fruit, Herbs & Vegetables of Italy- might be considered the first (unintended) example of customised diets for breast cancer prevention based on individual genetic make-up (i.e., nutraceuticals against human breast carcinomas bearing HER2 oncogene amplification/overexpression). First, the so-called salad vegetables dietary pattern (i.e., a high consumption of raw vegetables and olive oil) appears to exert a protective effect mostly confined to the HER2-positive breast cancer subtype, with no significant influence on the occurrence of HER2-negative breast cancers. Second, all the main olive oil constituents (i.e., the omega-9 monounsaturated fatty acid oleic acid and polyphenolic compounds such as the secoiridoid oleuropein or the lignan 1-[+]-acetoxypinoresinol) dramatically reduce HER2 expression and specifically induce apoptotic cell death in cultured HER2- positive breast cancer cells, with marginal effects against HER2-negative cells. Third, an olive oil-rich diet negatively influences experimental mammary tumorigenesis in rats likewise decreasing HER2 expression levels. If early 1600s Castelvetro's salads can be used as dietary protocols capable to protecting women against biologically aggressive HER2-positive breast cancer subtypes is an intriguing prospect that warrants to be evaluated in human pilot studies in the future. Here, at least, we would like to recognise Giacomo Castelvetro as the father of modern nutritional genomics in oncology.