Distinct Breast Cancer Subtypes Research Articles

Failure patterns and survival outcomes in triple negative breast cancer (TNBC): a 15 year comparison of 448 non-Hispanic black and white women.

Purpose Triple negative breast cancer (TNBC) is a distinct subtype of breast cancer with unique pathologic, molecular and clinical behavior. It occurs more frequently in young blacks and has been reported to have a shorter disease-free interval. We undertook this study to analyze the demographic characteristics, failure patterns, and survival outcomes in this disease.MethodsA total of 448 non-Hispanic black and white women were identified over a 15 year period from 1996 to 2011. Demographic and clinical information including age, race, menopausal status, stage, tumor characteristics, and treatments were collected. Fisher’s exact test and multivariable Cox regression were used to compare failure patterns and survival outcomes between races.Results49 % (n = 223) were black. 59 % patients were between 41 and 60 years, with 18 % ≤40 years. 57 % were premenopausal and 89 % had grade 3 tumors. Stage II (47 %) was most frequent stage at diagnosis followed by stage III (28 %). 32 % had lymphovascular invasion. Adjusting for age, stage, and grade, there was no difference in survival outcomes (OS, DFS, LFFS, and DFFS) between the two races. 62 (14 %) patients failed locally either in ipsilateral breast or chest wall, and 19 (4 %) failed in the regional lymphatics. Lung (18 %) was the most frequent distant failure site with <12 % each failing in brain, liver and bones.ConclusionFailure patterns and survival outcomes did not differ by race in this large collection of TNBC cases. Lung was the predominate site of distant failure followed by brain, bone, and liver. Few patients failed in the regional lymphatics.

A novel subtype classification and risk of breast cancer by histone modification profiling.

Breast cancer has been classified into several intrinsic molecular subtypes on the basis of genetic and epigenetic factors. However, knowledge about histone modifications that contribute to the classification and development of biologically distinct breast cancer subtypes remains limited. Here we compared the genome-wide binding patterns of H3K4me3 and H3K27me3 between human mammary epithelial cells and three breast cancer cell lines representing the luminal, HER2, and basal subtypes. We characterized thousands of unique binding events as well as bivalent chromatin signatures unique to each cancer subtype, which were involved in different epigenetic regulation programs and signaling pathways in breast cancer progression. Genes linked to the unique histone mark features exhibited subtype-specific expression patterns, both in cancer cell lines and primary tumors, some of which were confirmed by qPCR in our primary cancer samples. Finally, histone mark-based gene classifiers were significantly correlated with relapse-free survival outcomes in patients. In summary, we have provided a valuable resource for the identification of novel biomarkers of subtype classification and clinical prognosis evaluation in breast cancers.

Recent developments and translational aspects in targeted therapy for metastatic breast cancer

Biologically distinct subtypes of metastatic breast cancer (MBC) have been defined by multiple efforts in recent years, showing broad heterogeneity at the molecular level of disease. Throughout this endeavour, oncogenic drivers within MBC were identified as potential therapeutic targets. With recent results from clinical trials targeting these well-known cancer-promoting pathways, this review is trying to elucidate as well as summarise current new therapeutic aspects in MBC and shed light on translational aspects within this entity.

Diagnostic Potential of lncRNAs in Cancer

For decades, diversification of cancers, in terms of origin of organs or cell types, allows precise understanding of genetic and epigenetic aberrations. Indeed, this approach has vastly improved cancer management for numerous cancer types. For instance, patients of breast cancer are benefitted by different management schemes according to distinct breast cancer subtypes (i.e. luminal A, luminal B, HER2 and basal-like). It is widely accepted that the feasibility of personalized medicine battling cancer is laid by exploiting the complete malignant/driver events within a particular cancer subtype. However, the direction of cancer research is tilted recently by a suggestion that cancer management can be improved by bringing out a bigger picture of the cancer population. Here, the idea of pan-cancer analysis is raised by The Cancer Genome Atlas (TCGA) program in which different cancer types are gathered together for analysis, as it is believed that diverging driver events among various cancer types most often generate converging genetic signatures and biological pathways during cancer development. The pan-cancer analysis project builds a joint data set from separate TCGA disease projects of multiple cancer types, which increases the statistical power to detect functional genomic determinants of disease. Subsequently, it allows the identification of both tissue-specific aspects of cancer and intrinsic molecular commonalities across tumor types (Cancer Genome Atlas Research Network et al., 2013). With this approach, several groups have already identified critical oncogenic signatures (Ciriello et al., 2013), mutation landscape (Kandoth et al., 2013) and microRNA-target interactions (Jacobsen et al., 2013) across diverse cancer types. In this issue of EBioMedicine, Ching et al. utilize the pan-cancer analysis to explore the diagnostic and prognostic potentials of long non-coding RNAs (lncRNAs), and subsequently reveal a panel of six lncRNAs as biomarkers for multiple cancer types (Ching et al., 2016). Previous work showed that lncRNAs show higher tissue specificity compared to mRNAs (Derrien et al., 2012). The work of Ching et al. supported this notion, and extended it by showing a reduction of specificity after malignant transformation, which is consistent with the de-differentiation status of cancers widely observed. They further show that lncRNA profiles in cancer cells are highly specific to distinct cancer types, which pinpoint the predicting values of lncRNA expressions during cancer management. Numerous studies have demonstrated the potential of different lncRNAs for cancer diagnosis and prognosis, but the investigations are mainly focusing on a single cancer subtypes. Ching et al. take a global approach and identify six pan-cancer lncRNAs that robustly and accurately predict a wide range of cancer types. The six pan-cancer lncRNAs are also meritorious in predicting overall survival and relapse free survival in different cancer types. This finding should have laid the foundation for the development of biomarker screening platform for non-invasive testing of cancer incidence and outcome. More importantly, the pan-cancer analysis approach makes the translation of the finding more convenient, and shortens the timeframe of clinical trials because of the availability of a larger pool of patients. This may be an important step towards a more practical health-checking module in the future. On the other hand, the role of the putative pan-cancer lncRNAs identified in this study is potentially pivotal to cancer biology. It is interesting that their analysis suggested that well characterized lncRNAs such as HOTAIR does not meet the stringent selection criteria as a pan-cancer lncRNA, despite its frequently association and overexpression in several cancer types such as breast cancer (Gupta et al., 2010), liver cancer (Yang et al., 2011), and pancreatic cancer (Kim et al., 2013). Given the consistent expression pattern of pan-cancer lncRNAs across cancer types, it is interesting to extrapolate the possible roles of every one of them. Although as biomarkers, the pan-cancer lncRNAs may not necessarily link to any driver event as passenger genetic alterations can still demonstrate good value of prediction. Ching et al. has shown that they may regulate critical pro-cancerous effects such as cell proliferation and cell migration. Future study is warranted to unveil the underlying reason for their consistency across different cancer types. It is also of interest to understand the difference between these pan-cancer lncRNAs with other cancer-associated lncRNAs regarding their roles in cancer. As pointed out by the pioneers of the pan-cancer project, there are still limitations of analysis across cancer types in terms of inconsistent data acquisition method, incompatible clinical data across cancer types and false-positive discovery due to loosen statistical analysis (Cancer Genome Atlas Research Network et al., 2013). Nonetheless, diversification of cancer is still proven to be the essential strategy in exploiting the uniqueness of every cancer types, as agreed by the Ching et al. As such, it is possible to pick the suitable candidates out of various targets for effective pharmaceutical intervention, as the current technology is limiting the selection of druggable targets.

Higher levels of TIMP-1 expression are associated with a poor prognosis in triple-negative breast cancer.

BackgroundTissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein that can directly regulate apoptosis and metastasis. In this study, we investigated the functional and molecular mechanisms by which TIMP-1 influences triple-negative breast cancer (TNBC).MethodsThe expression level of TIMP-1 in breast cancer tissues was analyzed using the ONCOMINE microarray database. The overall survival of patients with distinct molecular subtypes of breast cancer stratified by TIMP-1 expression levels was evaluated using Kaplan–Meier analysis. Bisulfate sequencing PCR (BSP) was used to analyze the methylation status of the TIMP-1 promoter. Real-time-PCR (RT-PCR), Western blot and ELISA assays were used to evaluate gene and protein expression in cell lines and human tissue specimens. In addition, TIMP-1 function was analyzed using a series of in vitro and in vivo assays with cells in which TIMP-1 was inhibited using RNAi or neutralizing antibodies.ResultsWe found that serum TIMP-1 levels were strongly enhanced in patients with TNBC and that elevated TIMP-1 levels were associated with a poor prognosis in TNBC. However, TIMP-1 levels were not significantly associated with overall survival in other subtypes of breast cancer or in the overall population of breast cancer patients. We also report the first evidence that the TIMP-1 promoter is hypomethylated in TNBC cell lines compared with non-TNBC cell lines, suggesting that aberrant TIMP-1 expression in TNBC results from reduced DNA methylation. RNAi-mediated silencing of TIMP-1 in TNBC cells induced cell cycle arrest at the G1 phase and reduced cyclin D1 expression. In addition, mechanistic analyses revealed that the p-Akt and p-NF-κB signaling pathways, but not the GSK-3β and MAPK1/2 pathways, are associated with TIMP-1 overexpression in TNBC cells. Moreover, neutralizing antibodies against TIMP-1 significantly decreased the rate of tumor growth in vivo.ConclusionsOur findings suggest that TIMP-1 is a biomarker indicative of a poor prognosis in TNBC patients and that targeting TIMP-1 may provide an attractive therapeutic intervention specifically for triple-negative breast cancer patients.

Association of adiposity, dysmetabolisms, and inflammation with aggressive breast cancer subtypes: a cross-sectional study.

Obesity and metabolic syndrome are risk and prognostic factors for breast cancer (BC) and are associated with chronic inflammation. We investigated the association between distinct BC subtypes and markers of adiposity, dysmetabolisms, and inflammation. We analyzed 1779 patients with primary invasive BC treated at a single institution, for whom anthropometric and clinical-pathological data were archived. BC subtypes were classified by immunohistochemical staining of ER, PR, HER2, and Ki67, and their relations with the study markers were assessed by multinomial logistic regression. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated taking luminal A as reference. All subtypes more aggressive than luminal A were significantly more frequent in younger (<45years) than older women. Before menopause, luminal B HER2-negative tumors were positively associated with large waist (OR 2.55, 95% CI 1.53-4.24) and insulin resistance (OR 1.90, 95% CI 1.05-3.41); luminal B HER2-positive tumors with large waist (OR 2.11, 95% CI 1.03-4.35) and triple-negative tumors with overweight (OR 3.04, 95% CI 1.43-6.43) and high C-reactive protein (p trend=0.026). In postmenopausal women aged <65, luminal B HER2-negative (OR 1.94, 95% CI 1.16-3.24) and luminal B HER2-positive tumors (OR 2.48, 95% CI 1.16-5.27) were positively related with metabolic syndrome. Dysmetabolisms and inflammation may be related to different BC subtypes. Before menopause, triple-negative cancers were related to obesity and chronic inflammation, and aggressive luminal subtypes to abdominal adiposity. After menopause, in women aged <65 these latter subtypes were related to metabolic syndrome. Control of adiposity and dysmetabolism can reduce the risk of aggressive BC subtypes, improving the prognosis.

High prevalence of luminal B breast cancer intrinsic subtype in Colombian women.

Breast cancer is the most frequent malignancy in women worldwide. Distinct intrinsic subtypes of breast cancer have different prognoses, and their relative prevalence varies significantly among ethnic groups. Little is known about the prevalence of breast cancer intrinsic subtypes and their association with clinicopathological data and genetic ancestry in Latin Americans. Immunohistochemistry surrogates from the 2013 St. Gallen International Expert Consensus were used to classify breast cancers in 301 patients from Colombia into intrinsic subtypes. We analyzed the distribution of subtypes by clinicopathological variables. Genetic ancestry was estimated from a panel of 80 ancestry informative markers. Luminal B breast cancer subtype was the most prevalent in our population (37.2%) followed by luminal A (26.3%), non-basal triple negative (NBTN) (11.6%), basal like (9%), human epidermal growth factor receptor 2 (HER2) enriched (8.6%) and unknown (7.3%). We found statistical significant differences in distribution between Colombian region (P = 0.007), age at diagnosis (P = 0.0139), grade (P < 0.001) and recurrence (P < 0.001) according to intrinsic subtype. Patients diagnosed with HER2-enriched, basal-like and NBTN breast cancer had the highest African ancestry. Future studies analyzing the molecular profiles of breast cancer in Colombian women will help us understand the molecular basis of this subtype distribution and compare the molecular characteristics of the different intrinsic subtypes in Colombian patients.

Insulin Receptor Substrate Adaptor Proteins Mediate Prognostic Gene Expression Profiles in Breast Cancer.

Therapies targeting the type I insulin-like growth factor receptor (IGF-1R) have not been developed with predictive biomarkers to identify tumors with receptor activation. We have previously shown that the insulin receptor substrate (IRS) adaptor proteins are necessary for linking IGF1R to downstream signaling pathways and the malignant phenotype in breast cancer cells. The purpose of this study was to identify gene expression profiles downstream of IGF1R and its two adaptor proteins. IRS-null breast cancer cells (T47D-YA) were engineered to express IRS-1 or IRS-2 alone and their ability to mediate IGF ligand-induced proliferation, motility, and gene expression determined. Global gene expression signatures reflecting IRS adaptor specific and primary vs. secondary ligand response were derived (Early IRS-1, Late IRS-1, Early IRS-2 and Late IRS-2) and functional pathway analysis examined. IRS isoforms mediated distinct gene expression profiles, functional pathways, and breast cancer subtype association. For example, IRS-1/2-induced TGFb2 expression and blockade of TGFb2 abrogated IGF-induced cell migration. In addition, the prognostic value of IRS proteins was significant in the luminal B breast tumor subtype. Univariate and multivariate analyses confirmed that IRS adaptor signatures correlated with poor outcome as measured by recurrence-free and overall survival. Thus, IRS adaptor protein expression is required for IGF ligand responses in breast cancer cells. IRS-specific gene signatures represent accurate surrogates of IGF activity and could predict response to anti-IGF therapy in breast cancer.

Overview of Genetically Engineered Mouse Models of Distinct Breast Cancer Subtypes.

Advances in the screening of new therapeutic options have significantly reduced the breast cancer death rate over the last decade. Despite these advances, breast cancer remains the second leading cause of cancer death among women. This is due in part to the complexity of the disease, which is characterized by multiple subtypes that are driven by different genetic mechanisms and that likely arise from different cell types of origin. Because these differences often drive treatment options and outcomes, it is important to select relevant preclinical model systems to study new therapeutic interventions and tumor biology. Described in this unit are the characteristics and applications of validated genetically engineered mouse models (GEMMs) of basal-like, luminal, and claudin-low human subtypes of breast cancer. These different subtypes have different clinical outcomes and require different treatment strategies. These GEMMs can be considered faithful surrogates of their human disease counterparts. They represent alternative preclinical tumor models to cell line and patient-derived xenografts for preclinical drug discovery and tumor biology studies.

Abstract P3-06-15: Notch3 as a predictor of GSI sensitivity in distinct subtypes of triple negative breast cancer

Abstract Background: Breast cancer is the second leading cause of cancer-associated death in women. Triple negative breast cancer (TNBC) accounts for 10-15% of breast cancer, lacks expression of ER, PR, and HER2 gene amplification. Due to lack of targeted therapy, TNBC has the worst prognosis. TNBC is a heterogeneous disease with six distinct subtypes. Current treatment includes combination of surgery, radiation therapy and chemotherapy. Notch signaling is increased in TNBC and predicts for worst overall outcome. The goal of the study is to identify novel Notch-biomarkers that predict sensitivity of subtypes of TNBC to Notch inhibition in both bulk and cancer stem cells. Methods: Two subtypes of TNBC, Mesenchymal stem-like MDA-MB-231 and basal like MDA-MB-468 cells were used. Notch signaling was evaluated using both RNA-sequencing and quantitative real time PCR (q-RTPCR). Cancer stem cell markers were evaluated using Aldefluor assay, CD44 high/ CD24 low expression, and mammosphere forming assay. To study the effect of chemotherapy and Notch, MDA-MB-231 and MDA-MB-468 cells were treated with carboplatin or a pan Notch inhibitor- gamma secretase inhibitor (GSI) or combination of both and cell viability was measured using the trypan blue exclusion test. Results: The RNA-seq and RT-PCR results showed that Notch3 is differentially expressed in the two cell lines. Notch3 was knocked down using siRNA and its effect on cell viability was assessed after GSI or carboplatin or combination treatment. MDA-MB-468 cells had higher Notch receptors and activity, most notably Notch3. MDA-MB-468 cells had high levels of Aldefluor whereas MDA-MB-231 cells showed higher levels of CD44 High/CD24 Low expression. MDA-MB-468 cells had higher mammosphere forming efficiency (MFE) compared to MDA-MB-231. Notch inhibition decreased MFE of MDA-MB-468 whereas it increased MFE of MDA-MB-231 cells. MDA-MB-468 cells have lower IC50 for carboplatin or GSI compared to MDA-MB-231 cells, as measured by cell viability assay. However, combination treatment lowered IC50 for GSI in MDA-MB-231 cells as compared to MDA-MB-468 cells. Since Notch3 levels were strikingly different between the two cell lines, we hypothesized that Notch3 levels are necessary for GSI sensitivity. Carboplatin treatment increased Notch3 in MDA-MB-231 cells and the increase in Notch3 could be the reason for lower IC50 of GSI during combination treatment as increased Notch3 would provide the substrate for gamma-secretase. In MDA-MB-468 cells, Notch3 knockdown significantly reduced cell viability and was similar to GSI, suggesting that GSI acts through Notch3. Furthermore, Notch3 knockdown and carboplatin treatment reduced viability comparable to carboplatin and GSI treatment, reinforcing that the GSI acts through Notch3 in TNBC. Using the Kaplan-Meier Plotter, high Notch3 predicted poor recurrence free survival post chemotherapy in patients with ER-, HER2-, basal breast cancer. Conclusions: GSI acts through Notch3 in two TNBC subtypes and combination of chemotherapy with Notch inhibition results in a better outcome as compared to either drug alone. Future experiments would elucidate the role of Notch3 inhibition in targeting cancer stem cells post chemotherapy treatment in different subtypes of TNBC. Citation Format: Shah D, Osipo C. Notch3 as a predictor of GSI sensitivity in distinct subtypes of triple negative breast cancer. [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 P3-06-15.

Abstract P1-08-09: Increased prevalence of luminal B subtype in Colombian women with breast cancer

Abstract Background: Breast cancer is the most frequent malignancy in women worldwide. Distinct intrinsic subtypes of breast cancer have different prognoses, and their relative prevalence varies significantly among ethnic groups. Hispanic/Latino (H/L) populations are a genetically admixed and heterogeneous group, with variable levels of European, Native American and African ancestries. Breast cancer in H/L patients is understudied from a molecular standpoint, and most studies reported so far include limited numbers of H/L patients and assign ethnicity based on self-reported data rather than ancestry. This is the first study to explore the prevalence of breast cancer intrinsic subtypes in Colombia and their association with clinicopathological data and genetic ancestry. Methods: Immunohistochemistry surrogates from the 2013 St. Gallen International Expert Consensus were applied to classify breast cancer into intrinsic subtypes in 301 patients diagnosed between 2008 and 2012 at the Colombian National Cancer Institute. We analyzed the distribution of subtypes by age, histologic type, node status, margins at surgery, AJCC stage, tumor size, Bloom-Richardson grade, histologic features, administration and response to neoadjuvant therapy, adjuvant therapy and recurrence. Genetic ancestry was estimated from a panel of 80 ancestry-informative markers (AIM). Results: Luminal B breast cancer subtype was the most prevalent in our population (47.5%), followed by luminal A (23.9%), non-basal triple negative (9.3%), basal-like (8.6%), HER2-enriched (8%), and unknown (2.6%). The average of age at diagnosis was 55 and the average tumor size was 4.08 cm. We found statistical significant differences in age at diagnosis, Bloom-Richardson grade, histologic features, adjuvant chemotherapy and recurrence according to intrinsic subtype. Consistent with North American and European observations, basal-like and non-basal triple negative were poorly differentiated tumors and more likely to be diagnosed at younger ages compared to luminal tumors. Patients diagnosed with HER2-enriched, basal and non-basal triple negative breast cancer had the highest African ancestry. Conclusions: Luminal B tumors, a high risk subset of ER-positive breast cancer, occur with remarkably higher prevalence in Colombian women with breast cancer compared to North American and European populations. Triple-negative subtypes and HER2-enriched tumors appeared to be more frequent among patients with African ancestry, as observed in North American cohorts. Future studies analyzing the molecular profiles of breast cancer in Colombian women will help us understand the molecular basis of this subtype distribution and compare the molecular characteristics of the different intrinsic subtypes in Colombian Hispanic/Latina patients. Citation Format: Serrano-Gomez SJ, Sanabria MC, Hernández-Suarez GA, Garcia O, Silva C, Romero A, Mejía JC, Fejerman L, Antonia T, Miele L, Zabaleta J. Increased prevalence of luminal B subtype in Colombian women with breast cancer. [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 P1-08-09.

Abstract PR06: Analysis of enhancer transcription reveals novel gene regulatory networks in breast cancer

Abstract Enhancer transcription is a defining feature of active enhancers. We and others have shown that enhancers that produce transcripts (so called “eRNAs”) are more likely to (1) be associated with active chromatin marks, such as H3K4me1 and K3K27ac, (2) loop to target gene promoters, and (3) be associated with target gene activation. Thus, enhancer transcription is a good predictor of active enhancers. In this regard, we have shown that enhancer transcription can be used in the absence of any other genomic information to predict enhancers. We have used Global Run-On coupled deep sequencing (GRO-seq) in 14 different breast cancer cell lines representing the five distinct molecular subtypes of breast cancer, coupled with a computational pipeline that we have developed, to predict enhancers based solely on enhancer transcription. We found both common and unique sites of enhancer transcription across the cell lines. In addition, we observed that enhancer transcription correlates with nearest gene transcription. Unsupervised hierarchical clustering of enhancer transcription was sufficient to segregate the breast cancer cell lines into their specific molecular subtypes. Transcription factor motif analysis performed at the sites of enhancer transcription identified transcription factors that may be important for the transcriptional programs of each cell type. Transcription factors whose motifs were uniquely enriched in a specific cell type were observed to be bound at the enhancers using locus-specific ChIP-qPCR assay. siRNA-mediated knockdown of the cognate transcription factors reduced enhancer transcription and cell proliferation in those cell types. The GRO-seq data were integrated with ChIP-seq data for several histone modifications typically enriched at promoters, gene bodies, enhancers, and repressive regions of the genome. The results from these analyses provide additional support for our enhancer identification pipeline. Taken together, our analyses have revealed novel gene regulatory networks that underlie breast cancer subtype-specific biology. This work was supported by a postdoctoral fellowship from the American Cancer Society - Lee National Denim Day Fellowship to H.L.F., grants from the NIH/NIDDK and CPRIT to W.L.K. and a grant from CPRIT to the LONESTAR Consortium. Citation Format: Hector L. Franco, Anusha Nagari, Yuanxin Xi, Wenqian Li, Dana Richardson, Kaori Tanaka, Jing Li, The CPRIT LONESTAR Consortium, Michelle C. Barton, Xiaobing Shi, Khandan Keyomarsi, Mark T. Bedford, Wei Li, Sharon Y.R. Dent, W. Lee Kraus. Analysis of enhancer transcription reveals novel gene regulatory networks in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR06.

Integration of genomic, transcriptomic and proteomic data identifies two biologically distinct subtypes of invasive lobular breast cancer.

Invasive lobular carcinoma (ILC) is the second most frequently occurring histological breast cancer subtype after invasive ductal carcinoma (IDC), accounting for around 10% of all breast cancers. The molecular processes that drive the development of ILC are still largely unknown. We have performed a comprehensive genomic, transcriptomic and proteomic analysis of a large ILC patient cohort and present here an integrated molecular portrait of ILC. Mutations in CDH1 and in the PI3K pathway are the most frequent molecular alterations in ILC. We identified two main subtypes of ILCs: (i) an immune related subtype with mRNA up-regulation of PD-L1, PD-1 and CTLA-4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone related subtype, associated with Epithelial to Mesenchymal Transition (EMT), and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using the somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization may help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies.

Impact of Breast Cancer Subtype Defined by Immunohistochemistry Hormone Receptor and HER2 Status on the Incidence of Immediate Postmastectomy Reconstruction.

Immediate postmastectomy reconstruction has become an increasingly popular choice for breast cancer patients recently. However, whether molecular subtype of cancer impacts the incidence of breast reconstruction is unclear. We aimed to investigate the association between breast cancer subtype defined by immunohistochemistry hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status and recent rates of immediate postmastectomy reconstruction in the United States.The National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database was used to evaluate stage I–III breast cancer patients with different subtypes who underwent either mastectomy alone or mastectomy plus reconstruction between 2010 and 2012. Univariate and multivariate analyses were conducted to identify factors influencing the incidence of immediate reconstruction.Of 47,123 women included, 33.1% (10,712/32,376) of HR+/HER2−, 33.1% (1912/5768) of HR+/HER2+, 29.6% (850/2875) of HR−/HER2+, and 27.7% (1689/6104) of triple negative breast cancer patients received immediate breast reconstruction (chi-square test, P < 0.001), respectively. Thus, HER2-overexpressing and triple negative breast cancer patients received significantly less breast reconstruction. After adjusting for demographic, socioeconomic, geographic, or clinicopathologic factors, HER2-overexpressing (OR 0.896, 95% CI 0.817–0.984) and triple negative (OR 0.806, 95% CI 0.751–0.866) breast cancer patients remained less likely to undergo immediate postmastectomy reconstruction compared with HR+/HER2− or HR+/HER2+ patients. No significant difference was found in the type of reconstruction among different subtypes. Subgroup analysis showed that the difference of breast reconstruction rates among distinct subtypes varied with different grade and stage groups, and the association between breast cancer subtype and the reconstruction rate was not significant in low grade and early stage patients.This population-based study determined that breast cancer subtype was an independent predictor for the utilization of immediate postmastectomy reconstruction. Patients with HER2-overexpressing or triple negative breast cancer subtype that has relatively higher risk of local recurrence, were less likely to receive immediate breast reconstruction than those with luminal tumors. Further studies are needed to disclose more underlying reasons of different reconstruction incidences for distinct subtypes of breast cancer.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

Molecular Subtype-Specific Expression of MicroRNA-29c in Breast Cancer Is Associated with CpG Dinucleotide Methylation of the Promoter

Basal-like breast cancer is a molecularly distinct subtype of breast cancer that is highly aggressive and has a poor prognosis. MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in basal-like breast cancer. We here report that epigenetic modifications at the miR-29c promoter, rather than copy number variation of the gene, may drive the lower expression of miR-29c in basal-like breast cancer. Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c. Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes. Furthermore, inhibition of CpG methylation with 5-aza-CdR increases miR-29c expression in basal-like breast cancer cells. Flourescent In Situ Hybridization (FISH) revealed chromosomal abnormalities at miR-29c loci in breast cancer cell lines, but with no correlation between copy number variation and expression of miR-29c. Our data demonstrated that dysregulation of miR-29c in basal-like breast cancer cells may be in part driven by methylation at CpG sites. Epigenetic control of the miR-29c promoter by epigenetic modifiers may provide a potential therapeutic target to overcome the aggressive behavior of these cancers.

ECO/siRNA nanoparticles and breast cancer metastasis.

ECO/siRNA nanoparticles and breast cancer metastasis.

Abstract 3331: Gene expression profiling after radiation in human breast cancer specimens and breast cancer cell lines

Abstract Background: Breast radiotherapy is currently “one size fits all” regardless of breast cancer subtype (eg. luminal, basal). Emerging clinical data suggests that these distinct subtypes of breast cancer have unique patterns of response to radiation. There is a clinical need to identify radiation response biomarkers in order to provide individualized and optimal radiotherapy. Here we report on gene expression profiles associated with radiation response from: i) a rare clinico-genomic data in women treated with preoperative breast radiation and, ii) data from a pilot study of 16 biologically diverse breast tumor cell lines. Methods: Microarray analysis was done using the Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA 2.0) and the Affymetrix HU133A2.0 arrays. RNA was harvested from paired pre- and post-radiation formalin-fixed paraffin-embedded breast cancer tissue in 26 patients treated with radiation prior to surgical resection. In addition, RNA was obtained from 16 biologically diverse breast cancer cell lines exposed to radiation treatment (5Gy and 0Gy). The genes with the highest coefficient of variation were selected and a paired sample t-test was used to evaluate the significance of these changes. We compared the top 100 significantly induced human genes with genes identified as differentially expressed in the 16 breast cancer cell lines. Findings of interest were validated using qPCR, IHC and wWestern blotting assays. Results: Fourteen genes had significant adjusted p-values for differential expression in the human data and were differentially expressed among the radiation responsive versus non-responsive cell lines. Differential expression of genes involved in: apoptosis, cell cycle and MAPK signaling pathways were observed in both the luminal human tumors and the largely luminal radiation responsive cell lines. Differential expression of FAS, a critical modulator of programmed cell death, was highly significant. Conclusion: The known function of FAS suggests a biologically plausible role in breast subtype specific radiation response. Induction of FAS is seen in human tissue, and preferentially in luminal breast cancer cell lines. Citation Format: Sharareh Siamakpour-Reihani, Wei Chen, Chen-Ting Lee, Yingchun Zhou, Kouros Owzar, Jen-Tsan Chi, Janet K. Horton. Gene expression profiling after radiation in human breast cancer specimens and breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3331. doi:10.1158/1538-7445.AM2015-3331

Aberrant DNA methylation impacts gene expression and prognosis in breast cancer subtypes.

DNA methylation has a substantial impact on gene expression, affecting the prognosis of breast cancer (BC) patients dependent on molecular subtypes. In this study, we investigated the prognostic relevance of the expression of genes reported as aberrantly methylated, and the link between gene expression and DNA methylation in BC subtypes. The prognostic value of the expression of 144 aberrantly methylated genes was evaluated in ER+/HER2-, HER2+, and ER-/HER2- molecular BC subtypes, in a meta-analysis of two large transcriptomic cohorts of BC patients (n = 1,938 and n = 1,640). The correlation between gene expression and DNA methylation in distinct gene regions was also investigated in an independent dataset of 104 BCs. Survival and Pearson correlation analyses were computed for each gene separately. The expression of 48 genes was significantly associated with BC prognosis (p < 0.05), and 32 of these prognostic genes exhibited a direct expression-methylation correlation. The expression of several immune-related genes, including CD3D and HLA-A, was associated with both relapse-free survival (HR = 0.42, p = 3.5E-06; HR = 0.35, p = 1.7E-08) and overall survival (HR = 0.50, p = 5.5E-04; HR = 0.68, p = 4.5E-02) in ER-/HER2- BCs. On the overall, the distribution of both positive and negative expression-methylation correlation in distinct gene regions have different effects on gene expression and prognosis in BC subtypes. This large-scale meta-analysis allowed the identification of several genes consistently associated with prognosis, whose DNA methylation could represent a promising biomarker for prognostication and clinical stratification of patients with distinct BC subtypes.

Abstract OT3-2-04: ADAPT - Adjuvant Dynamic marker-Adjusted Personalized Therapy trial optimizing risk assessment and therapy response prediction in early breast cancer

Abstract Background: Early therapy response is currently not regarded for further treatment decisions as standard of care in the treatment of breast cancer (BC). Predictive markers for the success of a certain therapy could support the physician’s choice of adequate and beneficial therapies by simultaneous reduction of unnecessary toxicity. Proliferation makers as Ki-67 seem to be a suitable tool, as dynamic changes of proliferation (as result of induction therapy) have been shown to be most important for outcome of neoadjuvant chemotherapy prediction in patients with pCR in distinct BC subtypes (luminal B, TNBC, HER2+). Methods: Trial design: ADAPT combines early assessment of prognosis by conventional markers (e.g. molecular classification, nodal status) with dynamic measurement of proliferation changes during a 3-week induction therapy, using baseline diagnostic core biopsy and a second biopsy after induction therapy. ADAPT consists of an umbrella trial and five different sub-trials (HR+/HER2-, HR+/HER2+, HR-/HER2+, HR-/HER2-, Elderly) and is set up as prospective, multi-center, controlled, non-blinded, randomized phase II/III trial. Subtype-specific treatment across the sub-trials is highly innovative and involves the following treatment strategies: • HR+/HER2-: endocrine therapy (ET) vs. chemotherapy (4xPac q2w – 4xEC q2w vs. 8xNab-Pac q1w – 4xEC q2w) + ET, depending on risk classification/early response. • HER2+/HR+: T-DM1 vs. T-DM1 + ET vs. trastuzumab + ET. • HER2+/HR-: Trastuzumab + Pertzumab ± Paclitaxel q1w. • TN: Nab-Paclitaxel + Gemcitabine vs. nab-Pac + Carboplatin. • Elderly: 2xMyocet + Cyclophosphamide q3w, depending on cPR/cCR or NC/toxicity the treatment will be continued for two more cycles or changed to 6xPac q1w. Adaptation/change in therapy regimens can be made by interim analysis after n=130 in each sub-trial. Eligibility criteria: Histologically confirmed unilateral primary invasive BC with known HR-/HER2-status (central pathology) for allocation to the respective sub-trial. Pts requiring chemo- or targeted (anti-HER2) therapy must have adequate laboratory values and organ function and must have no contraindications for the planned treatment. Primary endpoints: Evaluation of dynamic test for outcome prediction/prospective comparison of 5yr EFS in responders (intermediate risk (RS 12-25) / good response to short-term ET in HR+/HER2- or pts with pCR in HER2+/TN BC) compared to low risk HR+/HER2- (RS≤11, N0-1) pts (control group). Statistical methods: Assumption across sub-protocols: adjuvant CTx can be spared in HR+/HER2- or pCR be achieved in HER2+/TN in expected 1120 (HR+/HER2-) or 170 (HER2+/TN) pts, respectively. Outcome will be compared to the control group (expected n=640 HR+/HER2- pts: low risk (by RS), i.e. no CTx). Assuming 94% 5yr survival in control group, one-sided test of non-inferiority at 95% CI will have 80% power for survival non-inferiority margin of 3.2% (i.e. 90.8% survival). Present and target accrual: By June 2014, 73 active sites have recruited 1820 pts for ADAPT HR+/HER2-. Target accrual is 4000 pts. 190 of 380 pts were successfully randomized for ADAPT HER2+/HR+. ADAPT HER2+/HR- has included 17 of 220 pts and ADAPT Triple Negative has recruited 150 of 336 pts. Citation Format: Ulrike Nitz, Oleg Gluz, Raquel von Schumann, Daniel Hofmann, Ronald E Kates, Sherko Kuemmel, Michael Braun, Claudia Schumacher, Benno Nuding, Bahriye Aktas, Helmut Forstbauer, Nicolai Maass, Mahdi Rezai, Stefan Kraemer, Mathias Warm, Rachel Wuerstlein, Nadia Harbeck. ADAPT - Adjuvant Dynamic marker-Adjusted Personalized Therapy trial optimizing risk assessment and therapy response prediction in early breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT3-2-04.