The promise of microarrays in the management and treatment of breast cancer

Breast cancer is the most common malignancy afflicting women from Western cultures. Developments in breast cancer molecular and cellular biology research have brought us closer to understanding the genetic basis of this disease. Recent advances in microarray technology hold the promise of further increasing our understanding of the complexity and heterogeneity of this disease, and providing new avenues for the prognostication and prediction of breast cancer outcomes. These new technologies have some limitations and have yet to be incorporated into clinical use, for both the diagnosis and treatment of women with breast cancer. The most recent application of microarray genomic technologies to studying breast cancer is the focus of this review

Abstract PD6-6: Estrogen receptor (ESR1) mutations confer resistance to hormone therapy using a common mechanism

Abstract Background: The idea that somatic estrogen receptor gene (ESR1) mutations could play an important role in the evolution of hormone-responsive breast cancers was proposed by us with our original identification of two ESR1 mutations at residues K303 and Y537. Technical issues with ESR1 mutation detection and the resulting paucity of reports in tumors led many to assume that ESR1 mutations were not there. However, with recent Next Generation Sequencing of metastatic tumors, mutation of ESR1 is now an accepted certainty. ESR1 mutant allele frequencies vary over a wide dynamic range, and are usually a minority population within tumors. Therefore how does a minor subclonal tumor population drive resistance in metastatic tumors? Methods: MCF-7 cells expressing endogeous wild-type ER was transduced with ESR1 mutants K303R, Y537N, Y537S, and D538G lentivirus and stable clones selected. ER transcriptional assays and growth in soft agar were performed. Digital drop (dd) PCR and primer extension snp detection were used to ascertain mutant:WT ESR1 allele frequency in cell lines, 200 primary tumors from patients treated with tamoxifen monotherapy, and 20 metastatic breast tumors. Results: Mutant ER constitutive transcriptional activity was fully antagonized by the antiestrogens tamoxifen or fulvestrant in MCF-7 stable transfectants. In contrast, soft agar growth of all ESR1 mutant-expressing cells was unexpectedly and completely resistant to the growth inhibitory effects of tamoxifen, although mutant-expressing cells were a minority subpopulation in the stable clones. Therefore, in cells with WT ER co-expression, the mutant resistant phenotype dominates. We found that phosphorylation of IGF1Rß; was constitutively increased in all ESR1-mutant expressing cells. Treatment with a specific IGF1Rß inhibitor in combination with tamoxifen drastically restored hormone sensitivity in cells expressing the ESR1 mutations. These results suggest a convergence in resistance mechanisms between the K303R and Y537 ESR1 mutation hot spots. We are exploring whether the dominant mutant ESR1 resistant phenotype occurs via activation of paracrine mediators, and have identified altered IGF-1 and interleukin 6 signaling in mutant-expressing cells. Mutation detection in a retrospective cohort of primary and metastatic breast tumors is ongoing and will be presented. Conclusions: We hypothesize that the selection of dominant-acting ESR1 mutations in tumors is a key event in breast cancer progression, potentially due to the selective pressure of antiestrogens. The dominant-resistant phenotype of ESR1 mutants in a majority WT background supports the subclonal evolution of ESR1 mutations in breast cancer recurrence. A common resistance mechanism (like consitutive IGF1Rß activation) should enable biologic targeting of ESR1 mutation-positive metastatic patients a feasible clinical goal. Support: NIH/NCI R01 CA72038 and CPRIT RP1210732 to SAWF. Citation Format: Luca Gelsomino, Guowei Gu, Yassine Rechoum, Sebastiano Ando', Suzanne AW Fuqua. Estrogen receptor (ESR1) mutations confer resistance to hormone therapy using a common mechanism [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 PD6-6.</jats:p

AR collaborates with ERα in aromatase inhibitor-resistant breast cancer

Androgen receptor (AR) is an attractive target in breast cancer because of its frequent expression in all the molecular subtypes, especially in estrogen receptor (ER)-positive luminal breast cancers. We have previously shown a role for AR overexpression in tamoxifen resistance. We engineered ER-positive MCF-7 cells to overexpress aromatase and AR (MCF-7 AR Arom cells) to explore the role of AR in aromatase inhibitor (AI) resistance. Androstendione (AD) was used as a substrate for aromatization to estrogen. The nonsteroidal AI anastrazole (Ana) inhibited AD-stimulated growth and ER transcriptional activity in MCF-7 Arom cells, but not in MCF-7 AR Arom cells. Enhanced activation of pIGF-1R and pAKT was found in AR-overexpressing cells, and their inhibitors restored sensitivity to Ana, suggesting that these pathways represent escape survival mechanisms. Sensitivity to Ana was restored with AR antagonists, or the antiestrogen fulvestrant. These results suggest that both AR and ERα must be blocked to restore sensitivity to hormonal therapies in AR-overexpressing ERα-positive breast cancers. AR contributed to ERα transcriptional activity in MCF-7 AR Arom cells, and AR and ERα co-localized in AD&nbsp;+&nbsp;Ana-treated cells, suggesting cooperation between the two receptors. AR-mediated resistance was associated with a failure to block ER transcriptional activity and enhanced up-regulation of AR and ER-responsive gene expression. Clinically, it may be necessary to block both AR and ERα in patients whose tumors express elevated levels of AR. In addition, inhibitors to the AKT/IGF-1R signaling pathways may provide alternative approaches to block escape pathways and restore hormone sensitivity in resistant breast tumors

Abstract 1996: Identification of four subgroups of Triple Negative Breast Cancer (TNBC) by genomic profiling.

Abstract Poorly defined molecular heterogeneity and a lack of effective molecular targets hinder advancements in the treatment of ER-/PR-/HER2- breast cancer (TNBC). Meta-analyses of public datasets have revealed new subgroups, but these studies are plagued by samples collected from multiple protocols and poor reproducibility. We aimed to identify stable subsets that could be treated with targeted therapy by employing integrated genomic profiling of a large number of TNBC cases (confirmed by IHC) from a single protocol before applying those subgroups to publicly available TNBC sets. We employed non-negative matrix factorization on two independent gene expression datasets (Discovery n = 84 and Validation n = 118) using the top 1000 variant genes in each set. Cophenetic, dispersion, and silhouette metrics revealed 4 clusters, with significant enrichment of differentially expressed (DE) genes in each subgroup among both sets (all comparisons Fisher Exact p &amp;lt; 2.2E-16). A parsimonious gene centroid signature of 480 total genes was selected based on Goeman's Global Test BH-adjusted p-values and fold change of only the Discovery Set. Subgroups were assigned by Pearson Correlation in the Discovery (Rand Index = 0.97) and Validation Sets (RI = 0.84), as well as an External Set made up of 214 IHC-confirmed cases of TNBCs, and another 5 publicly available studies with clinical outcome data for TNBCs. Ingenuity Pathway Analysis was carried out separately on DE genes from each dataset (BH adjusted p &amp;lt; 0.01). There was overwhelming statistical agreement for all subgroups between the Discovery, Validation, and External Sets. Subgroup 1 was defined by Intermediate Grade LumB tumors and overexpression of ESR1, HER-2, and AR, with activated downstream signaling of ESR1 despite being ER- by IHC. In subgroup 2, CDKN2A and TP53 appeared activated while MYC was inhibited. Dozens of immune cell signaling pathways were downregulated in the third subgroup, a basal-like set of TNBCs, which was driven by inhibition of cytokine gamma-IFN. Disease-free and Overall Survival was the worst for subgroup 3 (log rank p = 0.041 and 0.039). The final subgroup (4) was found to be p53 inactivated, a known feature of TNBC but specific to this second basal-like subgroup. Analysis of 84 and 58 cases with corresponding copy number profiling data from the Discovery and Validation Sets revealed subgroup specific copy number changes in addition to known TNBC patterns. Most notably, loss of chr 6 was unique to subgroup 1, while all other tumors shared common loss of chr 5. We have described four molecular phenotypes of TNBC in two independent sets from the same protocol, and a third set composed of public data. These subgroups have strong agreement of DE genes, pathways, and regulators, and are additionally supported by DNA events and prognostic differences between subgroups. We suggest several promising druggable targets based on overexpressed genes in each subgroup. Citation Format: Matthew D. Burstein, Anna Tsimelzon, Kyle R. Covington, Suzanne AW Fuqua, Jenny C. Chang, Susan G. Hilsenbeck, C Kent Osborne, Gordon B. Mills, Ching C. Lau, Powel H. Brown. Identification of four subgroups of Triple Negative Breast Cancer (TNBC) by genomic profiling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1996. doi:10.1158/1538-7445.AM2013-1996</jats:p

Abstract S4-03: Identification of a notch-driven breast cancer stem cell gene signature for anti-notch therapy in an ER+ presurgical window model

Abstract Background: Resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors, AI) for ER+ breast cancer is a major cause of mortality and new treatment paradigms are needed. Cancer stem cells drive breast cancer growth and are resistant to standard therapy. Notch signaling aids survival of these resistant stem cells and is inhibited by gamma-secretase inhibitors (GSI). We showed combining GSI with ET in mice caused shrinkage of breast cancer tumors. A presurgical window biomarker modulation model was used to confirm this discovery in humans. Methods: The GSI MK-0752 was added to ET in patients before definitive surgery (ClinTrials.gov NCT00756717). There were 3 biopsies: day 0 (prestudy), day 14 (after ET alone), and day 25 at definitive surgery (after continued ET plus MK-0752, 350 mg orally 3d on, 4d off, 3d on). Biopsies were analyzed for genes increased or decreased by GSI, to confirm that Notch and cancer stem cell pathways were inhibited. Real-time PCR was used to validate expression of genes identified in pathway analyses of microarray datasets generated from the biopsies. Mammosphere-forming assays were performed to confirm that ET+GSI impacts breast cancer stem cells. The qRT-PCR data were evaluated using ANOVA with repeated measures and ANOVA was performed on mammosphere results. Results: The accrual goal was met and therapy well-tolerated in 20 evaluable women (PSABCS 2011, abs# S1-5). Of 33 genes identified by analysis of expression microarrays, 19 genes (FDR&amp;lt;8%) were impacted significantly by GSI+ ET (3 increased, 16 decreased) compared to initial biopsy and/or ET alone. Genes with increased expression were DAXX, NOXA (both pro-apoptotic) and LNFG (tumor suppressor). Six of 16 genes that decreased (NOTCH1, NOTCH4, HEYL, HES1, HES5, and HEY2) are Notch pathway-associated genes. The GSI decreased expression of 3 genes from cell cycle and proliferative pathways (Ki67, CCND1, CCNA2) and inhibited 2 genes expressed in cancer stem cells (RUNX1 and ALDH1A1). Five genes directly/indirectly regulated by Notch were decreased by GSI (RICTOR, RPTOR, MMP7, ADAM19, and PRH). Estrogen deprivation for 3 days, mimicking short exposure to an AI, increased mammosphere-forming ability of ER+ breast cancer cells more than 2 fold. The GSI MRK-003 blocked this mammosphere formation by 95%-98%. Conclusions: A 7-day course of the GSI MK-0752 added to ET in the presurgical window had significant biomarker responses: decrease in Notch signaling, cancer stem cell genes, proliferation-associated genes, the mTORC1 and 2 complex genes RICTOR and RPTOR, metalloproteinases that promote metastasis, and PRH; as well as increase in 3 key genes that promote apoptosis and tumor suppression. These results suggest that 1) GSI inhibited the intended Notch pathway, 2) putative breast cancer stems cells can be targeted by this strategy, and 3) the biomarkers identified create a gene signature for anti-Notch therapy in ER+ breast cancer. Validation of efficacy of the GSI+ET therapy combination and this gene signature in a clinical trial is planned. Support: Breast Cancer Research Foundation (research grant), Merck Oncology (drug/arrays), Swim Across America (clinical trial costs), and DOD BC073237 (KRC). Citation Format: Kathy S Albain, Andrei Y Zlobin, Kyle R Covington, Brian T Gallahger, Susan G Hilsenbeck, Cheryl M Czerlanis, Shelly Lo, Patricia A Robinson, Ellen R Gaynor, Constantine Godellas, Davide Bova, Kathy Czaplicki, Barbara Busby, Patrick J Stiff, Suzanne AW Fuqua, Lucio Miele, Clodia Osipo. Identification of a notch-driven breast cancer stem cell gene signature for anti-notch therapy in an ER+ presurgical window model [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 S4-03.</jats:p

Abstract P5-05-03: Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib

Abstract Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response rate (pCR) was 27%. To investigate resistance mechanisms our lab developed 10 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell line panel and a subset of pre-treatment baseline specimens from TBCRC006. Methods: Parental (P) lines and LR/TR/LTR derivatives of 9 HER2+ BC cell line models were profiled with whole exome and RNA sequencing. Mutations detected in R lines but not in same-model P lines were identified. cDNAs were assessed by targeted Sanger sequencing. Single cells of the BT474AZ-LR line were cloned and their cDNAs were sequenced. Mutant-specific Q-PCR was designed to sensitively quantify mutations. Whole exome sequencing (minimum depth 100X) of 17 baseline tumor/normal pairs from TBCRC006 were performed on Illumina HiSeq. Results: We found and validated the HER2 L755S mutation in the BT474ATCC-LTR line and the BT474AZ-LR line (∼30% of DNA/RNA/cDNA in BT474AZ-LR), in which the HER pathway was reactivated to cause resistance. Overexpression of this mutation was previously shown to induce L resistance in HER2-negative BC cell lines, suggesting a role as an acquired L/LT resistance driver in HER2+ BC. Sanger sequencing of BT474AZ-LR single cell clones found the HER2 L755S mutation in every clone but only in ∼30% of the HER2 copies. Using sensitive mutant-specific Q-PCR, we found statistically higher levels of HER2 L755S expression in BT474ATCC-P and BT474AZ-P compared to parentals of other HER2+ BC cell lines (UACC812/AU565/SKBR3/SUM190). These data suggest that this mutation exists subclonally within BT474 parental lines and was selected to become the more dominant population in the two resistant lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of both BT474ATCC-LTR/AZ-LR cells (IC50: Afa 0.02µM vs. L 3 µM). Western blots confirmed inhibition of the HER and downstream Akt and MAPK signaling in the LR cells by Afa. Sequencing of TBCRC006 baseline samples found the HER2 L755S mutation in 1/17 subjects. This patient did not achieve pCR after neoadjuvant LT treatment. The variant was present in 2% of the reads, indicating it as a subclonal event in this patient’s baseline tumor. Conclusion: Acquired resistance in two of our BT474 LR/LTR lines is due to selection of HER2 L755S subclones present in the parental cell population. The higher HER2 L755S levels detected in BT474 parentals compared with other HER2+ BC parental lines, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura M Heiser, Nicholas Wang, Kyle R Covington, Edward S Chen, Alexander Renwick, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne AW Fuqua, Gary C Chamness, Chad Shaw, David A Wheeler, Joe W Gray, Susan G Hilsenbeck, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib [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 P5-05-03.</jats:p