High Estrogen Receptor Research Articles

Meta-STEPP: subpopulation treatment effect pattern plot for individual patient data meta-analysis.

We have developed a method, called Meta-STEPP (subpopulation treatment effect pattern plot for meta-analysis), to explore treatment effect heterogeneity across covariate values in the meta-analysis setting for time-to-event data when the covariate of interest is continuous. Meta-STEPP forms overlapping subpopulations from individual patient data containing similar numbers of events with increasing covariate values, estimates subpopulation treatment effects using standard fixed-effects meta-analysis methodology, displays the estimated subpopulation treatment effect as a function of the covariate values, and provides a statistical test to detect possibly complex treatment-covariate interactions. Simulation studies show that this test has adequate type-I error rate recovery as well as power when reasonable window sizes are chosen. When applied to eight breast cancer trials, Meta-STEPP suggests that chemotherapy is less effective for tumors with high estrogen receptor expression compared with those with low expression. Copyright © 2016 John Wiley & Sons, Ltd.

Abstract B17: High expression of the transcription factor Kaiso correlates with aggressive breast cancers (BC) and racial disparity in BC outcomes in women of African ancestry

Abstract The highly metastatic triple negative breast cancers (TNBC) lack expression of estrogen-receptor (ER), progesterone-receptor (PR) and human epidermal growth factor receptor-2 (HER2), and thus have limited patient treatment options and prognosis. Intriguingly TNBC is most prevalent in young women of African Ancestry (WAA) who, despite having lower BC incidence rates than Caucasian women, have higher mortality rates. This racial disparity appears unrelated to socio-economic status and increasing evidence hints at an ancestral genetic predisposition to TNBC development and high mortality rates. Recently, a significant correlation was found between nuclear expression of the transcription factor Kaiso, ERα negativity, basal/TNBCs and invasive ductal carcinoma in African American (AA) women. Emerging evidence from our lab also found that high Kaiso expression is associated with shorter metastasis-free survival in aggressive breast cancers. Based on these findings we hypothesized that Kaiso may play a crucial role in the metastatic spread of advanced breast cancers, and perhaps contribute to the racial disparity in TNBC prevalence and BC outcomes in WAA. To gain insight into Kaiso's role in aggressive breast cancers and the racial disparity associated with BC outcomes, we generated stable Kaiso-depleted TNBC cells (MDA-231) and then performed xenograft studies in immuno-compromised mice. Kaiso-depleted MDA-231 cells exhibited no or negligible metastases to the lungs and liver, compared to control MDA-231 cells, which formed extensive metastases in the lungs and liver of immuno-compromised mice. We also analysed Kaiso expression levels and subcellular localization in a tissue microarray (TMA) comprised of breast tumor tissues from ~100 Caucasian and ~100 AA women. Intriguingly, we observed a significant correlation between high nuclear Kaiso expression and ER(-) status in WAA, but surprisingly we found that high nuclear Kaiso expression is associated with ER(+) status and better overall survival in women of European descent. Collectively our findings implicate high Kaiso expression in breast cancer aggressiveness and suggest a context-dependent role for Kaiso in BC outcomes in women of different ethnicities. Ongoing studies are focused on clarifying the role of Kaiso in TNBC outcomes in WAA, and will incorporate analysis of a clinically annotated TMA comprised of ~200 TNBC tissues obtained from Barbadian and Nigerian women in conjunction with mechanistic studies to elucidate the signaling pathways involved in Kaiso-mediated tumor metastasis. Our findings may unravel a therapeutic role for Kaiso as a useful biomarker for the prognosis of TNBCs especially in WAA. Citation Format: Blessing I. Bassey-Archibong, Lyndsay G. A. Rayner, Kevin Gardner, Juliet Daniel. High expression of the transcription factor Kaiso correlates with aggressive breast cancers (BC) and racial disparity in BC outcomes in women of African ancestry. [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 B17.

Abstract P5-15-06: 10-year follow-up of adjuvant ovarian suppression in high risk premenopausal breast cancer

Abstract Background: A large randomized study has shown the value of temporary ovarian suppression during the administration of chemotherapy in the treatment of high risk estrogen receptor positive (ER+) breast cancer (BC) (Pagani et al. N Engl J Med 2014). In another study ovarian suppression added to chemotherapy appeared to protect ovarian function (Moore et al. N Engl J Med 2014) and improve the expected outcome of estrogen receptor negative (ER-) BC. Nevertheless, both these studies have a median follow-up time limited to 68 months and 49 months, respectively. Here we present the data of a large non randomized phase II study of adjuvant ovarian suppression in ER+ and ER- breast cancer with a median follow-up of 120 months (range 90-220 months). The primary end point was the ovarian function preservation rate, secondary end points were disease-free survival (DFS) and overall survival (OS). Methods: Between 06-1997 and 06-2007, 200 premenopausal, high risk early BC patients entered the study. All patients received the LH-RH analogue before starting chemotherapy. Breast conserving and radical surgery were performed in 74% and 26% of patients, respectively. Systemic therapy was tailored to the biological characteristics of each patient, and followed by radiation therapy and hormonal therapy in ER+ tumors. Results: The median patient's age was 43 years (range 26-45). The mean number of positive axillary nodes was 3.2 (range 1-25). Seventy-one % of patients were ER+ and/or progesterone receptor positive (PGR+), 29% were ER - and PGR-. The median KI-67 was 30% (range 15% -100%). Twenty-one % of patients were c-ErbB-2 positive. After a median follow up of 120 months (range 90-220), normal menses returned in 90% of patients younger than 40 years and in 56% of patients older than 40 years. The 10 and 15-year DFS rate were 85.5% and 71%, respectively, while the 10 and 15-year OS rate were 91%, and 71%, respectively. The standard pattern of toxicity of chemotherapy was observed. Hot flashes and G1 osteopenia occurring after LH-RH analogue administration were temporary and subsided after the cessation of therapy. Conclusions: LH-RH administration, concurrent with chemotherapy is tolerable and effective. Five full term pregnancies were documented. A favourable impact on the expected DFS and OS was observed. ER- patients had late new primaries, but no recurrence after 5 years, while ER+ patients had disease recurrence even after 13 years. Citation Format: Recchia F, Candeloro G, Rosselli M, Bratta M, Rea S. 10-year follow-up of adjuvant ovarian suppression in high risk premenopausal 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 P5-15-06.

Abstract A43: Context-dependent growth effects of progestins in estrogen receptor-positive breast cancer patient-derived xenografts (PDX)

Abstract Introduction: Progesterone (P4) and progesterone receptors (PR) play elusive roles in breast cancer. Synthetic progestins in hormone replacement therapy increase breast tumorigenesis, yet these same progestins at high doses can be an effective therapy for some advanced breast tumors. The literature supports that progestins can either stimulate or inhibit breast cancer cell growth, and an increasing body of evidence supports P4 promotes expansion of breast cancer stem cells. Interplay between PR and estrogen receptors (ER) is emerging as important in dictating growth and response to treatment. There is historical precedence for PR interference of ER dependent gene transcription; recent reports identify that PR modulate ER DNA binding events. Given the importance of PR as a biomarker and potential therapeutic target, and the uncertainty surrounding its context-dependent actions, we aimed resolve this using breast cancer PDX models. The purpose of this study was to evaluate the effects of natural and synthetic progestins on growth and estrogen-dependent gene expression in luminal breast tumors, and to define contexts that dictate their divergent effects. Experimental procedures: Six luminal breast cancer PDX developed at UCD-AMC were used for these studies. Most profiled as Luminal B subtype. Tumors generally fell into two types: a pure luminal phenotype and a mixed luminal/basal phenotype based on cytokeratin expression. Tumors varied in expression of ER, PR, and androgen receptors (AR) from low (<10% positive cells), moderate (30-50%), to high (>90%). Tumors were grown in vivo under conditions of continuous placebo, 17β-estradiol (estrogen), estrogen plus progesterone (P4), or estrogen plus the synthetic progestin medroxyprogesterone acetate (MPA) for several months and growth parameters monitored. Receptor content was measure by immunohistochemistry and western blots. Gene expression profiles of select tumors were determined using Affymetrix® GeneChip human gene microarrays and/or RNAseq and results analyzed using Partek Genomics Suite™ software, and Metacore™ pathway analysis. Summary: In two pure luminal PDX with high receptor levels (ER, PR, AR) both P4 and MPA significantly inhibited estrogen-dependent growth (growth-inhibited, GI). Two PDX (one pure luminal, one mixed) with moderate ER and low PR and AR showed no significant effect of P4 or MPA on estrogen-dependent growth (growth neutral, GN). One mixed luminal/basal PDX with low ER and PR and moderate AR showed no effect with P4, whereas MPA simulated tumor growth above that with estrogen alone (growth stimulated, GS). Study of one PDX is ongoing. In the GI tumors both P4 and MPA reversed expression of ~30% of estrogen regulated genes. In particular these tumors had high estrogen-dependent expression of the Notch pathway, which was blocked by both P4 and MPA. Progestins did not alter estrogen-dependent genes in the GN or GS tumors, which did not include the Notch pathway. MPA stimulated a unique set of genes in the GS tumor, and was able to mobilize AR to the nucleus whereas P4 did not. Conclusion: Progestins have mixed effects on breast cancer PDX tumor growth. Tumors that have high ER and PR and are highly estrogen-dependent are more likely to exhibit strong growth inhibition by progestins (both P4 and MPA). Tumors with mixed luminal/basal phenotype that have less ER and PR and are moderately estrogen-dependent are less likely to be progestin-inhibited. In tumors with low ER and PR but high AR content MPA can act as an AR agonist to stimulate growth. Tumor-specific downstream estrogen-dependent pathways such as Notch may indicate whether progestins are therapeutic. Additional tumors are needed to confirm these results with the long term goal of developing a molecular assay to define those tumors that may benefit from progestin treatment. Citation Format: Jessica Finlay-Schultz, Heather M. Brechbuhl, Rachel B. Rosen, Austin E. Gillen, Britta M. Jacobsen, Peter Kabos, Carol A. Sartorius. Context-dependent growth effects of progestins in estrogen receptor-positive breast cancer patient-derived xenografts (PDX). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A43.

Abstract A40: ESR1 gene alterations in the metastatic breast cancer with resistance to aromatase inhibitor

Abstract Background: Estrogen receptor (ER) positive breast cancer can be treated by endocrine therapy; however a certain population of ER-positive patients becomes resistant to endocrine therapy after long-term estrogen deprivation treatment. ESR1 gene alterations, mutations, translocation and amplification, have been reported as one of the reasons. Patients and Methods: We successfully obtained metastatic tissue samples from 30 patients with resistance to aromatase inhibitor (AI), and 7 patients with resistance to ethinylestradiol (EE2). ER and PgR expression by immunohistochemistry, and ESR1 gene amplification using FISH and copy number variation (CNV) by PCR, and ESR1 gene mutation including Y537N, Y537C, Y 537S and D538G by droplet digital PCR, were examined, in order to evaluate the endocrine responsiveness. Results: High expression of ER (median; 90%) and low expression of PgR (median 5%) were seen in majority of the patients treated by AI. Signal enhancement of DNA-FISH (> 2.0 fold) was seen in 2 (8%) of 25 patients, and signal gain (1.0< ≤2.0 fold) was seen in 11 (44%) of 25 patients. The increasing of DNA-CNV of ESR1 gene was seen in 3 cases of the 25 examined cases. There was no relationship between DNA-FISH and CNV of ESR1. High frequent ESR1 mutations were seen in 7 (28%) at Y537N, 4 (16%) at Y537C, 11 (44%) at Y 537S, and 2 (8%) at D538G. Double mutation, Y537N/S, was seen in 6 (24%) patients, who tended to unresponsive to subsequent endocrine therapy. After EE2 treatment, ER expression was decreased and PgR expression was increased, but there was no relationship between EE2 treatment and ESR1 gene alteration. Conclusion: In the patients with resistance to AI, high ER / low PgR expression was frequently seen, which was no correlation to ESR1 DNA-amplification. The long non-coding RNA in ESR1 gene locus might be related to this discrepancy (Tomita et al; Nat Commun. 2015). Furthermore, accumulation of ESR1 gene mutations tended to be related to endocrine unresponsiveness. Citation Format: Hirotaka Iwase, Takashi Takeshita, Mutsuko Yamamoto-Ibusuki, Touko Inao, Aiko sueta, Saori Fujiwara. ESR1 gene alterations in the metastatic breast cancer with resistance to aromatase inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A40.

Gene-expression signature functional annotation of breast cancer tumours in function of age.

BackgroundBreast cancer biological characteristics change as age advances. Today, there is a lack of knowledge regarding age-specific molecular alterations that characterize breast tumours, notably in elderly patients. The vast majority of studies that aimed at exploring breast cancer in function of age are based on clinico-pathological data. Gene-expression signatures (GES), which in some ways capture biological information in a non-reductionist manner, represent powerful tools able to explore tumour heterogeneity.MethodsTwenty-five GES were used for functional annotation of breast tumours in function of age: five for molecular subtyping, seven for immune response, three for metabolism, seven for critical pathways in cancer and three for prognosis. Affymetrix® genomics datasets were exclusively used to avoid cross-platform normalization issues. Available corresponding clinico-pathological data were also retrieved and analysed.ResultsFifteen publicly available datasets were pooled for a total of 2378 breast cancer patients (whole cohort), out of whom 1413 were of Caucasian origin. Three age groups were defined: ≤ 40 years (AG1), > 40 to < 70 years (AG2) and ≥ 70 years (AG3). We confirmed that age influenced the incidence of molecular subtypes. We found a significant growing incidence of luminal B and a decreasing kinetics for basal-like in function of age. We showed that AG3 luminal B tumours were less aggressive than AG1 luminal B tumours based on different GES (iron metabolism, mitochondrial oxidative phosphorylation and reactive stroma), recurrence score prognostic GES and histological grade (SBR). Contrary to tumours of young patients, tumours of elderly patients concentrated favourable GES scores: high oestrogen receptor and mitochondrial oxidative phosphorylation, low proliferation, basal-like, glycolysis, chromosomal instability and iron metabolism, and low GES prognostic scores (van’t Veer 70-GES, genomic grade index and recurrence score).ConclusionsFunctional annotation of breast tumours by means of 25 GES demonstrated a decreasing aggressiveness of breast tumours in function of age. This strategy, which can be strengthened by increasing the number of representative GES to gain more insight into biological systems involved in this disease, provides a framework to develop rational therapeutic strategies in function of age.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0153-6) contains supplementary material, which is available to authorized users.

A60 - Next Generation Sequencing mutational analysis in “Triple Positive” breast cancer

Background: Breast cancer is a heterogeneous disease, for histological types and molecular classifications. HER2 protein overexpression or gene amplification is reported in 15-20% of primary breast carcinoma and 50% of these are also hormonal receptors positive. The co-expression of these receptors could activate different transduction pathways and molecular characterization of these tumors has not yet been widely explored. Material and methods: We retrospectively analyzed biologic and clinical data of 15 patients (pts) with primary triple positive (Estrogen = 1%, Progesterone = 1% and HER2 IHC 3+ or SISH amplified) breast cancer, diagnosed from 2005 to 2008 at Ferrara University Hospital. Tumor sample were analyzed with nex generation sequencing (NGS) and the results were correlated with the pts clinical parameters. The gene analyzed with NGS were: ABL1, ALK, CDH1, CDKN2A, ErbB2, FBXW7, FGFR3, GNAQ, JAK3, KDR, KIT, MET, mTOR, TP53, PIK3CA, PTEN STK11. We considered significant the mutations present in more than 15% of reads, and used computer prediction programs (PolyPhen2 algorithm) and information in literature and mutations databases (Cosmic) to define the clinical significance of mutations. Results: We have found 14 significant mutations in 10 pts: 7 in PIK3CA, 2 in TP53, 1 in ABL1, 1 in CDKN2A, 1 in ErbB2, 1 in KDR and 1 in MET. One pts had 3 different mutations, 2 pts showed 2 mutations and 7 pts had a single mutation. The PIK3CA mutation (6 pts, 40%) was numerically the most frequent in pts with postmenopausal status (83.4% vs 16.6%), with stage II or III (83.4% vs 16.6%), with high proliferative activity (83.4% MIB1 = 20% vs 16.6% < 20%) and with high estrogen receptor positivity (= 50%) (83.4% vs 16.6%). All pts were treated according to guidelines of the period. At a median follow-up of 85.4 months, only 3 pts relapsed, one with single TP53 mutation (PFS 14.3 months), one with ABL1 single mutation (PFS 48.2 months) and one without significant mutation (PFS 24.6 months). Conclusions: As reported in Genoma Cancer Atlas, PIK3CA and TP53 mutations are the most frequent in our Luminal B HER2 enriched breast cancer pts. In our series PIK3CA mutations were more frequent compared to literature, probably due to the particular characteristics of triple positive pts. No clear relationship between recurrence and type of mutation can be confirmed.

Breast Carcinoma; Human Epidermal Growth Factor Receptor-2 (HER-2) and Grading Correlation

Introduction: Overexpression of Human Epidermal Growth factor Receptor-2 (HER-2) is one of the most important prognostic and predictive factors of breast cancer, observed in 25% - 30% of breast carcinoma patients leading to poor prognosis and feasible anti HER-2 antibody drugs. The objective of this study was to evaluate the HER-2 frequency in target population and its correlation with histologic grade as well as tumor pathology, estrogen receptor (ER) and P53 in our patients. Methods: A total of 300 cases (all female) aged 24- 80 year, were randomly selected from patients who were admitted in two of the Tehran University of Medical Sciences affiliated centers (Emam Khomeini Cancer Institute and Shariati hospital) over a 2-year period (2013-2014). Assessment of tumors for HER-2, P53, ER, pathological type and histologic grade was performed. HER-2 over expression defined as three plus (+++) in immunohistochemistry (IHC). Results: The mean age was 49.6±11 years. HER-2 over expression was seen in 34% of the patients. Significant correlations were found between HER-2+, P53+ and high histologic grade and ER (P<0.05). However, there was no significant correlation between HER-2 and pathologic type of tumors in our study. Conclusion: Co-expression of several poor prognostic biomarkers in breast cancer (HER-2 +, P53 +, ER- , high grade) may predict more aggressive phenotype, worse disease and lower overall survival in these patients.

Clinicopathological Characteristics and Survival Outcomes of Invasive Cribriform Carcinoma of Breast: A SEER Population-Based Study.

Invasive cribriform carcinoma (ICC) is a rare histologic subtype of breast cancer. We aimed to investigate the clinicopathological characteristics and survival outcomes of ICC.Using the Surveillance, Epidemiology, and End Results (SEER) database, we identified 233,337 female patients diagnosed with ICC (n = 618) or infiltrating ductal carcinoma (IDC) (n = 232,719). Univariate and multivariate survival analyses were utilized to calculate and compare disease-specific survival (DSS) and overall survival (OS). A 1:1 paired match was carried out on age, tumor stage, tumor grade, estrogen receptor (ER) status, and progesterone receptor (PR) status. Baseline characteristics and survival outcomes were also analyzed in ER-positive tumors. Subgroup analyses summarized the hazard ratio (HR) of IDC versus ICC using a forest plot.ICCs presented smaller size, lower grade, higher ER and PR positive rate, less nodal metastasis, and were less likely to be treated with mastectomy compared to IDCs. Five-year DSS rates were significantly better for patients with ICC than for patients with IDC (98.8% vs. 93%, P < 0.001). Five-year OS rates were 95.3% versus 90.1% (P < 0.001). After adjustment for common clinicopathological factors in the multivariate analysis, patients with ICC showed limited DSS advantage over the IDC group (HR = 0.75, 95% CI: 0.38-1.51, P = 0.421). No significant difference in DSS nor OS was observed in matched groups between ICC and IDC. Analysis among ER-positive patients revealed similar prognostic factors as among all patients. Survival analysis in different tumor grade subgroups showed no significant difference between ICC and IDC.ICCs have unique clinicopathological characteristics, higher rates of breast-conserving surgery, and more favorable prognosis compared to the overall IDC population. Difference in tumor grade between the 2 groups may partially explain the different outcome. Improved clinical and biological understanding of ICC might lead to more individualized and tailored therapy for breast cancer patients.

Abstract 2837: A surrogate gene expression signature of tumor infiltrating lymphocytes (TILs) predicts degree of benefit from trastuzumab added to standard adjuvant chemotherapy in NSABP (NRG) trial B-31 for HER2+ breast cancer

Abstract Background: Tumor infiltrating lymphocytes (TILs) have demonstrated to be predictive of trastuzumab as well as chemotherapy benefit in adjuvant FinHER and neoadjuvant GeparQuattro phase III trials of trastuzumab in HER2-positive breast cancer patients (pts). Since TILs assessment is subject to inter-observer variations, we attempted to develop a surrogate gene expression signature of TILs and evaluate it as a potential predictive marker for the degree of benefit from trastuzumab plus standard adjuvant chemotherapy in NSABP B-31. Methods: Two hundred cases selected from the discovery cohort of B-31 with Affymetrix gene expression data were assessed for the percentage of TILs using International TILs Working Group guidelines. Gene set enrichment analysis (GSEA) was done to identify functionally enriched biologic pathways. Genes strongly associated with high TILs were identified (N = 119). Hierarchical clustering analysis of TILs signature was performed in Affymetrix gene expression data set from 731 cases randomly selected from B-31. Proportional hazard models were used to test the heterogeneity of degree of benefit from trastuzumab subgroups identified by TILs signature with disease-free survival as the end point. Results: Among 200 cases, 34 were classified as morphologic high (&amp;gt;50%) TILs. GSEA revealed that tumors with high TILs expressed up-regulation of genes in several immune activation pathways involving T and B cells. Tumors with low TILs had up-regulation of muscular, fibrous tissue, extracellular matrix, and estrogen receptor (ER) gene sets. ER status by immunohistochemistry (IHC) was also inversely associated with TILs (p = 0.002). Hierarchical clustering of genes most strongly associated with TILs identified a small subset (N = 86, 12%) out of 731 pts with high expression of TILs-associated genes and more benefit from trastuzumab (HR = 0.06, CI 0.01-0.47, p = 0.007) versus samples with low or intermediate TILs (HR = 0.57, CI 0.43-0.76, p&amp;lt;0.001) (interaction p = 0.03). Conclusions: High TILs are associated with high immune, low stromal, and low ER gene expression levels. Identification of a subgroup with increased benefit from trastuzumab in breast cancer confirms previous studies on TILs as another complementary predictive marker for trastuzumab benefit in HER2-positive breast cancer. Furthermore, our study provides a comprehensive gene expression analysis and its relationship to TILs in breast cancer. While the clinical utility of TILs or its gene expression signature is limited because even low TILs are associated with significant benefit from trastuzumab, they may prove to be useful as biomarkers for immunotherapy. Support NCI U10-CA-12027, -69651, -37377, -69974; PA U24-CA-114732; PA DOH disclaims responsibility for analysis, interpretations, or conclusions. Citation Format: Seong-Rim Kim, Patrick G. Gavin, Katherine L. Pogue-Geile, Nan Song, Melanie Finnigan, Hanna Bandos, Jong-Hyeon Jeong, Priya Rastogi, Edward H. Romond, Louis Fehrenbacher, Eleftherios P. Mamounas, Sandra M. Swain, D. Lawrence Wickerham, Charles E. Geyer, Joseph P. Costantino, Norman Wolmark. A surrogate gene expression signature of tumor infiltrating lymphocytes (TILs) predicts degree of benefit from trastuzumab added to standard adjuvant chemotherapy in NSABP (NRG) trial B-31 for HER2+ breast cancer. [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 2837. doi:10.1158/1538-7445.AM2015-2837

Abstract 4996: Expression of the estrogen receptor, progesterone receptor and PTEN in endometrial cancers from women with type II diabetes mellitus

Abstract Objectives: The anti-diabetic drug, metformin, has been found to decrease proliferation, reduce estrogen receptor (ER) expression and inhibit downstream targets of the mTOR pathway in a pre-operative window study in endometrial cancer patients. Thus, we compared the endometrial cancers between patients with Type II diabetes taking metformin versus those not on metformin, in regards to expression of ER, progesterone receptor (PR) and PTEN. Methods: Patients with Type II diabetes who underwent surgical staging for endometrial cancer at our institution were identified. These patients were divided into two cohorts based on whether or not they were on metformin. The use of insulin and other anti-diabetic agents was recorded. Using triplicate cores from surgical specimens, a tissue microarray was constructed from formalin-fixed paraffin-embedded hysterectomy specimens. ER, PR and PTEN expression was measured by immunohistochemistry in both the epithelial and stromal components of the tumors. H scores were calculated, averaged among the triplicate cores, and compared between the two groups using Mann-Whitney U test and negative binomial regression analysis. Results: Triplicate cores were obtained from 162 endometrial cancers from patients with Type II diabetes. Of the 162 patients, 102 (63%) were taking metformin and 60 (37%) were not on metformin. Median BMI for metformin users and non-users was 40 and 37, respectively (p = 0.04). There was no significant difference in tumor stage, grade or histology between the two groups. There was no association between metformin use and PR or PTEN expression. There was a trend towards higher ER expression in the non-metformin users, which was statistically significant for stromal cells (p = 0.04) but not for epithelial cells (p = 0.13). However, when adjusting for age, BMI, race, tumor grade and stage, there was no association between metformin use and ER expression. The median H score for ER did differ between tumors from patients on metformin only (median 26) versus insulin only (median 91), but this was not statistically significant. Conclusions: Expression of ER, PR and PTEN in endometrial cancers did not differ amongst patients with Type II diabetes regardless of whether their diabetes treatment regimen involved metformin or insulin. Further studies are underway to compare the endometrial cancers of metformin users versus non-users in regards to other insulin and mTOR signaling targets. Citation Format: Dario R. Roque, Arthur M. Dizon, Brooke Rambally, Siobhan O'Connor, Paola A. Gehrig, Sheri A. Denslow, Victoria L. Bae-Jump. Expression of the estrogen receptor, progesterone receptor and PTEN in endometrial cancers from women with type II diabetes mellitus. [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 4996. doi:10.1158/1538-7445.AM2015-4996

Trinucleotide repeat expansions in human breast cancer-susceptibility genes: relevant targets for aspirin chemoprevention?

Defining novel molecular mechanisms pertinent to aspirin chemoprevention of breast cancer (BC) and to explain controversial epidemiological results in this regard. Literature search in relevant databases with the following key words; aspirin, nucleotide repeat expansions, breast cancer. Human genome contains nucleotide repeat expansions and exon-1 of the androgen receptor gene AR contains a CAG string with an average of 20 repeats. Longer AR CAG repeats associate with lower AR protein functioning leading relatively higher estrogen receptor signals and higher risk of hormone receptor-positive BC. Nucleotide repeat expansions also exist in E2F4 and POLG genes in BC. In cell culture models, aspirin reduces CAG.CTG expansions in kidney cells and restores myogenic differentiation in cells obtained from tissues with myotonic dystrophy, a disorder caused by large CTG expansions. We hypothesize that aspirin reduction of trinucleotide repeat expansions in breast cancer-susceptibility genes may be one of the relevant mechanisms of its chemopreventive effects.

Estrogen Receptor Expression in 21-Gene Recurrence Score Predicts Increased Late Recurrence for Estrogen-Positive/HER2-Negative Breast Cancer.

To identify the individual genes or gene modules that lead to the OncoptypeDx 21-gene recurrence score's reduced performance after 5 years and thereby identify indices of residual risk that may guide selection of patients for extended adjuvant therapy. We conducted a retrospective assessment of the relationship between (i) the individual genes and gene modules of the Recurrence Score and (ii) early (0-5 years) and late (5-10 years) recurrence rates in 1,125 postmenopausal patients with primary estrogen receptor-positive breast cancer treated with anastrozole or tamoxifen in the Arimidex, Tamoxifen, Alone or Combined (ATAC) randomized clinical trial. In the HER2-negative population (n = 1,009), estimates of recurrence risk were similar between years 0-5 and 5-10 for proliferation and invasion modules but markedly different for the estrogen module and genes within it (all split at the median): for low estrogen module, annual recurrence rates were similar across the two time windows (2.06% vs. 2.46%, respectively); for high estrogen module, annual rates were 1.14% versus 2.72%, respectively (P interaction = 0.004). Estrogen receptor transcript levels showed inverse prediction across the time windows: HR, 0.88 (0.73-1.07) and 1.19 (0.99-1.43), respectively (P interaction = 0.03). Similar time-, module-, and estrogen-dependent relationships were seen for distant recurrence. Patients with tumors with high estrogen receptor transcript levels benefit most from 5 years' endocrine therapy but show increased recurrence rates after 5 years and may benefit from extended therapy. Improved prognostic profiles may be created by considering period of treatment and follow-up time.

Abstract P4-04-06: CD146 positive and negative stroma direct breast tumor estrogen receptor levels, therapeutic response and metastatic potential

Abstract Background: Cellular heterogeneity within all breast cancer subtypes remains a major cause of treatment failure and development of metastatic disease. Currently, both preclinical studies and drug development efforts focus almost exclusively on the epithelial component of breast cancers. Despite strong preclinical data novel therapies often fail in clinical testing. We propose that this failure is, in part, due to taking tumor cells out of their context and using pre-clinical models that fail to capture the complexity of human disease. We hypothesize that tumors hijack normal components of the tissue microenvironment and use it to their advantage. Here we demonstrate that similar to the normal hematopoetic niche, two major subtypes of breast cancer stroma can be defined by CD146 expression. We further show that the ratio of the stromal subtypes alters the response to therapy and increases the metastatic potential of breast cancer cells (BCC). Results: Tumor associated stroma, from all breast cancer subtypes, contains a mixture of CD146+ and CD146- fibroblasts. We isolated and derived pure human CD146+ and CD146- clonal lines. Both subtypes expressed markers of activated fibroblasts and clustered by gene expression profiling with normal stromal cell lines HS27A (CD146+) or HS5 (CD146-) according to CD146 expression. Although both stromal subtypes were derived from tumor associated tissue, CD146+ breast cancer stroma clustered with normal breast associated stroma and correlated with good clinical outcome (Finak et. al. Nature Med 2008). CD146- stroma clustered with breast cancer associated stroma and predicted worse outcome. Using cell line and patient-derived xenograft models of estrogen receptor (ER) positive breast cancer, we demonstrated that CD146+ compared to CD146- stroma supported significantly higher ER expression in BCCs. BCC co-cultured with CD146+ stroma responded more robustly to estrogen treatment and anti-endocrine therapy with tamoxifen. Next we used expression profiling data to predict stromal influence on treatment response. CD146+ stroma expressed 3-fold more TGFβ than CD146- stroma. Inhibiting TGFβ decreased proliferation 2-fold in BCCs grown in media conditioned by CD146+ stroma, but not by CD146- stroma. Conversely, HBEGF expression was 3-fold higher in CD146- stroma compared to CD146+ stroma. Inhibiting EGFR decreased proliferation 1.5-fold in BCCs grown in conditioned by CD146- stroma, but not by CD146+ stroma. In addition, intracardiac injection of stroma resulted in distant metastases in our primary orthotopic PDX model of breast cancer. Lastly we confirmed our preclinical observation using a small set of clinical samples. Patients with high CD146+ to CD146- stromal ratio had better clinical outcomes than patients with high CD146- to CD146+ stromal ratio. Conclusion: We conclude that stromal subtypes defined by CD146 expression direct the heterogeneity of ER expression, response to therapy and the metastatic potential of breast cancer cells. Citation Format: Heather M Brechbuhl, Jessica J Finlay-Schultz, Tomomi M Yamamoto, Austin E Gillen, Anthony Elias, Carol A Sartorius, Peter Kabos. CD146 positive and negative stroma direct breast tumor estrogen receptor levels, therapeutic response and metastatic potential [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 P4-04-06.

Abstract P6-08-06: The multidisciplinary application of genomics in clinical practice (MAGIC) survey: Identification of early stage hormone receptor-positive (HR+), HER2– breast cancer (BC) patients for whom multigene assays may be valuable

Abstract Background Treatment recommendations for early stage HR+, HER2– BC patients depend on many factors. The MAGIC survey evaluated which criteria clinicians use regarding the need for adjuvant chemotherapy (AdjCT) and showed that there was substantial heterogeneity across clinicians and countries in treatment decisions (Aapro et al, EBCC 2014, abstract 24). Multigene assays (MGA) help to make more-informed decisions by providing prognostic and predictive information beyond traditional parameters, but are not always needed. The data presented here show for which BC patient profiles there is a high heterogeneity in treatment recommendations. We suggest that MGAs may be useful to guide treatment recommendations in these cases. Methods From August 2013 until January 2014, physicians with ≥5 years’ experience in BC treatment and participating in multidisciplinary teams were invited for the online MAGIC survey. The survey evaluated respondent characteristics and registered treatment recommendations for randomly generated early BC patient profiles (n=672). A conjoint analysis was used to assess which patient attributes were considered for treatment decisions. Results The survey was completed by 911 physicians from 52 countries, of whom 72% had &amp;gt;10 years’ experience. Their treatment recommendations showed that for BC patient profiles with only high-risk or only low-risk characteristics, there was a high consensus to recommend AdjCT or no AdjCT (endocrine treatment alone); 42% of the profiles had &amp;gt;75% probability of being recommended AdjCT and 6% had &amp;gt;75% chance of being recommended no AdjCT. If interactions between patient characteristics were not considered, age was ranked as the most important patient characteristic for AdjCT decisions, followed by tumor grade, tumor size, nodal status, and expression of Ki67, estrogen receptor (ER), and progesterone receptor. The combination of patient attributes and their interactions were, as expected, of importance; some node-positive patients or patients with a Grade 3 tumor had &amp;gt;75% probability to be recommended no AdjCT (eg, older patients or patients with a small [&amp;lt;2 cm] tumor). Conversely, some patients with small, Grade 1 tumors had &amp;gt;75% probability to be recommended AdjCT (eg, young or node-positive patients). In total, 104 patient profiles (15%) were identified for which treatment recommendations were highly heterogeneous, with a probability of &amp;lt;50% for both an AdjCT treatment recommendation and no AdjCT as a treatment recommendation. These patient profiles tended to have the following characteristics: &amp;gt;50 years old, tumor size &amp;lt;3 cm, Grade 1 or 2 tumor, high ER expression, and Ki67 expression &amp;lt;20%. Conclusions There was substantial heterogeneity in treatment recommendations and an overall tendency to give chemo-endocrine rather than endocrine treatment alone. The highest uncertainty in treatment decisions was seen in patients with intermediate risk by clinical and pathological parameters. This opens questions concerning treatment decisions and in such cases MGAs may be most useful. Citation Format: Matti Aapro, Juan Enrique Bargallo Rocha, Michele De Laurentiis, Roberto Elizalde, László Landherr, Barbro Linderholm, Terry Mamounas, Christos Markopoulos, Miguel Martin, Patrick Neven, Alexander Petrovsky, Dan Rea, Roman Rouzier, Vincent Smit, Christer Svedman, Christoph Thomssen. The multidisciplinary application of genomics in clinical practice (MAGIC) survey: Identification of early stage hormone receptor-positive (HR+), HER2– breast cancer (BC) patients for whom multigene assays may be valuable [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 P6-08-06.

A Validated Model for Identifying Patients Unlikely to Benefit From the 21-Gene Recurrence Score Assay

BackgroundPredicting recurrence risk and chemotherapy benefit in early-stage breast cancer can be challenging, and Oncotype DX (ODX) is often used to gain insight. However, it is still unclear whether ODX can benefit in all cases. To clarify ODX's usefulness we sought to develop a model using readily available pathologic markers to help clinicians make that determination. Patients and MethodsClinical pathologic data from 221 hormone receptor-positive, HER2-negative invasive breast cancer patients was used to create a model. The model was then validated on a second institution's set of 319 patients. ResultsThe model has 2 simple rules: low grade and positive progesterone receptor tumors (LG+PR) are low risk, and high grade or low estrogen receptor (ER) (ER < 20%) tumors (HG/LER) are high risk. The TAILORx (Trial Assigning Individualized Options for Treatment (Rx)) trial thresholds of Recurrence Score (RS) ≤ 10, when chemotherapy is of little benefit, and RS ≥ 26 when chemotherapy might be beneficial were used to judge model performance. Impressively, the misclassifications of an HG/LER patient who has an RS ≤ 10 were 0% and 2%, and for LG+PR patients who had an RS ≥ 26 were 0% and 2.6%. In the validation set, 28% (66 of 232) of the indeterminate group (neither in the HG/LER nor the LG+PR groups) had an RS ≤ 10 or an RS ≥ 26; this group might clinically benefit from ODX. ConclusionA simple 2-rule model based on readily available pathologic data was developed and validated, which categorized patients into high and low risk for recurrence. Identification of patients who are unlikely to benefit from ODX testing could result in significant cost avoidance.

High Affinity Fluorescent Ligands for the Estrogen Receptor

AbstractFluorescent binders of the estrogen receptor (ER) are used in binding assays and in detection or imaging studies. However, fluorescence labelling of ER ligands usually leads to substantial decreases in binding affinity. In this study, we describe the development of high affinity fluorescent ER ligands. Cyanine dyes of the MiDye series were directly attached to the SERMs 4‐hydroxytamoxifen (OHT) and raloxifene (Ral); linkers were deliberately omitted. This approach yielded conjugates with ERα binding affinities superior to the natural ligand estradiol. The OHT‐ and Ral‐MiDye conjugates emitted in the 600–800 nm range. First round staining experiments showed that the conjugates, but not the dyes alone, accumulate in cells expressing estrogen‐binding receptors.

17β-estradiol ameliorates oxygen-induced retinopathy in the early hyperoxic phase

Retinopathy of prematurity (ROP) is a major and leading cause of blindness in premature infants. It has been realized that early treatment for ROP is important. However, all the early treatments of ROP are focusing on peripheral retinal ablation which does not surmount the limit of extinguishing retinal neovascularization and protecting the retinas of children with ROP from the injury of ablation. In this study, we investigated the morphological changes of retina and oxidative stress alterations in the early phase of oxygen-induced retinopathy (OIR) and tested the effects of 17β-estradiol (17β-E2), a nonselective estrogen receptor (ER) agonist, on early phase OIR development. We found that large central capillary-free areas were induced in the retinas of pups exposed to hyperoxia on postnatal day 9 (P9), whereas vascularization was almost complete in the retinas of pups exposed to normoxia at the same age. The concentrations of malondiadehyde (MDA), an end-product of oxidative stress, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major enzyme producing free radicals, as well as the activity of NADPH oxidase were significantly elevated in the retinas of pups exposed to hyperoxia on P9 and postnatal day 13 (P13) compared to those in age matched pups exposed to normoxia. Treatment with 17β-E2 decreased not only the percentage of the central capillary-free area to total retina area but also the concentrations of MDA and NADPH oxidase as well as the activity of NADPH oxidase in a dose-dependent manner in pups exposed to hyperoxia on p9 and P13. The concentration of VEGF was significantly decreased on P9 but increased on P14 in the retinas of pups exposed to hyperoxia, whereas it was significantly elevated on P9 but decreased on P14 in the retinas of pups treated with 17β-E2. The effect of 17β-E2 could be reversed by the co-treatment with ICI182780, a high affinity estrogen receptor antagonist, which suggested that 17β-E2 might exert its effect on early hyperoxic phase of OIR through estrogen receptor. Our results suggest that treatment with antioxidant drugs at early hyperoxic phase of ROP even before the appearance of retinal neovascularization may be more effective than their application to ROP at late phase, which may abolish the deleterious factors that contribute to retinal neovascularization and promote retinal blood vessels to develop healthily.

Activation of mTOR/S6K But Not MAPK Pathways Might Be Associated With High Ki-67, ER+, and HER2− Breast Cancer

BackgroundEvaluation of luminal A and luminal B characteristics of estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer is considered important. Although the phosphoinositide 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways are thought to be involved in the luminal B subtype, the details of their contribution to breast cancer remain unclear. Materials and MethodsWe determined the activation of these pathways (phosphorylated MAPK [pMAPK] and protein S6 [pS6; a downstream molecule of PI3K/Akt/mammalian target of rapamycin (mTOR)/S6 kinase (S6K)]) in 108 ER+, HER2− breast cancer cases with high and low Ki-67 expression. The ER, progesterone receptor (PgR), Ki-67, p53 expression levels were also determined immunohistochemically. The cutoff value for Ki-67 was set at 15%. ResultsA significantly greater percentage of cancer cases with high Ki-67 expression showed pS6 positivity than did those with low Ki-67 expression (53.2% vs. 19.7%; P = .0003). No significant differences were found between the cases with high and low expression levels were detected for p53 (23.4% vs. 11.5%; P = .12) or pMAPK (36.2% vs. 34.4%; P = .85) positivity. Multivariate analysis showed that pS6 positivity (odds ratio 5.16, 95% confidence interval 1.95-13.63; P = .0009), nuclear grade 2 and 3, and low PgR expression (≤ 20%) were independently associated with the high Ki-67 subset. ConclusionFrom our findings, we have concluded that the pS6 expression level is associated with the characteristics of breast cancer with high Ki-67 expression. Because these associations were observed, irrespective of menopausal status, the biologic difference seems to be less affected by estrogen signaling than by activation of S6 protein, especially in terms of proliferation. Our findings have also indicated that targeting the mTOR/S6K pathway might be a useful strategy for the treatment of ER+/HER2− breast cancer with high Ki-67 expression.

Abstract 987: Comprehensive characterization of urothelial bladder cancer: a TCGA Project update

Abstract Urothelial carcinoma (UC) is a major cause of morbidity and mortality for which there are no approved molecularly targeted agents and few good treatment options beyond cisplatin-based chemotherapy. As part of The Cancer Genome Atlas (TCGA) Project, we analyzed 131 chemotherapy-naive, muscle-invasive UC tumors for somatic mutations, DNA copy number variants (CNVs), mRNA and microRNA expression, protein expression and phosphorylation, DNA methylation, transcript splicing, gene fusion, viral integration, pathway perturbation, clinical correlates, and histopathology (TCGA Research Network, Nature, in press). Whole-exome sequencing showed 29 recurrently mutated genes. Potential therapeutic targets include altered PIK3CA, ERBB2, FGFR3, TSC1, and ERBB3, plus mutated chromatin-regulating genes MLL, MLL2, MLL3, CREBBP, CHD7, SRCAP, ARID1A, KDM6A (UTX), and EP300. There were 22 arm-level CNVs and 27 focally amplified or deleted regions. CDKN2A was deleted in 47%. Low-pass whole genome sequencing identified FGFR3-TACC3 fusions. Viral DNA was identified in 6% (CMV, HHV6B, HPV16, BK polyoma), and viral transcripts were identified in 4% (CMV, BK polyoma, HPV16). . mRNA-seq identified 4 tumor clusters. Cluster I shows papillary morphology and FGFR3 dysregulation. Clusters I and II express high HER2 (ERBB2) and estrogen receptor beta signaling signature, sharing features with Luminal A breast cancer. Cluster III shows similarities to Basal-like breast and squamous cell head and neck carcinomas. . Integrated analyses confirm alteration of multiple pathways, including cell cycle regulation (93%), kinase and PI3-K signaling (72%), and epigenetic regulation (histone-modifiers: 89%; SWI/SNF nucleosome remodeling complex: 64%). Recurrent alterations in the PI3-kinase/AKT/mTOR pathway (42%) and RTK/RAS pathway (44%) are potentially actionable. . Overall, this study and others have identified multiple druggable targets in UC. FGFR3 is activated by mutation, gene fusion, and overexpression, suggesting clinical trials of FGFR3 inhibitors. PI3-kinase/mTOR/AKT/TSC1 pathway alterations are frequent, and mutation in TSC1 has been associated with response to mTOR inhibitors. ERBB2 amplifications and activating mutations may be targetable with agents such as trastuzumab, trastuzumab-DM1, lapatinib, and neratinib. The frequent alterations in epigenetic regulatory pathways suggest trials of agents such as the bromodomain inhibitors. The project is now being updated on the basis of data on 117 additional tumors. Citation Format: John N. Weinstein, Jaegil Kim, Chad J. Creighton, Rehan Akbani, Katherine A. Hoadley, William Y. Kim, Margaret B. Morgan, Toshinori Hinoue, Andrew Cherniack, Xiaoping Su, Andrew J. Mungall, Michael C. Ryan, Jonathan E. Rosenberg, Dean F. Bajorin, Bogdan Czerniak, Donna Hansel, Victor E. Reuter, Brian D. Robinson, Hikmat A. Al-Ahmadie, Jeffrey S. Damrauer, Wei Zhang, Yuexin Liu, Dmitry Gordenin, Joshua M. Stuart, Nikolaus Schultz, Gordon Robertson, Raju Kucherlapati, Peter W. Laird, Gordon B. Mills, David J. Kwiatkowski, Seth P. Lerner, representing TCGA's Bladder Cancer Working Group. Comprehensive characterization of urothelial bladder cancer: a TCGA Project update. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 987. doi:10.1158/1538-7445.AM2014-987