Citations

540 Background: Risk of death from other malignancies (OM) and other causes (OC) than breast cancer (BC) increases with age. Effects of baseline factors on type of death were assessed with competing risks analyses. Methods: In NCIC CTG MA.17, 5,187 women free of recurrent breast cancer after 5 years of tamoxifen were randomized to letrozole (L, 2,593 women) or placebo (P, 2,594 women). The primary endpoint was disease free survival (DFS), and secondary, overall survival (OS). Follow-up was to October 9, 2005: median 3.9 years, range <0.1 to 7.0 years. Effects of competing risks were examined for endpoints of BC, OM, and OC for 11 baseline trial factors: treatment, age, menopausal status, duration of prior tamoxifen, adjuvant radiotherapy, bone fracture, osteoporosis, cardiovascular disease, hormone receptor status, nodal status, adjuvant chemotherapy. Lagakos’ hierarchical method (Lagakos, Appl. Statist. 1978; 27:235–241) was used to test for differential effects of baseline factors on type of death (BC, OM, OC). Results: Rate of censoring was 97.8%, with 256 deaths (BC, 102; OM, 50; OC, 100; unknown, 4). Non-breast cancer deaths accounted for 60% of known deaths; 72%, for those ≥70 years; and 48%, for those <70 years. Two baseline factors differentially affected type of death. Women with cardiovascular disease were more likely to die from OC (p=0.02), while those with osteoporosis were more likely to die of OM (p=0.03). Age and nodal status had directionally similar effects. Older women had shorter survival from all 3 causes of death (p=0.01). Lymph node positivity was associated with worse survival (p=0.003). Conclusions: Extended L provides similar proportional benefit in improving DFS for all ages of women (Muss ref abstract SABCS 2006). However, the magnitude of competing non-breast cancer, and non-treatment related, causes of death needs to be considered more frequently, since with early detection and improved therapies, breast cancer patients may increasingly be expected to survive their disease to die from another cause. The novel association between baseline osteoporosis and other malignancies is being explored quantitatively. No significant financial relationships to disclose.

Abstract Purpose: To determine if the degree of estrogen suppression with aromatase inhibitors (AI: anastrozole, exemestane, letrozole) is associated with efficacy in early-stage breast cancer, and to examine for differences in the mechanism of action between the three AIs. Experimental Design: Matched case–control studies [247 matched sets from MA.27 (anastrozole vs. exemestane) and PreFace (letrozole) trials] were undertaken to assess whether estrone (E1) or estradiol (E2) concentrations after 6 months of adjuvant therapy were associated with risk of an early breast cancer event (EBCE). Preclinical laboratory studies included luciferase activity, cell proliferation, radio-labeled ligand estrogen receptor binding, surface plasmon resonance ligand receptor binding, and nuclear magnetic resonance assays. Results: Women with E1 ≥1.3 pg/mL and E2 ≥0.5 pg/mL after 6 months of AI treatment had a 2.2-fold increase in risk (P = 0.0005) of an EBCE, and in the anastrozole subgroup, the increase in risk of an EBCE was 3.0-fold (P = 0.001). Preclinical laboratory studies examined mechanisms of action in addition to aromatase inhibition and showed that only anastrozole could directly bind to estrogen receptor α (ERα), activate estrogen response element-dependent transcription, and stimulate growth of an aromatase-deficient CYP19A1−/− T47D breast cancer cell line. Conclusions: This matched case–control clinical study revealed that levels of estrone and estradiol above identified thresholds after 6 months of adjuvant anastrozole treatment were associated with increased risk of an EBCE. Preclinical laboratory studies revealed that anastrozole, but not exemestane or letrozole, is a ligand for ERα. These findings represent potential steps towards individualized anastrozole therapy.

Abstract A micro version of a nuclear binding assay to assess the biological activity of receptors for steroid hormones was developed for application to small (needle) biopsies of human tumors for the purpose of predicting responses to steroid therapy. This easier assay requires 10-fold less tissue than the original nuclear binding assay described for progesterone receptors in the avian oviduct, endometrium, and endometrial carcinomas (Spelsberg TC, et al., Endocrinology 1987;121:631). We describe the application of this micro assay to normal avian oviduct and cancers of the human breast, and we demonstrate a tissue specificity and saturation of nuclear binding. The micro assay reliably measured as little as 0.5 mg equivalents of tissue per assay tube. Results for breast tumors determined to be estrogen-receptor-positive by the standard dextran-coated charcoal method were also determined with this nuclear binding assay. As described previously for progesterone receptors in endometrial carcinomas, some receptor-positive breast biopsies displayed negligible capacity for nuclear binding. Therefore, with the present assay we have identified nonfunctional receptors in these biopsies, which may be useful for accurate prediction of patients' responses to therapy with hormones.

Abstract Background Adjuvant bisphosphonates, when given in a low-estrogen environment, can decrease breast cancer recurrence and death. Treatment guidelines include recommendations for adjuvant bisphosphonates in postmenopausal patients. SWOG/Alliance/Canadian Cancer Trials Group/ECOG-ACRIN/NRG Oncology study S0307 compared the efficacy of three bisphosphonates in early-stage breast cancer. Methods Patients with stage I–III breast cancer were randomly assigned to 3 years of intravenous zoledronic acid, oral clodronate, or oral ibandronate. The primary endpoint was disease-free survival (DFS) with overall survival as a secondary outcome. All statistical tests were two-sided. Results A total of 6097 patients enrolled. Median age was 52.7 years. Prior to being randomly assigned, 73.2% patients indicated preference for oral vs intravenous formulation. DFS did not differ across arms in a log-rank test (P = .49); 5-year DFS was 88.3% (zoledronic acid: 95% confidence interval [CI] = 86.9% to 89.6%), 87.6% (clodronate: 95% CI = 86.1% to 88.9%), and 87.4% (ibandronate: 95% CI = 85.6% to 88.9%). Additionally, 5-year overall survival did not differ between arms (log rank P = .50) and was 92.6% (zoledronic acid: 95% CI = 91.4% to 93.6%), 92.4% (clodronate: 95% CI = 91.2% to 93.5%), and 92.9% (ibandronate: 95% CI = 91.5% to 94.1%). Bone as first site of recurrence did not differ between arms (P = .93). Analyses based on age and tumor subtypes showed no treatment differences. Grade 3/4 toxicity was 8.8% (zoledronic acid), 8.3% (clodronate), and 10.5% (ibandronate). Osteonecrosis of the jaw was highest for zoledronic acid (1.26%) compared with clodronate (0.36%) and ibandronate (0.77%). Conclusions We found no evidence of differences in efficacy by type of bisphosphonate, either in overall analysis or subgroups. Despite an increased rate of osteonecrosis of the jaw with zoledronic acid, overall toxicity grade differed little across arms. Given that patients expressed preference for oral formulation, efforts to make oral agents available in the United States should be considered.

Abstract Background: In early stage, ERα-positive breast cancer, the concurrent use of endocrine therapy and chemotherapy has not been shown to be superior to sequential use. We hypothesized that genetic biomarkers can aid in selecting patients who would benefit from chemo-endocrine therapy. Our previous genome-wide association study (GWAS) and functional studies revealed that a single nucleotide polymorphism (SNP) in ZNF423, rs9940645, determines tamoxifen response. Specifically, 4-hydroxytamoxifen (4-OH-TAM) increases the gene expression of ZNF423, a transcription factor for BRCA1, in cells that are carry the rs9940645 variant, but not the wildtype, genotype. In this study, we set out to identify additional genes regulated by ZNF423, which, upon identifying a relationship between ZNF423 and cell cycle genes, led us to investigate taxane response in a SNP- and tamoxifen-dependent fashion. Method: Gene correlation analysis using The Cancer Genome Atlas (TCGA) breast cancer dataset was used to identify genes with expression correlated with that of ZNF423. Quantitative reverse transcription PCR, chromatin immunoprecipitation, and luciferase reporter assays were used to validate the gene associations. To test ZNF423 rs9940645-specific phenotypes, we used ZR75-1 cells, which are homozygous for the variant SNP, and ZR75-1 cells that were generated to be homozygous wild type (WT) using CRISPR-Cas9 technology, in addition to Hs578T breast cells with ERα overexpression (WT) and HCC1500 cells (variant). Cell cycle was assessed by propidium iodide staining. Cytotoxicity was assessed using a colorimetric assay. Results: Mitosis-related genes VRK1 and PBK, which encode histone H3 kinases, were experimentally validated to be regulated by ZNF423. Specifically, ZNF423 knockdown resulted in decreased VRK1 and PBK expression and activity. Additionally, ZNF423 knockdown resulted in enhanced docetaxel-induced G2/M arrest and cytotoxicity which could be rescued by VRK1 or PBK overexpression. Lastly, breast cancer cells carrying the rs9940645 variant SNP genotype had increased G2-M arrest and decreased cell viability when treated with docetaxel in combination with estradiol and 4-OH-TAM. Conclusion: We identified ZNF423regulated genes involved in the G2/M phase of the cell cycle. We found that 4-OH-TAM sensitized ERα-positive breast cancer cells to docetaxel treatment in a ZNF423 SNP-dependent manner. Our findings suggest that patients with rs9940645 variant genotype may benefit from concurrent tamoxifen endocrine therapy and docetaxel chemotherapy. This would impact a substantial proportion of patients given the minor allele frequency of 0.47 for this SNP. Citation Format: Gen Wang, Sisi QIN, Jacqueline Zayas, James N. Ingle, Mohan Liu, Richard Weinshilboum, Kunwei Shen, Liewei Wang. 4-Hydroxytamoxifen enhances sensitivity of estrogen receptor α-positive breast cancer to docetaxel in an estrogen and ZNF423 SNP-dependent fashion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2940.

552 Background: Bisphosphonates reduce the risk of bone metastases in postmenopausal women with early-stage breast cancer but carry the risk of osteonecrosis of the jaw (ONJ). We used the data collected in the S0307 trial to describe factors associated with provoked and unprovoked ONJ. Methods: In S0307, 6097 patients with Stage I-III breast cancer who had surgery were randomized to receive zoledronic acid (ZA) 4mg IV monthly for 6 months, then every 3 months, clodronate (CL) 1600mg daily, or ibandronate (IB) 50mg daily for three years, with no difference in bone metastases or disease-free survival. Patients completed dental procedures prior to and had a dental exam at enrollment. Pearson’s Chi-squared and Student’s T-test were used to test differences in categorical and continuous variables, respectively; logistic regression was used test independent association. Results: Of 5836 evaluable women, 48 developed ONJ, which was associated with bisphosphonate type (28/2124 (1.26%) for ZA, 8/2185 (0.36%) for CL and 12/1527 (0.77%) for IB (p = 0.002). Median time to onset of ONJ was 24.9 (1.4-66.6) for ZA, 41.2 months (range 33.8-67.4) for CL, 23.9 (2.1-75.3) for IB (p = 0.0447). Infection was present in 21 (43.8%) and absent in 20. ONJ was considered unprovoked in 20 (41.7%) and provoked by dental extraction in 20 (41.7%), periodontal disease in 14 (29.2%), denture trauma in 6 (12.5%), other dental surgery in 3 (6.3%). ONJ was associated with dental calculus (OR 2.03 (95% CI: 1.08-3.81), gingivitis (OR 2.11, 95% CI: 1.12-3.98), and periodontal disease (OR 2.87, 95% CI 1.45-5.53) that were moderate/severe and > 4mm periodontitis (OR 2.20, 95% CI 1.18-4.08). Patients with provoked and unprovoked ONJ had similar amounts of dental disease and lesion location. ONJ was not associated with dentures, plaque, chemotherapy, corticosteroids or renal adverse events. In multivariate analysis (limited by small sample), only bisphosphonate type was associated with ONJ. Conclusions: ONJ prevalence was low, likely due to patients completing dental procedures before enrollment. ZA carried a higher risk of ONJ (though current adjuvant dosing interval recommendations are less frequent), with time to onset similar to IB. Oral CL and IB (not currently available in the United States) are thus likely more appropriate for patients with poor dental health. Clinical trial information: NCT00127205.

Abstract Breast cancer is a heterogeneous disease, and unsupervised clustering approaches using gene expression data have identified 3-6 distinct subtypes of triple negative breast cancer (TNBC). A genomically and clinically distinct subtype of TNBC is referred to as LAR (Luminal Androgen Receptor). Tumors with this subtype typically express high levels of the AR and exhibit alterations within genes involved in the PI3K pathway (e.g. PIK3CA mutations). Prospective studies are underway using drugs that target the AR alone or in combination with PI3K and CDK 4/6 inhibitors. Given the importance of accurately identifying this subtype, we sought to develop an online tool that uses submitted gene expression data to confidently characterize LAR samples by corroborating the classification with previously published clustering approaches. Methods: We have investigated TNBC RNA-Seq data from The Cancer Genome Atlas (TCGA) breast cancer study (N=123 samples) by cluster analysis. Analysis of the average silhouette width in both biased and unbiased K-means clustering approaches demonstrated LAR and basal as two distinct and significant clusters. A shrunken centroid model of 426 differentially expressed genes, named as CABAL (Clustering Among BAsal and Luminal androgen receptor), was constructed by comparing LAR and basal subtypes. Results: We applied the CABAL model to classify the four TNBC microarray datasets that were previously used in clustering experiments as well as an independent RNA-Seq data cohort. Non-negative matrix factorization (NMF) and fuzzy clustering were applied to the samples (N=1046). Clustering similarity among the methods was assessed with the adjusted rand index, and CABAL demonstrated significant similarity with both fuzzy and NMF clustering methods. Similarly, hierarchical clustering analysis performed on the pooled cohort of 1046 samples recapitulated the CABAL classification with an area under the receiver operating curve of 0.91. Conclusions: Confident and robust identification of samples with the LAR phenotype is paramount in the assessment of clinical associations and therapeutic efficacy. To facilitate LAR identification, we have provided a web-based prediction tool of the CABAL classification, integrated with the NMF and fuzzy clustering results to identify candidate LAR samples. The end user is provided with the pair-wise adjusted rand indexes, thus reinforcing in the clustering characterizations. Further, our online LAR depiction tool provides a set of graphical and tabular summaries, which will be illustrated, while providing additional molecular characterizations of the PAM50 and Metabric classifications. The availability of this tool could advance the genomic research and treatment of TNBC patients. Citation Format: Thompson KJ, Alaparthi T, Sinnwell JP, Carlson EE, Tang X, Bockol M, Vedell PT, Ingle JN, Suman V, Weinshilboum RM, Wang L, Boughey JC, Kalari KR, Goetz MP. Molecular subtyping of androgen receptor-positive patients using gene expression profiles [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-03-04.

Abstract Background: The LAR subtype is a genomically distinct subset of TNBC. Using a large cohort of non-metastatic TNBC patients (pts) with long term follow-up, we sought to further characterize the clinicopathologic features and outcomes of LAR vs non-LAR TNBC. Methods: From a cohort of 9982 women with surgically-treated non-metastatic breast cancer, 605 met criteria for TNBC (ER/PR&lt;1% and HER2-negative) by central pathology. RNA extracted from 304 FFPE tumor specimens using the HighPure RNA extraction kit was subjected to TruSeq RNA Access library preparation and sequencing on a HiSeq2500. Adequate RNA was available for 283 pts. Tumors were classified as LAR or non-LAR using a shrunken centroid model, CABAL (Clustering Among BAsal and Luminal androgen receptor). In addition to previously described analyses [Leon-Ferre et al, Breast Cancer Res Treat 2017], immunohistochemical (IHC) androgen receptor (AR) staining was performed and the impact of various parameters on invasive disease-free survival (IDFS) and overall survival (OS) was assessed using Cox proportional hazards models. Results: 58 (20%) tumors were classified as LAR and 225 (80%) as non-LAR. Compared to non-LAR, LAR pts were older (mean age 65 vs 54) and more often postmenopausal (79%vs53%), both p=0.01. Apocrine histology was more common among LAR tumors (21%vs0%), which were also lower grade (grade3: 69%vs95%) and had lower Ki-67 (Ki-67&gt;15%: 64%vs82%), all p&lt;0.01. Additionally, LAR tumors had lower median stromal tumor infiltrating lymphocytes (TILs, 20%vs25%) and were less frequently lymphocyte-predominant [≥50% stromal or intratumoral TILs (19%vs32%)], although neither reached statistical significance. AR IHC was available for 223 of 283 tumors. Median AR IHC score in LAR was 65% (range 0-100%) vs 0% (range 0-90%) in non-LAR. T/N stage, surgery type, and receipt of adjuvant chemotherapy (AdjCT) or radiotherapy were similar between LAR and non-LAR. LAR pts had shorter IDFS and OS compared to non-LAR (5.6 vs 11.8 yrs and 10.8 vs 20.8 yrs, respectively), although this did not reach statistical significance. Test of proportional hazard assumption was not significant for IDFS or OS (p = 0.30 and 0.09). IDFS estimates were numerically higher in LAR vs non-LAR (80.2%vs70.5%,p = 0.92) at 3yrs post-diagnosis; whereas the opposite was true (40.9%vs55.6%,p = 0.07) after 10yrs. OS estimates at 3 and 5yrs were similar between LAR and non-LAR, but at 10yrs OS was inferior in LAR (40.9%vs66.4%,p = 0.24). In a univariate analysis including both LAR and non-LAR, older age, higher N stage, lower TILs and absence of AdjCT were associated with poorer IDFS and OS. In a multivariate analysis, higher N stage and absence of AdjCT remained associated with both poorer IDFS and OS; while lower stromal TILs were associated with poorer IDFS (p=0.01), and with a trend towards poorer OS (p=0.07). Conclusions: LAR TNBCs occurred in older women, were lower grade, and had lower TIL density than nonLAR tumors. While significant differences in IDFS or OS were not demonstrated, LAR pts exhibited a numerically lower risk of a disease event at 3yrs, but higher risk by 10yrs compared to nonLAR pts. In the entire cohort, higher N stage, absence of AdjCT and lower TILs were independently associated with poorer outcomes. Citation Format: Leon-Ferre RA, Polley M-Y, Liu H, Kalari KR, Boughey JC, Liu MC, Cafourek V, Negron V, Ingle JN, Thompson KJ, Tang X, Barman P, Carlson E, Visscher DW, Carter JC, Couch FJ, Goetz MP. Characteristics, outcomes and prognostic factors of luminal androgen receptor (LAR) triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-08-01.