Goss, Paul E.

Abstract Background: Adjuvant breast cancer therapy is informed by whether a tumour is positive or negative for the biomarkers ER, PgR, and HER2, often without regard to level of positivity. Quantitation has been proposed to improve therapeutic management. Adjunctive statistical standardization has been proposed to improve inter-laboratory comparability of biomarkers results. Methods: This primary report utilized adjunctive statistical standardization of machine-quantitated image analysis biomarker assessments. CCTG MA.27 (NCT00066573) is an adjuvant phase III trial of exemestane versus anastrozole in postmenopausal women with ER+ and/or PgR+ tumours. IHC ER, PgR, and HER2 were centrally assessed, with FISH (HER2;HER2/CEP17) determinations for equivocal IHC HER2. HSCOREs were statistically standardized to a mean of 0, standard deviation of 1 following Box-Cox variance stabilization transformations of square for ER and natural logarithm for PgR (0.1 was added to 0 HSCOREs). The primary endpoint was STEEP distant disease-free survival (DDFS) at the longest trial follow-up of median 4.1 years. Survival was described with Kaplan-Meier plots. The univariate Wilcoxon (Peto-Prentice) test statistic was used with usual designation of negative/positive (0; >0), and standardized cut-points at standard deviations about mean of 0(<-1; (-1,0]; (0,1]; >1). Cox multivariate regressions adjusted for age, T and N stage, grade, lymphovascular invasion, treatment, and baseline patient demographics, utilized likelihood ratio tests. Nominal significance was p=0.05. Results: Of the 7576 women accrued, 3048 had machine-quantitated image analysis results: 2900 (95%) for ER; 2726 (89%) for PgR. Only 8 women were ASCO/CAP ER- (HSCORE 0); PgR HSCORE was 0 for 533. Statistically standardized units differentiated DDFS ER levels (p< 0.001) and PgR levels (p< 0.001). In adjusted multivariate analyses, higher ER HSCORE was associated with better DDFS (p=0.05) with weak evidence of an association (p=0.11) for standardized HSCORE, and no significant association (respectively, p=0.28, p=0.54) in models with PgR. Higher PgR was associated with better DDFS (p=0.001) in all multivariate assessments, including those with ER. Conclusions: DDFS was superior for patients with higher ER and PgR standardized units compared with those with HSCOREs <-1. Adjunctive statistical standardization, similar to that mandated for clinical practice by the World Health Organization for BMD, should improve inter-laboratory comparability of biomarker results for similar patient populations. Biomarker N DDFS DDFS 5-year (%) 95% CI ER total 2900 ER <-1 506 86 (82, 91) ER (-1, 0] 934 94 (92, 96) ER ( 0, 1] 919 94 (92, 96) ER >1 541 96 (93, 98) PgR total 2726 PgR <-1 734 89 (86, 92) PgR (-1, 0] 439 92 (89, 95) PgR ( 0, 1] 967 95 (93, 96) PgR >1.0 586 98 (97,100) Citation Format: Judy-Anne Chapman, Jane Bayani, Sandip SenGupta, John MS Bartlett, Tammy Piper, Mary Anne Quintayo, Shakeel Virk, Paul Goss, James Ingle, Matthew Ellis, George Sledge Jr, George Budd, Manuela Rabaglio, Rafat Ansari, Richard Tozer, David D'Souza, Haji Chalchal, Silvana Spadafora, Vered Stearns, Edith A. Perez, Karen Gelmon, Timothy Whelan, Catherine Elliott, Lois Shepherd, Bingshu Chen, Karen Taylor. Adjunctive statistical standardization of quantitated machine image analysis of Estrogen and Progesterone Receptors: CCTG MA.27 trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-27-10.

Aromatase inhibitors (AIs) are the treatment of choice for hormone receptor–positive early breast cancer in postmenopausal women. None of the third‐generation AIs are superior to the others in terms of efficacy. We attempted to identify genetic factors that could differentiate between the effectiveness of adjuvant anastrozole and exemestane by examining single‐nucleotide polymorphism (SNP)‐treatment interaction in 4,465 patients. A group of SNPs were found to be differentially associated between anastrozole and exemestane regarding outcomes. However, they showed no association with outcome in the combined analysis. We followed up common SNPs near LY75 and GPR160 that could differentiate anastrozole from exemestane efficacy. LY75 and GPR160 participate in epithelial‐to‐mesenchymal transition and growth pathways, in both cases with SNP‐dependent variation in regulation. Collectively, these studies identified SNPs that differentiate the efficacy of anastrozole and exemestane and they suggest additional genetic biomarkers for possible use in selecting an AI for a given patient.

Abstract Purpose: To determine genetic variants that could differentiate aromatase inhibitors (AIs) efficacy in early stage breast cancer. Experimental Design: We performed a stratified cox-proportional hazards analysis utilizing stratification factors and other covariates examining Breast Cancer Free Interval (BCFI) SNP-treatment interaction in Caucasian patients entered on the MA.27 trial. This analysis involved 4465 patients (2226 on anastrozole and 2239 on exemestane arm) including 245 (121 on anastrozole and 124 events on exemestane arm) with a breast event. Preclinical laboratory studies included luciferase activity, chromatin immunoprecipitation (ChIP) assay, and cell migration assays. Results: We identified 887 SNPs with a p-value <1E-4 that could differentiate anastrozole from exemestane efficacy. We next used GTEx databases to determine whether these SNPs might be cis-eQTLs with nearby genes, and found that 95 SNPs were eQTL with 14 genes. Functional validation of SNP effect in these 14 genes on response to anastrozole and exemestane revealed that 3 SNPs showed genotype-dependent differences between anastrozole and exemestane. Two of the three SNPs, rs1877193 and rs6735923 located upstream of LY75 gene, were associated with higher LY75 gene expression. Our drug-SNP interaction GWAS showed that both SNPs were associated with better BCFI for exemestane compared to anastrozole (HR= 0.447, 0.458). The SNP rs62293499 located downstream of the GPR160 gene was also associated with better BCFI for exemestane compared to anastrozole (HR=0.433). Interestingly, all 3 SNPs were associated with worse outcome (shorter BCFI) in anastrozole treated patients (HR=1.39~1.58), but with longer BCFI in the exemestane treated patients (HR=0.58~0.71) based on our GWAS results in MA.27 trial. However, all 3 SNPs showed no association with BCFI if the two treatment arms, anastrozole and exemestane, were combined (HR=0.92~1.08). Consistently, cells with Ly75 or GPR160 SNP variant genotypes were more sensitive to exemestane compared to anastrozole. Mechanistically, the 3 SNPs regulated estrogen receptor-dependant LY75 and GPR160 expression. LY75 suppression induced epithelial-to-mesenchymal transition (EMT) in breast cancer cell lines, accompanied by increased migratory capacity in vitro. LY75 knockdown also resulted in predominant downregulation of functional pathways such as cell proliferation, while pathways associated with mesenchymal stimulation were generally increased. GPR160 knockdown also resulted in downregulation of cell proliferation pathways. Conclusions: This SNP genotype and AI treatment interaction clinical study revealed unique genetic variants that differentiate anastrozole and exemestane efficacy. The signals were lost in the GWAS analysis when anastrozole and exemestane were combined. Preclinical laboratory studies revealed novel functions of LY75 and GPR160 in breast cancer. These findings represent potential steps towards individualized AI therapy. Citation Format: Junmei Cairns, James N. Ingle, Krishna R. Kalari, Lois E. Shepherd, Matthew J. Ellis, Paul E. Goss, Poulami Barman, Erin E. Carlson, Matthew P. Goetz, Richard M. Weinshilboum, Liewei Wang. The interaction between SNP genotype and aromatase inhibitor treatment response in early breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-103.

Aromatase inhibitors (AIs) reduce breast cancer recurrence and prolong survival, but up to 30% of patients exhibit recurrence. Using a genome-wide association study of patients entered on MA.27, a phase III randomized trial of anastrozole versus exemestane, we identified a single nucleotide polymorphism (SNP) in CUB And Sushi multiple domains 1 ( CSMD1) associated with breast cancer–free interval, with the variant allele associated with fewer distant recurrences. Mechanistically, CSMD1 regulates CYP19 expression in an SNP- and drug-dependent fashion, and this regulation is different among 3 AIs: anastrozole, exemestane, and letrozole. Overexpression of CSMD1 sensitized AI-resistant cells to anastrozole but not to the other 2 AIs. The SNP in CSMD1 that was associated with increased CSMD1 and CYP19 expression levels increased anastrozole sensitivity, but not letrozole or exemestane sensitivity. Anastrozole degrades estrogen receptor α (ERα), especially in the presence of estradiol (E2). ER + breast cancer organoids and AI- or fulvestrant-resistant breast cancer cells were more sensitive to anastrozole plus E2 than to AI alone. Our findings suggest that the CSMD1 SNP might help to predict AI response, and anastrozole plus E2 serves as a potential new therapeutic strategy for patients with AI- or fulvestrant-resistant breast cancers.

Objectives Based on our previous findings that postmenopausal women with estrone (E1) and estradiol (E2) concentrations at or above 1.3 pg/ml and 0.5 pg/ml, respectively, after 6 months of adjuvant anastrozole therapy had a three-fold risk of recurrence, we aimed to identify a single-nucleotide polymorphism (SNP)-based model that would predict elevated E1 and E2 and then validate it in an independent dataset. Patients and methods The test set consisted of 322 women from the M3 study and the validation set consisted of 152 patients from MA.27. All patients were treated with adjuvant anastrozole, had on-anastrozole E1 and E2 concentrations and genome-wide genotyping. Results SNPs were identified from the M3 genome-wide association study. The best model to predict the E1-E2 phenotype with high balanced accuracy was a support vector machine model using clinical factors plus 46 SNPs. We did not have an independent cohort that is similar to the M3 study with clinical, E1-E2 phenotypes and genotype data to test our model. Hence, we chose a nested matched case-control cohort (MA.27 study) for testing. Our E1-E2 model was not validated but we found the MA.27 validation cohort was both clinically and genomically different. Conclusions We identified a SNP-based model that had excellent performance characteristics for predicting the phenotype of elevated E1 and E2 in women treated with anastrozole. This model was not validated in an independent dataset but that dataset was clinically and genomically substantially different. The model will need validation in a prospective study.

Abstract Objective: The aim of the study was to quantify baseline estradiol (E2) and estrone (E1) concentrations according to selected patient characteristics in a substudy nested within the MAP.3 chemoprevention trial. Methods: E2 and E1 levels were measured in 4,068 postmenopausal women using liquid chromatography-tandem mass spectrometry. Distributions were described by age, years since menopause, race, body mass index (BMI), smoking status, and use and duration of hormone therapy using the Kruskal-Wallis test. Multivariable linear regression was also used to identify characteristics associated with estrogen levels. Results: After truncation at the 97.5th percentile, the mean (SD)/median (IQR) values for E2 and E1 were 5.41 (4.67)/4.0 (2.4-6.7) pg/mL and 24.7 (14.1)/21 (15-31) pg/mL, respectively. E2 and E1 were strongly correlated (Pearson correlation [ r ] = 0.8, P < 0.01). The largest variation in E2 and E1 levels was by BMI; mean E2 and E1 levels were 3.5 and 19.1 pg/mL, respectively for women with BMI less than 25 and 7.5 and 30.6 pg/mL, respectively, for women with BMI greater than 30. E2 and E1 varied by age, BMI, smoking status, and prior hormone therapy in multivariable models ( P < 0.01). Conclusions: There was large interindividual variability observed for E2 and E1 that varied significantly by participant characteristics, but with small absolute differences except in the case of BMI. Although the majority of participant characteristics were independently associated with E1 and E2, together, these factors only explained about 20% of the variation in E1 and E2 levels.

550 Background: 5187 postmenopausal women were originally randomized to NCIC CTG MA.17 to receive letrozole (LET) or placebo (PLAC) after 5 years of tamoxifen. The hazard ratio (HR) for disease-free survival (DFS) was 0.58 (0.450.76, p=0.00004) after a median follow-up of 30 months (mo). The trial was unblinded in October 2003 after the first interim analysis. Women randomized to PLAC were offered LET at the time of unblinding. The goal of this analysis was to determine whether women switching from PLAC to LET benefit in terms of disease outcome and to evaluate treatment related toxicities. Methods: LET and PLAC-LET have been compared to PLAC, based on the hazard ratio and adjusting for baseline patient and disease variables including, among others, tumor size, nodal status and prior adjuvant chemotherapy. Results: Information about their follow-up treatment after unblinding was available on 2268 women originally assigned to PLAC and who were free of recurrence and alive at the time of unblinding. Among them, 1655 crossed over from PLAC to LET while 613 elected no treatment. With 54 mo f/up the HR for DFS was 0.31 (0.18, 0.55: p<0.0001) favoring patients who crossed over to LET compared to those who stayed on no treatment. The treatment switch was well tolerated with no significant difference in bone fractures or cardiovascular events. An updated analysis of DFS, DDFS and OS by nodal and tumor receptor status, prior chemotherapy, menopausal status at the start of tamoxifen, and duration of prior tamoxifen therapy will be presented at the meeting. Conclusion: Women with hormone dependent breast cancer prescribed LET after a prolonged delay from completing tamoxifen experienced a significant improvement in outcome (DFS, DDFS, OS) and should be considered for this therapy. [Table: see text]

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.