Citations

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.

Abstract Background: Obesity is a well established risk factor not only for increased prostate cancer incidence, but for poorer survival from prostate cancer. However, there is limited knowledge on the role of the circulating lipidome in prostate cancer. In order to target lipid metabolism optimally, it is critical to understand the spectrum of changes in circulating lipid profiles and the association with clinical outcome across the natural history of prostate cancer. Aim: To assess the relationship between the plasma lipidome and clinical outcome in localized and metastatic prostate cancer. Methods: Liquid chromatography-tandem mass spectrometry was used to quantitate over 500 lipid species in plasma samples from 3 cohorts (1) 389 men with localized prostate cancer, (2) 44 men with metastatic hormone-sensitive prostate cancer (mHSPC), and (3) 137 men with metastatic castration-resistant prostate cancer (CRPC). Associations between circulating lipids with metastatic relapse, androgen-deprivation therapy (ADT) failure or overall survival for each relevant disease stage were examined by latent class analysis and cox regression. Results: Circulating lipid profiles displaying elevated levels of ceramides were associated with metastatic relapse in localized prostate cancer (HR 5.8, 95% CI 3.0-11, P 1 × 10−6), early ADT failure in mHSPC (HR 2.4, 95% CI 1.1-5.3, P 0.03), and shorter overall survival in CRPC (HR 2.5, 95% CI 1.7-3.7, P 2 × 10−6). ADT failure in mHSPC and shorter overall survival in CRPC were also associated with elevated levels of other sphingolipids (sphingomyelins, hexosylceramides). The prognostic significance of the high risk lipid profiles in localized prostate cancer was independent of clinicopathological characteristics (lipid profile HR 4.7, 95%CI 2.4-9.3, P 7 × 10−6) when modeled with Gleason score (P<0.0001) and pathological stage (P<0.0001). Furthermore, the circulating lipid profiles were independent of metabolic factors (localized prostate cancer lipid profile HR 8.2, 95% CI 2.6-25, P 0.0003 when modeled with diabetes, statin, hypertension, body mass index [BMI]; CRPC lipid profile HR 2.6, 95% CI 1.7-3.8, P x10−6 when modeled with BMI). Conclusion: Elevated circulating ceramides are associated with poorer clinical outcomes across the natural history of prostate cancer (from localized to metastatic hormone-sensitive to metastatic castration resistant prostate cancer). This demonstrates that aberrant lipid metabolism in the patients occurs early in prostate cancer and could be therapeutically targeted in prospective clinical trials to improve prostate cancer outcomes. Citation Format: Lisa Glen Horvath, Hui-Ming Lin, Blossom Mak, Kate Mahon, Nicole Yeung, Maria Docanto, Peter Sutherland, andrew Shepherd, Winston Tan, Arun Azad, Manish Kohli, Peter Meikle, Lisa Butler. Lipidomic analysis of circulating lipids across the natural history of prostate cancer identifies aberrant ceramide metabolism [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 4761.

Abstract Introduction Elevated mammographic density (MD) is a strong and independent risk factor for breast cancer, though underlying mechanisms are unclear. Prior studies have suggested that increased cumulative exposure to sex-steroid hormones and growth factors may impact both MD and MD-related breast cancer risk; however, most studies have only evaluated individual hormones. In this study, we simultaneously explored the relationship between 29 circulating hormones and growth factors with MD among postmenopausal women undergoing diagnostic breast biopsy. Methods We used data from 89 postmenopausal women, aged 44-65, who had complete measurements of 29 serum hormones from a single pre-biopsy blood draw (i.e., sex-steroid hormones: 15 estrogen/estrogen metabolites, 7 progesterone/progesterone metabolites; and non-sex hormones: insulin-like growth factor I and binding proteins (IGFBPs) 2-7). Volumetric MD (% fibroglandular volume) was assessed in pre-biopsy digital mammograms using single X-ray absorptiometry. Sufficient dimension reduction methods were used to compute a composite marker score that accommodates correlations among hormones and their relationship to MD. Backward elimination was applied to select log-transformed hormones contributing to the score at α=0.05; multivariable linear regression was used to further examine associations between selected hormones and MD within a single model, adjusting for age and BMI. Results Pearson correlations between hormones were moderate-to-strong. Dimension reduction methods identified 6 hormones as associated with MD (p<0.05): estradiol, 2-hydroxyestradiol, 2-methoxyestrone, 16-ketoestradiol, IGFBP-2, and 17α-hydroxyprogesterone, in models that considered MD as either continuous or dichotomous (>median vs. ≤median); BMI was consistently identified as the most significant predictor of MD (P <0.0001). In a multivariable linear regression model that included these 6 hormones, in addition to age and BMI, higher levels of 2-methoxyestrone, IGFBP-2, and 17α-hydroxyprogesterone were significantly associated with increased MD, whereas higher levels of age and BMI were significantly associated with lower MD. Conclusions We identified 6 out of 29 correlated hormones that were significantly associated with MD among postmenopausal women. For several, the directions of their associations with MD were comparable to those observed in prior studies that have separately evaluated these hormones in relation to breast cancer risk. We plan to further explore how circulating hormones concurrently affect MD in premenopausal women, accounting for menstrual cycle phase. Incorporation of dimension reduction methods in studies of multiple correlated hormones can help uncover new etiologic insights into the role of hormones and growth factors in MD and breast carcinogenesis and inform strategies for prevention. Citation Format: Shaoqi Fan, Ruth M. Pfeiffer, Manila Hada, Roni T. Falk, Maeve Mullooly, Hannah Oh, Berta Geller, Pamela Vacek, Donald Weave, John Shepherd, Jeff Wang, Sally Herschorn, Louise A. Brinton, Xia Xu, Mark E. Sherman, Britton Trabert, Gretchen L. Gierach. Associations of circulating hormones with mammographic density in postmenopausal women referred to diagnostic breast biopsy [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 3488.

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 Background Metformin has been associated with lower breast cancer (BC) risk and improved outcomes in observational studies. Multiple biologic mechanisms have been proposed, including a recent report of altered sex hormones. We evaluated the effect of metformin on sex hormones in MA.32, a phase III trial of nondiabetic BC subjects who were randomly assigned to metformin or placebo. Methods We studied the subgroup of postmenopausal hormone receptor-negative BC subjects not receiving endocrine treatment who provided fasting blood at baseline and at 6 months after being randomly assigned. Sex hormone-binding globulin, bioavailable testosterone, and estradiol levels were assayed using electrochemiluminescence immunoassay. Change from baseline to 6 months between study arms was compared using Wilcoxon sum rank tests and regression models. Results 312 women were eligible (141 metformin vs 171 placebo); the majority of subjects in each arm had T1/2, N0, HER2-negative BC and had received (neo)adjuvant chemotherapy. Mean age was 58.1 (SD=6.9) vs 57.5 (SD=7.9) years, mean body mass index (BMI) was 27.3 (SD=5.5) vs 28.9 (SD=6.4) kg/m2 for metformin vs placebo, respectively. Median estradiol decreased between baseline and 6 months on metformin vs placebo (−5.7 vs 0 pmol/L; P < .001) in univariable analysis and after controlling for baseline BMI and BMI change (P < .001). There was no change in sex hormone-binding globulin or bioavailable testosterone. Conclusion Metformin lowered estradiol levels, independent of BMI. This observation suggests a new metformin effect that has potential relevance to estrogen sensitive cancers.

Background In humans, maximal exercise capacity is reduced during hypoxic environmental conditions. In response to hypoxia, the hemoglobin binding affinity is decreased, promoting oxygen off‐loading at the tissue level and causing a right‐shift of the oxyhemoglobin dissociation curve (ODC). However, comparative biology demonstrates many species (e.g. Bar‐headed geese), who are adapted to hypoxia instead have high‐affinity hemoglobin (left‐shifted ODC). Therefore we studied unique human participants with hemoglobinopathies that result in high affinity hemoglobin (HAH) to investigate the role of oxygen affinity in exercise performance and tissue oxygenation during hypoxic conditions. Methods HAH participants (n=5, 1M/4F, Age= 34±11 yr, P 50 =16±1 mmHg) and matched control participants (n=12, 5M/7F, age=39±12 yr, and P 50 =26±1 mmHg) completed two incremental exercise tests to exhaustion on a cycle ergometer under two conditions: 1) room air (F i O 2 = 0.21, NORM), and 2) normobaric hypoxia gas mixture (F i O 2 = 0.15, HYP), separated by a 45 minute break. The vastus lateralis (VL) muscle was studied using frequency‐domain near infrared spectroscopy (NIRS) to measure concentrations of oxygenated ([OxyHb+Mb]), deoxygenated ([DeoxyHb+Mb]) hemoglobin and myoglobin, and tissue saturation (S t O 2 ). Data were analyzed using a three‐way (Inspirate [HYP, NORM], Hb type [Normal, HAH], exercise intensity [Rest, 40, 60, 80, 100% normoxic PPO]) univariate analysis of variance. Results HAH had an attenuated decline in exercise tolerance (peak power output at maximal exertion) in HYP compared to controls (HAH: 2.9±6.4% decline vs CTL: 11.7±5.4% decline, P<0.05). There was a significant effect of Hb type on [DeoxyHb+Mb] (P=0.048) suggesting less O 2 offloading in HAH compared to controls in the VL at rest and throughout graded exercise in both NORM and HYP. There were no significant effects of inspirate or exercise intensity on [DeoxyHb+Mb], and no significant differences in [OxyHb+Mb] between HAH and controls. There was a significant effect of Hb type on S t O 2 in NORM controls (P<0.01) However, during hypoxic exercise S t O 2 was the same in both HAH and controls except at end exercise (HAH 64.1±6.4% vs CTL: 62.1±4.2%, P<0.05). Conclusion HAH individuals have less oxygen off‐loading at rest and during exercise but higher tissue saturation during exercise in NORM compared to controls. Our results suggest that the similar levels of exercise capacity in hypoxia for HAH are due, at least in part, to maintenance of microvascular extraction similar to that during normoxic exercise. Future studies utilizing direct measurements of skeletal muscle blood flow during exercise are needed to understand integrative exercise in HAH patients. Support or Funding Information This work was funded by NIH R‐35‐HL139854 and the Mayo Foundation. Muscle oxygenation during graded exercise tests in normoxia and hypoxia. Data are presented as means ± standard error. Abbreviations: [OxyHb+Mb], concentration of oxygenated hemoglobin+myoglobin; [DeoxyHb+MB], concentration of deoxygenated hemoglobin+ myoglobin; PPO, peak power output. † denotes significant main effect of Hb types across all workloads. * denotes significant difference from CTL at same %PPO. Figure 1