Brinton, Louise A.

Abstract Background: The influence of sex hormone and insulin/insulin-like growth factor (IGF) axis signaling on endometrial cancer recurrence is unknown. We evaluated these pathways in a prospective cohort of Gynecologic Oncology Group (GOG)0210 trial endometrial adenocarcinoma patients. Methods: Stage II–IV patients (N = 816) were included in this study. Pretreatment specimens were tested for tumor mRNA and protein expression of IGF1, IGF2, IGF-binding proteins (IGFBP)-1 and -3, insulin (IR) and IGF-I receptors (IGF1R), phosphorylated IR/IGF1R (pIGF1R/pIR), and estrogen (ER) and progesterone receptors (PR) using qPCR and IHC. Serum concentrations of insulin, IGF-I, IGFBP-3, estradiol, estrone, and sex hormone binding globulin were measured. HRs and 95% confidence intervals (CI) for progression-free survival were calculated from Cox models adjusting for age, stage, and grade. Results: Recurrence occurred in 280 (34%) cases during a median of 4.6 years of follow-up. ER positivity (HR, 0.67; 95% CI, 0.47–0.95), IR positivity (HR, 0.53; 95% CI, 0.29–0.98), and circulating IGF-I (highest vs. lowest quartile: HR, 0.66; 95% CI, 0.47–0.92) were inversely associated with recurrence risk. Circulating estradiol (highest vs. lowest tertile: HR, 1.55; 95% CI, 1.02–2.36) and pIGF1R/pIR positivity (HR, 1.40; 95% CI, 1.02–1.92) were associated with increased recurrence risk. Conclusions: Circulating estradiol and tumor tissue phosphorylated (activated) IGR1R/IR were independently associated with higher risk of recurrence in patients with endometrial cancer. Impact: This study may inform future clinical trials of endocrine-targeted adjuvant therapies in patients with endometrial cancer that could include baseline assessment of serum and tissue biomarkers of estradiol and insulin signaling pathways.

Abstract Background: Accumulating evidence suggests a relationship between endometrial cancer and ovarian cancer. Independent genome-wide association studies (GWAS) for endometrial cancer and ovarian cancer have identified 16 and 27 risk regions, respectively, four of which overlap between the two cancers. We aimed to identify joint endometrial and ovarian cancer risk loci by performing a meta-analysis of GWAS summary statistics from these two cancers. Methods: Using LDScore regression, we explored the genetic correlation between endometrial cancer and ovarian cancer. To identify loci associated with the risk of both cancers, we implemented a pipeline of statistical genetic analyses (i.e., inverse-variance meta-analysis, colocalization, and M-values) and performed analyses stratified by subtype. Candidate target genes were then prioritized using functional genomic data. Results: Genetic correlation analysis revealed significant genetic correlation between the two cancers (rG = 0.43, P = 2.66 × 10−5). We found seven loci associated with risk for both cancers (PBonferroni < 2.4 × 10−9). In addition, four novel subgenome-wide regions at 7p22.2, 7q22.1, 9p12, and 11q13.3 were identified (P < 5 × 10−7). Promoter-associated HiChIP chromatin loops from immortalized endometrium and ovarian cell lines and expression quantitative trait loci data highlighted candidate target genes for further investigation. Conclusions: Using cross-cancer GWAS meta-analysis, we have identified several joint endometrial and ovarian cancer risk loci and candidate target genes for future functional analysis. Impact: Our research highlights the shared genetic relationship between endometrial cancer and ovarian cancer. Further studies in larger sample sets are required to confirm our findings.

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.

Abstract Blood lipids have been associated with the development of a range of cancers, including breast, lung and colorectal cancer. For endometrial cancer, observational studies have reported inconsistent associations between blood lipids and cancer risk. To reduce biases from unmeasured confounding, we performed a bidirectional, two‐sample Mendelian randomization analysis to investigate the relationship between levels of three blood lipids (low‐density lipoprotein [LDL] and high‐density lipoprotein [HDL] cholesterol, and triglycerides) and endometrial cancer risk. Genetic variants associated with each of these blood lipid levels ( P < 5 × 10 −8 ) were identified as instrumental variables, and assessed using genome‐wide association study data from the Endometrial Cancer Association Consortium (12 906 cases and 108 979 controls) and the Global Lipids Genetic Consortium (n = 188 578). Mendelian randomization analyses found genetically raised LDL cholesterol levels to be associated with lower risks of endometrial cancer of all histologies combined, and of endometrioid and non‐endometrioid subtypes. Conversely, higher genetically predicted HDL cholesterol levels were associated with increased risk of non‐endometrioid endometrial cancer. After accounting for the potential confounding role of obesity (as measured by genetic variants associated with body mass index), the association between genetically predicted increased LDL cholesterol levels and lower endometrial cancer risk remained significant, especially for non‐endometrioid endometrial cancer. There was no evidence to support a role for triglycerides in endometrial cancer development. Our study supports a role for LDL and HDL cholesterol in the development of non‐endometrioid endometrial cancer. Further studies are required to understand the mechanisms underlying these findings.

Abstract Purpose: Gene expression–based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with &gt;95% accuracy that was maintained in all analytic and biological validations. Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271

The association of progesterone/progesterone metabolites with elevated mammographic breast density (MBD) and delayed age-related terminal duct lobular unit (TDLU) involution, strong breast cancer risk factors, has received limited attention. Using a reliable liquid chromatography-tandem mass-spectrometry assay, we quantified serum progesterone/progesterone metabolites and explored cross-sectional relationships with MBD and TDLU involution among women, ages 40–65, undergoing diagnostic breast biopsy. Quantitative MBD measures were estimated in pre-biopsy digital mammograms. TDLU involution was quantified in diagnostic biopsies. Adjusted partial correlations and trends across MBD/TDLU categories were calculated. Pregnenolone was positively associated with percent MBD-area (MBD-A, rho: 0.30; p-trend = 0.01) among premenopausal luteal phase women. Progesterone tended to be positively associated with percent MBD-A among luteal phase (rho: 0.26; p-trend = 0.07) and postmenopausal (rho: 0.17; p-trend = 0.04) women. Consistent with experimental data, implicating an elevated 5α-pregnanes/3α-dihydroprogesterone (5αP/3αHP) metabolite ratio in breast cancer, higher 5αP/3αHP was associated with elevated percent MBD-A among luteal phase (rho: 0.29; p-trend = 0.08), but not postmenopausal women. This exploratory analysis provided some evidence that endogenous progesterone and progesterone metabolites might be correlated with MBD, a strong breast cancer risk factor, in both pre- and postmenopausal women undergoing breast biopsy. Additional studies are needed to understand the role of progesterone/progesterone metabolites in breast tissue composition and breast cancer risk.

Mammographic breast density (MD) reflects breast fibroglandular content. Its decline following adjuvant tamoxifen treated, estrogen receptor (ER)-positive breast cancer has been associated with improved outcomes. Breast cancers arise from structures termed lobules, and lower MD is associated with increased age-related lobule involution. We assessed whether pre-treatment involution influenced associations between MD decline and risk of breast cancer-specific death. ER-positive tamoxifen treated patients diagnosed at Kaiser Permanente Northwest (1990–2008) were defined as cases who died of breast cancer (n = 54) and matched controls (remained alive over similar follow-up; n = 180). Lobule involution was assessed by examining terminal duct lobular units (TDLUs) in benign tissues surrounding cancers as TDLU count/mm2, median span and acini count/TDLU. MD (%) was measured in the unaffected breast at baseline (median 6-months before) and follow-up (median 12-months after tamoxifen initiation). TDLU measures and baseline MD were positively associated among controls (p < 0.05). In multivariable regression models, MD decline (≥10%) was associated with reduced risk of breast cancer-specific death before (odds ratio (OR): 0.41, 95% CI: 0.18–0.92) and after (OR: 0.41, 95% CI: 0.18–0.94) adjustment for TDLU count/mm2, TDLU span (OR: 0.34, 95% CI: 0.14–0.84), and acini count/TDLU (OR: 0.33, 95% CI: 0.13–0.81). MD decline following adjuvant tamoxifen is associated with reduced risk of breast cancer-specific death, irrespective of pre-treatment lobule involution.

Abstract Background: Mammographic breast density (MBD) is a strong breast cancer (BC) risk factor, but its biologic underpinnings are poorly understood. Use of estrogen plus progestin menopausal hormone therapy is linked to increased MBD and BC risk. Experimental data suggest that ratios of tumor promoting (5α-dihydroprogesterone [5αP]) and anti-tumorigenic (3α-dihydroprogesterone [3αP]) progesterone metabolite levels may be related to BC risk. However, relationships of endogenous progesterone and its metabolites with MBD and BC risk have not been established. Accordingly, we assessed levels of circulating progesterone and its metabolites with MBD. Methods: In this cross-sectional study, serum progesterone and its metabolites were quantified using a novel liquid chromatography-tandem mass spectrometry assay in 103 postmenopausal and 52 premenopausal (luteal menstrual cycle phase) women, ages 40-65, undergoing diagnostic image-guided ipsilateral breast biopsy. MBD was measured as percent fibroglandular volume (MBD-V) on pre-biopsy digital mammograms using single X-ray absorptiometry. Square-root transformed MBD-V was examined across tertile categories of progesterone/progesterone metabolites using age and body mass index (BMI)-adjusted linear regression models. Results: Concentrations of the hormones were as follows among postmenopausal women: progesterone [mean: 12.6 pmol/L (range: 5.2-45.8)], 3αP [5.6 pmol/L (1.4-18.8)], 5αP [100 pmol/L (16.7-388)], and 5αP/3αP ratio [26.1 (2.1-150)]; and among luteal phase premenopausal women: progesterone [2063 pmol/L (13.6-7098), 3αP [12.7 pmol/L (2.4-64.4)], 5αP [243 pmol/L (25.3-774)], 5αP/3αP ratio [25.9 (2.3-73.7)]. Among postmenopausal women, progesterone was positively associated with MBD-V (Tertile 3 vs. 1: β=0.68, p-trend=0.02). A similar borderline positive association was observed among premenopausal women (β=0.74, p-trend=0.10). Additional adjustment for circulating estradiol did not substantively alter observed associations. Levels of 3αP, 5αP and the 5αP/3αP ratio were not associated with MBD-V among pre- or postmenopausal women. Conclusions: Concentrations of progesterone and it metabolites show substantial inter-woman variation. We observed a positive association between endogenous progesterone and MBD-V among both postmenopausal and premenopausal luteal phase women. We did not observe an association with the ratio of 5αP to 3αP levels and MBD-V. These findings suggest the need for additional studies to understand the biological basis of the role of progesterone and its metabolites in MBD and BC risk. Citation Format: Manila Hada, Hannah Oh, Sharon Fan, Roni T. Falk, Berta Geller, Pamela Vacek, Donald Weaver, John Shepherd, Jeff Wang, Bo Fan, Amir P. Mahmoudzadeh, Serghei Malkov, Sally Herschorn, Louise A. Brinton, Xia Xu, Mark E. Sherman, Britton Trabert, Gretchen L. Gierach. Relationship of serum progesterone and progesterone metabolites with mammographic density [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 588.

Abstract Purpose: Ovarian cancer is a heterogeneous disease that can be divided into multiple subtypes with variable etiology, pathogenesis, and prognosis. We analyzed DNA methylation profiling data to identify biologic subgroups of ovarian cancer and study their relationship with histologic subtypes, copy number variation, RNA expression data, and outcomes. Experimental Design: A total of 162 paraffin-embedded ovarian epithelial tumor tissues, including the five major epithelial ovarian tumor subtypes (high- and low-grade serous, endometrioid, mucinous, and clear cell) and tumors of low malignant potential were selected from two different sources: The Polish Ovarian Cancer study, and the Surveillance, Epidemiology, and End Results Residual Tissue Repository (SEER RTR). Analyses were restricted to Caucasian women. Methylation profiling was conducted using the Illumina 450K methylation array. For 45 tumors array copy number data were available. NanoString gene expression data for 39 genes were available for 61 high-grade serous carcinomas (HGSC). Results: Consensus nonnegative matrix factorization clustering of the 1,000 most variable CpG sites showed four major clusters among all epithelial ovarian cancers. We observed statistically significant differences in survival (log-rank test, P = 9.1 × 10−7) and genomic instability across these clusters. Within HGSC, clustering showed three subgroups with survival differences (log-rank test, P = 0.002). Comparing models with and without methylation subgroups in addition to previously identified gene expression subtypes suggested that the methylation subgroups added significant survival information (P = 0.007). Conclusions: DNA methylation profiling of ovarian cancer identified novel molecular subgroups that had significant survival difference and provided insights into the molecular underpinnings of ovarian cancer. See related commentary by Ishak et al., p. 5729

Abstract Background: Insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 play important roles in carcinogenesis, particularly for breast cancer. However, little is known about whether the IGF system influences histologic characteristics of normal glandular tissue and whether relationships vary by race. Lesser degrees of age-related terminal duct lobular unit (TDLU) involution, as reflected by higher numbers of TDLUs and acini per TDLU, have been associated with higher breast cancer risk. We examined the associations of IGF measures with TDLU involution of normal breast using standardized TDLU measures. Methods: Among 715 Caucasian and 283 African American (AA) women with normal breast tissue samples from the Komen Tissue Bank, serum concentrations of IGF-I and IGFBP-3 were quantified using enzyme-linked immunosorbent assay (ELISA). Hematoxilyn and eosin-stained tissue sections were assessed for numbers of TDLUs (“TDLU count”) and acini/TDLU. Zero-inflated Poisson regression models with a robust variance estimator were used to estimate associations of IGF-I, IGFBP-3, and IGF-I:IGFBP-3 molar ratio (tertiles) with TDLU count by race and menopausal status, adjusting for potential confounders. We also tested for interactions by race using likelihood ratio tests. Results: AA (vs. Caucasian) women had higher age-adjusted mean levels of serum IGF-I (137 vs. 131 ng/mL, p=0.07) and lower levels of IGFBP-3 (4165 vs. 4684 ng/mL, p&lt;0.0001); the differences persisted after adjustment for additional covariates including BMI and parity/age at first birth. Postmenopausal IGFBP-3 was inversely associated with TDLU count among both AA (RR T3vs.T1=0.49, 95% CI=0.28-0.84, p-trend=0.04) and Caucasian (RR T3vs.T1=0.63, 95% CI=0.41-0.99, p-trend=0.04) women. In premenopausal women, higher IGF-I:IGFBP-3 ratios were associated with higher TDLU count in Caucasians (OR T3vs.T1=1.33, 95% CI=1.01-1.31, p-trend=0.04) but not in AA (OR T3vs.T1=0.65, 95% CI=0.42-1.00, p-trend=0.05) women. There was no statistically significant interaction by race (p-interaction≥0.10). Conclusions: Our data suggest the potential role of the IGF system, particularly IGFBP-3, in TDLU involution of the normal breast among both Caucasian and AA women. Citation Format: Hannah OH, Ruth M. Pfeiffer, Roni T. Falk, Hisani N. Horne, Jackie Xiang, Michael Pollak, Louise A. Brinton, Anna Maria V. Storniolo, Mark E. Sherman, Gretchen L. Gierach, Jonine D. Figueroa. Serum insulin-like growth factor (IGF)-I and IGF binding protein-3 in relation to terminal duct lobular unit involution in Caucasian and African American women: The Susan G. Komen Tissue Bank [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A36.