Fan, Shaoqi

Abstract Studies have identified genetic and epidemiologic factors associated with mammographic density (MD) phenotypes. However, MD-associated genetic variants only account for a small proportion of the total estimated heritability. Interrogating interactions between genetic and epidemiologic factors could potentially identify additional MD-associated loci, expand our understanding of the genetic basis of MD phenotypes, and clarify how epidemiologic factors modulate relationships between genetic variants and MD. We conducted six separate genome-wide, gene–environment (GxE) interaction analyses, applying 2 degrees of freedom (df) and 1df interaction tests, for each of three MD phenotypes (percent density, dense area (DA), and nondense area (NDA)). The six epidemiologic factors considered were height, ever parous, parity, ever menopausal hormone therapy, ever breastfeeding, and months of breastfeeding. We included European ancestry participants from multiple studies within the Markers of Density consortium and the Breast Cancer Association Consortium (n = 4895-16 218 depending on specific analyses). We identified 11 loci with genome-wide significant (P < 5 × 10−8) interaction tests including two novel common genetic signals interacting with parity (8p21.2) and ever breastfeeding (19p13.2) for NDA. Our results suggest that epidemiologic risk factors might influence relationships between common genetic variants and MD phenotypes at particular genomic loci.

Purpose: Women whose mammographic breast density declines within 12–18 months of initiating tamoxifen for chemoprevention or adjuvant treatment show improved therapeutic responses compared with those whose density is unchanged. We tested whether measuring changes in sound speed (a surrogate of breast density) using ultrasound tomography (UST) could enable rapid identification of favorable responses to tamoxifen. Methods: We evaluated serial density measures at baseline and at 1 to 3, 4 to 6, and 12+ months among 74 women (aged 30–70 years) following initiation of tamoxifen for clinical indications, including an elevated risk of breast cancer (20%) and diagnoses of in situ (39%) or invasive (40%) breast carcinoma, enrolled at Karmanos Cancer Institute and Henry Ford Health System (Detroit, MI, USA). For comparison, we evaluated an untreated group with screen negative mammography and frequency-matched on age, race, and menopausal status (n = 150), at baseline and 12 months. Paired t-tests were used to assess differences in UST sound speed over time and between tamoxifen-treated and untreated patients. Results: Sound speed declined steadily over the 12 month period among patients receiving tamoxifen (mean (SD): −3.0 (8.2) m/s; p = 0.001), whereas density remained unchanged in the untreated group (mean (SD): 0.4 (7.1) m/s; p = 0.75 (relative change between groups: p = 0.0009)). In the tamoxifen group, we observed significant sound speed reductions as early as 4–6 months after tamoxifen initiation (mean (SD): −2.1 (6.8) m/s; p = 0.008). Sound speed reductions were greatest among premenopausal patients (P-interaction = 0.0002) and those in the middle and upper tertiles of baseline sound speed (P-interaction = 0.002). Conclusions: UST can image rapid declines in sound speed following initiation of tamoxifen. Given that sound speed and mammographic density are correlated, we propose that UST breast imaging may capture early responses to tamoxifen, which in turn may have utility in predicting therapeutic efficacy.

Abstract Background: Tamoxifen therapy has been shown to have greater therapeutic benefit among women whose breast density declines subsequent to treatment than among women whose density does not decline. Although limited data suggests that endocrine symptoms after tamoxifen initiation may be associated with improved breast cancer outcomes, it is unknown whether these symptoms are associated with reductions in breast density. We therefore evaluated endocrine symptoms and change in breast density in a 12-month longitudinal study of women undergoing tamoxifen therapy for clinical indications. Methods: Cohort members (N=74) were aged 30-74 years in the Ultrasound Study of Tamoxifen at Karmanos Cancer Institute and Henry Ford Health Systems (Detroit, MI). Endocrine symptoms and breast density were both assessed prior to tamoxifen initiation (T0) and at 1-3 months (T1), 4-6 months (T2), and approximately 12 months (T3) post-tamoxifen initiation. Treatment-associated endocrine symptoms included treatment-emergent or increasing vasomotor and/or joint symptoms. Endocrine symptom severity was also assessed with a Likert scale for symptom frequency and categorized as no symptoms (score=0), low/moderate symptoms (score=1-5), and high symptoms (score=6+). Sound speed (m/s), a surrogate of volumetric breast density, was measured with whole breast ultrasound tomography. Change in breast density was calculated as density at T1-T3 minus density at T0. We used multivariable linear regression to estimate mean change in density by endocrine symptoms adjusting for age, race, menopausal status, body mass index, and baseline sound speed. Generalized estimating equations were used to account for within-subject correlations over time. Results: Women with treatment-associated endocrine symptoms had an overall greater mean reduction in breast density compared with women without symptoms (mean change [95% CI]: -1.97 m/s [-3.80, -0.13]; -0.27 m/s [- 3.50, 2.95], respectively; p=0.22). Longitudinal trends in breast density significantly differed for women with versus without treatment-associated endocrine symptoms (p-interaction=0.02). Significant declines in breast density over time were observed among women with treatment-associated endocrine symptoms (p-trend=0.005), but not among women without symptoms (p-trend=0.16). Similar trends in breast density decline were observed among women with higher symptom severity (p-trends for no symptoms=0.53; low/moderate symptoms=0.04; high symptoms=0.008). Conclusions: These findings suggest that treatment-associated endocrine symptoms may be associated with a significant decline in breast density after tamoxifen initiation. Further studies are needed to assess whether these observations can predict clinical outcome, and if confirmed, both treatment-associated endocrine symptoms and observed reductions in breast density may be useful for patients and providers to improve adherence. Citation Format: Cody Ramin, Ruth Pfeiffer, Shaoqi Fan, Maeve Mullooly, Roni T. Falk, Mark A. Sak, Michael S. Simon, David H. Gorski, Haythem Ali, Peter Littrup, Nebojsa Duric, Mark E. Sherman, Gretchen L. Gierach. Treatment-associated endocrine symptoms and change in ultrasound tomography measures of breast density after tamoxifen therapy [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 5805.

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.

Mammographic percent density (MPD) is an independent risk factor for developing breast cancer, but its inclusion in clinical risk models provides only modest improvements in individualized risk prediction, and MPD is not typically assessed in younger women because of ionizing radiation concerns. Previous studies have shown that tissue sound speed, derived from whole breast ultrasound tomography (UST), a non-ionizing modality, is a potential surrogate marker of breast density, but prior to this study, sound speed has not been directly linked to breast cancer risk. To that end, we explored the relation of sound speed and MPD with breast cancer risk in a case-control study, including 61 cases with recent breast cancer diagnoses and a comparison group of 165 women, frequency matched to cases on age, race, and menopausal status, and with a recent negative mammogram and no personal history of breast cancer. Multivariable odds ratios (ORs) and 95% confidence intervals (CIs) were estimated for the relation of quartiles of MPD and sound speed with breast cancer risk adjusted for matching factors. Elevated MPD was associated with increased breast cancer risk, although the trend did not reach statistical significance (OR per quartile = 1.27, 95% CI: 0.95, 1.70; ptrend = 0.10). In contrast, elevated sound speed was significantly associated with breast cancer risk in a dose–response fashion (OR per quartile = 1.83, 95% CI: 1.32, 2.54; ptrend = 0.0003). The OR trend for sound speed was statistically significantly different from that observed for MPD (p = 0.005). These findings suggest that whole breast sound speed may be more strongly associated with breast cancer risk than MPD and offer future opportunities for refining the magnitude and precision of risk associations in larger, population-based studies, including women younger than usual screening ages.

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.

Abstract Delayed terminal duct lobular unit (TDLU) involution is associated with elevated mammographic breast density (MD). Both are independent breast cancer risk factors among women with benign breast disease (BBD). Prior digital analyses of normal breast tissues revealed that epithelial nuclear density (END) and TDLU involution are inversely correlated. Accordingly, we examined associations of END, TDLU involution, and MD in BBD clinical biopsies. This study included digitized images of 262 representative image-guided hematoxylin and eosin–stained biopsies from 224 women diagnosed with BBD, enrolled within the cross-sectional BREAST-Stamp project that were visually assessed for TDLU involution (TDLU count/100 mm2, median TDLU span and median acini count per TDLU). A digital algorithm estimated nuclei count per unit epithelial area, or END. Single X-ray absorptiometry of prebiopsy ipsilateral craniocaudal digital mammograms measured global and localized MD surrounding the biopsy region. Adjusted ordinal logistic regression models assessed relationships between tertiles of TDLU and END measures. Analysis of covariance examined mean differences in MD across END tertiles. TDLU measures were positively associated with increasing END tertiles [TDLU count/100 mm2, ORT3vsT1: 3.42, 95% confidence interval (CI), 1.87–6.28; acini count/TDLUT3vsT1, OR: 2.40, 95% CI, 1.39–4.15]. END was significantly associated with localized, but not, global MD. Relationships were most apparent among patients with nonproliferative BBD. These findings suggest that quantitative END reflects different but complementary information to the histologic information captured by visual TDLU and radiologic MD measures and merits continued evaluation in assessing cellularity of breast parenchyma to understand the etiology of BBD.

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 Introduction: Defining the histologic correlates of mammographic breast density (MD) may provide insights into why elevated MD is related to increased breast cancer risk. Studies suggest that reduced terminal ductal lobular (TDLU) involution is associated with elevated MD, and both are independent breast cancer risk factors among women who have undergone a biopsy for benign breast disease (BBD). Prior digital histologic analyses of normal breast tissues revealed epithelial nuclear density (END) and TDLU involution are correlated. Accordingly, we examined associations of END with TDLU involution and MD in clinical biopsies. Methods: We analyzed 262 image-guided breast biopsies diagnosed as BBD from 224 women. TDLU involution was visually assessed as TDLU count/mm2 and TDLU span (inversely related to level of involution) in background normal tissue, evaluated using digitized images. The Genie Classifier (Aperio) was applied to images to estimate nuclei count per unit epithelial area, (END). Single X-ray Absorptiometry of pre-biopsy craniocaudal digital mammograms was applied to measure global MD (percent fibroglandular volume (%FGV)). Analysis of covariance, adjusted for age and body mass index, examined mean END differences across tertiles of TDLU/MD measures. Analyses were conducted at the biopsy level using SAS PROC GENMOD to account for within-woman correlations. All tests were two-tailed. Results: Overall, 67% of BBD biopsies were proliferative. Higher END was observed among proliferative than non-proliferative BBD (median END: 10,187 vs. 9,953 respectively; p=0.04). Among all women, END significantly increased with increasing tertiles of TDLU measures (p-trends: TDLU count/100mm2=0.0001, TDLU span=0.046). Whereas TDLU metrics were positively associated with %FGV, no relationship was observed between END and %FGV. In analyses stratified by BBD severity, however, END and %FGV were positively associated among women with non-proliferative disease (p-trend=0.04), findings not observed with proliferative disease. Conclusions: Automated END and visually assessed TDLU involution metrics were positively associated with each other and with MD. However, associations were diluted for proliferative lesions, suggesting that applying automated digital pathology tools to unsegmented digital images of whole sections of BBD biopsies does not demonstrate the same associations with MD as visual assessment of TDLU involution. Citation Format: Maeve Mullooly, Samantha Puvanesarajah, Shaoqi Fan, Ruth M. Pfeiffer, Linnea Olsson, Manila Hada, Erin L. Kirk, Pamela M. Vacek, Donald L. Weaver, John A. Shepherd, Amir P. Mahmoudzadeh, Jeff Wang, Stephen M. Hewitt, Sally D. Herschorn, Mark E. Sherman, Melissa A. Troester, Gretchen L. Gierach. Utilizing digital pathology to understand breast epithelial characteristics of benign breast disease among women undergoing diagnostic image-guided breast biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3260.