Pfeiffer, Ruth M.

Terminal duct lobular units (TDLUs) are the predominant source of future breast cancers, and lack of TDLU involution (higher TDLU counts, higher acini count per TDLU and the product of the two) is a breast cancer risk factor. Numerous breast cancer susceptibility single nucleotide polymorphisms (SNPs) have been identified, but whether they are associated with TDLU involution is unknown. In a pooled analysis of 872 women from two studies, we investigated 62 established breast cancer SNPs and relationships with TDLU involution. Poisson regression models with robust variance were used to calculate adjusted per‐allele relative risks (with the non‐breast cancer risk allele as the referent) and 95% confidence intervals between TDLU measures and each SNP. All statistical tests were two‐sided; P < 0.05 was considered statistically significant. Overall, 36 SNPs (58.1%) were related to higher TDLU counts although this was not statistically significant (p = 0.25). Six of the 62 SNPs (9.7%) were nominally associated with at least one TDLU measure: rs616488 (PEX14), rs11242675 (FOXQ1) and rs6001930 (MKL1) were associated with higher TDLU count (p = 0.047, 0.045 and 0.031, respectively); rs1353747 (PDE4D) and rs6472903 (8q21.11) were associated with higher acini count per TDLU (p = 0.007 and 0.027, respectively); and rs1353747 (PDE4D) and rs204247 (RANBP9) were associated with the product of TDLU and acini counts (p = 0.024 and 0.017, respectively). Our findings suggest breast cancer SNPs may not strongly influence TDLU involution. Agnostic genome‐wide association studies of TDLU involution may provide new insights on its biologic underpinnings and breast cancer susceptibility.

Abstract Purpose: Breast biopsies with benign findings (benign breast disease [BBD]) outnumber biopsies revealing in-situ or invasive cancer 4:1, providing an opportunity to identify histopathological features that predict breast cancer risk. Greater degrees of involution of terminal duct lobular units (TDLUs), the structures within the breast that produce milk and the primary source of breast cancer precursors, have been inversely associated with breast cancer risk among women with BBD. We recently developed measures to quantitate levels of TDLU involution, which demonstrate high inter/intra pathologist reproducibility. Here we assessed whether TDLU counts/100mm2 and median TDLU span (microns), two measures inversely related to degree of TDLU involution, are associated with subsequent breast cancer risk among women with BBD. Methods: From the Mayo BBD cohort (n = 9,376), we evaluated benign biopsies from 99 women who later developed breast cancer (cases) and 145 age-matched controls who did not develop breast cancer. Digitized images of biopsy sections were reviewed to enumerate TDLUs/mm2 and measure median TDLU span (microns) for up to ten normal TDLUs. Breast cancer risk factors were available from questionnaires or medical records. To assess associations with breast cancer risk, subjects were categorized into quartile levels of TDLU counts and median TDLU spans, based on data from controls. Odds ratios (ORs) and 95% confidence intervals (CI) were estimated using multivariable conditional logistic regression models, adjusted for histologic type of BBD, family history of breast cancer and menopausal hormone use. Results: Compared to controls, women who developed breast cancer had higher median number of TDLU counts/100mm2 (28 vs 20, p = 0.03) and larger TDLU spans (300 vs 267 microns, p = 0.14). In multivariable models higher TDLU counts (quartile4 vs. quartile1, OR = 2.44, 95%CI = 0.96-6.19, ptrend = 0.02) and larger TDLU span measures (quartile4 vs. quartile1, OR = 2.83, 95%CI = 1.13-7.06, ptrend = 0.03) were associated with subsequent diagnosis of breast cancer. Combinatorial metrics of TDLU counts with median TDLU span measures identified women at higher risk; specifically, women above the median for both TDLU span and TDLU counts had an OR = 3.75 (95%CI = 1.40-10.00, ptrend = 0.008), compared with women below the median for TDLU span and TDLU counts. Conclusion: These data show that lack of TDLU involution, as measured by increased persistence of TDLU counts, larger median TDLU spans and cross-classification using these measures was associated with increased breast cancer risk among women with BBD, extending prior work in this cohort based on other TDLU involution metrics. Future studies to identify determinants of TDLU involution, its association with breast cancer risk and its potential as an intermediate endpoint in prevention studies warrant consideration. Citation Format: Jonine D. Figueroa, Ruth Pfeiffer, Maya Palakal, Amy C. Degnim, Derek Radisky, Lynn C. Hartmann, Marlene Frost, Melody L. Stallings Mann, Louise A. Brinton, Daphne Papathomas, Daniel Visscher, Mark E. Sherman. Standardized measures of lobular involution and subsequent breast cancer risk among women with benign breast disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4682. doi:10.1158/1538-7445.AM2015-4682

Abstract Introduction: Increased levels of circulating estrogens are associated with elevated risks of postmenopausal breast cancer. Previously, we showed that levels of sex steroid hormones in breast adipose tissue are positively correlated with circulating hormones. Further evaluation of hormone levels in breast tissues may help to explain the relationship between estrogens and breast cancer risk. Specifically, data suggest that focal breast lesions consisting of macrophages encircling dying adipocytes, referred to as “crown-like structures (CLS)”, are associated with increased aromatase activity, which could increase conversion of androgens to estrogens locally in the breast. Therefore, we assessed whether the presence of CLS affects the correlations between blood and breast adipose tissue hormone levels. Methods: Participants included 84 postmenopausal women with invasive breast cancer in the Polish Breast Cancer Study, a population based case-control study conducted from 2000 to 2003. We previously reported sex steroid hormone levels in cancer-free breast adipose tissue removed as part of breast excisions performed for cancer. Here, using immunohistochemical stains, we evaluated the number of CD68 positive macrophages and the presence of full CLS (i.e., complete adipocyte encirclement) in benign breast tissue sections prepared from surgical pathology specimens containing breast cancer. The relationship between the number of CD68 positive macrophages per unit area of fat and hormone levels was examined using Spearman rank correlations (rs). We assessed potential effect modification of the association between blood and tissue hormone levels by presence or absence of CLS by including an interaction term in linear regression models. Results: The number of CD68 positive macrophages per unit area of fat was not significantly correlated with levels of estrone, estradiol, androstenedione or testosterone measured in blood (rs = 0.001, -0.04, -0.07, and -0.09, respectively; p&gt;0.05) or breast adipose tissue (rs = -0.15, 0.06, -0.06, and -0.08, respectively; p&gt;0.05). Correlations between serum and adipose tissue hormones did not statistically significantly differ between women with CLS (n = 30) compared to those without CLS (n = 54). CLS were found more frequently in breast adipose tissue from overweight (i.e., women with a body mass index (BMI) between 25 and 29.9) (43%) and obese women (BMI &gt;30) (30%) versus normal weight women, with (BMI between 18.5 and 24.9), (27%); (p = 0.04, χ2 test). The number of CD68 positive macrophages per unit area of fat was not significantly related to BMI (p = 0.31). Conclusion: Among postmenopausal women with invasive breast cancer, the presence of CLS was more common among overweight and obese women, but CLS did not influence the correlation between circulating and breast adipose tissue sex steroid hormone levels. Citation Format: Maeve Mullooly, Hannah P. Yang, Roni T. Falk, Sarah Nyante, Renata Cora, Ruth M. Pfeiffer, Derek C. Radisky, Daniel W. Visscher, Lynn C. Hartmann, Amy C. Degnim, Frank Z. Stanczyk, Jonine F. Figueroa, Montserrat Garcia-Closas, Jolanta Lissowska, Melissa T. Troester, Louise A. Brinton, Mark E. Sherman, Gretchen L. Gierach. Investigation of the relationship between crown-like structures and adipose tissue hormone levels among postmenopausal women with breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2767. doi:10.1158/1538-7445.AM2015-2767

Abstract Background: Terminal duct lobular units (TDLU) are the predominant source of breast cancers. Lesser degrees of age-related TDLU involution have been associated with increased breast cancer risk, but factors that influence involution are largely unknown. We assessed whether circulating hormones, implicated in breast cancer risk, are associated with levels of TDLU involution using data from the Susan G. Komen Tissue Bank (KTB) at the Indiana University Simon Cancer Center (2009–2011). Methods: We evaluated three highly reproducible measures of TDLU involution, using normal breast tissue samples from the KTB (n = 390): TDLU counts, median TDLU span, and median acini counts per TDLU. RRs (for continuous measures), ORs (for categorical measures), 95% confidence intervals (95% CI), and Ptrends were calculated to assess the association between tertiles of estradiol, testosterone, sex hormone–binding globulin (SHBG), progesterone, and prolactin with TDLU measures. All models were stratified by menopausal status and adjusted for confounders. Results: Among premenopausal women, higher prolactin levels were associated with higher TDLU counts (RRT3vsT1:1.18; 95% CI: 1.07–1.31; Ptrend = 0.0005), but higher progesterone was associated with lower TDLU counts (RRT3vsT1: 0.80; 95% CI: 0.72–0.89; Ptrend &lt; 0.0001). Among postmenopausal women, higher levels of estradiol (RRT3vsT1:1.61; 95% CI: 1.32–1.97; Ptrend &lt; 0.0001) and testosterone (RRT3vsT1: 1.32; 95% CI: 1.09–1.59; Ptrend = 0.0043) were associated with higher TDLU counts. Conclusions: These data suggest that select hormones may influence breast cancer risk potentially through delaying TDLU involution. Impact: Increased understanding of the relationship between circulating markers and TDLU involution may offer new insights into breast carcinogenesis. Cancer Epidemiol Biomarkers Prev; 23(12); 2765–73. ©2014 AACR.

Abstract Background: Terminal ductal lobular units (TDLUs), the functional units of the breast for lactation, are the structures from which most breast cancers arise. TDLU involution, reflecting atrophy and loss of TDLUs, has been related to lower subsequent breast cancer risk. The predictors of such breast tissue aging, however, are not well defined. Given that estrogens and other hormones play a central role in the etiology of postmenopausal breast cancers, we conducted a study to assess the relationship between four hormones and sex hormone-binding globulin (SHBG) with markers of TDLU involution. Methods: Subjects included healthy women who volunteered between 2009 and 2011 to participate in the Komen Tissue Bank Study. Participants completed a risk factor questionnaire, provided a blood sample, and donated breast tissue for research. Our analysis was restricted to postmenopausal women who were not currently taking menopausal hormones (n=153). A pathologist, masked to patient data, assessed tissue sections for the presence of TDLUs and, among sections containing TDLUs (n=88), measured TDLU diameters for up to ten TDLUs per section. Both presence of TDLUs and TDLU diameter are inversely related to involution. Chemiluminescent immunometric assays were used to measure serum levels of estradiol (E2), SHBG, follicle-stimulating hormone (FSH), and prolactin. Percent free E2 was calculated from measured E2 and SHBG levels. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the presence of TDLUs (none or any) associated with tertiles of serum hormone and SHBG levels. Multivariable ordinal logistic regression was used to calculate ORs, 95% CIs, and p-trend for TDLU diameter categorized as tertiles (≥218.4, 218.4-281, and ≥281 microns) with serum hormone and SHBG levels. All models were adjusted for potential confounding factors. Results: The study population was 87.6% Caucasian with a mean age of 59.5 years, and &gt;2/3 of the women were overweight or obese. Increasing percent free E2 was significantly associated with larger TDLU diameter (OR&gt;2.53 vs. ≥1.99: 5.58, 95% CI: 1.48-21.07; p-trend=0.01), whereas increasing SHBG was associated with smaller TDLU diameter (OR&gt;73nmol/L vs. ≥38 nmol/L: 0.19, 95% CI: 0.05-0.74; p-trend=0.02). Total E2 (p-trend=0.49), FSH (p-trend=0.85), and prolactin (p-trend=0.58) were not associated with TDLU diameter. We did not find significant relationships between the presence of TDLUs and any of the serum levels. Our observed relationships were not modified by age or other breast cancer risk factors, including BMI, years since menopause, and history of bilateral oophorectomy. Conclusion: Based on limited data, these findings suggest that sex hormones may play a role in TDLU involution. Thus, TDLU involution may represent one mechanism by which hormone levels influence breast cancer risk. TDLU measurements may therefore be helpful in assessing breast cancer risk as a marker of breast tissue aging. Analysis of the relationships between hormones and TDLU involution among premenopausal women may provide additional insights into early events in breast carcinogenesis. Citation Format: Zeina G. Khodr, Mark E. Sherman, Ruth M. Pfeiffer, Gretchen L. Gierach, Louise A. Brinton, Roni T. Falk, Deesha Patel, Laura Linville, Susan E. Clare, Daniel Visscher, Carolyn Mies, Stephen Hewitt, Anna Maria Storniolo, Jonine D. Figueroa. Endogenous hormone levels and lobular involution of the breast among healthy, postmenopausal women in the Komen Tissue Bank Study. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B89.

Abstract Background: Terminal duct lobular units (TDLUs) are the anatomical source of most breast cancers. TDLU involution, manifested as atrophy and loss of TDLUs, is associated with reduced breast cancer risk. We hypothesize that breast cancer susceptibility loci are associated with TDLU involution. Accordingly, we evaluated relationships between 25 established breast cancer susceptibility markers and their association with TDLU involution in the Susan G. Komen for the Cure® Tissue Bank (KTB). Methods: Subjects were women ages 18-84 (N=680), who had no personal history of any cancer, were not currently taking menopausal hormones, completed a risk factor questionnaire, provided an Oragene® saliva sample, and donated a 10-guage needle tissue core from the upper outer breast quadrant for research. We genotyped 25 known single nucleotide polymorphisms (SNPs) associated with breast cancer risk using TaqMan® assays. Pathologists masked to risk data classified three features inversely related to TDLU involution using one histological section of breast tissue/subject: number of TDLUs per section (TDLU density), TDLU diameters and number of acini per TDLU. SNPS were compared to metrics of TDLU involution categorized in tertiles: TDLU density (0-1, 2-8, 9+), mean TDLU diameter (53-291, 292-415, 416-1143 microns), and mean number of acini per TDLU (2-10, 11-20, 21+). We estimated age-adjusted per-allele odds ratios (ORs) and 95% confidence intervals (CIs) using ordinal logistic regression models with TDLU measures as the outcome and SNPs as the explanatory variables. Associations with TDLU density were based on up to 680 women with genotype data, while associations with diameter and acini measures were based on up to 469 women with observed TDLUs. Results: The rs3803662 (TOX3/TNRC9) SNP risk allele showed an association with increased TDLU density, larger TDLUs, and more acini in TDLUs (TDLU density per-allele OR=1.29 95%CI=1.04-1.60; TDLU diameter OR=1.32 95%CI=1.00-1.74; and TDLU acini OR=1.34 95%CI=1.02-1.77), consistent with previous observations of significant associations seen for this SNP with mammographic density (a well-established risk factor for breast cancer), breast cancer risk, and survival of breast cancer. In addition, we observed associations for four other SNPs with TDLU density (rs311499, rs10483813, rs2981582, rs3817198), for one other SNP with mean TDLU diameter (rs11249433), and for three other SNPs with mean TDLU acini number (rs704010, rs10941679, rs11249433). Conclusion: These data suggest that breast cancer susceptibility SNPs may be associated with TDLU involution levels, and could represent a mechanism by which they influence breast cancer risk. These findings warrant replication in other studies and provide support for the concept that levels of TDLU involution represent an intermediate marker of breast cancer risk. Citation Format: Jonine D. Figueroa, Gretchen L. Gierach, Ruth Pfeiffer, Stephen Chanock, Louise Brinton, Deesha A. Patel, Daniel Visscher, Carolyn Mies, Stephen Hewitt, Susan Clare, Anna Maria Storniolo, Mark Sherman. Associations between breast cancer susceptibility markers and terminal duct lobular unit involution in normal breast tissues among women from the Susan G. Komen for the Cure® Tissue Bank. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 58.

Abstract Little is known about the effects of many recognized breast cancer risk factors on the normal breast. We hypothesize that risk factors affect the number and morphometry of terminal ductal lobular units (TDLUs), the microanatomical structure from which most breast cancers arise. To assess this hypothesis, we evaluated relationships between breast cancer risk factors and TDLU number, TDLU diameter, and acini per TDLU in breast tissue donated by healthy volunteers. Subjects included 786 healthy women ages 18-84 years without a personal history of any cancer, who provided a risk factor questionnaire and donated a 10-guage needle core from the upper outer breast quadrant. A pathologist, masked to subject data, assessed TDLU number per section, TDLU diameter and acini per TDLU using digitized images of hematoxylin and eosin stained sections of tissue. Seventy-two randomly selected images were examined by two additional pathologists. We generated ordered categorical variables for the number of TDLUs per section (0-1, 2-8, 9+), median TDLU diameter (53-262, 263-377, 378-1375 microns) and median number of acini per TDLU (2-10, 11-20, 21+). We computed Spearman correlations between measures, and intraobserver and interobserver agreement. Associations between breast cancer risk factors and TDLU measures were assessed by estimating odds ratios (OR) and 95% confidence intervals (CI) using ordinal logistic regression models, adjusted for age. TDLU diameter and acini count were highly correlated (rho =0.71); TDLU number per section was less strongly related to the diameter (rho= 0.18) and acini measures (rho=0.20). Inter and intraobserver agreement for TDLU measures were highly correlated (rho range 0.68-0.98). All TDLU measurements were significantly lower among women age 50 years and older compared with younger women. Compared with Caucasians, African American women had more TDLUs per section (ORadj=2.03, 95% CI= 1.06-3.89). Greater numbers of TDLUs were also related to having a family history of two or more relatives with breast cancer compared with none (ORadj=2.01, 95% CI=0.91-4.46); and for parous compared with nulliparous women (ORadj=1.61, 95% CI=1.16-2.25). In contrast, parity was associated with reduced median number of acini per TDLU (ORadj=0.60, 95% CI = 0.40-0.90) and median diameter (ORadj=0.64, 95% CI = 0.42-0.96) compared with nulliparity. Our data suggest that TDLUs are more frequent among women with a positive family history of breast cancer or African Americans. Parity, an established protective factor for breast cancer risk, was related to increased numbers of TDLUs, but fewer acini per TDLU, suggesting that TDLUs may increase with pregnancy and then involute. Our data, along with published results linking TDLU involution with a reduction in breast cancer risk, suggest TDLU morphometry may reflect early biologic changes marking breast cancer susceptibility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4465. doi:1538-7445.AM2012-4465

Abstract Obesity and black race have been associated with excess risk of multiple myeloma. The association of obesity with monoclonal gammopathy of undetermined significance (MGUS) is unknown. Further, it is not known whether the increased risk of multiple myeloma and MGUS in blacks is related to socioeconomic status, genetic susceptibility, or both. We screened 1000 black and 996 white women (range, 40-79 years) of similar socioeconomic status for MGUS; the aim of the study was to assess MGUS risk in relation to obesity and race. A total of 39 (3.9%) blacks and 21 (2.1%) whites had MGUS. On multivariate analysis, obesity (odds ratio [OR] = 1.8; P = .04), black race (OR = 1.8; P = .04), and increasing age (> 55 vs < 43 years; OR = 2.5; P = .03) were independently associated with an excess risk of MGUS. Our findings support the hypothesis that obesity is etiologically linked to myelomagenesis. The 2-fold excess of MGUS among blacks compared with whites of similar socioeconomic status supports a role for susceptibility genes in MGUS.

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma-cell proliferative disorder associated with a life-long risk of progression to multiple myeloma (MM). It is not known whether MM is always preceded by a premalignant asymptomatic MGUS stage. Among 77 469 healthy adults enrolled in the nationwide population-based prospective Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial, we identified 71 subjects who developed MM during the course of the study in whom serially collected (up to 6) prediagnostic serum samples obtained 2 to 9.8 years prior to MM diagnosis were available. Using assays for monoclonal (M)–proteins (electrophoresis/immunofixation) and kappa-lambda free light chains (FLCs), we determined longitudinally the prevalence of MGUS and characterized patterns of monoclonal immunoglobulin abnormalities prior to MM diagnosis. MGUS was present in 100.0% (87.2%-100.0%), 98.3% (90.8%-100.0%), 97.9% (88.9%-100.0%), 94.6% (81.8%-99.3%), 100.0% (86.3%-100.0%), 93.3% (68.1%-99.8%), and 82.4% (56.6%-96.2%) at 2, 3, 4, 5, 6, 7, and 8+ years prior to MM diagnosis, respectively. In approximately half the study population, the M-protein concentration and involved FLC-ratio levels showed a yearly increase prior to MM diagnosis. In the present study, an asymptomatic MGUS stage consistently preceded MM. Novel molecular markers are needed to better predict progression to MM in patients with MGUS.