Introduction Benign breast disease (BBD) and high mammographic breast density (MBD) are prevalent and independent risk factors for invasive breast cancer. It has been suggested that temporal changes in MBD may impact future invasive breast cancer risk, but this has not been studied among women with BBD. Methods We undertook a nested case–control study within a cohort of 15,395 women with BBD in Kaiser Permanente Northwest (KPNW; 1970–2012, followed through mid-2015). Cases ( n = 261) developed invasive breast cancer > 1 year after BBD diagnosis, whereas controls ( n = 249) did not have breast cancer by the case diagnosis date. Cases and controls were individually matched on BBD diagnosis age and plan membership duration. Standardized %MBD change (per 2 years), categorized as stable/any increase (≥ 0%), minimal decrease of less than 5% or a decrease greater than or equal to 5%, was determined from baseline and follow-up mammograms. Associations between MBD change and breast cancer risk were examined using adjusted unconditional logistic regression. Results Overall, 64.5% ( n = 329) of BBD patients had non-proliferative and 35.5% ( n = 181) had proliferative disease with/without atypia. Women with an MBD decrease (≤ − 5%) were less likely to develop breast cancer (Odds Ratio (OR) 0.64; 95% Confidence Interval (CI) 0.38, 1.07) compared with women with minimal decreases. Associations were stronger among women ≥ 50 years at BBD diagnosis (OR 0.48; 95% CI 0.25, 0.92) and with proliferative BBD (OR 0.32; 95% CI 0.11, 0.99). Discussion Assessment of temporal MBD changes may inform risk monitoring among women with BBD, and strategies to actively reduce MBD may help decrease future breast cancer risk.

PDF file - 219K. Please Note: Supplementary Table 1 was updated 06/20/12. Please see the published correction notice for details of modifications to the article made in order to correct author error.

Supplementary Figure S1. Percent density from baseline to T5 is shown for each individual patient. Supplementary Figure S2. Percent density from baseline to T5 and change in density compared to the previous mammogram at T1 to T5. Supplementary Figure S3. Percent density from baseline to T5. Supplementary Figure S4. Percent density from baseline to T5 and change in density compared to the previous mammogram at T1 to T5. Supplementary Figure S5. Percent density from baseline to T5 and change in density compared to the previous mammogram at T1 to T5

BACKGROUND:Mammographic breast density (MBD) decline post-tamoxifen initiation is a favorable prognostic factor in estrogen receptor (ER)-positive breast cancer (BC) and has potential utility as a biomarker of tamoxifen response. However, the prognostic value of MBD decline may vary by molecular characteristics among ER-positive patients.METHODS:We investigated associations between MBD decline (≥10% vs <10%) and breast cancer-specific mortality (BCSM) among ER-positive breast cancer patients aged 36-87 years at diagnosis treated with tamoxifen at Kaiser Permanente Northwest (1990-2008). Patients who died of BC (case patients; n = 62) were compared with those who did not (control patients; n = 215) overall and by tumor molecular characteristics (immunohistochemistry [IHC]-based subtype [luminal A-like: ER-positive/progesterone receptor [PR]-positive/HER2-negative/low Ki67; luminal B-like: ER-positive and 1 or more of PR-negative, HER2-positive, high Ki67] and modified IHC [mIHC]-based recurrence score of ER/PR/Ki67). Percent MBD was measured in the unaffected breast at baseline mammogram (mean = 6 months before tamoxifen initiation) and follow-up (mean = 12 months post-tamoxifen initiation). Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were computed from logistic regression models. All statistical tests were 2-sided.RESULTS:MBD decline was statistically significantly associated with reduced risk of BCSM overall (OR = 0.38, 95% CI = 0.15 to 0.92). This association was, however, stronger among women with aggressive tumor characteristics including luminal B-like (OR = 0.17, 95% CI = 0.04 to 0.73) vs A-like (OR = 0.74, 95% CI = 0.19 to 2.92); large (OR = 0.26, 95% CI = 0.08 to 0.78) vs small (OR = 0.41, 95% CI = 0.04 to 3.79) tumors; PR-negative (OR = 0.02, 95% CI = 0.001 to 0.37) vs PR-positive (OR = 0.50, 95% CI = 0.18 to 1.40) disease; and high (OR = 0.25, 95% CI = 0.07 to 0.93) vs low (OR = 0.44, 95% CI = 0.10 to 2.09) mIHC3 score.CONCLUSION:The findings support MBD decline as a prognostic marker of tamoxifen response among patients with aggressive ER-positive BC phenotypes, for whom understanding treatment effectiveness is critical.

Double-edged “Soil”: Stromal Microenvironment in Breast Cancer Development Mustapha Abubakar, MD, PhD1; Shaoqi Fan, MPH1; Máire A. Duggan, MD, FRCPC2; Ruth M. Pfeiffer, PhD1; Scott Lawrence, M.S.3; Kathryn Richert-Boe, MD4; Andrew G. Glass, MD4; Teresa M. Kimes, MS4; Jonine D. Figueroa, PhD, MPH5; Thomas E. Rohan, MBBS, PhD6; Gretchen L. Gierach, PhD, MPH1. Affiliations 1Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institute of Health (NIH), USA2Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, T2N2Y9, Alberta, Canada3Molecular and Digital Pathology Laboratory, Cancer Genomics Research Laboratory, Leidos Biomedical Research, Inc., Frederick, MD 217024Kaiser Permanente Center for Health Research, Portland, Oregon 5Usher Institute of Population Health Sciences and Informatics, The University of Edinburgh, Scotland, UK6Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York, 10461 Deceased Background Over a century after Paget’s postulation of the “seed” and “soil” hypothesis of breast cancer metastasis, understanding of the role of the “soil” (i.e., local or distant stromal microenvironment) in supporting and promoting the growth and dissemination of “seed” tumor cells has increased considerably. However, the role of the stromal microenvironment in early stages of breast cancer development, including tissue origins of etiologic heterogeneity, remains poorly understood. To date, the prevailing model of breast cancer development involves a linear sequence of epithelial changes that begin from normal epithelium, progress to flat epithelial atypia (FEA), develop into atypical ductal hyperplasia, transform into ductal carcinoma in situ (DCIS), and culminate in the emergence of invasive breast cancer. An alternative model proposes the evolution of FEA from usual ductal hyperplasia, but this is not supported by a clear genetic link between the two. Notably, established models of breast cancer development are predicated almost exclusively on sequences of epithelial changes. Although recent efforts have shed light on the significance of stromal microenvironment in DCIS to invasive breast cancer progression, its role in breast cancer development following benign breast disease (BBD) is less well-studied. MethodsIn this case-control study, nested within a cohort of 15,395 BBD patients from Kaiser Permanente Northwest (1970-2012) who were followed for subsequent development of invasive breast cancer, we evaluated archival diagnostic, formalin-fixed and paraffin-embedded tissue blocks using high-accuracy machine learning algorithms for the detailed characterization of stromal microenvironment on digitized H&E-stained breast biopsy sections. Stromal phenotypes, including total stromal-to-epithelial ratio (TSER), dense stromal-to-epithelial ratio (DSER), loose stromal-to-epithelial ratio (LSER) and stromal cellular density (SCD) were defined based on the distributions of total stromal area, dense (mostly fibrous/collagenized, inter-lobular) stroma, loose (mostly pale, intra-lobular or remodeled) stroma, and stromal cellularity, respectively. Relationships between stromal features and invasive breast cancer incidence through 2012 were assessed in multivariable conditional logistic regression models adjusted for BBD histological classification, body mass index, menopausal status/menopausal hormone therapy use, parity and age at first live birth (AFLB), family history of breast cancer (FHBC), oophorectomy, and mammographic density. Analyses were performed overall and by BBD histologic classification. We also evaluated associations between stromal features and breast cancer risk according to tumor characteristics that define divergent etiologic pathways, namely estrogen receptor (ER) expression and histologic grade. Results The current analysis is comprised of 486 cases and 487 controls, representing 95% of the case-control study population, for whom digitized H&E-stained sections were suitable for image analysis. The median age at diagnosis was 51.4 (range=18-86) years. ~55% of the participants were either overweight (30.1%) or obese (24.5%) at BBD diagnosis, and 69% had non-proliferative disease (NPD), 28% proliferative disease (PD) without atypia, and 3% atypical hyperplasia (AH). 13% of BBD biopsies contained simple fibroadenoma, ~2% complex fibroadenoma, 8% sclerosing adenosis, 5% radial scar, and 14% columnar cell lesions. The median (range) values (%) of total, dense, and loose connective tissue stroma were 39.3% (0.6-89.9%), 25.1% (0.1-84.5%), and 8.6% (0.2-59.0%), respectively. On average, BBD lesions contained ~6 times more stroma than epithelium. Average TSER, DSER, LSER, and SCD were 6.3, 4.4, 1.8, and 7.5%, respectively. Overall, increasing TSER was associated with decreasing risk of breast cancer [OR(95% CI)Q4 vs Q1=0.51(0.32, 0.82); p-trend=0.009]. The protective effect of TSER was, however, stronger in relation to DSER [OR(95% CI)Q4 vs Q1=0.48(0.29, 0.79); p-trend=0.007] than LSER [OR(95% CI)Q4 vs Q1=0.84(0.52, 1.36); p-trend=0.70]. Conversely, increasing SCD was statistically significantly associated with increasing breast cancer risk [OR(95% CI)Q4 vs Q1=2.21 (1.38, 3.56); p-trend=0.001]. Although findings were stronger among patients with NPD than PD, there was no heterogeneity in the association by BBD histology. Of the stromal features, DSER and SCD were most predictive of breast cancer risk but these were not independent of one another. To test their joint association with risk, we combined categories [low (<25th percentile), intermediate (25th–75th percentile), and high (>75th percentile) for each variable] in a composite, stromal disruption (SD), variable as follows: 1) no SD (high DSER and low SCD); 2) minimal disruption (high DSER and intermediate SCD, or vice versa); 3) moderate SD (intermediate DSER and high SCD, or vice versa); and 4) substantial SD (low DSER and high SCD). BBD patients with moderate [OR(95% CI)=1.74(1.01, 2.99)] or substantial [OR(95% CI)=2.70(1.51, 4.84)] SD were at statistically significantly elevated risk of breast cancer than those with no SD. Younger women, those with proliferative BBD, parous and AFLB <30 years, positive FHBC, absent involution, and being postmenopausal were statistically significantly more likely to develop BBDs with substantial SD than those with no SD. Substantial SD was associated with elevated risk of both ER+ [OR(95% CI)=1.97(1.16, 3.36)] and ER- [OR(95% CI)=2.09(0.77, 5.69)] breast cancer. In terms of grade, substantial SD was more strongly associated with risks of high [OR(95% CI)=3.17(1.28, 7.85)] and intermediate [OR(95% CI)=2.30(1.10, 4.83)] than low [OR(95% CI)=1.61(0.74, 3.50)] grade tumors overall (p-heterogeneity=0.44). This association was stronger among patients with NPD [OR(95% CI) substantial SD vs no SD=5.75(2.04, 16.09); 2.83(1.15, 6.97); and 1.36(0.53, 3.46) for high, intermediate, and low grade tumors, respectively (p-heterogeneity=0.03)]. Because AH has been implicated in the development of ER+/low grade but not ER+/high grade tumors, we further evaluated the role of AH and SD in ER+ breast cancer risk: contrasting patterns were observed in associations between AH, substantial SD, and risk of ER+ tumors defined by levels of histologic grade. While AH more strongly predisposed to risk of low [OR(95% CI)=6.32(1.09, 20.08)] than high [OR(95% CI)=1.04(0.10, 11.24)] grade ER+ tumors, substantial SD more strongly predisposed to risk of high [OR(95% CI)=5.28(1.54, 18.10)] than low [OR(95% CI)=1.52(0.76, 3.06)] grade ER+ tumors.Conclusion Components of the stromal microenvironment in BBD showed disparate associations with breast cancer risk factors and risk of subsequent invasive breast cancer. In particular, increasing ratio of dense (mostly fibrous/collagenized and inter-lobular), but not loose (mostly pale/myxoid, intra-lobular, remodeled), connective tissue stroma to epithelium was strongly associated with reduced risk of breast cancer. Conversely, increasing stromal cellularity was associated with increasing risk of breast cancer. In combination, decreasing amounts of dense stroma and concomitant increase in loose stroma, epithelial volume, and stromal cellularity resulted in a stromal disruption phenotype that was strongly associated with increased breast cancer risk overall, but particularly of aggressive high grade tumors. These results were independent of BBD histologic diagnosis. Many of the observed risk factor associations with stromal microenvironment features were consistent with their breast cancer risk relationships, suggesting that stromal changes may reflect cumulative exposure to breast cancer risk factors. These findings provide new etiologic insights into stromal role in breast cancer risk, including tissue origins of breast cancer etiologic heterogeneity, with the potential to aid risk stratification and clinical decision-making for BBD patients. Citation Format: Mustapha Abubakar, Shaoqi Fan, Maire A. Duggan, Ruth M. Pfeiffer, Scott Lawrence, Kathryn Richert-Boe, Andrew Glass, Teresa M. Kimes, Jonine D. Figueroa, Thomas E. Rohan, Gretchen L. Gierach. Double-edged “soil”: Stromal microenvironment in breast cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY25-02.

Background Benign breast disease (BBD) is a strong breast cancer risk factor, but identifying patients that might develop invasive breast cancer remains a challenge. Methods By applying machine-learning to digitized hematoxylin and eosin-stained biopsies and computer-assisted thresholding to mammograms obtained circa BBD diagnosis, we generated quantitative tissue composition metrics and determined their association with future invasive breast cancer diagnosis. Archival breast biopsies and mammograms were obtained for women (18-86 years of age) in a case-control study, nested within a cohort of 15 395 BBD patients from Kaiser Permanente Northwest (1970-2012), followed through mid-2015. Patients who developed incident invasive breast cancer (ie, cases; n = 514) and those who did not (ie, controls; n = 514) were matched on BBD diagnosis age and plan membership duration. All statistical tests were 2-sided. Results Increasing epithelial area on the BBD biopsy was associated with increasing breast cancer risk (odds ratio [OR](Q4 vs Q1) = 1.85, 95% confidence interval [CI] = 1.13 to 3.04; P-trend = .02). Conversely, increasing stroma was associated with decreased risk in nonproliferative, but not proliferative, BBD (P-heterogeneity = .002). Increasing epithelium-to-stroma proportion (ORQ4 vs Q1 = 2.06, 95% CI =1.28 to 3.33; P-trend = .002) and percent mammographic density (MBD) (ORQ4 vs Q1 = 2.20, 95% CI = 1.20 to 4.03; P-trend = .01) were independently and strongly predictive of increased breast cancer risk. In combination, women with high epithelium-to-stroma proportion and high MBD had substantially higher risk than those with low epithelium-to-stroma proportion and low MBD (OR = 2.27, 95% CI = 1.27 to 4.06; P-trend = .005), particularly among women with nonproliferative (P-trend = .01) vs proliferative (P-trend = .33) BBD. Conclusion Among BBD patients, increasing epithelium-to-stroma proportion on BBD biopsies and percent MBD at BBD diagnosis were independently and jointly associated with increasing breast cancer risk. These findings were particularly striking for women with nonproliferative disease (comprising approximately 70% of all BBD patients), for whom relevant predictive biomarkers are lacking.

Abstract Background Among women diagnosed with invasive breast cancer, 30% have a prior diagnosis of benign breast disease (BBD). Thus, it is important to identify factors among BBD patients that elevate invasive cancer risk. In the general population, risk factors differ in their associations by clinical pathologic features; however, whether women with BBD show etiologic heterogeneity in the types of breast cancers they develop remains unknown. Methods Using a nested case-control study of BBD and breast cancer risk conducted in a community healthcare plan (Kaiser Permanente Northwest), we assessed relationships of histologic features in BBD biopsies and patient characteristics with subsequent breast cancer risk and tested for heterogeneity of associations by estrogen receptor (ER) status, tumor grade, and size. The study included 514 invasive breast cancer cases (median follow-up of 9 years post-BBD diagnosis) and 514 matched controls, diagnosed with proliferative or non-proliferative BBD between 1971 and 2006, with follow-up through mid-2015. Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained using multivariable polytomous logistic regression models. Results Breast cancers were predominantly ER-positive (86%), well or moderately differentiated (73%), small (74% < 20 mm), and stage I/II (91%). Compared to patients with non-proliferative BBD, proliferative BBD with atypia conferred increased risk for ER-positive cancer (OR = 5.48, 95% CI = 2.14–14.01) with only one ER-negative case, P-heterogeneity = 0.45. The presence of columnar cell lesions (CCLs) at BBD diagnosis was associated with a 1.5-fold increase in the risk of both ER-positive and ER-negative tumors, with a 2-fold increase (95% CI = 1.21–3.58) observed among postmenopausal women (56%), independent of proliferative BBD status with and without atypia. We did not identify statistically significant differences in risk factor associations by tumor grade or size. Conclusion Most tumors that developed after a BBD diagnosis in this cohort were highly treatable low-stage ER-positive tumors. CCL in BBD biopsies may be associated with moderately increased risk, independent of BBD histology, and irrespective of ER status.

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.

Purpose It is clinically challenging to integrate genomic-classifier results that report a numeric risk of recurrence into treatment recommendations for localized prostate cancer, which are founded in the framework of risk groups. We aimed to develop a novel clinical-genomic risk grouping system that can readily be incorporated into treatment guidelines for localized prostate cancer. Materials and Methods Two multicenter cohorts (n = 991) were used for training and validation of the clinical-genomic risk groups, and two additional cohorts (n = 5,937) were used for reclassification analyses. Competing risks analysis was used to estimate the risk of distant metastasis. Time-dependent c-indices were constructed to compare clinicopathologic risk models with the clinical-genomic risk groups. Results With a median follow-up of 8 years for patients in the training cohort, 10-year distant metastasis rates for National Comprehensive Cancer Network (NCCN) low, favorable-intermediate, unfavorable-intermediate, and high-risk were 7.3%, 9.2%, 38.0%, and 39.5%, respectively. In contrast, the three-tier clinical-genomic risk groups had 10-year distant metastasis rates of 3.5%, 29.4%, and 54.6%, for low-, intermediate-, and high-risk, respectively, which were consistent in the validation cohort (0%, 25.9%, and 55.2%, respectively). C-indices for the clinical-genomic risk grouping system (0.84; 95% CI, 0.61 to 0.93) were improved over NCCN (0.73; 95% CI, 0.60 to 0.86) and Cancer of the Prostate Risk Assessment (0.74; 95% CI, 0.65 to 0.84), and 30% of patients using NCCN low/intermediate/high would be reclassified by the new three-tier system and 67% of patients would be reclassified from NCCN six-tier (very-low- to very-high-risk) by the new six-tier system. Conclusion A commercially available genomic classifier in combination with standard clinicopathologic variables can generate a simple-to-use clinical-genomic risk grouping that more accurately identifies patients at low, intermediate, and high risk for metastasis and can be easily incorporated into current guidelines to better risk-stratify patients.

2 Background: Significant genomic diversity exists in the androgen receptor ( AR) and its activity (AR-A) in mCRPC. In localized prostate cancer, the biologic, prognostic, and therapeutic clinical implications of AR-A heterogeneity have yet to be interrogated Methods: Genome-wide expression profiles of FFPE RP or biopsy tumor samples were evaluated from a prospective registry cohort (n = 5,239, NCT02609269) and six retrospective institutional cohorts (n = 1,170). AR-A was calculated based on expression of 9 targets of AR.. Results: Utilizing 6,409 localized prostate adenocarcinomas with full transcriptomic data, we found there was marked inter-individual transcriptomic diversity in AR and AR-A expression, and weak correlation between them (r = 0.08 to 0.36 based on cohort), in contrast to mCRPC that has a strong correlation between AR and AR-A expression (r = 0.76). Additionally, serum PSA had no correlation to intratumoral AR-A (r = 0.06). Unsupervised hierarchical clustering identified a distinct subclass of low AR-A prostate tumors, which had increased markers of immunogenicity (decreased T-regs and MDSCs, and increased CD3 effector T-cells), increased neuroendocrine marker expression ( NCAM1, ENO2, and SCG2), and decreased DNA repair pathway expression (all p < 0.001). Clinically, low AR-A tumors had more rapid development of metastatic disease in three independent cohorts, were more prone to develop resistance to hormonal therapy and develop CRPC, and were found at an increased frequency in African-American men. Interrogating in vitro drug sensitivity analyses utilizing the NCI-60 panel, low AR-A tumors appear more sensitive to platinum chemotherapy and PARP inhibition, and less sensitive to hormone therapy and taxanes. Conclusions: The diversity in AR-signaling in localized prostate cancer represents important biological heterogeneity that is both prognostic and predictive of treatment response. These findings are provocative in that low AR-A tumors may be more susceptible to immunotherapy, PARP inhibition, platinum chemotherapy, and/or radiotherapy. Patients with low AR-A tumors warrant dedicated biomarker enhanced clinical trials.

MicroRNAs are endogenous, small non-coding RNAs that control gene expression by directing their target mRNAs for degradation and/or posttranscriptional repression. Abnormal expression of microRNAs is thought to contribute to the development and progression of cancer. A history of benign breast disease (BBD) is associated with increased risk of subsequent breast cancer. However, no large-scale study has examined the association between microRNA expression in BBD tissue and risk of subsequent invasive breast cancer (IBC). We conducted discovery and validation case-control studies nested in a cohort of 15,395 women diagnosed with BBD in a large health plan between 1971 and 2006 and followed to mid-2015. Cases were women with BBD who developed subsequent IBC; controls were matched 1:1 to cases on age, age at diagnosis of BBD, and duration of plan membership. The discovery stage (316 case-control pairs) entailed use of the Illumina MicroRNA Expression Profiling Assay (in duplicate) to identify breast cancer-associated microRNAs. MicroRNAs identified at this stage were ranked by the strength of the correlation between Illumina array and quantitative PCR results for 15 case-control pairs. The top ranked 14 microRNAs entered the validation stage (165 case-control pairs) which was conducted using quantitative PCR (in triplicate). In both stages, linear regression was used to evaluate the association between the mean expression level of each microRNA (response variable) and case-control status (independent variable); paired t-tests were also used in the validation stage. None of the 14 validation stage microRNAs was associated with breast cancer risk. The results of this study suggest that microRNA expression in benign breast tissue does not influence the risk of subsequent IBC.

Background Insights into the molecular pathogenesis of breast cancer might come from molecular analysis of tissue from early stages of the disease. Methods We conducted a case–control study nested in a cohort of women who had biopsy-confirmed benign breast disease (BBD) diagnosed between 1971 and 2006 at Kaiser Permanente Northwest and who were followed to mid-2015 to ascertain subsequent invasive breast cancer (IBC); cases ( n = 218) were women with BBD who developed subsequent IBC and controls, individually matched (1:1) to cases, were women with BBD who did not develop IBC in the same follow-up interval as that for the corresponding case. Targeted sequence capture and sequencing were performed for 83 genes of importance in breast cancer. Results There were no significant case–control differences in mutation burden overall, for non-silent mutations, for individual genes, or with respect either to the nature of the gene mutations or to mutational enrichment at the pathway level. For seven subjects with DNA from the BBD and ipsilateral IBC, virtually no mutations were shared. Conclusions This study, the first to use a targeted multi-gene sequencing approach on early breast cancer precursor lesions to investigate the genomic basis of the disease, showed that somatic mutations detected in BBD tissue were not associated with breast cancer risk.

The third category for extent of involution in Table 4 was published incorrectly in the original publication. The correct classification is ≥ 75% and the corrected Table 4 is given in the Correction article.

IMPORTANCE Within 10 years after breast cancer diagnosis, roughly 5% of patients develop contralateral breast cancer (CBC). Randomized trials have found that therapy including tamoxifen citrate and aromatase inhibitors (AIs) reduces CBC risk. But little is known about the magnitude and duration of protective associations within the context of real-world clinical management settings, where varying durations of and gaps in treatment are common.OBJECTIVE To determine the association between adjuvant tamoxifen and AI therapy and CBC risk within a general community setting.DESIGN, SETTING, AND PARTICIPANTS A retrospective cohort study of CBC risk among 7541 patients diagnosed with a first primary unilateral invasive breast cancer at Kaiser Permanente Institute for Health Research (Colorado) or Kaiser Permanente Northwest Center for Health Research (Oregon) between January 1, 1990, and December 31, 2008. Data were analyzed from 1 year after diagnosis of the first breast cancer through the earliest of the following events: CBC diagnosis, other second cancer diagnosis, death, last tumor registry follow-up, exit from the Kaiser Permanente health care plan, or end of study follow-up (December 31, 2010, for Oregon and December 31, 2011, for Colorado).EXPOSURES Adjuvant tamoxifen use and AI therapy were treated as time-dependent exposures, assessed using electronic prescription records.MAIN OUTCOMES AND MEASURES Incident CBC based on long-term systematic follow-up.RESULTS Among 7541 women with invasive breast cancer, median age at initial breast cancer diagnosis was 60.6 years (age range, 24.9-84.9 years). Women were predominantly (92.9% [7009 of 7541]) of white race. During a median of 6.3 years (range, 1-20.9 years) of follow-up, 248 women developed CBC (45 in situ and 203 invasive). Contralateral breast cancer risk decreased significantly with increasing tamoxifen therapy duration. In current users, the relative risk (RR) per year of tamoxifen use was 0.76 (95% CI, 0.64-0.89), with an estimated 66%(RR, 0.34; 95% CI, 0.29-0.40) RR reduction for 4 years of use compared with nonusers. Risk reductions were slightly smaller for past users but were still significant at least 5 years after stopping tamoxifen therapy (RR per year of use, 0.85; 95% CI, 0.71-0.995). In addition, AI use without tamoxifen therapy was associated with reduced CBC risk (RR for AI users compared with nonusers, 0.48; 95% CI, 0.22-0.97). Risk reductions were most apparent among women whose primary and CBCs were estrogen receptor positive.CONCLUSIONS AND RELEVANCE Tamoxifen therapy was associated with reduced CBC risk during treatment and after its cessation, with risk progressively decreasing as tamoxifen therapy duration increased. Among those surviving at least 5 years, tamoxifen use for at least 4 years was estimated to prevent 3 CBCs per 100 women by 10 years after an estrogen receptor-positive first breast cancer, an absolute risk reduction that is consistent with findings from clinical trials. If adjuvant endocrine therapy is indicated for breast cancer treatment, these findings in concert with trial data suggest that women should be encouraged to complete the full course.

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

Abstract Tamoxifen-associated mammographic density (MD) reductions are linked to improved breast cancer survival. We evaluated MD at six time points to determine the timing of greatest reduction following tamoxifen initiation. We sampled 40 Kaiser Permanente Northwest estrogen receptor (ER)–positive breast cancer patients from a prior study of MD change, according to tamoxifen use duration and age at diagnosis: <4 years tamoxifen and ≤50 years (N = 6) or >50 years (N = 10) old; ≥4 years tamoxifen and ≤50 years (N = 13) or >50 years (N = 11) old. A single reader evaluated percent MD in the contralateral breast on baseline (pre-diagnosis) and five approximately yearly post-diagnostic (T1 to T5) mammograms. Mean MD change was calculated. Interactions with age (≤50 and >50 years), tamoxifen duration (<4 and ≥4 years), and baseline MD (tertiles) were tested in linear regression models. Overall, the largest MD decline occurred by T1 (mean 4.5%) with little additional decline by T5. Declines differed by tertile of baseline MD (Pinteraction < 0.01). In the highest tertile, the largest reduction occurred by T1 (mean 14.9%), with an additional reduction of 3.6% by T5. Changes were smaller in the middle and lowest baseline MD tertiles, with cumulative reductions of 3.0% and 0.4% from baseline to T5, respectively. There were no differences by age (Pinteraction = 0.36) or tamoxifen duration (Pinteraction = 0.42). Among ER-positive patients treated with tamoxifen and surviving ≥5 years, most of the MD reduction occurred within approximately 12 months of tamoxifen initiation, suggesting that MD measurement at a single time point following tamoxifen initiation can identify patients with substantial density declines. Cancer Epidemiol Biomarkers Prev; 25(1); 212–6. ©2015 AACR.

s Hum Hered 2016;81:51–61 DOI: 10.1159/000447110 52 observed some significant differences. We used permutationbased tests to assess evidence that non-random sets of RVs are associated with transmission to the affected child. We will expand this approach to consider the linkage disequilibrium structure and further characterize functionality of RVs, which may offer a viable method for resolving causal variants that could explain associations found in GWAS.