Abstract SY25-02: Double-edged “soil”: Stromal microenvironment in breast cancer development
Authors: Abubakar Mustapha , Fan Shaoqi , Duggan Maire A. , Pfeiffer Ruth M. , Lawrence Scott , Richert-Boe Kathryn , Glass Andrew , Kimes Teresa M. , Figueroa Jonine D. , Rohan Thomas E. , Gierach Gretchen L. 
Published: June 2022

Abstract
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 (&lt;25th percentile), intermediate (25th–75th percentile), and high (&gt;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 &lt;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.