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Background: Immunotherapy is a rapidly evolving treatment option in breast cancer; However, the breast cancer immune microenvironment is understudied in Black and younger ( Methods: We used histologic and RNA-based immunoprofiling methods to characterize the breast cancer immune landscape in 1,952 tumors from the Carolina Breast Cancer Study (CBCS), a population-based study that oversampled Black (n = 1,030) and young women (n = 1,039). We evaluated immune response leveraging markers for 10 immune cell populations, compared profiles to those in The Cancer Genome Atlas (TCGA) Project [n = 1,095 tumors, Black (n = 183), and young women (n = 295)], and evaluated in association with clinical and demographic variables, including recurrence. Results: Consensus clustering identified three immune clusters in CBCS (adaptive-enriched, innate-enriched, or immune-quiet) that varied in frequency by race, age, tumor grade and subtype; however, only two clusters were identified in TCGA, which were predominantly comprised of adaptive-enriched and innate-enriched tumors. In CBCS, the strongest adaptive immune response was observed for basal-like, HER2-positive (HER2+), triple-negative breast cancer (TNBC), and high-grade tumors. Younger patients had higher proportions of adaptive-enriched tumors, particularly among estrogen receptor (ER)-negative (ER−) cases. Black patients had higher frequencies of both adaptive-enriched and innate-enriched tumors. Immune clusters were associated with recurrence among ER− tumors, with adaptive-enriched showing the best and innate-enriched showing the poorest 5-year recurrence-free survival. Conclusions: These data suggest that immune microenvironments are intricately related to race, age, tumor subtype, and grade. Impact: Given higher mortality among Black and young women, more defined immune classification using cell-type–specific panels could help explain higher recurrence and ultimately lead to targetable interventions.

Background Breast cancer subtype can be classified using standard clinical markers (estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2)), supplemented with additional markers. However, automated biomarker scoring and classification schemes have not been standardized. The aim of this study was to optimize tumor classification using automated methods in order to describe subtype frequency in the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. Methods Using immunohistochemistry (IHC), we quantified the expression of ER, PR, HER2, the proliferation marker Ki67, and two basal-like biomarkers, epidermal growth factor receptor (EGFR) and cytokeratin (CK)5/6, in 1381 invasive breast tumors from African American women. RNA-based (prediction analysis of microarray 50 (PAM50)) subtype, available for 574 (42%) cases, was used to optimize classification. Subtype frequency was calculated, and associations between subtype and tumor characteristics were estimated using logistic regression. Results Relative to ER, PR and HER2 from medical records, central IHC staining and the addition of Ki67 or combined tumor grade improved accuracy for classifying PAM50-based luminal subtypes. Few triple negative cases (

Background: African American breast cancer patients have lower frequency of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 (HER2)-negative disease and higher subtype-specific mortality. Racial differences in molecular subtype within clinically defined subgroups are not well understood.Methods: Using data and biospecimens from the population-based Carolina Breast Cancer Study (CBCS) Phase 3 (2008-2013), we classified 980 invasive breast cancers using RNA expression-based PAM50 subtype and recurrence (ROR) score that reflects proliferation and tumor size. Molecular subtypes (Luminal A, Luminal B, HER2-enriched, and Basal-like) and ROR scores (high vs low/medium) were compared by race (blacks vs whites) and age (≤50 years vs > 50 years) using chi-square tests and analysis of variance tests.Results: Black women of all ages had a statistically significantly lower frequency of Luminal A breast cancer (25.4% and 33.6% in blacks vs 42.8% and 52.1% in whites; younger and older, respectively). All other subtype frequencies were higher in black women (case-only odds ratio [OR] = 3.11, 95% confidence interval [CI] = 2.22 to 4.37, for Basal-like; OR = 1.45, 95% CI = 1.02 to 2.06, for Luminal B; OR = 2.04, 95% CI = 1.33 to 3.13, for HER2-enriched). Among clinically HR+/HER2- cases, Luminal A subtype was less common and ROR scores were statistically significantly higher among black women.Conclusions: Multigene assays highlight racial disparities in tumor subtype distribution that persist even in clinically defined subgroups. Differences in tumor biology (eg, HER2-enriched status) may be targetable to reduce disparities among clinically ER+/HER2- cases.

Complete age-related regression of mammary epithelium, often termed postmenopausal involution, is associated with decreased breast cancer risk. However, most studies have qualitatively assessed involution. We quantitatively analyzed epithelium, stroma, and adipose tissue from histologically normal breast tissue of 454 patients in the Normal Breast Study. High-resolution digital images of normal breast hematoxylin and eosin-stained slides were partitioned into epithelium, adipose tissue, and nonfatty stroma. Percentage area and nuclei per unit area (nuclear density) were calculated for each component. Quantitative data were evaluated in association with age using linear regression and cubic spline models. Stromal area decreased (P = 0.0002), and adipose tissue area increased (P < 0.0001), with an approximate 0.7% change in area for each component, until age 55 years when these area measures reached a steady state. Although epithelial area did not show linear changes with age, epithelial nuclear density decreased linearly beginning in the third decade of life. No significant age-related trends were observed for stromal or adipose nuclear density. Digital image analysis offers a high-throughput method for quantitatively measuring tissue morphometry and for objectively assessing age-related changes in adipose tissue, stroma, and epithelium. Epithelial nuclear density is a quantitative measure of age-related breast involution that begins to decline in the early premenopausal period.

Using a murine model of basal-like breast cancer, we tested the hypothesis that chronic obesity, an established breast cancer risk and progression factor in women, induces mammary gland epigenetic reprogramming and increases mammary tumor growth. Moreover, we assessed whether the obesity-induced epigenetic and protumor effects are reversed by weight normalization. Ovariectomized female C57BL/6 mice were fed a control diet or diet-induced obesity (DIO) regimen for 17 weeks, resulting in a normal weight or obese phenotype, respectively. Mice on the DIO regimen were then randomized to continue the DIO diet or were switched to the control diet, resulting in formerly obese (FOb) mice with weights comparable with control mice. At week 24, all mice were orthotopically injected with MMTV-Wnt-1 mouse mammary tumor cells. Mean tumor volume, serum IL6 levels, expression of proinflammatory genes in the mammary fat pad, and mammary DNA methylation profiles were similar in DIO and FOb mice and higher than in controls. Many of the genes found to have obesity-associated hypermethylation in mice were also found to be hypermethylated in the normal breast tissue of obese versus nonobese human subjects, and nearly all of these concordant genes remained hypermethylated after significant weight loss in the FOb mice. Our findings suggest that weight normalization may not be sufficient to reverse the effects of chronic obesity on epigenetic reprogramming and inflammatory signals in the microenvironment that are associated with breast cancer progression. Cancer Prev Res; 9(5); 339–48. ©2016 AACR.

Introduction Epidemiological studies linking dietary fat intake and obesity to breast cancer risk have produced inconsistent results. This may be due to the difficulty of dissociating fat intake from obesity, and/or the lack of defined periods of exposure in these studies. The pubertal mammary gland is highly sensitive to cancer-causing agents. We assessed how high fat diet (HFD) affects inflammation, proliferative, and developmental events in the pubertal gland, since dysregulation of these can promote mammary tumorigenesis. To test the effect of HFD initiated during puberty on tumorigenesis, we utilized BALB/c mice, for which HFD neither induces obesity nor metabolic syndrome, allowing dissociation of HFD effects from other conditions associated with HFD. Methods Pubertal BALB/c mice were fed a low fat diet (12% kcal fat) or a HFD (60% kcal fat), and subjected to carcinogen 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumorigenesis. Results HFD elevated mammary gland expression of inflammatory and growth factor genes at 3 and 4 weeks of diet. Receptor activator of nuclear factor kappa-B ligand (RANKL), robustly induced at 4 weeks, has direct mitogenic activity in mammary epithelial cells and, as a potent inducer of NF-κB activity, may induce inflammatory genes. Three weeks of HFD induced a transient influx of eosinophils into the mammary gland, consistent with elevated inflammatory factors. At 10 weeks, prior to the appearance of palpable tumors, there were increased numbers of abnormal mammary epithelial lesions, enhanced cellular proliferation, increased growth factors, chemokines associated with immune-suppressive regulatory T cells, increased vascularization, and elevated M2 macrophages. HFD dramatically reduced tumor latency. Early developing tumors were more proliferative and were associated with increased levels of tumor-related growth factors, including increased plasma levels of HGF in tumor-bearing animals. Early HFD tumors also had increased vascularization, and more intra-tumor and stromal M2 macrophages. Conclusions Taken together in this non-obesogenic context, HFD promotion of inflammatory processes, as well as local and systemically increased growth factor expression, are likely responsible for the enhanced tumorigenesis. It is noteworthy that although DMBA mutagenesis is virtually random in its targeting of genes in tumorigenesis, the short latency tumors arising in animals on HFD showed a unique gene expression profile, highlighting the potent overarching influence of HFD.