Clinical, Epidemiologic, and Pathologic Significance of ERBB2-Low Expression in Breast Cancer.

Importance: It is unclear whether breast cancer (BC) with low ERBB2 expression (ERBB2-low) is a distinct clinical, pathological, and epidemiological entity from BC classified as no ERBB2 expression (ERBB2-negative). Objective: To evaluate the clinical, pathological, and epidemiologic features of BC with ERBB2-low expression compared with ERBB2-negative BC in a large population study. Design, Setting, and Participants: This cohort study was conducted as part of the Pathways Study, a prospective, racially and ethnically diverse cohort study of women with BC enrolled between 2006 and 2013 in Kaiser Permanente Northern California (KPNC). The hematoxylin and eosin slides underwent centralized pathology review, including the percentage of tumor infiltrating lymphocytes (TILs). Breast biomarker results were extracted from pathology reports, and women were included if they had a documented ERBB2 value that was not classified ERBB2-positive. Data were analyzed from February 2023 through January 2024. Exposure: Clinical and tumor characteristics associated with BC and ERBB2-low or ERBB2-negative status. Main Outcome and Measures: ERBB2-low was defined as immunohistochemistry score of 1+ or 2+ (negative by in situ hybridization); ERBB2-negative was defined as immunohistochemistry score of 0+. Other data were collected by self-report or extraction from electronic health records, including BC risk factors, tumor characteristics, treatment modality, and survival outcomes, with recurrence-free survival (RFS) as the primary outcome and overall survival (OS) and BC-specific mortality (BCSM) as secondary outcomes. The clinical, pathological, and epidemiological variables were compared between ERBB2-low and ERBB2-negative BC. Results: Of 2200 eligible patients (all female; with mean [SD] age, 60.4 [11.9] years), 1295 (57.2%) had tumors that were ERBB2-low. Hormone receptors were positive in 1956 patients (88.9%). The sample included 291 Asian patients (13.2%), 166 Black patients (7.5%), 253 Hispanic patients (11.5%), 1439 White patients (65.4%), and 51 patients (2.3%) who identified as other race or ethnicity (eg, American Indian or Alaska Native and Pacific Islander). Within the hormone receptor-negative group, patients whose tumors had ERBB2-low staining, compared with those with ERBB2-negative tumors, had better OS (hazard ratio [HR], 0.54; 95% CI, 0.33-0.91; P = .02), RFS (HR, 0.53; 95% CI, 0.30-0.95; P = .03), and BCSM (HR, 0.43; 95% CI, 0.22-0.84; P = .01). In multivariable survival analysis stratified by hormone receptor status and adjusted for key covariates, patients with ERBB2-low and hormone receptor-negative tumors had lower overall mortality (HR, 0.48; 95% CI, 0.27-0.83; P = .009), RFS (HR, 0.45; 95% CI, 0.24-0.86; P = .02), and BCSM (subdistribution HR, 0.21; 95% CI, 0.10-0.46; P < .001) compared with patients with ERBB2-negative and hormone receptor-negative tumors. Within the hormone receptor-negative subtype, patients with ERBB2-low and high TILs tumors had better survival across all 3 outcomes compared with patients with ERBB2-negative and low TILs tumors. Additionally, patients with ERBB2-low and low TILs tumors had better BCSM (subdistribution HR, 0.36; 95% CI, 0.14-0.92; P = .03). Conclusions and Relevance: These findings suggest that there were clinical, pathological, and epidemiological differences between ERBB2-low and ERBB2-negative BC, raising the possibility that ERBB2-low might be a unique biologic entity.

Neighborhood Disadvantage, African Genetic Ancestry, Cancer Subtype, and Mortality Among Breast Cancer Survivors.

Importance: Racial disparities in breast cancer (BC) survival arise from multilevel causes, which may exert influence at different stages of BC progression. Clarifying the importance of genetic and social factors could help prioritize interventions. Objective: To jointly examine associations between African genetic ancestry, social environment, and mortality from any cause and BC in Black BC survivors. Design, Setting, and Participants: This population-based cohort study enrolled self-identified Black women aged 20 to 75 years with histologically confirmed BC from June 2005 to May 2019 and followed them up until death or censoring in September 2021. Participants lived in 10 New Jersey counties. Data were analyzed between December 2022 and April 2023. Exposures: A neighborhood socioeconomic status (nSES) index composed of census tract measures (education, income, wealth, employment status, and occupation) was linked to residential addresses at diagnosis. Percentage African ancestry was estimated using the ADMIXTURE program. Main Outcomes and Measures: Sequentially adjusted (age adjusted: age and interview year; fully adjusted: age adjusted with individual SES, lifestyle factors, and comorbidities) logistic regression models were fit to estimate associations with tumor subtypes (estrogen receptor-negative [ER-] vs estrogen receptor-positive [ER+]; triple-negative breast cancer [TNBC] vs luminal A), and Cox models were fit for associations with all-cause mortality (ACM) and breast cancer-specific mortality (BCSM). Models for BCSM were fit using Fine-Gray competing risks models, and robust standard errors were used to account for census tract-level clustering. Results: Among 1575 participants, median (IQR) African ancestry was 85% (76%-90%), and median (IQR) age was 55 (46-63) years. A 10-percentage point increase in African ancestry was associated with higher odds of ER- vs ER+ (adjusted odds ratio [aOR], 1.08; 95% CI, 0.98-1.18) and TNBC vs luminal (aOR, 1.15; 95% CI, 1.02-1.31) tumors, but not with ACM or BCSM. A 1-IQR increase in nSES was associated with lower ACM (adjusted hazard ratio [aHR], 0.76; 95% CI, 0.63-0.93), and the HR for BCSM was less than 1 but not statistically significant (aHR, 0.81; 95% CI, 0.62-1.04) in age-adjusted models, but associations attenuated following further adjustment for potential mediators (individual SES, lifestyles, comorbidities). Conclusions and Relevance: In this cohort study of Black female BC survivors, higher African ancestry was associated with aggressive tumor subtypes. Compared with genetic ancestry, mediating pathways related to social environments may be more important for survival in these patients.

Effect of neighborhood deprivation index on breast cancer survival in the United States.

PURPOSE: To analyze the association between the Neighborhood Deprivation Index (NDI) and clinical outcomes of locoregional breast cancer (BC). METHODS: Surveillance, Epidemiology and End Results (SEER) database is queried to evaluate overall survival (OS) and disease-specific survival (DSS) of early- stage BC patients diagnosed between 2010 and 2016. Cox multivariate regression was performed to measure the association between NDI (Quintiles corresponding to most deprivation (Q1), above average deprivation (Q2), average deprivation (Q3), below average deprivation (Q4), least deprivation (Q5)) and OS/DSS. RESULTS: Of the 88,572 locoregional BC patients, 27.4% (n = 24,307) were in the Q1 quintile, 26.5% (n = 23,447) were in the Q3 quintile, 17% (n = 15,035) were in the Q2 quintile, 13.5% (n = 11,945) were in the Q4 quintile, and 15.6% (n = 13,838) were in the Q5 quintile. There was a predominance of racial minorities in the Q1 and Q2 quintiles with Black women being 13-15% and Hispanic women being 15% compared to only 8% Black women and 6% Hispanic women in the Q5 quintile (p < 0.001). In multivariate analysis, in the overall cohort, those who live in Q2 and Q1 quintile have inferior OS and DSS compared to those who live in Q5 quintile (OS:- Q2: Hazard Ratio (HR) 1.28, Q1: HR 1.2; DSS:- Q2: HR 1.33, Q1: HR 1.25, all p < 0.001). CONCLUSION: Locoregional BC patients from areas with worse NDI have poor OS and DSS. Investments to improve the socioeconomic status of areas with high deprivation may help to reduce healthcare disparities and improve breast cancer outcomes.

Effect of Neighborhood Deprivation Index on Breast Cancer Survival in the United States.

Purpose To analyze the association between the Neighborhood Deprivation Index (NDI) and clinical outcomes of early-stage breast cancer (BC). Methods Surveillance, Epidemiology and End Results (SEER) database is queried to evaluate overall survival (OS) and disease-specific survival (DSS) of early- stage BC patients diagnosed between 2010-2016. Cox multivariate regression was performed to measure the association between NDI (Quintiles corresponding to most deprivation (Q1), above average deprivation (Q2), average deprivation (Q3), below average deprivation (Q4), least deprivation (Q5)) and OS/DSS. Results Of the 88,572 early-stage BC patients, 27.4% (n = 24,307) were in the Q1 quintile, 26.5% (n = 23,447) were in the Q3 quintile, 17% (n = 15,035) were in the Q2 quintile, 13.5% (n = 11,945) were in the Q4 quintile, and 15.6% (n = 13,838) were in the Q5 quintile. There was a predominance of racial minorities in the Q1 and Q2 quintiles with Black women being 13-15% and Hispanic women being 15% compared to only 8% Black women and 6% Hispanic women in the Q5 quintile (p < 0.001). In multivariate analysis, in the overall cohort, those who live in Q2 and Q1 quintile have inferior OS and DSS compared to those who live in Q5 quintile (OS:- Q2: Hazard Ratio (HR) 1.28, Q1: HR 1.2; DSS:- Q2: HR 1.33, Q1: HR 1.25, all p < 0.001). Conclusion Early-stage BC patients from areas with worse NDI have poor OS and DSS. Investments to improve the socioeconomic status of areas with high deprivation may help to reduce healthcare disparities and improve breast cancer outcomes.

Association Between Recreational Physical Activity and mTOR Signaling Pathway Protein Expression in Breast Tumor Tissue.

Physical activity (PA) is associated with decreased signaling in the mTOR pathway in animal models of mammary cancer, which may indicate favorable outcomes. We examined the association between PA and protein expression in the mTOR signaling pathway in breast tumor tissue. Data on 739 patients with breast cancer, among which 125 patients had adjacent-normal tissue, with tumor expression for mTOR, phosphorylated (p)-mTOR, p-AKT, and p-P70S6K were analyzed. Self-reported recreational PA levels during the year prior to diagnosis were classified using the Centers for Disease Control and Prevention guideline as sufficient (for moderate or vigorous) PA or insufficient PA (any PA but not meeting the guideline) or no PA. We performed linear models for mTOR protein and two-part gamma hurdle models for phosphorylated proteins. Overall, 34.8% of women reported sufficient PA; 14.2%, insufficient PA; 51.0%, no PA. Sufficient (vs. no) PA was associated with higher expression for p-P70S6K [35.8% increase; 95% confidence interval (CI), 2.6-80.2] and total phosphoprotein (28.5% increase; 95% CI, 5.8-56.3) among tumors with positive expression. In analyses stratified by PA intensity, sufficient versus no vigorous PA was also associated with higher expression levels of mTOR (beta = 17.7; 95% CI, 1.1-34.3) and total phosphoprotein (28.6% higher; 95% CI, 1.4-65.0 among women with positive expression) in tumors. The study found that guideline-concordant PA levels were associated with increased mTOR signaling pathway activity in breast tumors. Studying PA in relation to mTOR signaling in humans may need to consider the complexity of the behavioral and biological factors. Significance: PA increases energy expenditure and limits energy utilization in the cell, which can influence the mTOR pathway that is central to sensing energy influx and regulating cell growth. We studied exercise-mediated mTOR pathway activities in breast tumor and adjacent-normal tissue. Despite the discrepancies between animal and human data and the limitations of our approach, the findings provide a foundation to study the mechanisms of PA and their clinical implications.

mTOR pathway gene expression in association with race and clinicopathological characteristics in Black and White breast cancer patients.

BACKGROUND: Aberrant activation of the mammalian Target of Rapamycin (mTOR) pathway has been linked to obesity and endocrine therapy resistance, factors that may contribute to Black-White disparities in breast cancer outcomes. We evaluated associations of race and clinicopathological characteristics with mRNA expression of key mTOR pathway genes in breast tumors. METHODS: Surgical tumor tissue blocks were collected from 367 newly diagnosed breast cancer patients (190 Black and 177 White). Gene expression of AKT1, EIF4EBP1, MTOR, RPS6KB2, and TSC1 were quantified by NanoString nCounter. Differential gene expression was assessed using linear regression on log2-transformed values. Gene expression and DNA methylation data from TCGA were used for validation and investigation of race-related differences. RESULTS: Compared to White women, Black women had relative under-expression of AKT1 (log2 fold-change = - 0.31, 95% CI - 0.44, - 0.18) and RPS6KB2 (log2 fold-change = - 0.11, 95% CI - 0.19, - 0.03). Higher vs. lower tumor grade was associated with relative over-expression of EIF4EBP1 and RPS6KB2, but with lower expression of TSC1. Compared to luminal tumors, triple-negative tumors had relative under-expression of TSC1 (log2 fold-change = - 0.42, 95% CI - 0.22, - 0.01). The results were similar in the TCGA breast cancer dataset. Post-hoc analyses identified differential CpG methylation within the AKT1 and RPS6KB2 locus between Black and White women. CONCLUSIONS: Over-expression of RPS6KB2 and EIF4EBP1 and under-expression of TSC1 might be indicators of more aggressive breast cancer phenotypes. Differential expression of AKT1 and RPS6KB2 by race warrants further investigation to elucidate their roles in racial disparities of treatment resistance and outcomes between Black and White women with breast cancer.

Identification of a Subset of Stage I Colorectal Cancer Patients With High Recurrence Risk.

BACKGROUND: A challenge in early-stage colorectal cancer (CRC) is identifying biomarkers that predict an increased risk for recurrence. A potential clinically adaptable biomarker is focal adhesion kinase (FAK), a tyrosine kinase that promotes invasion and metastasis. METHODS: An initial, single-institution, 298-patient cohort with all stages of CRC and long-term follow-up was assessed for FAK with tissue microarrays using immunohistochemistry. FAK expression was scored and dichotomized into high and low. Subsequently, a validation cohort of 517 early-stage CRCs from a separate institution was evaluated. All statistical tests were 2-sided. RESULTS: FAK overexpression did not correlate with any known histologic feature and was an early event in CRC, increasing from normal colon to stage I, and stage I to II, but not different at higher stages. High FAK was associated with decreased 10-year recurrence-free survival (RFS) among stage I patients (70.2% for high FAK vs 94.1% for low, P = .02), but not among higher stages in the initial cohort. The same finding was seen in the validation cohort (73.1% for high FAK vs 93.1% for low, P = .004). Multivariable survival analysis for stage I patients showed only two statistically significant factors predicting RFS: FAK (hazard ratio = 5.27, 95% confidence interval = 1.81 to 15.33, P = .002) and perineural invasion (hazard ratio = 7.38, 95% confidence interval = 1.01 to 53.96, P = .049). FAK was the only statistically significant factor in multivariable analysis across RFS, overall, and disease-specific survivals. CONCLUSIONS: High FAK expression identified a subset of stage I CRC patients with high incidence of recurrence and reduced survival, suggesting that FAK has important prognostic value. These patients would immediately benefit from more rigorous surveillance protocols for recurrent disease.

Multiplexed digital spatial profiling of invasive breast tumors from Black and White women.

The NanoString GeoMx digital spatial profiling is a new multiplexed platform that quantifies the abundance of tumor- and immune-related proteins in a spatially resolved manner. We performed DSP for the simultaneous assessment of 52 analytes within spatially resolved tissue compartments defined by pan-cytokeratin expression. We compared protein targets between 94 African American/Black and 65 European American/White cases, tumor and stromal tissue compartments, estrogen receptor alpha (ER)-positive and ER-negative cases, and explored potential biomarkers of survival. Of 33 analytes with robust signal for analysis, results were highly replicable. For a subset of markers, correlative analyses between DSP analytes and traditional immunohistochemistry scores revealed moderate to very strong associations between the two platforms. Similarly, DSP analytes and gene expression scores were concordant for 21 of 25 markers with overlap between the two datasets. Several analytes varied by ER status, and across the 25 immune markers surveyed, 14 had a significant inverse association with ER expression. B7 homolog 3 (B7-H3; encoded by CD276) was the only analyte to show a significant difference by race, being lower in both the tumor and stromal compartments in Black women. DSP markers that were associated with survival included CD8, CD25, CD56, CD127, EpCAM, ER, Ki-67, and STING. We conclude that DSP is an efficient tool for screening tumor- and immune-related markers in a simultaneous fashion and yields results that are concordant with established immune profiling assays. DSP immune analytes were inversely associated with ER expression, in agreement with a substantial body of previous work that documents higher immune infiltration in ER-negative breast cancers. This technology revealed that scores of the B7-H3 protein were significantly lower in breast cancers from Black women compared with White women, an intriguing finding that requires replication in independent and racially diverse female populations.

Genetic Variants in COX2 and ALOX Genes and Breast Cancer Risk in White and Black Women.

COX and ALOX genes are involved in inflammatory processes and that may be related to breast cancer risk differentially between White and Black women. We evaluated distributions of genetic variants involved in COX2 and ALOX-related pathways and examined their associations with breast cancer risk among 1,275 White and 1,299 Black cases and controls who participated in the Women's Circle of Health Study. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multivariable-adjusted logistic regression models. Our results showed differential associations of certain genetic variants with breast cancer according to menopausal and ER status in either White or Black women. In White women, an increased risk of breast cancer was observed for COX2-rs689470 (OR: 2.02, P = 0.01) in the dominant model, and was strongest among postmenopausal women (OR: 2.72, P = 0.02) and for estrogen receptor positive (ER+) breast cancers (OR: 2.60, P = 0.001). A reduced risk was observed for ALOX5-rs7099874 (OR: 0.75, P = 0.01) in the dominant model, and was stronger among postmenopausal women (OR: 0.68, P = 0.03) and for ER+ cancer (OR: 0.66, P = 0.001). Four SNPs (rs3840880, rs1126667, rs434473, rs1042357) in the ALOX12 gene were found in high LD (r2 >0.98) in White women and were similarly associated with reduced risk of breast cancer, with a stronger association among postmenopausal women and for ER- cancer. Among Black women, increased risk was observed for ALOX5-rs1369214 (OR: 1.44, P = 0.003) in the recessive model and was stronger among premenopausal women (OR: 1.57, P = 0.03) and for ER+ cancer (OR: 1.53, P = 0.003). Our study suggests that genetic variants of COX2 and ALOX genes are associated with breast cancer, and that these associations and genotype distributions differ in subgroups defined by menopausal and ER status between White and Black women. Findings may provide insights into the etiology of breast cancer and areas for further research into reasons for breast cancer differences between races.

Body fatness and breast cancer risk in relation to phosphorylated mTOR expression in a sample of predominately Black women.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway promoted by positive energy imbalance and insulin-like growth factors can be a mechanism by which obesity influences breast cancer risk. We evaluated the associations of body fatness with the risk of breast cancer varied with phosphorylated (p)-mTOR protein expression, an indication of the pathway activation. METHODS: Women with newly diagnosed breast cancer (n = 715; 574 [80%] Black and 141 [20%] White) and non-cancer controls (n = 1983; 1280 [64%] Black and 713 [36%] White) were selected from the Women's Circle of Health Study. Surgical tumor samples among the cases were immunostained for p-mTOR (Ser2448) and classified as p-mTOR-overexpressed, if the expression level ≥ 75th percentile, or p-mTOR-negative/low otherwise. Anthropometrics were measured by trained staff, and body composition was determined by bioelectrical impedance analysis. Odds ratios (ORs) of p-mTOR-overexpressed tumors and p-mTOR-negative/low tumors compared to controls were estimated using polytomous logistic regression. The differences in the associations by the p-mTOR expression status were assessed by tests for heterogeneity. RESULTS: Cases with p-mTOR-overexpressed tumors, but not cases with p-mTOR-negative/low tumors, compared to controls were more likely to have higher body mass index (BMI), percent body fat, and fat mass index (P-heterogeneity < 0.05), although the OR estimates were not significant. For the measurement of central adiposity, cases with p-mTOR overexpressed tumors had a higher odds of being at the Q3 (OR = 2.52, 95% CI = 1.46 to 4.34) and Q4 (OR = 1.99, 95% CI = 1.12 to 3.50) of waist circumference (WC) compared to controls. Similarly, cases with p-mTOR overexpressed tumors had a higher odds of being at the Q3 (OR = 1.82, 95% CI = 1.11 to 2.98) and Q4 (OR = 1.81, 95% CI = 1.11 to 2.98) of WHR compared to controls. These associations of WC and waist-to-hip ratio (WHR) did not differ by tumor p-mTOR status (P-heterogeneity = 0.27 and 0.48, respectively). CONCLUSIONS: Our findings suggest that in this population composed of predominately Black women, body fatness is associated with breast cancer differently for p-mTOR overexpression and p-mTOR negative/low expression. Whether mTOR plays a role in the obesity and breast cancer association warrants confirmation by prospective studies.

Epidemiology of Basal-like and Luminal Breast Cancers among Black Women in the AMBER Consortium.

BACKGROUND: Evidence suggests etiologic heterogeneity among breast cancer subtypes. Previous studies with six-marker IHC classification of intrinsic subtypes included small numbers of black women. METHODS: Using centralized laboratory results for estrogen receptor (ER), progesterone receptor, HER2, proliferation marker, Ki-67, EGFR, and cytokeratin (CK)5/6, we estimated case-only and case-control ORs for established breast cancer risk factors among cases (n = 2,354) and controls (n = 2,932) in the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. ORs were estimated by ER status and intrinsic subtype using adjusted logistic regression. RESULTS: Case-only analyses by ER status showed etiologic heterogeneity by age at menarche, parity (vs. nulliparity), and age at first birth. In case-control analyses for intrinsic subtype, increased body mass index and waist-to-hip ratio (WHR) were associated with increased risk of luminal A subtype, whereas older age at menarche and parity, regardless of breastfeeding, were associated with reduced risk. For basal-like cancers, parity without breastfeeding and increasing WHR were associated with increased risk, whereas breastfeeding and age ≥25 years at first birth were associated with reduced risk among parous women. Basal-like and ER-/HER2+ subtypes had earlier age-at-incidence distribution relative to luminal subtypes. CONCLUSIONS: Breast cancer subtypes showed distinct etiologic profiles in the AMBER consortium, a study of more than 5,000 black women with centrally assessed tumor biospecimens. IMPACT: Among black women, high WHR and parity without breastfeeding are emerging as important intervention points to reduce the incidence of basal-like breast cancer.

Gene expression of adipokines and adipokine receptors in the tumor microenvironment: associations of lower expression with more aggressive breast tumor features.

PURPOSE: Limited epidemiologic data are available on the expression of adipokines leptin (LEP) and adiponectin (ADIPOQ) and adipokine receptors (LEPR, ADIPOR1, ADIPOR2) in the breast tumor microenvironment (TME). The associations of gene expression of these biomarkers with tumor clinicopathology are not well understood. METHODS: NanoString multiplexed assays were used to assess the gene expression levels of LEP, LEPR, ADIPOQ, ADIPOR1, and ADIPOR2 within tumor tissues among 162 Black and 55 White women with newly diagnosed breast cancer. Multivariate mixed effects models were used to estimate associations of gene expression with breast tumor clinicopathology (overall and separately among Blacks). RESULTS: Black race was associated with lower gene expression of LEPR (P = 0.002) and ADIPOR1 (P = 0.01). Lower LEP, LEPR, and ADIPOQ gene expression were associated with higher tumor grade (P = 0.0007, P < 0.0001, and P < 0.0001, respectively) and larger tumor size (P < 0.0001, P = 0.0005, and P < 0.0001, respectively). Lower ADIPOQ expression was associated with ER- status (P = 0.0005), and HER2-enriched (HER2-E; P = 0.0003) and triple-negative (TN; P = 0.002) subtypes. Lower ADIPOR2 expression was associated with Ki67+ status (P = 0.0002), ER- status (P < 0.0001), PR- status (P < 0.0001), and TN subtype (P = 0.0002). Associations of lower adipokine and adipokine receptor gene expression with ER-, HER2-E, and TN subtypes were confirmed using data from The Cancer Genome Atlas (P-values < 0.005). CONCLUSION: These findings suggest that lower expression of ADIPOQ, ADIPOR2, LEP, and LEPR in the breast TME might be indicators of more aggressive breast cancer phenotypes. Validation of these findings are warranted to elucidate the role of the adipokines and adipokine receptors in long-term breast cancer prognosis.

Body fatness and mTOR pathway activation of breast cancer in the Women's Circle of Health Study.

Energy imbalance has an important role in breast cancer prognosis. Hyperactive mechanistic Target of Rapamycin (mTOR) pathway is associated with breast tumor growth, but the extent to which body fatness is associated with mTOR pathway activities in breast cancer is unclear. We performed immunostaining for mTOR, phosphorylated (p)-mTOR, p-AKT, and p-p70S6K in tumor tissue from 590 women (464 African Americans/Blacks and 126 Whites) with newly diagnosed invasive breast cancer in the Women's Circle of Health Study. Anthropometric measures were taken by study staff, and body composition was measured by bioelectrical impedance analysis. Linear regressions were used to estimate percent differences in protein expression between categories of body mass index (BMI), waist circumference, waist/hip ratio, fat mass, fat mass index, and percent body fat. We observed that BMI ≥ 35.0 vs. <25 kg/m2 was associated with 108.3% (95% CI = 16.9%-270.9%) and 101.8% (95% CI = 17.0%-248.8%) higher expression in p-mTOR and normalized p-mTOR, i.e., p-mTOR/mTOR, respectively. Quartiles 4 vs. 1 of waist/hip ratio was associated with 41.8% (95% CI = 5.81%-89.9%) higher mTOR expression. Similar associations were observed for the body fat measurements, particularly in patients with estrogen receptor-negative (ER-) tumors, but not in those with ER+ tumors, although the differences in associations were not significant. This tumor-based study found positive associations between body fatness and mTOR pathway activation, evident by a p-mTOR expression, in breast cancer. Our findings suggest that mTOR inhibition can be a treatment strategy to prevent the recurrence of these tumors in obese individuals.

Racial differences in CD8+ T cell infiltration in breast tumors from Black and White women.

BACKGROUND: African American/Black women with breast cancer have poorer survival than White women, and this disparity persists even after adjusting for non-biological factors. Differences in tumor immune biology have been reported between Black and White women, and the tumor immune milieu could potentially drive racial differences in breast cancer etiology and outcome. METHODS: We examined the association of CD8+ cytotoxic T cells with clinical-pathological variables in the Women's Circle of Health Study (WCHS) population of predominantly Black breast cancer patients. We evaluated 688 invasive breast tumor samples (550 Black, 138 White) using immunohistochemical staining of tissue microarray slides. CD8+ T cells were scored for each patient tumor sample with digital image analysis. RESULTS: Black women had a significantly higher percentage of high-grade, estrogen receptor (ER)-negative, and triple-negative tumors than White women and significantly higher CD8+ T cell density (median 87.6/mm2 vs. 53.1/mm2; p < 0.001). Within the overall population and in the population of Black women only, CD8+ T cell density was significantly higher in younger patients and patients with high-grade and ER/PR-negative tumors. No significant associations were observed between CD8+ T cell density and overall survival or breast cancer-specific survival in the overall population, or when Black patients were analyzed as a separate group. However, when stratified by subtype, Black women with triple-negative breast cancer and high CD8+ T cell density showed a trend towards better overall survival in comparison with patients with low CD8+ T cell density (HR = 0.51; 95% CI 0.25-1.04). CONCLUSIONS: Our data raise the possibility that distinct mechanisms of immune cell action may occur in different racial groups.

Somatic mutations of triple-negative breast cancer: a comparison between Black and White women.

PURPOSE: Understanding the contribution of tumor genome biology to racial disparities of triple-negative breast cancer (TNBC) is important for narrowing the cancer mortality gap between Black and White women. METHODS: We evaluated tumor somatic mutations using targeted sequencing of a customized panel of 151 genes and 15 copy number variations (CNVs) within a population of 133 TNBC patients, including 71 Black and 62 White women. RESULTS: The overall mutational burden between Black and White women with TNBC was not significantly different, with a median of 5 somatic changes per patient (point mutations and CNVs combined) for the customized panel (range 1-31 for Blacks vs. 1-26 for Whites; p = 0.76). Of the 151 genes examined, none were mutated at a significantly higher frequency in Black than in White cases, whereas two genes were mutated at a higher frequency in White cases-PIK3CA and NCOR1. No significant difference in the frequency of CNVs was observed between Black and White women with TNBC in our study population. CONCLUSION: Of gene mutations and CNVs in TNBC tumors from Black and White women, only PIK3CA and NCOR1 had significantly different, although slight, frequencies by race. These results indicate that overall differences observed in the mutation spectra between Black and White women with breast cancer are likely due to the differential distributions of breast cancer subtypes by race.

Immunohistochemical analysis of adipokine and adipokine receptor expression in the breast tumor microenvironment: associations of lower leptin receptor expression with estrogen receptor-negative status and triple-negative subtype.

BACKGROUND: The molecular mechanisms underlying the association between increased adiposity and aggressive breast cancer phenotypes remain unclear, but likely involve the adipokines, leptin (LEP) and adiponectin (ADIPOQ), and their receptors (LEPR, ADIPOR1, ADIPOR2). METHODS: We used immunohistochemistry (IHC) to assess LEP, LEPR, ADIPOQ, ADIPOR1, and ADIPOR2 expression in breast tumor tissue microarrays among a sample of 720 women recently diagnosed with breast cancer (540 of whom self-identified as Black). We scored IHC expression quantitatively, using digital pathology analysis. We abstracted data on tumor grade, tumor size, tumor stage, lymph node status, Ki67, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) from pathology records, and used ER, PR, and HER2 expression data to classify breast cancer subtype. We used multivariable mixed effects models to estimate associations of IHC expression with tumor clinicopathology, in the overall sample and separately among Blacks. RESULTS: Larger proportions of Black than White women were overweight or obese and had more aggressive tumor features. Older age, Black race, postmenopausal status, and higher body mass index were associated with higher LEPR IHC expression. In multivariable models, lower LEPR IHC expression was associated with ER-negative status and triple-negative subtype (P < 0.0001) in the overall sample and among Black women only. LEP, ADIPOQ, ADIPOR1, and ADIPOR2 IHC expression were not significantly associated with breast tumor clinicopathology. CONCLUSIONS: Lower LEPR IHC expression within the breast tumor microenvironment might contribute mechanistically to inter-individual variation in aggressive breast cancer clinicopathology, particularly ER-negative status and triple-negative subtype.

The Women's Circle of Health Follow-Up Study: a population-based longitudinal study of Black breast cancer survivors in New Jersey.

PURPOSE: The Women's Circle of Health Follow-Up Study is an ongoing longitudinal study of African American/Black breast cancer survivors in New Jersey, specifically designed to evaluate the impact of obesity and related comorbidities on breast cancer survival and health-related quality-of-life in this understudied population. Here, we describe our recruitment and data collection methods and compare characteristics of the overall cohort and the subcohort with follow-up data. METHODS: Newly diagnosed breast cancer cases have been recruited into the study since 2006. Pre-diagnosis data on relevant factors and a saliva sample are collected during an in-person interview within 12 months from diagnosis. In 2013, we began active follow up by recontacting participants annually, including two home visits at approximately 2 and 3 years post-diagnosis, during which blood samples are collected. Mortality outcomes (all-cause and breast cancer-specific mortality) are ascertained through linkage with New Jersey State Cancer Registry files. We expect to assemble a cohort of over 2000 Black breast cancer survivors with at least 800 of them having detailed post-diagnosis data. RESULTS: Distribution of sociodemographic characteristics, body mass index, comorbidities, clinicopathologic characteristics, and treatment modalities were very similar between those in the full cohort and the subset with follow-up data and blood samples. Obesity (> 50%), hypertension (> 58%), and diabetes (22%) were common in this population. CONCLUSIONS AND IMPLICATIONS FOR CANCER SURVIVORS: This ongoing longitudinal study represents a unique resource to better understand breast cancer outcomes, patient-reported symptoms, and health-related quality of life among Black breast cancer survivors.

Storage Conditions and Immunoreactivity of Breast Cancer Subtyping Markers in Tissue Microarray Sections.

Loss of immunoreactivity in tissue sections has been shown to occur when slide sections are stored at room temperature for prolonged periods of time. We conducted a systematic investigation to determine the extent of staining loss in various storage conditions to determine an optimal storage method. We investigated 6 antibodies that are commonly used for breast cancer subtyping in research studies with immunohistochemistry (ER, PR, HER2, CK5/6, EGFR, and Ki67) in formalin-fixed paraffin-embedded breast tissue microarrays consisting of 148 patients. Tissue microarrays were sectioned at various time points: fresh, 1 week, 1 month, 6 months, and 12 months before staining. Slides sectioned at each time point were stored in 5 storage conditions: desiccator, paraffin dipped, 4°C, -20°C, and -80°C. Immunohistochemistry scores were assessed over time with McNemar Test and Bowker Test of Symmetry. Desiccator storage was the only storage condition that did not show any loss in immunoreactivity for any antibody or time point in our study. Paraffin coated slides were the most difficult storage method operationally and also showed the most loss in immunoreactivity. Storing sections in a desiccator was the most effective method for minimizing immunoreactivity loss. Cold storage at 4°C is an intermediate option that is not as protective as a desiccator, but offers the advantage of being accessible to virtually all research labs.

FOXA1 Protein Expression in ER+ and ER- Breast Cancer in Relation to Parity and Breastfeeding in Black and White Women.

BACKGROUND: Forkhead box protein A1 (FOXA1) promotes luminal differentiation, and hypermethylation of the gene can be a mechanism of developing estrogen receptor-negative (ER-) breast cancer. We examined FOXA1 in breast tumor and adjacent normal tissue in relation to reproductive factors, particularly higher parity and no breastfeeding, that are associated with ER- tumors. METHODS: We performed IHC for FOXA1 in breast tumors (n = 1,329) and adjacent normal tissues (n = 298) in the Women's Circle of Health Study (949 Blacks and 380 Whites). Protein expression levels were summarized by histology (H) scores. Generalized linear models were used to assess FOXA1 protein expression in relation to reproductive factors by ER status. RESULTS: ER-positive (ER+) versus ER- tumors had higher FOXA1 protein expression (P < 0.001). FOXA1 expression was higher in tumor versus paired adjacent normal tissue in women with ER+ or non-triple-negative cancer (both P < 0.001), but not in those with ER- or triple-negative cancer. Higher number of births (1, 2, and 3+) was associated with lower FOXA1 protein expression in ER+ tumors [differences in H score, or ß = -8.5; 95% confidence interval (CI), -15.1 to -2.0], particularly among parous women who never breastfed (ß = -10.4; 95% CI, -19.7 to -1.0), but not among those who breastfed (ß = -7.5; 95% CI, -16.9 to 1.8). The associations for ER- tumors were similar, although they were not statistically significant. CONCLUSIONS: In this tumor-based study, higher parity was associated with lower FOXA1 expression in ER+ tumors, and breastfeeding may ameliorate the influence. IMPACT: These findings contribute to our understanding of FOXA1 methylation and breast cancer etiology.

Unbiased Lipidomic Profiling of Triple-Negative Breast Cancer Tissues Reveals the Association of Sphingomyelin Levels with Patient Disease-Free Survival.

The reprogramming of lipid metabolism is a hallmark of many cancers that has been shown to promote breast cancer progression. While several lipid signatures associated with breast cancer aggressiveness have been identified, a comprehensive lipidomic analysis specifically targeting the triple-negative subtype of breast cancer (TNBC) may be required to identify novel biomarkers and therapeutic targets for this most aggressive subtype of breast cancer that still lacks effective therapies. In this current study, our global LC-MS-based lipidomics platform was able to measure 684 named lipids across 15 lipid classes in 70 TNBC tumors. Multivariate survival analysis found that higher levels of sphingomyelins were significantly associated with better disease-free survival in TNBC patients. Furthermore, analysis of publicly available gene expression datasets identified that decreased production of ceramides and increased accumulation of sphingoid base intermediates by metabolic enzymes were associated with better survival outcomes in TNBC patients. Our LC-MS lipidomics profiling of TNBC tumors has, for the first time, identified sphingomyelins as a potential prognostic marker and implicated enzymes involved in sphingolipid metabolism as candidate therapeutic targets that warrant further investigation.