Xuezheng Sun

Relationship of Mammographic Density and Gene Expression: Analysis of Normal Breast Tissue Surrounding Breast Cancer

Purpose: Previous studies of breast tissue gene expression have shown that the extratumoral microenvironment has substantial variability across individuals, some of which can be attributed to epidemiologic factors. To evaluate how mammographic density and breast tissue composition relate to extratumoral microenvironment gene expression, we used data on 121 patients with breast cancer from the population-based Polish Womens Breast Cancer Study. Experimental Design: Breast cancer cases were classified on the basis of a previously reported, biologically defined extratumoral gene expression signature with two subtypes: an Active subtype, which is associated with high expression of genes related to fibrosis and wound response, and an Inactive subtype, which has high expression of cellular adhesion genes. Mammographic density of the contralateral breast was assessed using pretreatment mammograms and a quantitative, reliable computer-assisted thresholding method. Breast tissue composition was evaluated on the basis of digital image analysis of tissue sections. Results: The Inactive extratumoral subtype was associated with significantly higher percentage mammographic density (PD) and dense area (DA) in univariate analysis (PD: P = 0.001; DA: P = 0.049) and in multivariable analyses adjusted for age and body mass index (PD: P = 0.004; DA: P = 0.049). Inactive/higher mammographic density tissue was characterized by a significantly higher percentage of stroma and a significantly lower percentage of adipose tissue, with no significant change in epithelial content. Analysis of published gene expression signatures suggested that Inactive/higher mammographic density tissue expressed increased estrogen response and decreased TGF-β signaling. Conclusions: By linking novel molecular phenotypes with mammographic density, our results indicate that mammographic density reflects broad transcriptional changes, including changes in both epithelia- and stroma-derived signaling. Clin Cancer Res; 19(18); 4972–82. ©2013 AACR.

Performance of three-biomarker immunohistochemistry for intrinsic breast cancer subtyping in the AMBER consortium

Background: Classification of breast cancer into intrinsic subtypes has clinical and epidemiologic importance. To examine accuracy of IHC-based methods for identifying intrinsic subtypes, a three-biomarker IHC panel was compared with the clinical record and RNA-based intrinsic (PAM50) subtypes. Methods: Automated scoring of estrogen receptor (ER), progesterone receptor (PR), and HER2 was performed on IHC-stained tissue microarrays comprising 1,920 cases from the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. Multiple cores (1–6/case) were collapsed to classify cases, and automated scoring was compared with the clinical record and to RNA-based subtyping. Results: Automated analysis of the three-biomarker IHC panel produced high agreement with the clinical record (93% for ER and HER2, and 88% for PR). Cases with low tumor cellularity and smaller core size had reduced agreement with the clinical record. IHC-based definitions had high agreement with the clinical record regardless of hormone receptor positivity threshold (1% vs. 10%), but a 10% threshold produced highest agreement with RNA-based intrinsic subtypes. Using a 10% threshold, IHC-based definitions identified the basal-like intrinsic subtype with high sensitivity (86%), although sensitivity was lower for luminal A, luminal B, and HER2-enriched subtypes (76%, 40%, and 37%, respectively). Conclusion: Three-biomarker IHC-based subtyping has reasonable accuracy for distinguishing basal-like from nonbasal-like, although additional biomarkers are required for accurate classification of luminal A, luminal B, and HER2-enriched cancers. Impact: Epidemiologic studies relying on three-biomarker IHC status for subtype classification should use caution when distinguishing luminal A from luminal B and when interpreting findings for HER2-enriched cancers. Cancer Epidemiol Biomarkers Prev; 25(3); 470–8. ©2015 AACR.

Tumor Intrinsic Subtype Is Reflected in Cancer-Adjacent Tissue

Introduction: Overall survival of early-stage breast cancer patients is similar for those who undergo breast-conserving therapy (BCT) and mastectomy; however, 10% to 15% of women undergoing BCT suffer ipsilateral breast tumor recurrence. The risk of recurrence may vary with breast cancer subtype. Understanding the gene expression of the cancer-adjacent tissue and the stromal response to specific tumor subtypes is important for developing clinical strategies to reduce recurrence risk. Methods: We utilized two independent datasets to study gene expression data in cancer-adjacent tissue from invasive breast cancer patients. Complementary in vitro cocultures were used to study cell–cell communication between fibroblasts and specific breast cancer subtypes. Results: Our results suggest that intrinsic tumor subtypes are reflected in histologically normal cancer-adjacent tissue. Gene expression of cancer-adjacent tissues shows that triple-negative (Claudin-low or basal-like) tumors exhibit increased expression of genes involved in inflammation and immune response. Although such changes could reflect distinct immune populations present in the microenvironment, altered immune response gene expression was also observed in cocultures in the absence of immune cell infiltrates, emphasizing that these inflammatory mediators are secreted by breast-specific cells. In addition, although triple-negative breast cancers are associated with upregulated immune response genes, luminal breast cancers are more commonly associated with estrogen-response pathways in adjacent tissues. Conclusions: Specific characteristics of breast cancers are reflected in the surrounding histologically normal tissue. This commonality between tumor and cancer-adjacent tissue may underlie second primaries and local recurrences. Impact: Biomarkers derived from cancer-adjacent tissue may be helpful in defining personalized surgical strategies or in predicting recurrence risk. Cancer Epidemiol Biomarkers Prev; 24(2); 406–14. ©2014 AACR.

Benign Breast Tissue Composition in Breast Cancer Patients: Association with Risk Factors, Clinical Variables, and Gene Expression

Background: Breast tissue composition (epithelium, non-fatty stroma, and adipose) changes qualitatively and quantitatively throughout the lifespan, and may mediate relationships between risk factors and breast cancer initiation. We sought to identify relationships between tissue composition, risk factors, tumor characteristics, and gene expression. Methods: Participants were 146 patients from the Polish Breast Cancer Study, with data on risk factor and clinicopathological characteristics. Benign breast tissue composition was evaluated using digital image analysis of histologic sections. Whole-genome microarrays were performed on the same tissue blocks. Results: Mean epithelial, non-fatty stromal, and adipose proportions were 8.4% (SD = 4.9%), 27.7% (SD = 24.0%), and 64.0% (SD = 24.0%), respectively. Among women <50 years old, stroma proportion decreased and adipose proportion increased with age, with approximately 2% difference per year (P < 0.01). The variation in epithelial proportion with age was modest (0.1% per year). Higher epithelial proportion was associated with obesity (7.6% in nonobese vs. 10.1% in obese; P = 0.02) and with poorly differentiated tumors (7.8% in well/moderate vs. 9.9% in poor; P = 0.05). Gene expression signatures associated with epithelial and stromal proportion were identified and validated. Stroma-associated genes were in metabolism and stem cell maintenance pathways, whereas epithelial genes were enriched for cytokine and immune response pathways. Conclusions: Breast tissue composition was associated with age, body mass index, and tumor grade, with consequences for breast gene expression. Impact: Breast tissue morphologic factors may influence breast cancer etiology. Composition and gene expression may act as biomarkers of breast cancer risk and progression. Cancer Epidemiol Biomarkers Prev; 23(12); 2810–8. ©2014 AACR.

Parity-related molecular signatures and breast cancer subtypes by estrogen receptor status

IntroductionRelationships of parity with breast cancer risk are complex. Parity is associated with decreased risk of postmenopausal hormone receptor–positive breast tumors, but may increase risk for basal-like breast cancers and early-onset tumors. Characterizing parity-related gene expression patterns in normal breast and breast tumor tissues may improve understanding of the biological mechanisms underlying this complex pattern of risk.MethodsWe developed a parity signature by analyzing microRNA microarray data from 130 reduction mammoplasty (RM) patients (54 nulliparous and 76 parous). This parity signature, together with published parity signatures, was evaluated in gene expression data from 150 paired tumors and adjacent benign breast tissues from the Polish Breast Cancer Study, both overall and by tumor estrogen receptor (ER) status.ResultsWe identified 251 genes significantly upregulated by parity status in RM patients (parous versus nulliparous; false discovery rate = 0.008), including genes in immune, inflammation and wound response pathways. This parity signature was significantly enriched in normal and tumor tissues of parous breast cancer patients, specifically in ER-positive tumors.ConclusionsOur data corroborate epidemiologic data, suggesting that the etiology and pathogenesis of breast cancers vary by ER status, which may have implications for developing prevention strategies for these tumors.

Racial Differences in PAM50 Subtypes in the Carolina Breast Cancer Study

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.

Association of Parity and Time since Last Birth with Breast Cancer Prognosis by Intrinsic Subtype

Background: Parity and time since last birth influence breast cancer risk and vary by intrinsic tumor subtype, but the independent effects of these factors on prognosis have received limited attention. Methods: Study participants were 1,140 invasive breast cancer patients from phases I and II of the population-based Carolina Breast Cancer Study, with tissue blocks available for subtyping using immunohistochemical markers. Breast cancer risk factors, including pregnancy history, were collected via in-person interviews administered shortly after diagnosis. Vital status was determined using the National Death Index. The association of parity and birth recency with breast cancer–specific and overall survival was assessed using Cox proportional hazards models. Results: During follow-up (median = 13.5 years), 450 patients died, 61% due to breast cancer (n = 276). High parity (3+ births) and recent birth (<5 years before diagnosis) were positively associated with breast cancer–specific mortality, independent of age, race, and selected socioeconomic factors [parity, reference = nulliparous, adjusted HR = 1.76; 95% confidence interval (CI) = 1.13–2.73; birth recency, reference = 10+ years, adjusted HR = 1.29; 95% CI, 0.79–2.11]. The associations were stronger among patients with luminal tumors and those surviving longer than 5 years. Conclusions: Parity and recent birth are associated with worse survival among breast cancer patients, particularly among luminal breast cancers and long-term survivors. Impact: The biologic effects of parity and birth recency may extend from etiology to tumor promotion and progression. Cancer Epidemiol Biomarkers Prev; 25(1); 60–67. ©2015 AACR.

Pollen concentration and asthma exacerbations in Wake County, North Carolina, 2006–2012

Pollen has been generally linked to an increased risk for asthma exacerbation. However, the delayed effect (lag), the length of effect duration, and the association heterogeneity by pollen types have not been well characterized. Short-term associations between ambient concentration of various pollen types (tree, grass, and weed) and emergency department (ED) visits for asthma were assessed using data in Wake County, North Carolina, during 2006-2012. Distributed lag nonlinear models (DLNM) were used to characterize the associations, while adjusting for air pollutants, meteorological, and temporal factors. A strong association between same-day tree pollen and asthma ED visits was detected. This association lasted four days, with a 4-day cumulative risk ratio (RR) up to 2.10 (3500 grains/m(3) vs. 0 grains/m(3), 95% confidence interval [CI]=1.21-3.65). The associations of asthma ED visits with weed pollen and grass pollen were weak, suggestively starting from lag 2 and lasting 3 days, with the strongest association a 3-day cumulative RR of 1.08 (32 grains/m(3) vs. 0 grains/m(3), 95% CI=1.01-1.15) and 1.05 (11 grains/m(3) vs. 0 grains/m(3), 95% CI=1.00-1.11). Our results indicate that the association of ambient pollen and asthma exacerbation vary by pollen type, both quantitatively and temporally. These findings have important implications for optimizing targeted allergic disease prevention and management, and helping understand the etiology of ambient exposure-induced allergic diseases.

Race-associated biological differences among luminal A and basal-like breast cancers in the Carolina Breast Cancer Study

BackgroundWe examined racial differences in the expression of eight genes and their associations with risk of recurrence among 478 white and 495 black women who participated in the Carolina Breast Cancer Study Phase 3.MethodsBreast tumor samples were analyzed for PAM50 subtype and for eight genes previously found to be differentially expressed by race and associated with breast cancer survival: ACOX2, MUC1, FAM177A1, GSTT2, PSPH, PSPHL, SQLE, and TYMS. The expression of these genes according to race was assessed using linear regression and each gene was evaluated in association with recurrence using Cox regression.ResultsCompared to white women, black women had lower expression of MUC1, a suspected good prognosis gene, and higher expression of GSTT2, PSPHL, SQLE, and TYMS, suspected poor prognosis genes, after adjustment for age and PAM50 subtype. High expression (greater than median versus less than or equal to median) of FAM177A1 and PSPH was associated with a 63% increase (hazard ratio (HR) = 1.63, 95% confidence interval (CI) = 1.09–2.46) and 76% increase (HR = 1.76, 95% CI = 1.15–2.68), respectively, in risk of recurrence after adjustment for age, race, PAM50 subtype, and ROR-PT score. Log2-transformed SQLE expression was associated with a 20% increase (HR = 1.20, 95% CI = 1.03–1.41) in recurrence risk after adjustment. A continuous multi-gene score comprised of eight genes was also associated with increased risk of recurrence among all women (HR = 1.11, 95% CI = 1.04–1.19) and among white (HR = 1.14, 95% CI = 1.03–1.27) and black (HR = 1.11, 95% CI = 1.02–1.20) women.ConclusionsRacial differences in gene expression may contribute to the survival disparity observed between black and white women diagnosed with breast cancer.

Pubertal and adult windows of susceptibility to a high animal fat diet in Trp53-null mammary tumorigenesis

Premenopausal breast cancer is associated with increased animal fat consumption among normal weight, but not overweight women (Farvid et al., 2014). Our previous findings in obesity-resistant BALB/c mice similarly showed promotion of carcinogen-induced mammary tumorigenesis by a diet high in saturated animal fat (HFD). This effect was specific to pubertal versus adult HFD. This study identifies the effects of HFD during puberty versus adulthood in Trp53-null transplant BALB/c mice and investigates its mechanism of enhancing tumorigenesis. Either pubertal or adult HFD is sufficient to increase incidence of Trp53-null mammary tumors. Puberty-restricted HFD exposure promoted tumor cell proliferation, increased angiogenesis, and increased recruitment of total and M2 macrophages in epithelial tumors. Adult-restricted exposure to HFD similarly increased proliferation, angiogenesis, recruitment of total and M2 macrophages, and additionally reduced apoptosis. Adult HFD also increased incidence of spindle cell carcinomas resembling claudin-low breast cancer, and thus adult HFD in the Trp53-null transplantation system may be a useful model for human claudin low breast cancer. Importantly, these results on Trp53-null and our prior studies on DMBA-induced mammary tumorigenesis demonstrate a pubertal window of susceptibility to the promotional effects of HFD, indicating the potential of early life dietary intervention to reduce breast cancer risk.