DNA methylation age in paired tumor and adjacent normal breast tissue in Chinese women with breast cancer.

BACKGROUND: Few studies have examined epigenetic age acceleration (AA), the difference between DNA methylation (DNAm) predicted age and chronological age, in relation to somatic genomic features in paired cancer and normal tissue, with less work done in non-European populations. In this study, we aimed to examine DNAm age and its associations with breast cancer risk factors, subtypes, somatic genomic profiles including mutation and copy number alterations and other aging markers in breast tissue of Chinese breast cancer (BC) patients from Hong Kong. METHODS: We performed genome-wide DNA methylation profiling of 196 tumor and 188 paired adjacent normal tissue collected from Chinese BC patients in Hong Kong (HKBC) using Illumina MethylationEPIC array. The DNAm age was calculated using Horvath's pan-tissue clock model. Somatic genomic features were based on data from RNA sequencing (RNASeq), whole-exome sequencing (WES), and whole-genome sequencing (WGS). Pearson's correlation (r), Kruskal-Wallis test, and regression models were used to estimate associations of DNAm AA with somatic features and breast cancer risk factors. RESULTS: DNAm age showed a stronger correlation with chronological age in normal (Pearson r = 0.78, P < 2.2e-16) than in tumor tissue (Pearson r = 0.31, P = 7.8e-06). Although overall DNAm age or AA did not vary significantly by tissue within the same individual, luminal A tumors exhibited increased DNAm AA (P = 0.004) while HER2-enriched/basal-like tumors exhibited markedly lower DNAm AA (P = < .0001) compared with paired normal tissue. Consistent with the subtype association, tumor DNAm AA was positively correlated with ESR1 (Pearson r = 0.39, P = 6.3e-06) and PGR (Pearson r = 0.36, P = 2.4e-05) gene expression. In line with this, we found that increasing DNAm AA was associated with higher body mass index (P = 0.039) and earlier age at menarche (P = 0.035), factors that are related to cumulative exposure to estrogen. In contrast, variables indicating extensive genomic instability, such as TP53 somatic mutations, high tumor mutation/copy number alteration burden, and homologous repair deficiency were associated with lower DNAm AA. CONCLUSIONS: Our findings provide additional insights into the complexity of breast tissue aging that is associated with the interaction of hormonal, genomic, and epigenetic mechanisms in an East Asian population.

Nitrated Polycyclic Aromatic Hydrocarbon (Nitro-PAH) Signatures and Somatic Mutations in Diesel Exhaust-Exposed Bladder Tumors.

BACKGROUND: Diesel exhaust is a complex mixture, including polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs (nitro-PAH), many of which are potent mutagens and possible bladder carcinogens. To explore the association between diesel exposure and bladder carcinogenesis, we examined the relationship between exposure and somatic mutations and mutational signatures in bladder tumors. METHODS: Targeted sequencing was conducted in bladder tumors from the New England Bladder Cancer Study. Using data on 797 cases and 1,418 controls, two-stage polytomous logistic regression was used to evaluate etiologic heterogeneity between bladder cancer subtypes and quantitative, lifetime estimates of respirable elemental carbon (REC), a surrogate for diesel exposure. Poisson regression was used to evaluate associations between REC and mutational signatures. RESULTS: We observed significant heterogeneity in the diesel-bladder cancer risk relationship, with a strong positive association among cases with high-grade, nonmuscle invasive TP53-mutated tumors compared with controls [ORTop Tertile vs.Unexposed, 4.8; 95% confidence interval (CI), 2.2-10.5; Ptrend < 0.001; Pheterogeneity = 0.002]. In muscle-invasive tumors, we observed a positive association between diesel exposure and the nitro-PAH signatures of 1,6-dintropyrene (RR, 1.93; 95% CI, 1.28-2.92) and 3-nitrobenzoic acid (RR, 1.97; 95% CI, 1.33-2.92). CONCLUSIONS: The relationship between diesel exhaust and bladder cancer was heterogeneous based on the presence of TP53 mutations in tumors, further supporting the link between PAH exposure and TP53 mutations in carcinogenesis. Future studies that can identify nitro-PAH signatures in exposed tumors are warranted to add human data supporting the link between diesel and bladder cancer. IMPACT: This study provides additional insight into the etiology and possible mechanisms related to diesel exhaust-induced bladder cancer.

Reproducibility and intratumoral heterogeneity of the PAM50 breast cancer assay.

BACKGROUND: The PAM50 assay is used routinely in clinical practice to determine breast cancer prognosis and management; however, research assessing how technical variation and intratumoral heterogeneity contribute to misclassification and reproducibility of these tests is limited. METHODS: We evaluated the impact of intratumoral heterogeneity on the reproducibility of results for the PAM50 assay by testing RNA extracted from formalin-fixed paraffin embedded breast cancer blocks sampled at distinct spatial locations. Samples were classified according to intrinsic subtype (Luminal A, Luminal B, HER2-enriched, Basal-like, or Normal-like) and risk of recurrence with proliferation score (ROR-P, high, medium, or low). Intratumoral heterogeneity and technical reproducibility (replicate assays on the same RNA) were assessed as percent categorical agreement between paired intratumoral and replicate samples. Euclidean distances between samples, calculated across the PAM50 genes and the ROR-P score, were compared for concordant vs. discordant samples. RESULTS: Technical replicates (N = 144) achieved 93% agreement for ROR-P group and 90% agreement on PAM50 subtype. For spatially distinct biological replicates (N = 40 intratumoral replicates), agreement was lower (81% for ROR-P and 76% for PAM50 subtype). The Euclidean distances between discordant technical replicates were bimodal, with discordant samples showing higher Euclidian distance and biologic heterogeneity. CONCLUSION: The PAM50 assay achieved very high technical reproducibility for breast cancer subtyping and ROR-P, but intratumoral heterogeneity is revealed by the assay in a small proportion of cases.

Moving Toward Findable, Accessible, Interoperable, Reusable Practices in Epidemiologic Research.

Data sharing is essential for reproducibility of epidemiologic research, replication of findings, pooled analyses in consortia efforts, and maximizing study value to address multiple research questions. However, barriers related to confidentiality, costs, and incentives often limit the extent and speed of data sharing. Epidemiological practices that follow Findable, Accessible, Interoperable, Reusable (FAIR) principles can address these barriers by making data resources findable with the necessary metadata, accessible to authorized users, and interoperable with other data, to optimize the reuse of resources with appropriate credit to its creators. We provide an overview of these principles and describe approaches for implementation in epidemiology. Increasing degrees of FAIRness can be achieved by moving data and code from on-site locations to remote, accessible ("Cloud") data servers, using machine-readable and nonproprietary files, and developing open-source code. Adoption of these practices will improve daily work and collaborative analyses and facilitate compliance with data sharing policies from funders and scientific journals. Achieving a high degree of FAIRness will require funding, training, organizational support, recognition, and incentives for sharing research resources, both data and code. However, these costs are outweighed by the benefits of making research more reproducible, impactful, and equitable by facilitating the reuse of precious research resources by the scientific community.

Prognostic significance of RNA-based TP53 pathway function among estrogen receptor positive and negative breast cancer cases.

TP53 and estrogen receptor (ER) are essential in breast cancer development and progression, but TP53 status (by DNA sequencing or protein expression) has been inconsistently associated with survival. We evaluated whether RNA-based TP53 classifiers are related to survival. Participants included 3213 women in the Carolina Breast Cancer Study (CBCS) with invasive breast cancer (stages I-III). Tumors were classified for TP53 status (mutant-like/wildtype-like) using an RNA signature. We used Cox proportional hazards models to estimate covariate-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for breast cancer-specific survival (BCSS) among ER- and TP53-defined subtypes. RNA-based results were compared to DNA- and IHC-based TP53 classification, as well as Basal-like versus non-Basal-like subtype. Findings from the diverse (50% Black), population-based CBCS were compared to those from the largely white METABRIC study. RNA-based TP53 mutant-like was associated with BCSS among both ER-negatives and ER-positives (HR (95% CI) = 5.38 (1.84-15.78) and 4.66 (1.79-12.15), respectively). Associations were attenuated when using DNA- or IHC-based TP53 classification. In METABRIC, few ER-negative tumors were TP53-wildtype-like, but TP53 status was a strong predictor of BCSS among ER-positives. In both populations, the effect of TP53 mutant-like status was similar to that for Basal-like subtype. RNA-based measures of TP53 status are strongly associated with BCSS and may have value among ER-negative cancers where few prognostic markers have been robustly validated. Given the role of TP53 in chemotherapeutic response, RNA-based TP53 as a prognostic biomarker could address an unmet need in breast cancer.

The Landscape of Immune Microenvironments in Racially Diverse Breast Cancer Patients.

BACKGROUND: Immunotherapy is a rapidly evolving treatment option in breast cancer; However, the breast cancer immune microenvironment is understudied in Black and younger (<50 years) patients. 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.

How to organise travel restrictions in the new future: lessons from the COVID-19 response in Hong Kong and Singapore.

It has been nearly 2 years since the first case of COVID-19 was reported. Governments worldwide have introduced numerous non-pharmaceutical interventions (NPIs) to combat this disease. Many of these NPIs were designed in response to initial outbreaks but are unsustainable in the long term. Governments are exploring how to adjust their current NPIs to resume normal activities while effectively protecting their population. As one of the most controversial NPIs, the implementation of travel restrictions varies across regions. Some governments have abandoned their previous travel restrictions because of the induced costs to society and on the economy. Other areas, including Hong Kong (Special Administrative Region of China) and Singapore, continue employing these NPIs as a long-term disease prevention tactic. However, the multidimensional impacts of travel restrictions require careful consideration of how to apply restrictions more appropriately. We have proposed an adapted framework to examine Hong Kong and Singapore's travel restrictions. We aimed to study these two regions' experiences in balancing disease control efforts with easing the burden on lives and livelihoods. Based on the experiences of Hong Kong and Singapore, we have outlined six policy recommendations to serve as the cornerstone for future research and policy practices.

Prospective evaluation of a breast-cancer risk model integrating classical risk factors and polygenic risk in 15 cohorts from six countries.

BACKGROUND: Rigorous evaluation of the calibration and discrimination of breast-cancer risk-prediction models in prospective cohorts is critical for applications under clinical guidelines. We comprehensively evaluated an integrated model incorporating classical risk factors and a 313-variant polygenic risk score (PRS) to predict breast-cancer risk. METHODS: Fifteen prospective cohorts from six countries with 239 340 women (7646 incident breast-cancer cases) of European ancestry aged 19-75 years were included. Calibration of 5-year risk was assessed by comparing expected and observed proportions of cases overall and within risk categories. Risk stratification for women of European ancestry aged 50-70 years in those countries was evaluated by the proportion of women and future cases crossing clinically relevant risk thresholds. RESULTS: Among women <50 years old, the median (range) expected-to-observed ratio for the integrated model across 15 cohorts was 0.9 (0.7-1.0) overall and 0.9 (0.7-1.4) at the highest-risk decile; among women ≥50 years old, these were 1.0 (0.7-1.3) and 1.2 (0.7-1.6), respectively. The proportion of women identified above a 3% 5-year risk threshold (used for recommending risk-reducing medications in the USA) ranged from 7.0% in Germany (∼841 000 of 12 million) to 17.7% in the USA (∼5.3 of 30 million). At this threshold, 14.7% of US women were reclassified by adding the PRS to classical risk factors, with identification of 12.2% of additional future cases. CONCLUSION: Integrating a 313-variant PRS with classical risk factors can improve the identification of European-ancestry women at elevated risk who could benefit from targeted risk-reducing strategies under current clinical guidelines.

TP53 Pathway Function, Estrogen Receptor Status, and Breast Cancer Risk Factors in the Carolina Breast Cancer Study.

BACKGROUND: TP53 and estrogen receptor (ER) both play essential roles in breast cancer development and progression, with recent research revealing cross-talk between TP53 and ER signaling pathways. Although many studies have demonstrated heterogeneity of risk factor associations across ER subtypes, associations by TP53 status have been inconsistent. METHODS: This case-case analysis included incident breast cancer cases (47% Black) from the Carolina Breast Cancer Study (1993-2013). Formalin-fixed paraffin-embedded tumor samples were classified for TP53 functional status (mutant-like/wild-type-like) using a validated RNA signature. For IHC-based TP53 status, mutant-like was classified as at least 10% positivity. We used two-stage polytomous logistic regression to evaluate risk factor heterogeneity due to RNA-based TP53 and/or ER, adjusting for each other and for PR, HER2, and grade. We then compared this with the results when using IHC-based TP53 classification. RESULTS: The RNA-based classifier identified 55% of tumors as TP53 wild-type-like and 45% as mutant-like. Several hormone-related factors (oral contraceptive use, menopausal status, age at menopause, and pre- and postmenopausal body mass index) were associated with TP53 mutant-like status, whereas reproductive factors (age at first birth and parity) and smoking were associated with ER status. Multiparity was associated with both TP53 and ER. When classifying TP53 status using IHC methods, no associations were observed with TP53. Associations observed with RNA-based TP53 remained after accounting for basal-like subtype. CONCLUSIONS: This case-case study found breast cancer risk factors associated with RNA-based TP53 and ER. IMPACT: RNA-based TP53 and ER represent an emerging etiologic schema of interest in breast cancer prevention research.

Comparative validation of the BOADICEA and Tyrer-Cuzick breast cancer risk models incorporating classical risk factors and polygenic risk in a population-based prospective cohort of women of European ancestry.

BACKGROUND: The Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) and the Tyrer-Cuzick breast cancer risk prediction models are commonly used in clinical practice and have recently been extended to include polygenic risk scores (PRS). In addition, BOADICEA has also been extended to include reproductive and lifestyle factors, which were already part of Tyrer-Cuzick model. We conducted a comparative prospective validation of these models after incorporating the recently developed 313-variant PRS. METHODS: Calibration and discrimination of 5-year absolute risk was assessed in a nested case-control sample of 1337 women of European ancestry (619 incident breast cancer cases) aged 23-75 years from the Generations Study. RESULTS: The extended BOADICEA model with reproductive/lifestyle factors and PRS was well calibrated across risk deciles; expected-to-observed ratio (E/O) at the highest risk decile :0.97 (95 % CI 0.51 - 1.86) for women younger than 50 years and 1.09 (0.66 - 1.80) for women 50 years or older. Adding reproductive/lifestyle factors and PRS to the BOADICEA model improved discrimination modestly in younger women (area under the curve (AUC) 69.7 % vs. 69.1%) and substantially in older women (AUC 64.6 % vs. 56.8%). The Tyrer-Cuzick model with PRS showed evidence of overestimation at the highest risk decile: E/O = 1.54(0.81 - 2.92) for younger and 1.73 (1.03 - 2.90) for older women. CONCLUSION: The extended BOADICEA model identified women in a European-ancestry population at elevated breast cancer risk more accurately than the Tyrer-Cuzick model with PRS. With the increasing availability of PRS, these analyses can inform choice of risk models incorporating PRS for risk stratified breast cancer prevention among women of European ancestry.

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.

Assessment of polygenic architecture and risk prediction based on common variants across fourteen cancers.

Genome-wide association studies (GWAS) have led to the identification of hundreds of susceptibility loci across cancers, but the impact of further studies remains uncertain. Here we analyse summary-level data from GWAS of European ancestry across fourteen cancer sites to estimate the number of common susceptibility variants (polygenicity) and underlying effect-size distribution. All cancers show a high degree of polygenicity, involving at a minimum of thousands of loci. We project that sample sizes required to explain 80% of GWAS heritability vary from 60,000 cases for testicular to over 1,000,000 cases for lung cancer. The maximum relative risk achievable for subjects at the 99th risk percentile of underlying polygenic risk scores (PRS), compared to average risk, ranges from 12 for testicular to 2.5 for ovarian cancer. We show that PRS have potential for risk stratification for cancers of breast, colon and prostate, but less so for others because of modest heritability and lower incidence.