Genome- and transcriptome-wide association studies of 386,000 Asian and European-ancestry women provide new insights into breast cancer genetics.

By combining data from 160,500 individuals with breast cancer and 226,196 controls of Asian and European ancestry, we conducted genome- and transcriptome-wide association studies of breast cancer. We identified 222 genetic risk loci and 137 genes that were associated with breast cancer risk at a p < 5.0 × 10-8 and a Bonferroni-corrected p < 4.6 × 10-6, respectively. Of them, 32 loci and 15 genes showed a significantly different association between ER-positive and ER-negative breast cancer after Bonferroni correction. Significant ancestral differences in risk variant allele frequencies and their association strengths with breast cancer risk were identified. Of the significant associations identified in this study, 17 loci and 14 genes are located 1Mb away from any of the previously reported breast cancer risk variants. Pathways analyses including 221 putative risk genes identified multiple signaling pathways that may play a significant role in the development of breast cancer. Our study provides a comprehensive understanding of and new biological insights into the genetics of this common malignancy.

Assertive community treatment for high-utilizing alcohol misuse patients: a before-and-after cohort study protocol.

BACKGROUND: The challenge posed by Alcohol-Related Frequent Attenders (ARFAs) in Emergency Departments (EDs) is growing in Singapore, marked by limited engagement with conventional addiction treatment pathways. Recognizing this gap, this study aims to explore the potential benefits of Assertive Community Treatment (ACT) - an innovative, community-centered, harm-reduction strategy-in mitigating the frequency of ED visits, curbing Emergency Medical Services (EMS) calls, and uplifting health outcomes across a quartet of Singaporean healthcare institutions. METHODS: Employing a prospective before-and-after cohort design, this investigation targeted ARFAs aged 21 years and above, fluent in English or Mandarin. Eligibility was determined by a history of at least five ED visits in the preceding year, with no fewer than two due to alcohol-related issues. The study contrasted health outcomes of patients integrated into the ACT care model versus their experiences under the exclusive provision of standard emergency care across Hospitals A, B, C and D. Following participants for half a year post-initial assessment, the evaluation metrics encompassed socio-demographic factors, ED, and EMS engagement frequencies, along with validated health assessment tools, namely Christo Inventory for Substance-misuse Services (CISS) scores, University of California, Los Angeles (UCLA) Loneliness scores, and Centre for Epidemiologic Studies Depression Scale Revised (CESD-R-10) scores. DISCUSSION: Confronted with intricate socio-economic and medical challenges, the ARFA cohort often grapples with heightened vulnerabilities in relation to alcohol misuse. Pioneering the exploration of ACT's efficacy with ARFAs in a Singaporean context, our research is anchored in a patient-centered approach, designed to comprehensively address these multifaceted clinical profiles. While challenges, like potential high attrition rates and sporadic data collection, are anticipated, the model's prospective contribution towards enhancing patient well-being and driving healthcare efficiencies in Singapore is substantial. Our findings have the potential to reshape healthcare strategies and policy recommendations. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04447079. Initiated on 25 June 2020.

Alterations to DNA methylation patterns induced by chemotherapy treatment are associated with negative impacts on the olfactory pathway.

BACKGROUND: Exposure to cytotoxic chemotherapy treatment may alter DNA methylation (DNAm) in breast cancer patients. METHODS: We performed DNAm analysis in 125 breast cancer patients with blood drawn before and after chemotherapy, using the Illumina MethylationEPIC array. DNAm changes of 588,798 individual CpGs (including 41,207 promoter regions) were evaluated using linear regression models adjusted for monocyte proportion. Gene set enrichment analyses (GSEA) were conducted to identify key Gene Ontology (GO) biological processes or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with chemotherapy. Results were validated in a separate cohort of breast cancer patients who were treated (n = 1273) and not treated (n = 872) by chemotherapy (1808 blood, 337 saliva). RESULTS: A total of 141 differentially methylated CpGs and 11 promoters were significantly associated with chemotherapy after multiple testing corrections in both the paired sample and single time point analyses. GSEA of promoter regions (pre-ranked by test statistics) identified six suppressed biological processes (p < 4.67e-8) related to sensory perception and detection of chemical stimuli, including smell perception (GO:0007606, GO:0007608, GO:0009593, GO:0050906, GO:0050907, and GO:0050911). The same six biological processes were significantly suppressed in the validation dataset (p < 9.02e-14). The KEGG pathway olfactory transduction (hsa04740) was also found to be significantly suppressed (ppaired-samples = 1.72e-9, psingle-timepoint-blood = 2.03e-15 and psingle-timepoint-saliva = 7.52e-56). CONCLUSION: The enrichment of imprinted genes within biological processes and pathways suggests a biological mechanism by which chemotherapy could affect the perception of smell.

Dynamic altruistic cooperation within breast tumors.

BACKGROUND: Social behaviors such as altruism, where one self-sacrifices for collective benefits, critically influence an organism's survival and responses to the environment. Such behaviors are widely exemplified in nature but have been underexplored in cancer cells which are conventionally seen as selfish competitive players. This multidisciplinary study explores altruism and its mechanism in breast cancer cells and its contribution to chemoresistance. METHODS: MicroRNA profiling was performed on circulating tumor cells collected from the blood of treated breast cancer patients. Cancer cell lines ectopically expressing candidate miRNA were used in co-culture experiments and treated with docetaxel. Ecological parameters like relative survival and relative fitness were assessed using flow cytometry. Functional studies and characterization performed in vitro and in vivo include proliferation, iTRAQ-mass spectrometry, RNA sequencing, inhibition by small molecules and antibodies, siRNA knockdown, CRISPR/dCas9 inhibition and fluorescence imaging of promoter reporter-expressing cells. Mathematical modeling based on evolutionary game theory was performed to simulate spatial organization of cancer cells. RESULTS: Opposing cancer processes underlie altruism: an oncogenic process involving secretion of IGFBP2 and CCL28 by the altruists to induce survival benefits in neighboring cells under taxane exposure, and a self-sacrificial tumor suppressive process impeding proliferation of altruists via cell cycle arrest. Both processes are regulated concurrently in the altruists by miR-125b, via differential NF-κB signaling specifically through IKKß. Altruistic cells persist in the tumor despite their self-sacrifice, as they can regenerate epigenetically from non-altruists via a KLF2/PCAF-mediated mechanism. The altruists maintain a sparse spatial organization by inhibiting surrounding cells from adopting the altruistic fate via a lateral inhibition mechanism involving a GAB1-PI3K-AKT-miR-125b signaling circuit. CONCLUSIONS: Our data reveal molecular mechanisms underlying manifestation, persistence and spatial spread of cancer cell altruism. A minor population behave altruistically at a cost to itself producing a collective benefit for the tumor, suggesting tumors to be dynamic social systems governed by the same rules of cooperation in social organisms. Understanding cancer cell altruism may lead to more holistic models of tumor evolution and drug response, as well as therapeutic paradigms that account for social interactions. Cancer cells constitute tractable experimental models for fields beyond oncology, like evolutionary ecology and game theory.

Evaluation of SNPs associated with mammographic density in European women with mammographic density in Asian women from South-East Asia.

PURPOSE: Mammographic density (MD), after accounting for age and body mass index (BMI), is a strong heritable risk factor for breast cancer. Genome-wide association studies (GWAS) have identified 64 SNPs in 55 independent loci associated with MD in women of European ancestry. Their associations with MD in Asian women, however, are largely unknown. METHOD: Using linear regression adjusting for age, BMI, and ancestry-informative principal components, we evaluated the associations of previously reported MD-associated SNPs with MD in a multi-ethnic cohort of Asian ancestry. Area and volumetric mammographic densities were determined using STRATUS (N = 2450) and Volpara™ (N = 2257). We also assessed the associations of these SNPs with breast cancer risk in an Asian population of 14,570 cases and 80,870 controls. RESULTS: Of the 61 SNPs available in our data, 21 were associated with MD at a nominal threshold of P value < 0.05, all in consistent directions with those reported in European ancestry populations. Of the remaining 40 variants with a P-value of association > 0.05, 29 variants showed consistent directions of association as those previously reported. We found that nine of the 21 MD-associated SNPs in this study were also associated with breast cancer risk in Asian women (P < 0.05), seven of which showed a direction of associations that was consistent with that reported for MD. CONCLUSION: Our study confirms the associations of 21 SNPs (19/55 or 34.5% out of all known MD loci identified in women of European ancestry) with area and/or volumetric densities in Asian women, and further supports the evidence of a shared genetic basis through common genetic variants for MD and breast cancer risk.

Breast cancer risk stratification using genetic and non-genetic risk assessment tools for 246,142 women in the UK Biobank.

PURPOSE: The benefit of using individual risk prediction tools to identify high-risk individuals for breast cancer (BC) screening is uncertain, despite the personalized approach of risk-based screening. METHODS: We studied the overlap of predicted high-risk individuals among 246,142 women enrolled in the UK Biobank. Risk predictors assessed include the Gail model (Gail), BC family history (FH, binary), BC polygenic risk score (PRS), and presence of loss-of-function (LoF) variants in BC predisposition genes. Youden J-index was used to select optimal thresholds for defining high-risk. RESULTS: In total, 147,399 were considered at high risk for developing BC within the next 2 years by at least 1 of the 4 risk prediction tools examined (Gail2-year > 0.5%: 47%, PRS2-yea r > 0.7%: 30%, FH: 6%, and LoF: 1%); 92,851 (38%) were flagged by only 1 risk predictor. The overlap between individuals flagged as high-risk because of genetic (PRS) and Gail model risk factors was 30%. The best-performing combinatorial model comprises a union of high-risk women identified by PRS, FH, and, LoF (AUC2-year [95% CI]: 62.2 [60.8 to 63.6]). Assigning individual weights to each risk prediction tool increased discriminatory ability. CONCLUSION: Risk-based BC screening may require a multipronged approach that includes PRS, predisposition genes, FH, and other recognized risk factors.

Characterisation of protein-truncating and missense variants in PALB2 in 15 768 women from Malaysia and Singapore.

BACKGROUND: Rare protein-truncating variants (PTVs) in partner and localiser of BRCA2 (PALB2) confer increased risk to breast cancer, but relatively few studies have reported the prevalence in South-East Asian populations. Here, we describe the prevalence of rare variants in PALB2 in a population-based study of 7840 breast cancer cases and 7928 healthy Chinese, Malay and Indian women from Malaysia and Singapore, and describe the functional impact of germline missense variants identified in this population. METHODS: Mutation testing was performed on germline DNA (n=15 768) using targeted sequencing panels. The functional impact of missense variants was tested in mouse embryonic stem cell based functional assays. RESULTS: PTVs in PALB2 were found in 0.73% of breast cancer patients and 0.14% of healthy individuals (OR=5.44; 95% CI 2.85 to 10.39, p<0.0001). In contrast, rare missense variants in PALB2 were not associated with increased risk of breast cancer. Whereas PTVs were associated with later stage of presentation and higher-grade tumours, no significant association was observed with missense variants in PALB2. However, two novel rare missense variants (p.L1027R and p.G1043V) produced unstable proteins and resulted in a decrease in homologous recombination-mediated repair of DNA double-strand breaks. CONCLUSION: Despite genetic and lifestyle differences between Asian and other populations, the population prevalence of PALB2 PTVs and associated relative risk of breast cancer, are similar to those reported in European populations.

Profiling Microbial Communities in Idiopathic Granulomatous Mastitis.

Idiopathic granulomatous mastitis (IGM) is a rare and benign inflammatory breast disease with ambiguous aetiology. Contrastingly, lactational mastitis (LM) is commonly diagnosed in breastfeeding women. To investigate IGM aetiology, we profiled the microbial flora of pus and skin in patients with IGM and LM. A total of 26 patients with IGM and 6 patients with LM were included in the study. The 16S rRNA sequencing libraries were constructed from 16S rRNA gene amplified from total DNA extracted from pus and skin swabs in patients with IGM and LM controls. Constructed libraries were multiplexed and paired-end sequenced on HiSeq4000. Metagenomic analysis was conducted using modified microbiome abundance analysis suite customised R-resource for paired pus and skin samples. Microbiome multivariable association analyses were performed using linear models. A total of 21 IGM and 3 LM paired pus and skin samples underwent metagenomic analysis. Bray−Curtis ecological dissimilarity distance showed dissimilarity across four sample types (IGM pus, IGM skin, LM pus, and LM skin; PERMANOVA, p < 0.001). No characteristic dominant genus was observed across the IGM samples. The IGM pus samples were more diverse than corresponding IGM skin samples (Shannon and Simpson index; Wilcoxon paired signed-rank tests, p = 0.022 and p = 0.07). Corynebacterium kroppenstedtii, reportedly associated with IGM in the literature, was higher in IGM pus samples than paired skin samples (Wilcoxon, p = 0.022). Three other species and nineteen genera were statistically significant in paired IGM pus−skin comparison after antibiotic treatment adjustment and multiple comparisons correction. Microbial profiles are unique between patients with IGM and LM. Inter-patient variability and polymicrobial IGM pus samples cannot implicate specific genus or species as an infectious cause for IGM.

The association of age at menarche and adult height with mammographic density in the International Consortium of Mammographic Density.

BACKGROUND: Early age at menarche and tall stature are associated with increased breast cancer risk. We examined whether these associations were also positively associated with mammographic density, a strong marker of breast cancer risk. METHODS: Participants were 10,681 breast-cancer-free women from 22 countries in the International Consortium of Mammographic Density, each with centrally assessed mammographic density and a common set of epidemiologic data. Study periods for the 27 studies ranged from 1987 to 2014. Multi-level linear regression models estimated changes in square-root per cent density (√PD) and dense area (√DA) associated with age at menarche and adult height in pooled analyses and population-specific meta-analyses. Models were adjusted for age at mammogram, body mass index, menopausal status, hormone therapy use, mammography view and type, mammographic density assessor, parity and height/age at menarche. RESULTS: In pooled analyses, later age at menarche was associated with higher per cent density (ß√PD = 0.023 SE = 0.008, P = 0.003) and larger dense area (ß√DA = 0.032 SE = 0.010, P = 0.002). Taller women had larger dense area (ß√DA = 0.069 SE = 0.028, P = 0.012) and higher per cent density (ß√PD = 0.044, SE = 0.023, P = 0.054), although the observed effect on per cent density depended upon the adjustment used for body size. Similar overall effect estimates were observed in meta-analyses across population groups. CONCLUSIONS: In one of the largest international studies to date, later age at menarche was positively associated with mammographic density. This is in contrast to its association with breast cancer risk, providing little evidence of mediation. Increased height was also positively associated with mammographic density, particularly dense area. These results suggest a complex relationship between growth and development, mammographic density and breast cancer risk. Future studies should evaluate the potential mediation of the breast cancer effects of taller stature through absolute breast density.

Development and validation of a circulating microRNA panel for the early detection of breast cancer.

BACKGROUND: Mammography is widely used for breast cancer screening but suffers from a high false-positive rate. Here, we perform the largest comprehensive, multi-center study to date involving diverse ethnic groups, for the identification of circulating miRNAs for breast cancer screening. METHODS: This study had a discovery phase (n = 289) and two validation phases (n = 374 and n = 379). Quantitative PCR profiling of 324 miRNAs was performed on serum samples from breast cancer (all stages) and healthy subjects to identify miRNA biomarkers. Two-fold cross-validation was used for building and optimising breast cancer-associated miRNA panels. An optimal panel was validated in cohorts with Caucasian and Asian samples. Diagnostic ability was evaluated using area under the curve (AUC) analysis. RESULTS: The study identified and validated 30 miRNAs dysregulated in breast cancer. An optimised eight-miRNA panel showed consistent performance in all cohorts and was successfully validated with AUC, accuracy, sensitivity, and specificity of 0.915, 82.3%, 72.2% and 91.5%, respectively. The prediction model detected breast cancer in both Caucasian and Asian populations with AUCs ranging from 0.880 to 0.973, including pre-malignant lesions (stage 0; AUC of 0.831) and early-stage (stages I-II) cancers (AUC of 0.916). CONCLUSIONS: Our panel can potentially be used for breast cancer screening, in conjunction with mammography.

Immunohistochemistry study of tumor vascular normalization and anti-angiogenic effects of sunitinib versus bevacizumab prior to dose-dense doxorubicin/cyclophosphamide chemotherapy in HER2-negative breast cancer.

PURPOSE: Tumor angiogenesis controlled predominantly by vascular endothelial growth factor and its receptor (VEGF-VEGFR) interaction plays a key role in the growth and propagation of cancer cells. However, the newly formed network of blood vessels is disorganized and leaky. Pre-treatment with anti-angiogenic agents can "normalize" the tumor vasculature allowing effective intra-tumoral delivery of standard chemotherapy. Immunohistochemistry (IHC) analysis was applied to investigate and compare the vascular normalization and anti-angiogenic effects of two commonly used anti-angiogenic agents, Sunitinib and Bevacizumab, administered prior to chemotherapy in HER2-negative breast cancer patients. METHODS: This prospective clinical trial enrolled 38 patients into a sunitinib cohort and 24 into a bevacizumab cohort. All received 4 cycles of doxorubicin/cyclophosphamide chemotherapy and pre-treatment with either sunitinib or bevacizumab. Tumor biopsies were obtained at baseline, after cycle 1 (C1) and cycle 4 (C4) of chemotherapy. IHC was performed to assess the tumor vascular normalization index (VNI), lymphatic vessel density (LVD), Ki67 proliferation index and expression of tumor VEGFR2. RESULTS: In comparison to Bevacizumab, Sunitinib led to a significant increase in VNI post-C1 and C4 (p < 0.001 and 0.001) along with decrease in LVD post-C1 (p = 0.017). Both drugs when combined with chemotherapy resulted in significant decline in tumor proliferation after C1 and C4 (baseline vs post-C4 Ki67 index p = 0.006 for Sunitinib vs p = 0.021 for Bevacizumab). Bevacizumab resulted in a significant decrease in VEGFR2 expression post-C1 (p = 0.004). CONCLUSION: Sunitinib, in comparison to Bevacizumab showed a greater effect on tumor vessel modulation and lymphangiogenesis suggesting that its administration prior to chemotherapy might result in improved drug delivery. TRIAL REGISTRY: ClinicalTrials.gov: NCT02790580 (first posted June 6, 2016).

Mammography screening is associated with more favourable breast cancer tumour characteristics and better overall survival: case-only analysis of 3739 Asian breast cancer patients.

BACKGROUND: Early detection of breast cancer (BC) through mammography screening (MAM) is known to reduce mortality. We examined the differential effect that mammography has on BC characteristics and overall survival and the sociodemographic determinants of MAM utilization in a multi-ethnic Asian population. METHODS: This study included 3739 BC patients from the Singapore Breast Cancer Cohort (2010-2018). Self-reported sociodemographic characteristics were collected using a structured questionnaire. Clinical data were obtained through medical records. Patients were classified as screeners (last screening mammogram ≤ 2 years before diagnosis), non-screeners (aware but did not attend or last screen > 2years), and those unaware of MAM. Associations between MAM behaviour (MB) and sociodemographic factors and MB and tumour characteristics were examined using multinomial regression. Ten-year overall survival was modelled using Cox regression. RESULTS: Patients unaware of screening were more likely diagnosed with late stage (ORstage III vs stage I (Ref) [95% CI]: 4.94 [3.45-7.07], p < 0.001), high grade (ORpoorly vs well-differentiated (reference): 1.53 [1.06-2.20], p = 0.022), nodal-positive, large size (OR>5cm vs ≤2cm (reference): 5.06 [3.10-8.25], p < 0.001), and HER2-positive tumours (ORHER2-negative vs HER2-positive (reference): 0.72 [0.53-0.97], p = 0.028). Similar trends were observed between screeners and non-screeners with smaller effect sizes. Overall survival was significantly shorter than screeners in the both groups (HRnon-screeners: 1.89 [1.22-2.94], p = 0.005; HRunaware: 2.90 [1.69-4.98], p < 0.001). Non-screeners and those unaware were less health conscious, older, of Malay ethnicity, less highly educated, of lower socioeconomic status, more frequently ever smokers, and less physically active. Among screeners, there were more reported personal histories of benign breast surgeries or gynaecological conditions and positive family history of breast cancer. CONCLUSIONS: Mammography attendance is associated with more favourable BC characteristics and overall survival. Disparities in the utility of MAM services suggest that different strategies may be needed to improve MAM uptake.

Overlap of high-risk individuals predicted by family history, and genetic and non-genetic breast cancer risk prediction models: implications for risk stratification.

BACKGROUND: Family history, and genetic and non-genetic risk factors can stratify women according to their individual risk of developing breast cancer. The extent of overlap between these risk predictors is not clear. METHODS: In this case-only analysis involving 7600 Asian breast cancer patients diagnosed between age 30 and 75 years, we examined identification of high-risk patients based on positive family history, the Gail model 5-year absolute risk [5yAR] above 1.3%, breast cancer predisposition genes (protein-truncating variants [PTV] in ATM, BRCA1, BRCA2, CHEK2, PALB2, BARD1, RAD51C, RAD51D, or TP53), and polygenic risk score (PRS) 5yAR above 1.3%. RESULTS: Correlation between 5yAR (at age of diagnosis) predicted by PRS and the Gail model was low (r=0.27). Fifty-three percent of breast cancer patients (n=4041) were considered high risk by one or more classification criteria. Positive family history, PTV carriership, PRS, or the Gail model identified 1247 (16%), 385 (5%), 2774 (36%), and 1592 (21%) patients who were considered at high risk, respectively. In a subset of 3227 women aged below 50 years, the four models studied identified 470 (15%), 213 (7%), 769 (24%), and 325 (10%) unique patients who were considered at high risk, respectively. For younger women, PRS and PTVs together identified 745 (59% of 1276) high-risk individuals who were not identified by the Gail model or family history. CONCLUSIONS: Family history and genetic and non-genetic risk stratification tools have the potential to complement one another to identify women at high risk.

Impact of contrast-enhanced mammography in surgical management of breast cancers for women with dense breasts: a dual-center, multi-disciplinary study in Asia.

OBJECTIVE: To evaluate the impact of pre-operative contrast-enhanced mammography (CEM) in breast cancer patients with dense breasts. METHODS: We conducted a retrospective review of 232 histologically proven breast cancers in 200 women (mean age: 53.4 years ± 10.2) who underwent pre-surgical CEM imaging across two Asian institutions (Singapore and Taiwan). Majority (95.5%) of patients had dense breast tissue (BI-RADS category C or D). Surgical decision was recorded in a simulated blinded multi-disciplinary team setting on two separate scenarios: (i) pre-CEM setting with standard imaging, and clinical and histopathological results; and (ii) post-CEM setting with new imaging and corresponding histological findings from CEM. Alterations in surgical plan (if any) because of CEM imaging were recorded. Predictors CEM of patients who benefitted from surgical plan alterations were evaluated using logistic regression. RESULTS: CEM resulted in altered surgical plans in 36 (18%) of 200 patients in this study. CEM discovered clinically significant larger tumor size or extent in 24 (12%) patients and additional tumors in 12 (6%) patients. CEM also detected additional benign/false-positive lesions in 13 (6.5%) of the 200 patients. Significant predictors of patients who benefitted from surgical alterations found on multivariate analysis were pre-CEM surgical decision for upfront breast conservation (OR, 7.7; 95% CI, 1.9-32.1; p = 0.005), architectural distortion on mammograms (OR, 7.6; 95% CI, 1.3-42.9; p = .022), and tumor size of ≥ 1.5 cm (OR, 1.5; 95% CI, 1.0-2.2; p = .034). CONCLUSION: CEM is an effective imaging technique for pre-surgical planning for Asian breast cancer patients with dense breasts. KEY POINTS: • CEM significantly altered surgical plans in 18% (nearly 1 in 5) of this Asian study cohort with dense breasts. • Significant patient and imaging predictors for surgical plan alteration include (i) patients considered for upfront breast-conserving surgery; (ii) architectural distortion lesions; and (iii) tumor size of ≥ 1.5 cm. • Additional false-positive/benign lesions detected through CEM were uncommon, affecting only 6.5% of the study cohort.

Multi-level determinants of breast cancer screening among Malay-Muslim women in Singapore: a sequential mixed-methods study.

OBJECTIVE: Ethnic disparity persists despite equal access to health care in Singapore, with Malay-Muslim women having the lowest mammogram uptake rate and highest breast cancer mortality rate. We sought to understand barriers to and facilitators for mammogram uptake in this community. METHODS: We used a sequential mixed-methods design to first explore reasons for screening and not screening for breast cancer, then determine factors associated with screening and regular screening in a survey. We used maximum variation sampling for semi-structured in-depth interviews to select screeners and non-screeners of diverse ages and educational levels. Twenty-three Malay-Muslim women aged 40-69 years old were interviewed. Themes were categorized using thematic analysis. For the survey, we applied the Health Belief Model, Social Ecological Model, as well as themes from the interviews and findings from previous studies on factors influencing screening in Muslim women to guide questionnaire design. We surveyed 271 Malay-Muslim women aged 50-69 years old in a nationally representative sample. Multivariable logistic regression was used to determine factors associated with ever gone for mammogram and regular mammogram uptake. RESULTS: Through in-depth-interviews, we found perceived benefits of saving lives and breasts from early detection, reminders from doctors and husbands, symptoms, perceived test from God, and personal responsibility to care for one's health facilitated screening. Barriers were perceived low susceptibility, inconvenience, cost, negative psychological effects, misinformation on mammogram triggering cancer cells, religious beliefs, perceived negative outcomes from mammography and distrust of doctor. From the survey, we found cues from health care professionals and needing symptoms before deciding to go for mammogram to be significantly associated with ever gone for mammogram and regular mammogram. Factors associated with ever gone for mammogram only included age, perceived benefits of saving lives from early detection, perceived importance of mammogram, Punishing Allah Reappraisal, and modesty. Factors associated with regular mammogram only included household income, perceived structural barriers to screening and perceived susceptibility to breast cancer. CONCLUSIONS: Mammogram uptake is affected by multiple levels of influence. Interventions to promote screening should be designed with multiple stakeholders including doctors, religious leaders and women who had attended screening.

Development and validation of a serum microRNA biomarker panel for detecting gastric cancer in a high-risk population.

OBJECTIVE: An unmet need exists for a non-invasive biomarker assay to aid gastric cancer diagnosis. We aimed to develop a serum microRNA (miRNA) panel for identifying patients with all stages of gastric cancer from a high-risk population. DESIGN: We conducted a three-phase, multicentre study comprising 5248 subjects from Singapore and Korea. Biomarker discovery and verification phases were done through comprehensive serum miRNA profiling and multivariant analysis of 578 miRNA candidates in retrospective cohorts of 682 subjects. A clinical assay was developed and validated in a prospective cohort of 4566 symptomatic subjects who underwent endoscopy. Assay performance was confirmed with histological diagnosis and compared with Helicobacter pylori (HP) serology, serum pepsinogens (PGs), 'ABC' method, carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9). Cost-effectiveness was analysed using a Markov decision model. RESULTS: We developed a clinical assay for detection of gastric cancer based on a 12-miRNA biomarker panel. The 12-miRNA panel had area under the curve (AUC)=0.93 (95% CI 0.90 to 0.95) and AUC=0.92 (95% CI 0.88 to 0.96) in the discovery and verification cohorts, respectively. In the prospective study, overall sensitivity was 87.0% (95% CI 79.4% to 92.5%) at specificity of 68.4% (95% CI 67.0% to 69.8%). AUC was 0.848 (95% CI 0.81 to 0.88), higher than HP serology (0.635), PG 1/2 ratio (0.641), PG index (0.576), ABC method (0.647), CEA (0.576) and CA19-9 (0.595). The number needed to screen is 489 annually. It is cost-effective for mass screening relative to current practice (incremental cost-effectiveness ratio=US$44 531/quality-of-life year). CONCLUSION: We developed and validated a serum 12-miRNA biomarker assay, which may be a cost-effective risk assessment for gastric cancer. TRIAL REGISTRATION NUMBER: This study is registered with ClinicalTrials.gov (Registration number: NCT04329299).

Epidemiological and ES cell-based functional evaluation of BRCA2 variants identified in families with breast cancer.

The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.

DNA methylation and breast cancer-associated variants.

BACKGROUND: A breast cancer polygenic risk score (PRS) comprising 313 common variants reliably predicts disease risk. We examined possible relationships between genetic variation, regulation, and expression to clarify the molecular alterations associated with these variants. METHODS: Genome-wide methylomic variation was quantified (MethylationEPIC) in Asian breast cancer patients (1152 buffy coats from peripheral whole blood). DNA methylation (DNAm) quantitative trait loci (mQTL) mapping was performed for 235 of the 313 variants with minor allele frequencies > 5%. Stability of identified mQTLs (p < 5e-8) across lifetime was examined using a public mQTL database. Identified mQTLs were also mapped to expression quantitative trait loci (eQTLs) in the Genotype-Tissue Expression Project and the eQTLGen Consortium. RESULTS: Breast cancer PRS was not associated with DNAm. A higher proportion of significant cis-mQTLs were observed. Of 822 significant cis-mQTLs (179 unique variants) identified in our dataset, 141 (59 unique variants) were significant (p < 5e-8) in a public mQTL database. Eighty-six percent (121/141) of the matched mQTLs were consistent at multiple time points (birth, childhood, adolescence, pregnancy, middle age, post-diagnosis, or treatment). Ninety-three variants associated with DNAm were also cis-eQTLs (35 variants not genome-wide significant). Multiple loci in the breast cancer PRS are associated with DNAm, contributing to the polygenic nature of the disease. These mQTLs are mostly stable over time. CONCLUSIONS: Consistent results from DNAm and expression data may reveal new candidate genes not previously associated with breast cancer.

Cost effectiveness analysis of a polygenic risk tailored breast cancer screening programme in Singapore.

BACKGROUND: This study aimed to evaluate the cost-effectiveness of a breast cancer screening programme that incorporates genetic testing using breast cancer associated single nucleotide polymorphisms (SNPs), against the current biennial mammogram-only screening programme to aid in its implementation into the current programme in Singapore. METHODS: A Markov model was used to compare the costs and health outcomes of the current screening programme, against a polygenic risk-tailored screening programme, which can advise a long-term screening strategy depending on the individual's polygenic risk. The model took the perspective of the healthcare system, with a time horizon of 40 years, following women from the age of 35 to 74. Epidemiological and cost data were taken from Asian studies, and an annual discount rate of 3% was used. The model outcome was the incremental cost-effectiveness ratio (ICER), calculated from the difference in costs per quality-adjusted life year (QALY). Scenarios with varying risk thresholds for each polygenic risk group were examined. One-way and probabilistic sensitivity analyses were performed to assess parameter uncertainty. RESULTS: The ICER for a polygenic risk-tailored breast cancer screening programme, compared with the current biennial mammogram-only screening programme, was - 3713.80 SGD/QALY, with incremental costs < 0 and incremental effects > 0. The scenario analysis of different polygenic risk cutoffs showed that the ICERs remain negative, with all ICERs falling within the south-east quadrant of the cost-effectiveness plane, indicating that tailored screening is more cost effective than mammogram-only screening, with lower costs and higher QALYs to be gained. This suggests that a polygenic risk-tailored breast cancer screening programme is cost effective, entailing lower cost than the current mammogram-only programme, while causing no additional harm to women. CONCLUSION: Results from this cost-effectiveness analysis show that polygenic risk-tailored screening is cost effective with an ICER of - 3713.80 SGD/QALY. Tailored screening remains cost effective even across varying percentile cutoffs for each risk group. While the results look promising for incorporating polygenic risk into the current breast cancer screening programme, further studies should be conducted to address various limitations.

Presentation of breast cancer, help seeking behaviour and experience of patients in their cancer journey in Singapore: a qualitative study.

BACKGROUND: Little is known about the presentation, help seeking behaviour for breast cancer in Singapore. Nor was there a study exploring the experience of patients in their breast cancer journey. METHODS: A qualitative interview study with thematic analysis, conducted with 36 patients. RESULTS: There is no clear pattern of presentation for breast cancer by cancer stage at diagnosis, age and ethnicity in the cancer journey of this group of patients. Patients were diagnosed with early to advanced stages cancer regardless of when they presented or took up treatment in their cancer journey. The reasons patients sought medical attention also did not appear to differ between the stages of cancer diagnosed, ethnic and age. Without setting a measure to define early and late presentation, we found that women shared similar experience in their breast cancer journey, regardless of age, ethnicity and stage of cancer at diagnosis. Poor knowledge of breast cancer (symptoms and causes); few practised regular BSE; denial of symptom; fear of hospitalisation, diagnosis and treatment; worries and stress over financial burden of treatment; misinformation in magazine and online sources; diet; stress; caring responsibility; support network; and use of alternative medicine before and after diagnosis were identified in patients' narratives. Strong social support; fear of being an emotional and financial burden for the family; and financial worries during treatment were also the recurring themes after diagnosis. CONCLUSION: A measure of breast cancer presentation - that accounts for the patient's experience in the cancer journey, the time interval and tumour biology - that is meaningful to patients, clinicians and researchers is needed. For research on late and delayed presentation, details on BSE practice - how often, when and was it done correctly - will improve the accuracy of time delay interval. For the public, concerted efforts to improve knowledge of breast cancer, survival and prognosis for early-diagnosed cancer, and the importance of regular and correct technique to perform BSE, are critical and urgent to address the rising breast cancer incidence in the country.