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.

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.

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.

Association between Breast Cancer Polygenic Risk Score and Chemotherapy-Induced Febrile Neutropenia: Null Results.

BACKGROUND: The hypothesis that breast cancer (BC) susceptibility variants are linked to chemotherapy-induced toxicity has been previously explored. Here, we investigated the association between a validated 313-marker-based BC polygenic risk score (PRS) and chemotherapy-induced neutropenia without fever and febrile neutropenia (FNc) in Asian BC patients. METHODS: This observational case-control study of Asian BC patients treated with chemotherapy included 161 FNc patients, 219 neutropenia patients, and 936 patients who did not develop neutropenia. A continuous PRS was calculated by summing weighted risk alleles associated with overall, estrogen receptor- (ER-) positive, and ER-negative BC risk. PRS distributions neutropenia or FNc cases were compared to controls who did not develop neutropenia using two-sample t-tests. Odds ratios (OR) and corresponding 95% confidence intervals were estimated for the associations between PRS (quartiles and per standard deviation (SD) increase) and neutropenia-related outcomes compared to controls. RESULTS: PRS distributions were not significantly different in any of the comparisons. Higher PRSoverall quartiles were negatively correlated with neutropenia or FNc. However, the associations were not statistically significant (PRS per SD increase OR neutropenia: 0.91 [0.79-1.06]; FNc: 0.87 [0.73-1.03]). No dose-dependent trend was observed for the ER-positive weighted PRS (PRSER-pos) and ER-negative weighted PRS (PRSER-neg). CONCLUSION: BC PRS was not strongly associated with chemotherapy-induced neutropenia or FNc.

Relevance of the MHC region for breast cancer susceptibility in Asians.

BACKGROUND: Human leukocyte antigen (HLA) genes play critical roles in immune surveillance, an important defence against tumors. Imputing HLA genotypes from existing single-nucleotide polymorphism datasets is low-cost and efficient. We investigate the relevance of the major histocompatibility complex region in breast cancer susceptibility, using imputed class I and II HLA alleles, in 25,484 women of Asian ancestry. METHODS: A total of 12,901 breast cancer cases and 12,583 controls from 12 case-control studies were included in our pooled analysis. HLA imputation was performed using SNP2HLA on 10,886 quality-controlled variants within the 15-55 Mb region on chromosome 6. HLA alleles (n = 175) with info scores greater than 0.8 and frequencies greater than 0.01 were included (resolution at two-digit level: 71; four-digit level: 104). We studied the associations between HLA alleles and breast cancer risk using logistic regression, adjusting for population structure and age. Associations between HLA alleles and the risk of subtypes of breast cancer (ER-positive, ER-negative, HER2-positive, HER2-negative, early-stage, and late-stage) were examined. RESULTS: We did not observe associations between any HLA allele and breast cancer risk at P < 5e-8; the smallest p value was observed for HLA-C*12:03 (OR = 1.29, P = 1.08e-3). Ninety-five percent of the effect sizes (OR) observed were between 0.90 and 1.23. Similar results were observed when different subtypes of breast cancer were studied (95% of ORs were between 0.85 and 1.18). CONCLUSIONS: No imputed HLA allele was associated with breast cancer risk in our large Asian study. Direct measurement of HLA gene expressions may be required to further explore the associations between HLA genes and breast cancer risk.

Associations between Pre-Diagnostic Physical Activity with Breast Cancer Characteristics and Survival.

Physical activity (PA) is known to reduce breast cancer (BC) risk and improve patient prognosis. However, the association between pre-diagnostic PA and the aggressiveness of BC is unclear. We investigated the associations between PA, BC tumour characteristics, and survival. This retrospective observational study included 7688 BC patients from the Singapore Breast Cancer Cohort (2010−2016). PA information from the questionnaire included intensity (light/moderate/vigorous) and duration (<1 h/1−2 h/>2 h per week). A PA score (1−5) incorporating intensity and duration was calculated. Associations between PA score and tumour characteristics such as stage, histological grade, nodal and hormone receptor status were examined using multinomial regression. Moreover, 10-year overall survival was estimated using Cox regression analysis in 6572 patients after excluding patients with invalid survival data and stage IV disease. Breast tumours associated with higher PA score were more likely to be non-invasive (ORinvasive vs. non-invasive(reference) [95% CI]: 0.71 [0.58−0.87], p-trend = 0.001), of lower grade (ORpoorly vs. well differentiated(reference): 0.69 [0.52−0.93], p = 0.014), ER-positive (ORER-negative vs. ER-positive(reference): 0.94 [0.89−1.00], p-trend = 0.049), PR-positive (ORPR-negative vs. PR-positive(reference): 0.82 [0.67−0.99], p = 0.041), HER2-negative (ORHER2-negative vs. HER2-positive(reference): 1.29 [1.02−1.62], p-trend = 0.002), and less likely to be of HER2-overexpressed subtype (ORHER2-overexpressed vs. Luminal A(reference): 0.89 [0.81−0.98], p-trend = 0.018). These associations (odds ratios) were more pronounced among post-menopausal patients. A higher PA score did not improve survival. Higher levels of pre-diagnostic PA were associated with less aggressive tumours in BC patients. This illustrated another benefit of PA in addition to its known role in BC risk reduction.

BREAst screening Tailored for HEr (BREATHE)-A study protocol on personalised risk-based breast cancer screening programme.

Routine mammography screening is currently the standard tool for finding cancers at an early stage, when treatment is most successful. Current breast screening programmes are one-size-fits-all which all women above a certain age threshold are encouraged to participate. However, breast cancer risk varies by individual. The BREAst screening Tailored for HEr (BREATHE) study aims to assess acceptability of a comprehensive risk-based personalised breast screening in Singapore. Advancing beyond the current age-based screening paradigm, BREATHE integrates both genetic and non-genetic breast cancer risk prediction tools to personalise screening recommendations. BREATHE is a cohort study targeting to recruit ~3,500 women. The first recruitment visit will include questionnaires and a buccal cheek swab. After receiving a tailored breast cancer risk report, participants will attend an in-person risk review, followed by a final session assessing the acceptability of our risk stratification programme. Risk prediction is based on: a) Gail model (non-genetic), b) mammographic density and recall, c) BOADICEA predictions (breast cancer predisposition genes), and d) breast cancer polygenic risk score. For national implementation of personalised risk-based breast screening, exploration of the acceptability within the target populace is critical, in addition to validated predication tools. To our knowledge, this is the first study to implement a comprehensive risk-based mammography screening programme in Asia. The BREATHE study will provide essential data for policy implementation which will transform the health system to deliver a better health and healthcare outcomes.

Integrative Profiling of T790M-Negative EGFR-Mutated NSCLC Reveals Pervasive Lineage Transition and Therapeutic Opportunities.

PURPOSE: Despite the established role of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC, drug resistance inevitably ensues, with a paucity of treatment options especially in EGFR T790M-negative resistance. EXPERIMENTAL DESIGN: We performed whole-exome and transcriptome analysis of 59 patients with first- and second-generation EGFR TKI-resistant metastatic EGFR-mutated NSCLC to characterize and compare molecular alterations mediating resistance in T790M-positive (T790M+) and -negative (T790M-) disease. RESULTS: Transcriptomic analysis revealed ubiquitous loss of adenocarcinoma lineage gene expression in T790M- tumors, orthogonally validated using multiplex IHC. There was enrichment of genomic features such as TP53 alterations, 3q chromosomal amplifications, whole-genome doubling and nonaging mutational signatures in T790M- tumors. Almost half of resistant tumors were further classified as immunehot, with clinical outcomes conditional on immune cell-infiltration state and T790M status. Finally, using a Bayesian statistical approach, we explored how T790M- and T790M+ disease might be predicted using comprehensive genomic and transcriptomic profiles of treatment-naïve patients. CONCLUSIONS: Our results illustrate the interplay between genetic alterations, cell lineage plasticity, and immune microenvironment in shaping divergent TKI resistance and outcome trajectories in EGFR-mutated NSCLC. Genomic and transcriptomic profiling may facilitate the design of bespoke therapeutic approaches tailored to a tumor's adaptive potential.

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.

Sequencing of E. coli strain UTI89 on multiple sequencing platforms.

OBJECTIVES: The availability of matched sequencing data for the same sample across different sequencing platforms is a necessity for validation and effective comparison of sequencing platforms. A commonly sequenced sample is the lab-adapted MG1655 strain of Escherichia coli; however, this strain is not fully representative of more complex and dynamic genomes of pathogenic E. coli strains. DATA DESCRIPTION: We present six new sequencing data sets for another E. coli strain, UTI89, which is an extraintestinal pathogenic strain isolated from a patient suffering from a urinary tract infection. We now provide matched whole genome sequencing data generated using the PacBio RSII, Oxford Nanopore MinION R9.4, Ion Torrent, ABI SOLiD, and Illumina NextSeq sequencers. Together with other publically available datasets, UTI89 has a nearly complete suite of data generated on most second- and third-generation sequencers. These data can be used as an additional validation set for new sequencing technologies and analytical methods. More than being another E. coli strain, however, UTI89 is pathogenic, with a 10% larger genome, additional pathogenicity islands, and a large plasmid, features that are common among other naturally occurring and disease-causing E. coli isolates. These data therefore provide a more medically relevant test set for development of algorithms.

Systems consequences of amplicon formation in human breast cancer.

Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer.

Targeted inactivation and identification of targets of the Gli2a transcription factor in the zebrafish.

Hedgehog (Hh) signaling is mediated by the Gli transcription factors and, in the zebrafish, plays an important role in patterning both the neural tube and myotome. Using a null allele of the gli2a gene induced by targeted mutagenesis, we show that Gli2a is completely dispensable in the fish but acts redundantly with Gli1 to regulate expression of known Hh targets, such as ptch2, prdm1a and eng2a, in the myotome and neural tube. To identify novel targets of Hh signaling, we performed chromatin immunoprecipitation sequencing (ChIP-seq) of whole embryo extracts. Samples were significantly enriched for 192 genomic regions, some of which are associated with four known Hh target genes, ptch1, ptch2, gli1 and olig2. Sequence analysis of these regions reveals a high level of conservation of Gli-binding sites from fish to mammals in some, but not all, cases. Expression analysis of other transcription units that are closely associated with peaks identified several putative targets not previously implicated as Hh targets, including myl10, hnmt, lrp4, efemp2, fras1, quo, and lamc1. Each of these genes shows loss of, or reduced expression in, embryos homozygous for an antimorphic allele of gli2a, you-too (yot), consistent with their being direct targets of Gli2a.