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Supplementary Data from 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 EGFRT790M-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.

BackgroundIdiopathic granulomatous mastitis (IGM) is a rare, chronic, 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 IGM and LM patients.MethodsIGM patients were recruited from 2018 to 2020; LM patients were recruited as controls. 16S rRNA sequencing libraries were constructed from 16S rRNA gene amplified from total DNA extracted from pus and skin swabs. 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.Results21 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, LM-skin; PERMANOVA, pCorynebacterium kroppenstedtii, reportedly associated with IGM in literature, was higher in IGM pus samples than paired skin sample (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.ConclusionsMicrobial profiles are unique between IGM and LM patients. Inter-patient variability and polymicrobial IGM pus samples cannot implicate specific genus or species as an infectious cause for IGM.

Background Exposure to cytotoxic chemotherapy treatment may alter DNA methylation (DNAm) in treated patients. Methods We performed DNAm analysis in 1,244 and 897 breast cancer patients treated and not treated by adjuvant chemotherapy using the Illumina MethylationEPIC array (1,804 blood, 337 saliva). DNAm changes of 620,095 individual CpGs and 41,581 promoters were evaluated using linear regression models, adjusting for age at diagnosis, ethnicity, years between sample collection and diagnosis and cell-type heterogeneity. Results from datasets normalized separately were combined by meta-analysis (random effects model). Gene set enrichment analyses were conducted to identify key processes or pathways associated with chemotherapy treatment. Results A total of 425 differentially methylated CpGs and 20 promoters were significantly associated with chemotherapy treatment (p< 5e-8). Enriched gene sets among 3,495 chemotherapy-associated promoters (unadjusted p< 0.05, preranked by Z scores) included three suppressed Gene Ontology (GO) terms that survived Bonferroni correction (GO:0002376, immune system process; GO:0009605, response to external stimulus; and GO:1903034: regulation of response to wounding). Using meta-analysis regression coefficients for all promoters as a ranking metric, olfactory transduction (KEGG, hsa04740) was found to be significantly suppressed (unadjusted p=6.38e-06, adjusted p=0.002). Taste transduction (hsa04742, unadjusted p=1.73e-03, adjusted p=0.565) was the next most significantly suppressed pathway. Conclusion The enrichment of imprinted genes within biological processes and pathways suggests a biological mechanism by which chemotherapy treatment could affect immune response, wound healing and changes in the perceptions of smell and taste. Citation Format: Peh Joo Ho, Alexis Jiaying Khng, Benita Kiat Tee Tan, Ern Yu Tan, Geok hoon Lim, Su-Ming Tan, Veronique Kiak Mien Tan, Elaine Hsuen Lim, Mikael Hartman, Jingmei Li. Alteration of DNA methylation landscape in breast patients treated with adjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-44.

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.

e22500 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 utilisation in a multi-ethnic Asian population. Methods: This study included 3,739 BC patients from the Singapore Breast Cancer Cohort (2010–2018). Self-reported socio-demographic characteristics were collected using a structured questionnaire. Clinical data were obtained through medical records. Patients were classified as screeners (last screening mammogram ≤two 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 tumors (ORHER2-negative VS -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(non-screeners HR: 1.89 [1.22-2.94], p = 0.005; unaware HR: 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.

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.

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 (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 (ORinvasivevs.non-invasive(reference) [95% CI]: 0.71 [0.58–0.87], p-trend = 0.001), of lower grade (ORpoorlyvs.well differentiated(reference): 0.69 [0.52–0.93], p = 0.014), ER-positive (ORER-negativevs.ER-positive(reference): 0.94 [0.89–1.00], p-trend = 0.049), PR-positive (ORPR-negativevs.PR-positive(reference): 0.82 [0.67–0.99], p = 0.041), HER2-negative (ORHER2-negativevs.HER2-positive(reference): 1.29 [1.02–1.62], p-trend = 0.002), and less likely to be of HER2-overexpressed subtype (ORHER2-overexpressedvs.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.