Beyond tobacco: genomic disparities in lung cancer between smokers and never-smokers.

BACKGROUND: Tobacco use is one of the main risk factors for Lung Cancer (LC) development. However, about 10-20% of those diagnosed with the disease are never-smokers. For Non-Small Cell Lung Cancer (NSCLC) there are clear differences in both the clinical presentation and the tumor genomic profiles between smokers and never-smokers. For example, the Lung Adenocarcinoma (LUAD) histological subtype in never-smokers is predominately found in young women of European, North American, and Asian descent. While the clinical presentation and tumor genomic profiles of smokers have been widely examined, never-smokers are usually underrepresented, especially those of a Latin American (LA) background. In this work, we characterize, for the first time, the difference in the genomic profiles between smokers and never-smokers LC patients from Chile. METHODS: We conduct a comparison by smoking status in the frequencies of genomic alterations (GAs) including somatic mutations and structural variants (fusions) in a total of 10 clinically relevant genes, including the eight most common actionable genes for LC (EGFR, KRAS, ALK, MET, BRAF, RET, ERBB2, and ROS1) and two established driver genes for malignancies other than LC (PIK3CA and MAP2K1). Study participants were grouped as either smokers (current and former, n = 473) or never-smokers (n = 200) according to self-report tobacco use at enrollment. RESULTS: Our findings indicate a higher overall GA frequency for never-smokers compared to smokers (58 vs. 45.7, p-value < 0.01) with the genes EGFR, KRAS, and PIK3CA displaying the highest prevalence while ERBB2, RET, and ROS1 the lowest. Never-smokers present higher frequencies in seven out of the 10 genes; however, smokers harbor a more complex genomic profile. The clearest differences between groups are seen for EGFR (15.6 vs. 21.5, p-value: < 0.01), PIK3CA (6.8 vs 9.5) and ALK (3.2 vs 7.5) in favor of never-smokers, and KRAS (16.3 vs. 11.5) and MAP2K1 (6.6 vs. 3.5) in favor of smokers. Alterations in these genes are comprised almost exclusively by somatic mutations in EGFR and mainly by fusions in ALK, and only by mutations in PIK3CA, KRAS and MAP2K1. CONCLUSIONS: We found clear differences in the genomic landscape by smoking status in LUAD patients from Chile, with potential implications for clinical management in these limited-resource settings.

Exome and Tissue-Associated Microbiota as Predictive Markers of Response to Neoadjuvant Treatment in Locally Advanced Rectal Cancer.

The clinical and pathological responses to multimodal neoadjuvant therapy in locally advanced rectal cancers (LARCs) remain unpredictable, and robust biomarkers are still lacking. Recent studies have shown that tumors present somatic molecular alterations related to better treatment response, and it is also clear that tumor-associated bacteria are modulators of chemotherapy and immunotherapy efficacy, therefore having implications for long-term survivorship and a good potential as the biomarkers of outcome. Here, we performed whole exome sequencing and 16S ribosomal RNA (rRNA) amplicon sequencing from 44 pre-treatment LARC biopsies from Argentinian and Brazilian patients, treated with neoadjuvant chemoradiotherapy or total neoadjuvant treatment, searching for predictive biomarkers of response (responders, n = 17; non-responders, n = 27). In general, the somatic landscape of LARC was not capable to predict a response; however, a significant enrichment in mutational signature SBS5 was observed in non-responders (p = 0.0021), as well as the co-occurrence of APC and FAT4 mutations (p < 0.05). Microbiota studies revealed a similar alpha and beta diversity of bacteria between response groups. Yet, the linear discriminant analysis (LDA) of effect size indicated an enrichment of Hungatella, Flavonifractor, and Methanosphaera (LDA score ≥3) in the pre-treatment biopsies of responders, while non-responders had a higher abundance of Enhydrobacter, Paraprevotella (LDA score ≥3) and Finegoldia (LDA score ≥4). Altogether, the evaluation of these biomarkers in pre-treatment biopsies could eventually predict a neoadjuvant treatment response, while in post-treatment samples, it could help in guiding non-operative treatment strategies.

Evaluation of Bacteria and Fungi DNA Abundance in Human Tissues.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

APOBEC-mediated DNA alterations: A possible new mechanism of carcinogenesis in EBV-positive gastric cancer.

Mechanisms of viral oncogenesis are diverse and include the off-target activity of enzymes expressed by the infected cells, which evolved to target viral genomes for controlling their infection. Among these enzymes, the single-strand DNA editing capability of APOBECs represent a well-conserved viral infection response that can also cause untoward mutations in the host DNA. Here we show, after evaluating somatic single-nucleotide variations and transcriptome data in 240 gastric cancer samples, a positive correlation between APOBEC3s mRNA-expression and the APOBEC-mutation signature, both increased in EBV+ tumors. The correlation was reinforced by the observation of APOBEC mutations preferentially occurring in the genomic loci of the most active transcripts. This EBV infection and APOBEC3 mutation-signature axis were confirmed in a validation cohort of 112 gastric cancer patients. Our findings suggest that APOBEC3 upregulation in EBV+ cancer may boost the mutation load, providing further clues to the mechanisms of EBV-induced gastric carcinogenesis. After further validation, this EBV-APOBEC axis may prove to be a secondary driving force in the mutational evolution of EBV+ gastric tumors, whose consequences in terms of prognosis and treatment implications should be vetted.

Genomics and epidemiology for gastric adenocarcinomas (GE4GAC): a Brazilian initiative to study gastric cancer

Gastric cancer (GC) is the fifth most common type of cancer worldwide with high incidences in Asia, Central, and South American countries. This patchy distribution means that GC studies are neglected by large research centers from developed countries. The need for further understanding of this complex disease, including the local importance of epidemiological factors and the rich ancestral admixture found in Brazil, stimulated the implementation of the GE4GAC project. GE4GAC aims to embrace epidemiological, clinical, molecular and microbiological data from Brazilian controls and patients with malignant and pre-malignant gastric disease. In this letter, we summarize the main goals of the project, including subject and sample accrual and current findings

Evidence for Mitochondrial Genome Methylation in the Yeast Candida albicans: A Potential Novel Epigenetic Mechanism Affecting Adaptation and Pathogenicity?

The commensal yeast Candida albicans is an opportunistic pathogen. In order to successfully colonize or infect the human body, the fungus must adapt to the host's environmental conditions, such as low oxygen tension (hypoxia), temperature (37°C), and the different carbon sources available. Previous studies demonstrated the adaptive importance of C. albicans genetic variability for its pathogenicity, although the contributions of epigenetic and the influence of environmental factors are not fully understood. Mitochondria play important roles in fungal energetic metabolism, regulation of nuclear epigenetic mechanisms and pathogenicity. However, the specific impact of inter-strain mitochondrial genome variability and mitochondrial epigenetics in pathogenicity is unclear. Here, we draw attention to this relevant organelle and its potential role in C. albicans pathogenicity and provide preliminary evidence, for the first time, for methylation of the yeast mitochondrial genome. Our results indicate that environmental conditions, such as continuous exposure for 12 weeks to hypoxia and 37°C, decrease the mitochondrial genome methylation in strains SC5314 and L757. However, the methylation decrease is quantitatively different in specific genome positions when strains SC5314 and L757 are compared. We hypothesize that this phenomenon can be promising for future research to understand how physical factors of the host affect the C. albicans mitochondrial genome and its possible impact on adaptation and pathogenicity.

Intraspecific comparative genomics of Candida albicans mitochondria reveals non-coding regions under neutral evolution.

The opportunistic fungal pathogen Candida albicans causes serious hematogenic hospital acquired candidiasis with worldwide impact on public health. Because of its importance as a nosocomial etiologic agent, C. albicans genome has been largely studied to identify intraspecific variation and several typing methods have been developed to distinguish closely related strains. Mitochondrial DNA can be useful for this purpose because, as compared to nuclear DNA, its higher mutational load and evolutionary rate readily reveals microvariants. Accordingly, we sequenced and assembled, with 8-fold coverage, the mitochondrial genomes of two C. albicans clinical isolates (L296 and L757) and compared these sequences with the genome sequence of reference strain SC5314. The genome alignment of 33,928 positions revealed 372 polymorphic sites being 230 in coding and 142 in non-coding regions. Three intergenic regions located between genes tRNAGly/COX1, NAD3/COB and ssurRNA/NAD4L, named IG1, IG2 and IG3, respectively, which showed high number of neutral substitutions, were amplified and sequenced from 18 clinical isolates from different locations in Latin America and 2 ATCC standard C. albicans strains. High variability of sequence and size were observed, ranging up to 56bp size difference and phylogenies based on IG1, IG2 and IG3 revealed three groups. Insertions of up to 49bp were observed exclusively in Argentinean strains relative to the other sequences which could suggest clustering by geographical polymorphism. Because of neutral evolution, high variability, easy isolation by PCR and full length sequencing these mitochondrial intergenic regions can contribute with a novel perspective in molecular studies of C. albicans isolates, complementing well established multilocus sequence typing methods.