Lower airway bacterial microbiome may influence recurrence after resection of early-stage non-small cell lung cancer.

OBJECTIVE: The lower airway bacterial microbiome influences carcinogenesis and response to immunotherapy in non-small cell lung cancer (NSCLC). We investigated the association of this microbiome with recurrence in early NSCLC. METHODS: Microbiomes of presurgery bronchoalveolar lavage (BAL) and saliva, and resected stage I NSCLC tumor and adjacent lung tissues of 48 patients were examined by 16S gene sequencing. Tumor gene expression was measured by RNA sequencing. RESULTS: Spatial relationships of the different biospecimen types was reflected in their microbiomes, with microbiomes of BAL intermediate to those of saliva and lung tissue. BAL and saliva microbiomes were less dissimilar in patients with high α-amylase levels in BAL, indicating oral aspiration as a source of lower airway microbiota. BAL microbiomes of patients with recurrence within 32 months of surgery differed from those without recurrence during ≥32 months of follow-up (n = 18 each), despite no difference for age, sex, smoking history, and tumor histology and grade. The recurrence-associated BAL microbiome signature was present in 16 of the 18 recurrence cases but in only two of the others. Signature presence was associated with shorter recurrence-free survival (log-rank test P < .001; hazard ratio = 14.5), and greater expression in tumors of genes for cell proliferation and epithelial mesenchymal transition. Immune cellular composition of the tumor microenvironment was not different between patients with and without the signature. CONCLUSIONS: Presurgery composition of lower airway microbiome may be associated with recurrence of early NSCLC. This association may reflect an influence of the microbiome on tumor biology.

Exploring the role of survivin in neuroendocrine neoplasms.

Neuroendocrine tumors (NETs) are a heterogenous group of tumors. While most NETs have excellent prognosis, certain subsets have aggressive biology and have limited treatment options. We explored the role of survivin in NET as a prognostic and potentially therapeutic marker. Tissue microarrays of 132 patients were stained for survivin using immunohistochemistry (IHC) and correlated with outcomes. Using genomic database, we then correlated survivin (BIRC5) mRNA expression with radiosensitivity index (RSI) in 52 samples of NET. Finally, we studied the effect of radiation on survivin expression in human cell lines and the impact of knock-down of BIRC5 on cell proliferation and radiation sensitivity. We found that survivin positivity by IHC correlated with a shorter survival (overall survival 8.5 years vs. 18.3 years, p < 0.001). There was a positive correlation between BIRC5 expression and RSI (r = 0.234, p < 0.0001). Radiation exposure increased BIRC5 gene expression in a human carcinoid cell line. Knockout of BIRC5 using siRNA reduced proliferation of neuroendocrine cells but did not increase radiation sensitivity. We conclude that survivin expression in NET correlates with an inferior survival and survivin expression in human carcinoid cell lines increases after exposure to ionizing radiation.

Sublethal Radiation Affects Antigen Processing and Presentation Genes to Enhance Immunogenicity of Cancer Cells.

While immunotherapy in cancer is designed to stimulate effector T cell response, tumor-associated antigens have to be presented on malignant cells at a sufficient level for recognition of cancer by T cells. Recent studies suggest that radiotherapy enhances the anti-cancer immune response and also improves the efficacy of immunotherapy. To understand the molecular basis of such observations, we examined the effect of ionizing X-rays on tumor antigens and their presentation in a set of nine human cell lines representing cancers of the esophagus, lung, and head and neck. A single dose of 7.5 or 15 Gy radiation enhanced the New York esophageal squamous cell carcinoma 1 (NY-ESO-1) tumor-antigen-mediated recognition of cancer cells by NY-ESO-1-specific CD8+ T cells. Irradiation led to significant enlargement of live cells after four days, and microscopy and flow cytometry revealed multinucleation and polyploidy in the cells because of dysregulated mitosis, which was also revealed in RNA-sequencing-based transcriptome profiles of cells. Transcriptome analyses also showed that while radiation had no universal effect on genes encoding tumor antigens, it upregulated the expression of numerous genes involved in antigen processing and presentation pathways in all cell lines. This effect may explain the immunostimulatory role of cancer radiotherapy.

Whole blood microRNA expression may not be useful for screening non-small cell lung cancer.

At least seven studies have suggested that microRNA levels in whole blood can be diagnostic for lung cancer. We conducted a large bi-institutional study to validate this. Qiagen® PAXgene™ Blood miRNA System was used to collect blood and extract RNA from it for 85 pathologic stage I-IV non-small cell lung cancer (NSCLC) cases and 76 clinically-relevant controls who had a benign pulmonary mass, or a high risk of developing lung cancer because of a history of cigarette smoking or age >60 years. Cases and controls were similar for age, gender, race, and blood hemoglobin and leukocyte but not platelet levels (0.23 and 0.26 million/µl, respectively; t test P = 0.01). Exiqon® MiRCURY™ microarrays were used to quantify microRNAs in RNA isolates. Quantification was also performed using Taqman™ microRNA reverse transcription (RT)-PCR assays for five microRNAs whose lung cancer-diagnostic potential had been suggested in seven published studies. Of the 1,941 human mature microRNAs detectable with the microarray platform, 598 (31%) were identified as expressed and reliably quantified among the study's subjects. However, none of the microRNAs was differentially expressed between cases and controls (P >0.05 at false discovery rate <5% in test using empirical Bayes-moderated t statistics). In classification analyses with leave-one-out internal cross-validation, cases and controls could be identified by microRNA expression with 47% and 50% accuracy with support vector machines and top-scoring pair methods, respectively. Cases and controls did not differ for RT-PCR-based measurements of any of the five microRNAs whose biomarker potential had been suggested by seven previous studies. Additionally, no difference for microRNA expression was noticed in microarray-based microRNA profiles of whole blood of 12 stage IA-IIIB NSCLC cases before and three-four weeks after tumor resection. These findings show that whole blood microRNA expression profiles lack diagnostic value for high-risk screening of NSCLC, though such value may exist for selective sub-groups of NSCLC and control populations.

APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages.

The extent, regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific C>U RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins, including many that are involved in pathogenesis of viral diseases. APOBEC3A, which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons, mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of C>U RNA editing in mammals.