Abstract B16: Histology and intervention related miRNA expression changes from baseline to follow-up paired bronchial biopsies of patient in the iloprost chemoprevention trial
Authors: Mascaux Celine , Keith Robert L. , Feser William J. , Lewis Marina T. , Baron Anna E. , Coldren Christopher D. , Merrick Dan T. , Kennedy Timothy C. , Eckelberger John I. , Franklin Wilbur A. , Bunn Paul A. , Miller York E. , Hirsch Fred R. 
Published: March 2013

Abstract
The iloprost lung cancer chemoprevention trial is the first to meet a primary endpoint of improvement in endobronchial histology, which is currently considered to be the best intermediate endpoint for chemoprevention (Keith et al, Cancer Prev Res, 2011). This placebo-controlled study offers the opportunity for discovering other surrogate endpoints and predictive biomarkers to incorporate into chemoprevention trials.
We compared miRNA expression in follow-up (FU) versus baseline biopsies in relation with histology change or not globally and stratified by treatment arm and smoking status.
In the 152 patients randomized in the iloprost trial, the paired baseline and FU biopsies were available in 125 patients: 40/35 current/former smokers in the iloprost arm and 25/25 current/former smokers in the placebo arm, respectively. We planned to analyze 500 biopsies: four biopsies from each of the 125 patients, 2 biopsies at baseline (worst and best diagnosis) and 2 biopsies at FU at the same site after six months of treatment with iloprost or placebo. We selected and analyzed 14 miRNAs differentially expressed during squamous cell lung carcinogenesis in our previous study (Mascaux et al, Eur Resp J, 2009).
In total, 496/500 biopsies with appropriate tissue were available. For every biopsy, total RNA was extracted from 8 adjacent 4 um cuts of formalin fixed paraffin embedded (FFPE) adjacent to the diagnostic section. A good reproducibility of the previously published data for the expression changes in the 14 miRNA accross lung preneoplasia stages was shown in the current study in baseline samples. This new study identifies significant changes in miR-34c expression between paired samples when histology is up- or downgraded. miR-34c is significantly down-regulated in paired biopsies when histology is up-graded and inversely. This correlation between miR-34c expression and histology changes is shown including all samples (r spearman correlation=0.23 p=0.0003), but also consistently in subgroups (iloprost.arm, current r=0.26, p=0.041 and former smokers r=0.24, p=0.066, placebo arm, current (r=0.23, p=0.046)). In contrast, miR-9 was significantly down-regulated in follow-up as compared with baseline biopsies, but was not correlated with histology changes. The down-regulation of miR-9 in follow-up versus baseline samples was found in all samples (t test, p&lt;0.0001) and was consistent in all and significant in most of subgroups (iloprost arm, all patients (p=0.0007) and current smokers (p=0.0023) and placebo arm, all patients (p=0.001), current smokers (p=0.047) and former smokers (p=0.0071).
In conclusion, miR-34c, a transcriptional target of p53 which is down-regulated by hypermethylation in lung cancer, is down-regulated when histology changes from the normal bronchial mucosa to high-grade bronchial lesions, and inversely, independently of treatment and smoking status. miR-34c expression is a good reflection of baseline histology and histology changes. Alternatively, miR-9 is systematically down-regulated in follow-up biopsies 6 months later at the same site independently of histology, treatment and tobacco status. This suggests that the down-regulation of miR-9 in the follow-up samples may be related to the biopsy: this miRNA could be involved in the tissue repair.