Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC.

Abstract. The genomic region containing PIK3CA was found to be amplified in esophageal squamous cell carcinoma (ESCC) tissue. PIK3CA at 3q26, which encodes the p110· catalytic subunit of phosphatidylinositol (PI) 3-kinase, is a unique intracellular signal transducer characterized by its lipid substrate specificity. In order to characterize PIK3CA in ESCC, we investigated hot-spot mutations in exons 1, 9 and 20, the copy number gain, the expression levels of mRNA and protein. Analysis in exon 9 of the PIK3CA gene revealed mutation in 7.7% (4 of 52) of ESCC samples. No mutation was detected in either exon 1 or exon 20. Copy number amplifications of PIK3CA were found in 12 of the 45 patients (26.7%). PIK3CA mRNAs were examined in 37 ESCC patients as determined by quantitative RT-PCR and the mean mRNA level of PIK3CA in ESCC tissues was 2.61-fold higher compared with that in corresponding non-tumorous esophageal epithelia (P<0.001). Immunohistochemically, positive immunoreaction for PIK3CA was detectable in 33 of 66 (50.0%) ESCC cases, while it was not detectable in the remaining 33 cases. Furthermore, comparing the cases with negative staining with those with positive staining for PIK3CA, the presence of node metastasis was significantly correlated with those with positive staining (P<0.05). This study is the first report providing comprehensive analysis of PIK3CA expression in ESCC. These results indicate that PIK3CA may play a crucial role in the development of ESCC and serve as an indicator for lymph node metastasis.

Prognostic tests for patients with early-stage lung cancer may provide needed guidance on postoperative surveillance and therapeutic decisions. We used a novel strategy to develop and validate a prognostic classifier for early-stage lung cancer. Specifically, we focused on 42 genes with roles in lung cancer or cancer prognosis. Expression of these biologically relevant genes and their association with relapse-free survival (RFS) were evaluated using microarray data from 148 patients with stage I lung adenocarcinoma. Seven genes associated with RFS were further examined by quantitative reverse transcription PCR in 291 lung adenocarcinoma tissues from Japan, the United States, and Norway. Only BRCA1, HIF1A, DLC1, and XPO1 were each significantly associated with prognosis in the Japan and US/Norway cohorts. A Cox regression-based classifier was developed using these four genes on the Japan cohort and validated in stage I lung adenocarcinoma from the US/Norway cohort and three publicly available lung adenocarcinoma expression profiling datasets. The results suggest that the classifier is robust across ethnically and geographically diverse populations regardless of the technology used to measure gene expression. We evaluated the combination of the four-gene classifier with miRNA miR-21 (MIR21) expression and found that the combination improved associations with prognosis, which were significant in stratified analyses on stage IA and stage IB patients. Thus, the four coding gene classifier, alone or with miR-21 expression, may provide a clinically useful tool to identify high-risk patients and guide recommendations regarding adjuvant therapy and postoperative surveillance of patients with stage I lung adenocarcinoma. Cancer Res; 73(13); 3821-32. Ó2013 AACR.