Background: microRNA (miRNA) expression profiles are being intensively investigated for their involvement in carcinogenesis. We evaluated the prognostic value of mature microRNA-21 (miR-21) and mature microRNA-205 (miR-205) overexpression in non–small cell lung cancer (NSCLC). Patients and methods: We studied 48 pairs of NSCLC fresh frozen tissue specimens collected at time of surgery and before chemotherapy. Highly specific amplification and quantification of mature miR-21 and mature miR-205 was achieved using looped real time RT-PCR. Results: miRNA expression, determined by real time RT-PCR, was defined by ΔΔCt measurements. We detected overexpression of mature miR-21 in 25 (52.0%) of the 48 NSCLC paired specimens and overexpression of miR-205 in 31 (64.6%). Overexpression was assessed after comparison of miRNA expression in NSCLC tissues and in their corresponding noncancerous tissues with respect to U6 expression. During the follow-up period, 29 of 48 (60.4%) patients relapsed, and 23 of 48 died (47.9%). Mature miR-21 was upregulated in 16 of 29 (55.2%) patients who relapsed and 15 of 23 (65.2%) patients who died. Mature miR-205 was overexpressed in 19 of 29 patients who relapsed (65.5%) and 15 of 23 patients who died (65.2%). Mature miR-21 overexpression correlated with overall survival (OS) of the patients (P = 0.027), whereas overexpression of mature miR-205 did not. Conclusions: Our results suggest that overexpression of mature miR-21 is an independent negative prognostic factor for OS in NSCLC patients.

BACKGROUND: Circulating tumor cell (CTC) analysis is a promising new diagnostic field for estimating the risk for metastatic relapse and metastatic progression in patients with cancer. CONTENT: Different analytical systems for CTC isolation and detection have been developed as immunocytochemical and molecular assays, most including separation steps by size or biological characteristics, such as expression of epithelial- or cancer-specific markers. Recent technical advancements in CTC detection and characterization include methods based on multiplex reverse-transcription quantitative PCR and approaches based on imaging and microfilter and microchip devices. New areas of research are directed toward developing novel assays for CTC molecular characterization. QC is an important issue for CTC analysis, and standardization of micrometastatic cell detection and characterization methodologies is important for the incorporation of CTCs into prospective clinical trials to test their clinical utility. The molecular characterization of CTCs can provide important information on the molecular and biological nature of these cells, such as the status of hormone receptors and epidermal and other growth factor receptor family members, and indications of stem-cell characteristics. This information is important for the identification of therapeutic targets and resistance mechanisms in CTCs as well as for the stratification of patients and real-time monitoring of systemic therapies. SUMMARY: CTC analysis can be used as a liquid biopsy approach for prognostic and predictive purposes in breast and other cancers. In this review we focus on state-of-the-art technology platforms for CTC isolation, imaging, and detection; QC of CTC analysis; and ongoing challenges for the molecular characterization of CTCs.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is crucial to identify novel diagnostic and therapeutic targets for individualized therapies. We developed a multiplexed PCR-coupled liquid bead array to detect the expression of multiple genes in CTCs. METHODS: mRNA isolated from immunomagnetically enriched CTCs was subjected to multiplex PCR for KRT19 (keratin 19; also known as CK19), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2], SCGB2A2 (secretoglobin, family 2A, member 2; also known as MGB1, mammaglobin A), MAGEA3 (melanoma antigen family A, 3), TWIST-1 [twist homolog 1 (Drosophila)], and HMBS (hydroxymethylbilane synthase; also known as PBGD). Biotinylated amplicons were hybridized against fluorescent microspheres carrying gene-specific capture probes and incubated with streptavidin–phycoerythrin. We quantified the captured labeled amplicons and decoded the beads by Luminex flow cytometry. The assay was validated for limit of detection, specificity, and comparison with reverse-transcription quantitative PCR (RT-qPCR), and its clinical performance was evaluated in 64 patients with operable breast cancer, 20 patients with metastasis, and 17 healthy individuals. RESULTS: The assay was specific for each gene in complex multiplexed formats and could detect the expression of each gene at the level of a single SK-BR-3 cell. The assay produced results comparable to those for RT-qPCR for each gene. None of the genes tested was detected in the CTC fraction of healthy donors. We detected KRT19, ERBB2, MAGEA3, SCGB2A2, and TWIST1 in 26.6%, 12.5%, 18.7%, 10.9%, and 31.2% of operable breast cancer patients, respectively, and detected the corresponding genes in 65%, 20%, 30%, 20%, and 20% of patients with verified metastasis, respectively. CONCLUSIONS: The expression of 6 genes in CTCs can be measured simultaneously and reliably, thereby saving precious sample and reducing the costs and time of analysis.