Feasibility of using low-volume tissue samples for gene expression profiling of advanced non-small cell lung cancers.

PURPOSE: The majority of patients with non-small cell lung cancer (NSCLC) present at an advanced clinical stage, when surgery is not a recommended therapeutic option. In such cases, tissues for molecular research are usually limited to the low-volume samples obtained at the time of diagnosis, usually via fine-needle aspiration (FNA). We tested the feasibility of performing gene expression profiling of advanced NSCLCs using amplified RNA from lung FNAs. EXPERIMENTAL DESIGN AND RESULTS: A total of 46 FNAs was tested, of which 18 yielded RNA of sufficient quality for microarray analysis. Expression profiles of these 18 samples were compared with profiles of 17 pairs of tumor and normal lung tissues that had been surgically obtained. Using a variety of unsupervised and supervised analytical approaches, we found that the FNA profiles were highly distinct from the normal samples and similar to the tumor profiles. CONCLUSIONS: We conclude that when RNA amplification is successful, gene expression profiles from NSCLC FNAs can determine malignancy and suggest that with additional refinement and standardization of sample collection and RNA amplification protocols, it will be possible to conduct additional and more detailed molecular analysis of advanced NSCLC using lung FNAs.

Using whole genome amplification (WGA) of low-volume biopsies to assess the prognostic role of EGFR, KRAS, p53, and CMET mutations in advanced-stage non-small cell lung cancer (NSCLC).

BACKGROUND: Progression of non-small cell lung cancer (NSCLC) from early- to late-stage may signify the accumulation of gene mutations. An advanced-stage tumor's mutation profile may also have prognostic value, guiding treatment decisions. Mutation detection of multiple genes is limited by the low amount of deoxyribonucleic acid extracted from low-volume diagnostic lung biopsies. We explored whole genome amplification (WGA) to enable multiple molecular analyses. METHODS: Eighty-eight advanced-stage NSCLC patients were enrolled. Their low-volume lung biopsies underwent WGA before direct sequencing for epidermal growth factor receptor (EGFR), KRAS (rat sarcoma virus), p53, and CMET (mesenchymal-epithelial transition factor) mutations. Overall survival impact was examined. Surgically-resected tumors from 133 early-stage NSCLC patients were sequenced for EGFR, KRAS and p53 mutations. We compared the mutation frequencies of both groups. RESULTS: It is feasible for low-volume lung biopsies to undergo WGA for mutational analysis. KRAS and CMET mutations have a deleterious effect on overall survival, hazard ratios 5.05 (p = 0.009) and 23.65 (p = 0.005), respectively. EGFR and p53 mutations, however, do not have a survival impact. There also does not seem to be significant differences in the frequency of mutations in EGFR, KRAS, and p53 between early- and advanced-stage disease: 20% versus 24% (p = 0.48), 29% versus 27% (p = 0.75), 10% versus 6% (p = 0.27), respectively. CONCLUSIONS: In advanced-stage NSCLC, KRAS, and CMET mutations suggest poor prognosis, whereas EGFR and p53 mutations do not seem to have survival impact. Mutations in EGFR, KRAS and p53 are unlikely to be responsible for the progression of NSCLC from early- to late-stage disease. WGA may be used to expand starting deoxyribonucleic acid from low-volume lung biopsies for further analysis of advanced-stage NSCLC.

An alternative approach to determining therapeutic choices in advanced non-small cell lung carcinoma (NSCLC): maximizing the diagnostic procedure and the use of low-volume lung biopsies.

BACKGROUND: Accurate mutational analysis, especially epidermal growth factor receptor (EGFR) mutations, of diagnostic biopsies from all Asian NSCLC patients is crucial to their clinical management, but faces problems. Here, we explore, within usual hospital constraints, the practicalities of incorporating mutational analysis in every newly diagnosed case of NSCLC, namely, maximizing tissue acquisition during the diagnostic procedure and determining the maximum quantity and quality of DNA sequence data available from these biopsies. METHODS: Sixty-eight Chinese patients were enrolled. Thirty-five underwent surgical resections for early-stage tumors. Thirty-three underwent diagnostic procedures, i.e., needle aspirates under bronchoscopic or computed tomographic/fluoroscopic guidance, or forceps biopsies via bronchoscopy. Separate samples for research purposes were obtained from these 33 patients during the diagnostic procedure. All samples were analyzed for mutations in EGFR exons 18 to 21, p53 exons 4 to 9, and Kras exon 2. RESULTS: No deaths occurred in this study. Success rates in obtaining sequence data from surgical samples versus low-volume samples for EGFR, p53, and Kras were 100% versus 85%, 100% versus 82%, and 100% versus 85%, respectively. Sequencing nine polymerase chain reaction products from each low-volume sample resulted in the exhaustion of all extracted DNA from three samples. CONCLUSIONS: Acquiring a separate low-volume lung biopsy sample for mutational analysis in lung cancer patients during the diagnostic procedure is feasible and may be a valuable complement to the usual diagnostic workflow in future.