A novel multiple FISH array for the detection of genetic aberrations in cancer

Abstract

Interphase multicolor fluorescence in situ hybridization (IM-FISH) has great promise for improving cancer diagnosis because it can directly visualize multiple changes in chromosomes and gene copy number on a cellto-cell basis. However, no more than four targets can be detected simultaneously by current commercially available IM-FISH protocols, and the DNA probes used are too large to detect the single-gene aberrations that characterize tumorigenesis. As a result, multiple FISH has a low sensitivity in detecting cancer cells. To overcome such limitations, we first developed specific genomic probes for the genes relevant to primary lung cancer. We next designed a multiple FISH array by arranging four different compositions of cocktails of four probes for each gene on a coverslip, which allowed four four-color FISH experiments to be performed in parallel on a single slide. We then tested the multiple FISH array on bronchial brushing samples from lung cancer patients to determine its ability to detect genetic abnormalities. A comparison of the data with the results of cytology and commercial four-color FISH suggested that the multiple FISH array had the highest sensitivity for cancer detection. The technique may thus be a powerful laboratory strategy for cancer prevention and early detection and for improved patient management.

A Panel of Sputum-Based Genomic Marker for Early Detection of Lung Cancer

“…Of the 6 genes, 3 genes (FHIT, HYAL2, and P16) were previously investigated in sputum of lung cancer patients for their genomic copy number alterations by our group (6,7,9,(14)(15)(16). The present observations of the genes are consistent with our earlier findings, confirming their potential role as biomarkers for early NSCLC.…”

“…Signals for each probe on the in situ minichip were analyzed blindly by investigators who did have knowledge of case-control status. The criteria for defining a gene with abnormal copy numbers and a positive cell with abnormal genetic changes were used as described in our previous reports (7,15). Briefly, more or less intense signals from the specific gene probe than from the corresponding CEP indicated a gain or loss of the gene.…”

“…The CEPs were used as internal controls for assessing changes of the target genes. In situ minichips consisting of the probes were made and then hybridized on the specimens as previously described (7,15). After washing, the slides were examined under microscopes equipped with appropriate filter sets (Leica Microsystems).…”

“…Collecting, processing, and cytologic studying sputum All participants in the 2 different medical centers underwent the same sputum collection using an induced protocol over the same interval of time (6,7,9,(14)(15)(16). Sputum was collected from lung cancer patients before they had received preoperative adjuvant chemotherapy or radiotherapy.…”

“…By using this approach, we concentrated the bronchial epithelial cells to 40% purity from 1.1% of the starting population, yielding enough bronchial epithelium for cytologic and molecular analyses of sputum (6). Furthermore, taking advantage of the developments in microarray, and based on the principle of in situ hybridization, we developed an in situ minichip assay that examined genomic copy number changes of multiple genes at once (7). In addition, using high-resolution comparative genomic hybridization microarrays, we identified a set of genetic signatures whose copy number changes were associated with early stage NSCLC (8).…”