MET, HGF, EGFR, and PXN gene copy number in lung cancer using DNA extracts from FFPE archival samples and prognostic significance.
Gene copy number analysis for some of the important molecules in lung tumorogenesis, such as MET, hepatocyte growth factor [(HGF), ligand for MET), epidermal growth factor receptor (EGFR), and paxillin (PXN), is likely to determine both the type of treatment and prognosis. Formalin-fixed paraffin-embedded (FFPE) archival tumor tissue samples are an excellent source for determining key molecular changes in the OncoGenome; however, existing extraction procedures yield relatively poor quality genomic DNA fragments. Although FISH is the method of choice for determining amplification of a gene, a more rapid quantitative poly-merase chain reaction (qPCR) technique to determine gene copy number can be used when reasonably good quality genomic DNA is available. We report here a relatively rapid method based on microwave/chelex-100 treatment that gives rise to genomic DNA fragments ranging from 1 to 12 Kb and beyond, thereby attesting to its superior quality. Genomic PCR for beta-globin gene gave reliable and reproducible results. The number of steps for extracting the DNA was kept to a minimum, and instead of precipitating the DNA, we preserved the genomic DNA extracts so as to prevent a loss in DNA yield. We found the extracts to be stable and amenable to qPCR and mutational analysis. Using lung adenocarcinoma FFPE samples and cell lines derived from lung adenocarcinomas, we demonstrated that the gene copy number for MET in lung adenocarcinoma tissue samples was preferentially increased over EGFR, HGF, and PXN and that it positively correlated with a better prognosis. In contrast, the genomic DNA extracted from 25 NSCLC cell lines gave a relatively higher gene copy number for all four genes evaluated. Our results indicate that the microwave/chelex-100-based method yields good-quality genomic DNA extracts that can be used for complex DNA analysis, such as determination of gene copy number. In addition, our data demonstrated that the adenocarcinoma cell lines potentially evolved under ex vivo conditions, and therefore, in genetic studies it is imperative to use primary tumors for generalized conclusions about lung tumors.
Kanteti
Rajani
R
Department of Medicine, Section of Hematology and Oncology University of Chicago, Chicago, IL, USA.
Yala
Soheil
S
Ferguson
Mark K
MK
Salgia
Ravi
R
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics*; D018628:Gene Dosage; D017228:Hepatocyte Growth Factor / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D051419:Paxillin / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011379:Prognosis; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics*
D017931:DNA Primers; D004273:DNA, Neoplasm; C496061:PXN protein, human; D051419:Paxillin; D017228:Hepatocyte Growth Factor; D066246:ErbB Receptors; D019859:Proto-Oncogene Proteins c-met