Characterization of genetic alterations in lung cancer

Lung cancer is the leading cause of cancer-related deaths in North America and Europe for both men and women. In order to improve the survival rate of lung cancer, early detection and treatment approaches need to be developed. Markers of genetic instability in preinvasive stages will facilitate treatment design. Conventional techniques used to identify genomic areas of instability such as microsatellite analysis and comparative genomic hybridization require amounts of DNA that exceed the yield from preinvasive lesions. In our laboratory, we have developed a PCR-based genome scanning method called scanning of microdissected archival lung lesions (SMALL)-PCR in order to detect recurrent genomic alterations. DNA was isolated from microdissected formalin fixed paraffin embedded squamous cell carcinomas, preinvasive stages and normal lung samples. Following whole genome screening with SMALL-PCR , altered genomic segments were cloned, sequenced and localized to chromosomal regions and the known genes within these regions were identified. One region identified by SMALL-PCR was investigated by fluorescent in situ hybridization and loss of heterozygosity to verify the findings of SMALL-PCR. The expression levels of selected genes were evaluated by reverse transcription-PCR using a panel of squamous cell lung carcinomas and paired normal samples from the same patient. Expression of a selected gene was evaluated in a developing mouse embryo as well as from lungs dissected from different embryonic stages. Attempts were made to investigate protein expression by immunohistochemistry. Sixty-four normal, preinvasive stages and tumour DNA samples originating from 16 patients were analyzed by SMALL-PCR. Scanning the genome using SMALL-PCR identified six recurrent chromosomal abnormalities detected in multiple patients. Four of the regions contain genes belonging to or interacting with the Wnt signaling pathway. Microsatellite analysis confirmed genomic instability at one of the regions, 11ql4.2 , discovered by SMALL-PCR. Expression analysis offzd4, a gene near the SMALL-PCR alteration, verified a statistically significant altered level of expression in tumour samples. Another region, 9q21.33 contained 2 genes that were evaluated for expression levels in tumour samples compared to normal samples, dapk and gasl were both differentially expressed when normalized against gapdh expression levels. Only dapk was differentially expressed in tumour samples when using B-actin for normalization. fzd4 expression was confirmed in the developing embryo at different time points. Expression offzd4 was also evident in lungs dissected from mice embryos as early as 11.5 days post coitum. Genome wide scanning using SMALL-PCR has identified recurring chromosomal alterations in premalignant and malignant lesions of the lung. These alterations point to the involvement of important developmental signaling pathways in the progression of lung cancer.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat