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(Chest. 2004;125:104S-105S.)
© 2004 American College of Chest Physicians

Construction and Optimization of Chromosome Arm-Specific Comparative Genomic Hybridization Arrays for Identifying Genetic Alterations in Preinvasive Lung Cancers*

Cathie Garnis, BSc; Bradley Coe, BSc; Laura-Jane Henderson, BSc; Adrian Ishkanian, MSc; Spencer Watson, BSc; Marco Marra, PhD; John Minna, MD; Stephen Lam, MD, FCCP; Calum MacAulay, PhD and Wan Lam, PhD

* From the British Columbia Cancer Research Centre (Mss. Garnis and Henderson, Mssrs. Coe, Ishkanian, and Watson, and Drs. Marra, S. Lam, MacAulay, and W. Lam), Vancouver, BC, Canada; and the Hamon Center for Therapeutic Oncology Research (Dr. Minna), University of Texas Southwestern Medical Center, Dallas, TX.

Correspondence to: Wan Lam, PhD, Senior Scientist, Cancer Genetics and Developmental Biology, British Columbia Cancer Research Centre, British Columbia Cancer Agency, 601 W 10th Ave, Vancouver, BC, Canada V5Z 1L3; e-mail: wanlam{at}bccancer.bc.ca

The accumulation of genetic alterations is known to parallel lung cancer development. Array-based comparative genomic hybridization (aCGH) offers the potential for screening DNA samples for the deletion and amplification of chromosomal regions at a resolution 10 to 50 times greater than that of conventional comparative genomic hybridization (CGH) methodologies.

The construction and use of high-resolution CGH arrays will facilitate the fine mapping of known regions and the discovery of localized genetic alterations in lung cancer. Our objectives were as follows: (1) to construct high-density bacterial artificial chromosome (BAC) CGH arrays spanning selected chromosome arms annotated with genetic markers, which will anchor our data to publicly available loss of heterozygosity studies; (2) to optimize aCGH for the limited quality and quantity of microdissected DNA; (3) and to determine the impact of cell heterogeneity on aCGH.

A tiling set of BAC clones was selected based on a physical map of each chromosome arm, which was assembled using both DNA sequence and BAC DNA fingerprint information. DNA was isolated from the selected clones in a 96-well format, and clone identity was confirmed by their fingerprint pattern. A linker-mediated polymerase chain reaction process was used to generate sufficient quantities of DNA for array construction. The amplified products were spotted in triplicate on amine-coated glass slides.

Arrays of amplified BAC DNA clones, spanning chromosome arms 1p, 3p, and 5p, have been constructed, and a whole genome array of > 30,000 loci is near completion. We have established experimental conditions for aCGH for clinical material, and we have assessed the impact of tissue heterogeneity and DNA quality/quantity in the generation of an accurate CGH profile. A panel of lung cancer cell lines and a collection of microdissected clinical specimens, which represent various developmental stages of squamous non-small cell lung cancer, have been analyzed to deduce the minimal regions of alteration.

High-resolution CGH array technology facilitates the fine mapping of known alterations and the discovery of novel alterations in lung cancer.


   Footnotes
 
Abbreviations: aCGH = array-based comparative genomic hybridization; CGH = comparative genomic hybridization





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