Lead articlesMolecular cytogenetic analysis of non-small cell lung carcinoma by spectral karyotyping and comparative genomic hybridization
Introduction
Lung cancer, the most common cause of cancer-related mortality in men and women in North America, has an overall 5-year survival rate of approximately 14% [1]. Approximately 80% of all lung cancers are classified as non-small cell lung carcinoma (NSCLC), which include adenocarcinoma (ADC), squamous cell carcinoma (SQCC), and large cell anaplastic subtypes.
In general, performing conventional cytogenetic analysis of solid tumors, such as NSCLC, have proven to be difficult and laborious. Good quality G-banded metaphase chromosomes are hard to prepare and karyotypes of lung tumors are complex with both structural and numerical changes, including many unidentifiable marker chromosomes. Cytogenetic analysis of short-term primary cultures and established cell lines in NSCLC have revealed a pattern of recurrent chromosomal aberrations, namely gains of 5p, 7, and losses of 3p, 9p, 17p, and the Y chromosome being most frequently reported 2, 3, 4, 5, 6. Similarly, allelic losses detected by loss of heterozygosity (LOH) studies in more than 600 NSCLC have identified several regions of consistent chromosomal deletion, namely at 2q, 3p, 5q, 8p, 9p, 11p, 13q, 17p, 17q, 18q, and 22q, which potentially contain tumor suppressor genes 7, 8, 9, 10, 11, 12, 13.
In the last several years, three molecular cytogenetic methods have greatly improved our abilities to study genomic change in solid tumors: fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY) 14, 15, 16, 17, 18. Interphase FISH is particularly useful for detecting specific aneusomies when metaphase preparations are not available. By interphase analysis, gains of chromosomes 6, 7, 12, and 17 and losses of chromosomes 9 and Y have been reported in NSCLC 19, 20. CGH is a generalized screening technique that identifies genomic imbalances (gains, losses, or amplification) and can be performed using DNA extracted from the tumor [16]. To date, there have been only five CGH studies of NSCLC, which identified non-random chromosomal gains or losses, primarily affecting chromosomes 3q, 5p, 7p, and 8q 21, 22, 23, 24, 25. The most recent molecular cytogenetic technique, spectral karyotyping (SKY), permits simultaneous hybridization of 24 fluorescently labeled human chromosome painting probes onto a tumor metaphase preparation [18]. SKY is particularly useful for identifying the chromosomes involved in complex rearrangements and markers of the type frequently encountered in NSCLC.
In this study, DNA from 8 primary tumor specimens and 15 cell lines were analyzed by CGH to detect genetic alterations. As a result of the study, copy number changes involving 8q24 were additionally studied by direct FISH analysis of cell lines to evaluate the status of the MYCC gene in NSCLC. A representative cell line of the major histological subtype of NSCLC, MGH7 (SQCC), and A549 (ADC), were analyzed by SKY to determine the identity and extent of the complex structural aberrations detected by G-banding. These results, in combination with the CGH findings, were used to determine whether there were sub-type specific changes.
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Primary tumors and cell lines
Twenty-three NSCLCs were studied by CGH. They consisted of 15 cell lines and 8 primary tumors. The samples included 9 SQCC, 10 ADC, 2 adenosquamous carcinomas (ADSC), and 2 large cell undifferentiated carcinomas (LCC). SQCC cell lines H157, H226, H520, and H1264; ADC cell lines, A549, H358; the ADC line, H125, and the LCC H661 were obtained from American Type Culture Collection (Rockville, MD, USA). The remaining cell lines, MGH4, MGH7, MGH8, MGH13, MGH24, and RVH6849 were established in our
Genetic aberrations in non-small cell lung carcinomas detected by comparative genomic hybridization
DNA copy number changes were detected in all 23 cases of NSCLC studied. The mean number of chromosomal imbalances per specimen was 11.4 (range, 1–26). The total number of chromosomal copy number changes in each specimen is summarized in Table 1. Gains predominated over losses with a ratio of 3:1. All chromosomes were involved in imbalances. A schematic representation of the results is shown in Fig. 1. The most frequently gained chromosomal arm was 5p (70%; 16/23). In four cases (MGH8, MGH24,
Discussion
In this study, a number of nonrandom chromosomal aberrations in NSCLC were identified, adding to the total number of CGH reports in this tumor type 21, 22, 23, 24, 25. Chromosomal regions that were frequently affected by DNA gains included 1q, 3q, 5p, 8q, 11q, 15q, 19q, and 20q. In keeping with previous reports, the most frequently gained chromosome arm in this series was 5p (70%), with three cases showing amplification of the whole p-arm and one case (MGH30) showing amplification in 5p15
Acknowledgments
We thank Sandra Johnson at the Banting Institute Cancer Cytogenetics Program and Ajay Pandita at the Ontario Cancer Institute for contributing their expertise in this project. This work was supported by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society.
Supported by the TGH-PMH thoracic Oncology Research Program and grants (J.S. and M.S.T.) from the Canadian Cancer Society and National Cancer Institute of Canada.
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