Comparison of genomic alterations by array comparative genomic hybridization between early-relapse and non-relapse non-small cell lung cancer patients

다국어 초록 (Multilingual Abstract)

Lung cancer is the leading cause of cancer death in developed countries and worldwide accounts for million deaths each year. Non-small cell lung cancers (NSCLCs) comprise 80% of all lung carcinomas, with adenocarcinomas (AdCCs) and squamous-cell carcinomas (SqCCs) representing the majority of these tumors. Although patients with early-stage NSCLC typically have better outcomes, 35% to 50% of the patient will relapse within five years after surgical treatment. The cytogenetic analyses of recurring genomic alterations play an important role in assessing pathogenesis and determining the prognosis of cancers. However, due to difficulties with culturing tumor cells and complexity of lesions, routine cytogenetic analyses of genomic alterations are not sufficient. Array comparative genomic hybridization (CGH) provides a method to quantitatively measure the changes of DNA copy number with an extremely high resolution and to map them directly onto the complete linear genome sequences. In this study, we used array CGH to assess genomic alterations in 21 AdCCs (11 early-relapse and 10 non-relapse) and 23 SqCCs (11 early-relapse and 12 non-relapse) fresh-frozen lung cancer tissues.
We identified genomic alterations that showed significant of different frequency between early-relapse and non-relapse AdCCs, including 5q21.3, 7p11.2, 9q34.11, 10p11.22, 11p13, 11p15.1, 11p15.4, 12p13.31, 16p13.12, 16q22.1, 17q11.2 and 21q22.11. Furthermore, genomic alterations of 6p12.1, 7p15.3-7p15.2 and 9q34.3 were different between the early-relapse and non-relapse SqCCs. Hierarchical clustering was performed using candidate clones, and gain and loss of selected clones were confirmed using FISH analysis. When the cutoff value was 4, the association study between candidate clones and relapse prediction revealed that early-relapse and non-relapse groups were most effectively separated, and sensitivity and specificity were 0.80 and 1.00, respectively. To develop the most effective relapsable signature for AdCCs, ROC curve and Kaplan-Meier survival analyses were performed. The resulting ROC curve showed an AUC of 0.894. Kaplan-Meier analysis showed a significant stratification in relapse-free survival of AdCCs. However, there was no association with SqCC patients.
To further confirm the results of array CGH, genomic alteration of selected-clones by array CGH analysis and copy number change of cancer related-candidate genes in AdCC and SqCC patients were compared by real-time qPCR, and array CGH and real-time qPCR data were found to correspond to delineated DNA copy number changes. However, mRNA expression of candidate genes was not correlated with the result of array CGH. In summary, many genomic alterations in NSCLC patients were observed using array CGH, and relapsable signature was identified in AdCC patients. This signature could be useful in stratifying patient groups according to relapse for adjuvant treatment after surgical treatment.

목차 (Table of Contents)

• 1. INTRODUCTION
• 1.1 Human lung cancer 1
• 1.2 Chromosomal alterations in human lung cancer 2
• 1.3 Screening tools of chromosomal alterations 3
• 1.4 Array CGH analysis for characterization of chromosomal alteration 5
• 1.5 Purpose of this study 6
• 2. MATERIALS AND METHODS
• 2-1 Subjects 8
• 2-2 Isolation of tissues genomic DNA 8
• 2-3 Array CGH analysis 9
• 2.4 Data analysis 11
• 2-5 Fluorescence in situ hybridization (FISH) analysis 12
• 2-6 Verification of DNA copy number change and mRNA expression using real-time qPCR 12
• 2-7 Statistical analysis 14
• 3. RESULTS
• 3-1 Genomic alterations in individual sample by array CGH 15
• 3-2 Identification of genomic alterations between early-relapse and non-relapse groups in NSCLC (AdCC and SqCC) patients 16
• 3-3 Validation of identified clones by FISH 17
• 3-4 Association between genomic alterations and recurrent relapse 18
• 3-5 Verification of genomic alterations and copy number change of candidate genes by real-time qPCR 19
• 3-6 Verification of mRNA expression of candidate genes by real-time RT-PCR 20
• 4. DISCUSSION 22
• 5. REFERENCE 62
• KOREAN ABSTRACT 74
• ACKNOWLEDGEMENT 77