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Background and objective It has been proven that allelic loss of chromosome region 3p occurs early and frequently in non-small-cell lung cancer, and numerous tumor suppressor genes at this locus may be targets of inactivation. The aim of this study is to investigate the relationship between loss of heterozygosity (LOH) of the allele genes on 3p and the diagnostic value of NSCLC. Methods A total of 32 NSCLC samples and 8 tissues of the pulmonary benign disease have been analyzed by fluorescent quantitative PCR. Results There were 25 of the 32 cases (78.125%) with NSCLC tumor had LOH on 3p. Among them, LOH detection was 14 cases (43.75%) at 3p25, 18 cases (56.25%) at 3p14 and 18 cases (56.25%) at 3p21.3, respectively, and was 18.75% (3p25)、31.25% (3p14)、50% (3p21.3) in the nucleated cell in peripheral blood, respectively. The total detection was 20 cases (65.625%). LOH detection in the nucleated cells in peripheral blood was correlation with the tumor tissues and the combined detection is better than single ones. Conclusion Our results showed that 3p LOH occur commonly in human NSCLC. The 3p LOH detection of the tumor tissue and the nucleated cell in peripheral blood is beneficial to the diagnosis of NSCLC. The combined LOH detection of 3 separate regions of 3p LOH is beneficial to increase the detection rate．