[Development and application of the method for controlling contamination of tumor samples with normal cells to detect deletion of P16 gene in primary lung carcinoma].

OBJECTIVE

The method is to be established to effectively promote the sensitivity of the detection of P16 gene deletion from tumor tissues mixed with normal cells and to determine the relationship between the gene alteration and the lung cancers.

METHODS

The normal DNA and that of MCF-7 cells with P16 gene deletion was proportionally mixed and the mixture was limitedly diluted. The exon2 of P16 gene and internal control gene fragment were amplified with template of various amounts of DNA. And polymerase chain reaction (PCR) technique was used in the amplification of exon2 of P16 gene with certain amount of lung cancer DNA as the templates. The amplified fragment was analyzed with SSCP.

RESULTS

Normal DNA did not contaminate P16 gene deletion when DNA template was 5 ng and normal cells less than 40%. No deletion and mutation of P16 gene was found in the small cell lung cancer and normal cancer-surrounding lung tissues, while P16 gene deletion and mutation were found respectively in 21 and 3 cases of 52 cases of non-small cell lung cancer. The rate of P16 gene deletion (72%) in lung cancer with lymphatic involvement was significantly higher than that in lung cancer without the metastasis (16.8%) (P < 0.05). It is found that P16 gene deletion was markedly associated with pathologic manifestation and prognosis of non-small cell lung cancer (P < 0.05).

CONCLUSIONS

Controlling of the amount of template DNA in PCR helps to promote the sensitivity of detection of P16 gene deletion. The abnormality of P16 gene might relate to the malignant development of non-small cell lung cancer.