The Study On The Relationship With Prognosis And Protein Expression Of P16-cylinD1-CDK4/6-pRb In Non-small-cell Lung Cancer

The p16-cyclinD1-CDK4/6-Rb pathway plays a very important role in the cell cycle regulation. Progression through the G1-S phase of the cell cycle is mediated by phosphorylation of the Rb resulting in the release of essential transcription factors such as E2F-1.The phosphorylation of Rb is regulated positively by cyclinDl/CDK4and negatively by CDK inhibitors, such as p16(CDKN2/MTS-1/INK4A). Rb and p16are known as tumor surpressors, and cyclinDl is regarded as oncoprotein. It has been shown that he p16-cyclinD-Rb pathway is closely related to tumorigenesis. Many tumor types display a high frequency of inactivation of at least one component of this pathway. The majority of the scientific studies that have analyzed the occurrence of cell cycle regulator aberrations within lung cancer have dealt with different tumors. Data concerning single class of sarcomas are very limited. In order to determine contributions of these three components to the progression of lung cancer, we studied here in84cases of lung cancer by analyzing protein alteration of p16, cyclinD1and Rb, and gene aberration of p16and CCND1. In addition, prognostic analysis was made on the basis of follow-up. The results were summarized as follows:1.Immunohistochemistry was performed to detect p16,cyclinDl and Rb expression. Aberrations involving the p16-cyclinD1-Rb pathway were observed in82/84(97.6%) cases. Abnormal expression frequency of p16, cyclinD1and Rb was38.7%,51.2%,54.8%,respectively.The p16-cyclinD-Rb pathway therefore emerges as the preferred target for molecular abnormalities in non-small-cell lung cancer. There was a significant inverse correlation between Rb and p16expression (P<0.01) and a direct correlation between Rb and cyclinDl expression (P<0.05). A clinico-pathologic survey indicated that negative expression of Rb was associated with poorly diferentiated lung cancer (P<0.05),suggesting the loss of Rb expression might play a role in malignant progression of lung cancer.2. The p16gene status of76cases of NSCLC was analysised by PCR-SSCP, MSP and DNA sequence.Of76cases of NSCLC,18(23.7%) showed5’CpG island Hypermethylation in p16gene;4(5.3%) showed point mutations(two Gâ†'T)missence mutation in exon2and two Gâ†'T mutation in exon-intron junction,as confirmed by DNA sequencing and2(2.6%) showed homozygous deletion. The total frequency of alteration in p16gene was31.6%(24/76).The results suggest both genetic and epigenetic events play an important role in oncogenic processes.3. The relationship between the CCND1gene amplification with non-small cell lung cancer was explored by PCR.CCND1gene amplification was detected in16/76cases(21.1%)by DPCR, accompanied by cyclinD1overexpression.However, there were still32/48cases (66.7%) of NSCLC, in which cyclinD1was overexpressed, showed no CCND1gene amplification.CCND1gene amplification was detected in14/34cases (41.2%)of Rb Positive NSCLC; CCND1gene amplification was detected in2/42cases of Rb negative NSCLC. There is statistically significant in the two groups(P<0.05).4. A Cox proportional hazard regression model was used for statistical evaluation of the prognostic factors; And Kaplan-Meier method was used to draw the survival curves, log-rank test was used to compare survival curves.The follow-up was available in43(51.2%) patients. Median survival time was24months.Age of the patient, volume of tumor, pathologic parameters,metastasis and abnormal cyclinD1expression were prognostic factors for overall survival. p16was an independent prognostic factors (P<0.01). Abnormal cyclinDl expression (P<0.05) andmetastasis(P<0.05),were determined to be significant prognostic factors for NSCLC.As the results mentioned above,we suggest that aberrations involving p16-cyclin D-pRB pathway play a very important role in pathogenesis of NSCLC.5’C pG-island hypermethylation in p16gene is a major way to inactive p16gene. CCND1gene amplification is common in NSCLC,which is one of the mechanisms leading to cyclinDl and may be responsible for the loss of cell cycle control in a fraction of NSCLC. p16expression, volume of the tumor and metastasis are valuable prognostic factors for NSCLC patients.