The Expression And Clinical Significance Of PTEN And PIK3CA In Lung Cancer

Background and ObjectiveLung cancer is increasingly detrimental to people's health. Since the mid-20th century, the incidence rates of lung cancer show a rising trend in most countries and the constant increasingly year by year, In the world, lung cancer ranks first in males and second or third in females in commonly seen malignant tumors.There are still no effective measures of early diagnosis and treatment for lung cancer.Therefore,deep significance to the early diagnosis, detection of metastasis and judgement of prognosis of lung cancer.Phosphatase and tensin homolog deleted on chromosome ten(PTEN)was a new tumor suppressor gene that was cloned in recent years.In 1997, Li and his partners isolated from 1 new tumor suppressor gene when they researched Loss of identity of chromosome 10q23 in primary breast cancer.In the same year, Stekd and other collaborators cloned from a tumor suppressor gene in the glioblastoma cell line,named for mutated in multiple advanced cancer 1(MMAC1).April In the same year,L i and his partners got one kind of tyrosine phosphatase when they studied protein tyrosine phosphatase of cells,this enzyme could reduced expression of TGFβ, and this enzyme is high expression in human epithelial cells, so named for TGFβregulated and ep ithelial cell enriched phosphatase (TEP1).In fact, three genes are the same gene, location 10q23, collectively referred to as PTEN.As the first of tumor suppressor gene with phosphatase activity of a phosphatase,PTEN acts on phosphatidyl inositol 3,4,5-triphosphate and focal adhesion kinase,result to their dephosphorylation.The function of serine kinase and threonine kinase were regulated,thus inhibit signaling pathways transduction that phosphoinositide 3-kinase and protein kinase B involved in.The result is a negative regulator of cell proliferation, induce apoptosis and inhibit cell invasion and metastasis. Currently, a variety of tumors were found with PTEN gene mutations, such as prostate cancer, breast cancer, endometrial cancer, lung cancer, bladder cancer, gastrointestinal tract tumors.PIK3CA is a newly oncogene in somatic cells. In 1994, PIK3CA was detected by in situ hybridizationsuch by Volinia and their collaborators, and its located in 3q26.3.PIK3CA encodes the p110αcatalytic subunit of phosphatidylinositol 3-kinases(PI3Ks),Activation of PI3Ks can phosphorylate phoshatidyplinositol 4,5-PIP2 to 3,4,5-PIP3.Phosphatidylinositol kinase-3 family is a kind of lipase,which function is to adjust signalling pathways, and related with tumor formation, cell proliferation and adhesion, live and migration, etc. The mutation of PIK3CA gene was found in many human tumors such as ovarian cancer, gastric cancer, colon cancer, head and neck squamous cell carcinoma, and so on.Previous researches on PTEN in lung carcinoma center on the expression of PTEN in different kinds of lung carcinomas, and studies on the expression by qualitative observation. Studies on quantitative analysis of PTEN in lung carcinomas haven't been investigated. With the development of molecular biology, people focus great attention on the tumorigenesis of cancers on the molecular level.Great attention is paid on the study of genes.Revealing of relationship between PTEN gene and development of lung cancer is helpful for the investigation of carcinogenesis of lung cancer.This study aimed to detect the expression of PTEN protein in normal lung tissues, benign diseases lung tissue and lung carcinomas, quantitative analysis of the intensities of PTEN protein were performed to reveal the expression characteristics and regular pattern of PTEN in lung cancer, detect the intensity of PTEN protein in the lung cancer and the significance in the diagnosis of lung cancer to provide subsidiary for the elucidation of tumor marker in the diagnosis of lung cancer. Polymerase chain reaction-single strand conformation polymorphism and DNA sequencing were used to analysis PIK3CA gene mutation and its relationship with the carcinogenesis of lung cancer.Test expression of PTEN protein and mutation of PIK3CA gene,discusses the role and mutual relationship in lung cancer.Meterials and MethodsⅠ.The quantitative analysis of PTEN expression in lung cancerExperimental materials and grouping:Tissues from the autopsy biopsy specimens of Nan fang Hospital Pathology Department between 2009-2010. Patients did not receive radiotherapy or chemotherapy before operation. Samples is as follows: 72cases of lung cancer patients, according to 2004 WHO/IASLC histopathology grade classification of lung cancer, lung cancer will be further divided into:25 cases of squamous cell carcinoma,42 cases of adenocarcinoma,3 cases of large cell carcinoma,2 cases of small cell carcinoma.26 cases lung cancer with lymph node metastasis,46 cases lung cancer with no lymph node metastasis.27 cases of central lung cancer,45cases of peripheral lung cancer.30 patients of TNM stageⅠ,30 cases of TNMⅡ,20 cases of TNMⅢ,2 cases of TNM IV.20 cases of adult group with benign lung diseases,8 cases of tuberculosis,6 cases of bronchiectasis,6 cases of bullae.21 cases of normal adult lungs.Experimental Methods:Detected the expression of PTEN protein in lung tissues of different types by immunohistochemical, application of Imageproplus image analysis system test PTEN protein-positive units (PU) value, than analyze the changings of PTEN expression intensity between different types of lung tissues,and analyze the relations between clinical and pathological features of lung cancer.Statistical analysis:SPSS 13.0 statistical analysis software application for processing. Comparison of samples averages were used to One-way ANOVA or Welch test; multiple comparisons between groups use LSD, S-N-K or Dunnett's test; Between the two groups use the independent-sample t-test. Matching samples compares use paired-sample t-test.Ⅱ.The diagnosis analysis of PTEN in lung cancerExperimental materials and grouping:See 1(The quantitative analysis of PTEN expression in lung cancer).Experimental Methods:application of immunohistochemical techniques and Imageproplus image analysis system detect the expression of PTEN protein in lung cancer tissue, normal adult lung tissue, benign diseases lung tissue,than combinate with diagnostic test assessment method to determine the diagnostic cut off value of PTEN, reveals the diagnosis and differential diagnosis of lung cancer in a supporting role.Statistical analysis:SPSS 13.0 statistical analysis software application for processing. Application of diagnostic test tables from different truncation values obtained and diagnostic parameters, combined with receiver operation characteristic curve(ROC curve)analysis PTEN protein cut off value. Ⅲ.The mutation analysis of PIK3CA in lung cancerExperimental Materials:See 1(The quantitative analysis of PTEN expression in lung cancer).Experimental Methods:Extract DNA from paraffin of normal adult lung tissue, benign diseases lung tissue and lung cancer tissue. Polymerase Chain Reaction-Single Strand Conformation Polymorphism and DNA sequencing analysis were used to detect PIK3CA gene mutation.There specific primers for exon 9 and 20 of PIK3CA gene were synthesized and used for PCR amplification.Statistical analysis:SPSS 13.0 statistical analysis software application for processing. Comparison of samples were used to Chi-square test.IV.Correlation analysis between PIK3CA and PTEN in lung cancerExperimental Materials:See 1(The quantitative analysis of PTEN expression in lung cancer).The inclusion criteria that samples were simultaneously amplified exon 9 and exon 20 was introduced.Composition of samples:Tissues from the autopsy biopsy specimens of Nan fang Hospital Pathology Department between 2009-2010. Patients did not receive radiotherapy or chemotherapy before operation. Samples is as follows:32 cases of lung cancer patients, according to 2004 WHO/IASLC histopathology grade classification of lung cancer, lung cancer will be further divided into:12 cases of squamous cell carcinoma,18 cases of adenocarcinoma,1 cases of large cell carcinoma,1cases of small cell carcinoma.11 cases lung cancer with lymph node metastasis,21 cases lung cancer with no lymph node metastasis.11 cases of central lung cancer,21cases of peripheral lung cancer.18 patients of TNM stage I,7 cases of TNMⅡ,7 cases of TNMⅢ,0 cases of TNMⅣ.14 cases of adult group with benign lung diseases,5 cases of tuberculosis,3 cases of bronchiectasis,6 cases of bullae.12 cases of normal adult lungs. Experimental Methods:Immunohistochemical methods and the PCR-SSCP were used to detect PTEN expression and PIK3CA mutation in normal adult lung tissue, benign diseases lung tissue and lung cancer tissue.Statistical analysis:SPSS13.0 statistical analysis software application for processing. Comparison of samples were used to Chi-square test. Correlation analysis used bivariate correlation analysis, Spearman correlation was Selected.ResultsⅠ.The quantitative analysis of PTEN expression in lung cancer1.PTEN PU value of lung cancer is less than PTEN PU values of normal adult lung tissue and benign disease lung tissue (P=0.000); PTEN PU value of benign disease lung tissue is less than PTEN PU values of normal adult lung tissue (P =0.000);PTEN PU value of squamous cell carcinoma,adenocarcinoma, large cell lung carcinoma and small cell lung carcinoma are basically the same (P=0.906);PTEN PU value of tuberculosis, bronchiectasis and bullae are basically the same (P=0.9738).2.PTEN PU value of Lung cancer and corresponding normal lung tissue, was significantly different (P= 0.000), PTEN PU value of normal lung tissues is higher than that in lung cancer; PTEN protein expression in the relationship between the two was not significant (P= 0.632).3. PTEN PU value of lung cancer cell nuclear is indifferent with sex (P=0.708), age (P=0.198), smoking history (P=0.924), tumor gross type (P=0.870), degree of differentiation of lung cancer (P=0.227), lymph node metastasis (P=0.658), distant metastasis (P=0.077) and TNM stage (P=0.543).Ⅱ.The diagnosis analysis of PTEN in lung cancer Application of immunohistochemical techniques and Imageproplus image analysis system detect the expression of PTEN protein in lung cancer, and receiver operation characteristic curve (ROC curve) was applied to analyze PTEN protein truncation value, When PTEN PU value less or equal to primary diagnosis of 20.95, a primary diagnosis of lung cancer may cannot be excluded If PTEN PU value less or equal to 1.00, can get a primary diagnosis of lung cancer.Ⅲ.The mutation analysis of PIK3CA in lung cancer1.Extract DNA from113 paraffin-embedded tissues of lung cancer, benign lung diseases and normal lung tissue,84 cases are successfully extracted, the success rate is 74.34%.2.PCR-SSCP technique was used to detect PIK3CA gene mutation in 84 cases lung cancer, benign lung diseases and normal lung tissue.After some times of silver staining,In 84 cases lung cancer, benign lung lesions And normal lung tissue,including 58 cases simultaneously amplified exon 9 and 20; 11 cases only amplified exon 9; 15 cases only amplified exon 20,abnormal migrating band were observed in fragment amplified from primer 9 of PIK3CA gene in 6 cases and from primer 20 of PIK3CA gene in 2 cases in lung carcinomas.No abnormal migrating band were observed in fragment amplified from primer 9 and 20 of PIK3CA gene in benign lung diseases and normal lung tissue.3.8 cases of PCR products were detected by DNA sequencing analysis,5 cases had single base substitution, Including:1 case in exon 9 from bases in A into C(AA:Glu into Ala),1 case base G into C(AA:Ser into Thr),2 cases bases A into G (AA:Glu into Gly).1 case in exon 20 form bases T become A(AA:Met into Ala). The other 3 cases did not appear to change DNA sequence.4.1n 69 cases successfully amplified PIK3CA exon 9, gene mutation was found in 4 cases of lung cancer, there were 1 case of squamous cell carcinoma,3 cases of adenocarcinoma. In 73 cases successfully amplified PIK3CA exon 20,none was found in lung cancer. Normal lung tissue and benign lesions Single base mutation were not detected in Inormal lung tissue and benign lung diseases, compared with lung cancer, the difference was statistically significant (P= 0.042).5.There was no correlation between PIK3CA gene mutation and patient's sex,age,smoking history,degree of differentiation of lung cancer, tumor gross type, degree of differentiation of lung cancer,lymph node metastasis, distant metastasis and TNM stage.Ⅳ.Correlation analysis between PIK3CA and PTEN in lung cancer1.PTEN expression of normal adult lung tissue,benign disease lung tissue and lung cancer was significantly different (P= 0.000),The formation of lung cancer was a significant negative correlation with the expression intensity of PTEN (r=-0.523,P=0.000).2.PIK3CA gene mutation of normal adult lung tissue,benign disease lung tissue and lung cancer was significantly different (P= 0.042),The formation of lung cancer was a significant positive correlation with the expression intensity of PTEN (r=-0.265, P=0.044).3.PTEN expression in lung was significantly negatively correlation with the PIK3CA gene mutation(r=-0.281,P=0.031).Conclusions1.The expression of PTEN is the same in different types of lung cancer;expression of PTEN in lung cancer cell nuclear is indifferent with sex, age,smoking history, tumor gross type, degree of differentiation of lung cancer, lymph node metastasis and TNM stage.2.When PTEN PU value less or equal to primary diagnosis of 20.95, a primary diagnosis of lung cancer may cannot be excluded If PTEN PU value less or equal to 1.00, can get a primary diagnosis of lung cancer.3.Compared normal lung tissues and benign lung diseases,PIK3CA gene mutations rate is significantly increased,which is that PIK3CA mutations may be associated with lung cancer.4.The expression of PTEN was a significant negative correlation with the PIK3CA gene mutation. In the formation of lung cancer, PIK3CA gene mutations and PTEN expression loss are mutually exclusive, PIK3CA gene mutations or PTEN abnormal expression related to the occurrence of lung cancer.