| Background and AimsMetastasis is the major cause of death in cancer patients, and there are currently no therapeutic agents available to prevent this disease.It would be useful to clarify the mechanism of metastasis for prediction or prevention of metastasis. Several model hypotheses try to explain the mechanism of metastasis, but there are some limitations in them. Modern molecular technologies and nanotechnology offer the possibilities of discovering new biomarkers for prevention metastasis and the feasibility of producing drugs on these new biomarkers. Current studies on the mechanism of metastasis are mostly based on cells in ex vivo experiments and animal models design, which are often artifical. Few studies focused on comparsion of primary cancer and their metastasis in a same patient. However, it is very important to further develop the theory of metastasis mechanism and discovering new target drugs.The papers that have been reported on comparison of primary cancer and matched metastasis maily focused on breast cancer and colon cancer. Few data have been published on primary lung cancer and their metastases.Lung cancer is one of the most common malignant tumors in the world, especially in China. About 70% of lung cancer patients are in late stages at the time of primary diagnose. Even if these patients who had been completely removed primary tumors, most of them would develop metastasis. Researchers should devote themselves to study lung cancer metastasis to resolve the puzzle of lung cancer treatment. Some key genes play important roles in many cancers. The mechanism of breast cancer had been clear clarified in some extent. The molecules that exert their effects on breast cancer metastasis maybe play the same role in lung cancer metastasis. Therefore, it may be helpful for research of lung cancer metastasis. Molecular target therapy is becoming available in treatment of lung cancer. Howerver, the efficiency of target therapy is related to their molecules status. Now the molecule status is mainly based on primary lung cancer tissue. Few data reveals whether changes in lung cancer metastasis tissues on expressions of these key genes. Some genes have been reported that their expressions are related to prognosis of lung cancer,such as VEGF, MET,HIF-1α,TGF-β1. These molecules inhibitors are in clinical trial or becoming possible target therapies in lung cancer. But we don't know whether expression on metastasis tissue. Doctors are often puzzled by diagnosed of pulmonary metastasis or double primay lung cancer in some patients. Reseachers hold the idea that the gene background between parimay cancer and metastasis are identical while it is heterogeneity among multiprimary lung cancer. Based on this, p53 mutation that often occur in lung cancer, had been used to discriminate pulmonary metasis from multiprimary lung cancer. However, there is heterogeneity of p53 mutation between breast cancer and matched metastasis. It is urgent to search for a more powerful tool to discriminate metastasis or primary cancer. Single nucleotide polymorphisms is the most advanced of polymorphisms, which may reflect heterogeneity of genetic background of individual. P53 72 codon poly-morphisms is reported to induce susceptibility of lung cancer. We hypothsized P53 72 codon polymorphisms can discriminate pulmonary metastasis from second primary lung cancer.Therefore, there are two major goals in this paper. One is to evaluate the similarities and differences on proteins expressions of CD133(Lung cancer stem cell marker), COX2, VEGF, MET, HIF-la, TNFa, TGF-betal and p53 between primary lung cancer and their metastases. The other is to discriminate pulmonary metastasis from multiprimary lung cancer through detection of p53 codon 72 SNP under RFSCP.Patients and Methods14 patients had been removed primay lung cancer tissues and metastasis tissues between 2002 and 2009.11 patients are diagnosed of metastasis.3 cases aren't certain according to current criteria on diagnosed of pulmonary metastasis. Their paired paraffin sections were collected to conduct the following procedure. 1. Paraffin sections were cut into microsections to conduct routine HE staining and SP immunohistochemistry.2.DNA were extracted from archived, paraffin-embedded sections under the guidelines of QiAGEN kits. The purification of DNA were examined on a special mechanism.3. PCR of p53 condon 72 was proceed on the valid DNA of samples.The productions were proceed to the method of restriction fragment length polymor-phism. The gene type were identified under a laser photo instrument.4. Non-parameter method were used to compare proteins expression insentity between paried tissues through Marginal Homogeneity test. P value<0.05 of two sides test were considered statistically significant.Results1. Clinical data Tumor grades are identical in 9 cases of 11 cases. Tumor grades of the other two cases were G1 and G2 in primary lung cancer but were only G2 in metastasis. The three cases who cann't be clearly diagnosed of metastasis showed masses again in pulmonary.2. Immunohistochemical ResultsVEGF:In the 11 paried primary and metastasis tissues, low expression was in 5 sections(22.7%), moderate expression in 14 sections(63.6%), high expression in 3 sections(13.6%). The expression scores of primary lung cancer were consistent with that of metastasis in 9 cases(81.8%). The differences don't show significantly statistical (P value=0.157>0.05).There are two cases who showed more intensity in metastases of lung and brain than in primary lung cancder. In the three cases who cann't be clearly diagnosed of metastasis, there are two cases concordance and one inconcordance.COX-2:In the 11 paried primary and metastasis tissues, the low, moderate and high expressions were respectively 9 sections(40.9%),9sections(40.9%),4 sections (18.2%). There is one cases incordance(9.1%). The differences don't show significantly statistical (P value=0.317>0.05). In the three cases who cann't be clearly diagnosed of metastasis, there are two cases concordance and one inconcordance. MET: In the 11 paried primary and metastasis tissues, the low, moderate and high expressions were respectively 3 samples (13.6%),6 samples (27.2%),13 samples(59.1%). There are two cases incordance(9.1%). The differences don't show significantly statistical (P value=0.157>0.05). In the three cases who cann't be clearly diagnosed of metastasis, there are two cases concordance and one inconcordance.HIF-la:In the 11 paried primary and metastasis tissues, the low, moderate and high expressions were respectively 4 samples (18.2%),15 samples(68.2%),3 samples (13.6%). There are two cases incordance(18.2%). The differences don't show significantly statistical (P value=0.180>0.05). In the three cases who cann't be clearly diagnosed of metastasis, they are concordance.CD 133:CD 133 showed membrane positive in only squamous tissues. The percent of positive cell was very low. CD 133 expression may be relation to the histological type.TGF-β1:The intensity was weak in tumor cell but strong in stromal cell. The positive cells are inflammations cells and fibroblast. TGF-β1 showed stronger in metastatis stromal than primay stromal, which maybe play a role in interaction of stromal and metastasis.TNFa: In the 11 paried primary and metastasis tissues, there is one case who show negative in both primary and metastasis lung cancer, the low, moderate and high expressions were respectively 6 samples (27.2%),11 samples(50%),3 samples (13.6%). There is one cases incordance(9.1%). The differences don't show significantly statistical (P value=0.317>0.05). In the three cases who cann't be clearly diagnosed of metastasis, they are concordance.P53:In the 11 paried primary and metastasis tissues, the low, moderate and high expressions were respectively 9 samples(40.9%),9 samples(40.9%),4 samples (18.2 %). There are three cases incordance(27.2%). The differences don't show significantly statistical (P value=0.564>0.05). In the three cases who cann't be clearly diagnosed of metastasis, there are one cases concordance and one inconcordance.3. The results of DNA extraction Three cases fail to extract poor DNA. The quality and purity of DNA in the other 11 cases can meet the demand of PCR.4. SNP of p53 condon 72:In the 9 cases who diganosed of primay and metastatis lung cancer, there is one cases inconcordance in gene type. The other three cases were all identical. Taking into account of positive control and metastatic control, they are paired primay and metastatis tissues.5. The proteins expressions in 14 cases. There are respectively inconcordance in 2 cases of COX2 expression,3 cases expression of MET,3 cases expression of VEGF,2 cases expression of HIF-la,1 cases of TNF-a,4cases of p53. Their P valles are more than 0.05.Conclusions:There are high concordance in selected protein expression and p53 SNP status between primay lung cancer and matched metastatis, suggest there are common clonal origin between them. However, there are still minor differences between them. The VEGF, COX2 expression in metastasis maybe depend on primay lung cancer tissue when their inhibitors were used in lung cancer. The Met, HIF-1αwere potential targets in prevent metastasis.P53 condon 72 polymorphism was a useful tool to discriminate pulmonary metastasis from multiprimay lung cancer.Although there is minor difference, high concordance of gene type prevails in between primay and metastasis. The variations exist between primay and metastasis, which may be caused by genome DNA instability of cancer cells which developed in the process of metastasis. |
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