Changes Of Mutations In Colorectal Cancer With Pulmonary Metastasis And Its Clinical Research

The morbidity and mortality of Colorectal carcinoma kept high.Its morbidity and mortality were located in the third and fourth worldwide.In China,due to the improvement of living standards and changes in living habits,the incidence of colorectal cancer in urban areas of China showed an increasing trend and younger characteristics.With the development of electronic endoscopy and tumor screening,the early diagnosis rate of colorectal cancer gradually increased.The prognosis of patients with colorectal cancer has improved significantly by early detection and intervention.However,almost all patients with cancer will eventually be transferred.In the event of distant metastases in patients with colorectal cancer,the survival rate dropped to around 10%.Therefore,It is important to study the Metastasis mechanism of colorectal cancer.It is helpful to monitor the progression of the disease,evaluate the prognosis of the patients and predict the curative effect.At the same time,it is also helpful to clarify the theory of tumor metastasis and find the target of tumor metastasis intervention.In addition to local regional lymph node metastasis,colorectal cancer is more prone to liver metastasis and lung metastasis.According to statistics,the incidence of lung metastases is about 14%.Liver is the first station of the intestinal blood reflux and the lung is the second station.Normally,lung metastasis of colorectal cancer commonly occurs subsequent to invasion of the liver.However,in some patients,colorectal cancer can not occur in the case of liver metastasis,directlyresulting in lung metastasisand even multiple lung metastases.Suggesting that the transfer of tumor cells to specific organs have a specific regular and molecular basis.To clarify these laws and in-depth excavation of these molecular basis for colorectal cancer patients with the design of the program has a positive significance.In this study,we will first determine the differencesof gene phenotype and mutation between primary and metastatic colorectal cancer,and then analyze the difference of gene mutation between primary and metastatic colorectal cancer.Finally,we will make simple functional verification and clinical analysis of the individual key genes found in the study.Our findings emphasize the importance of studying the natural process of metastasis and the impact of treatment on this process.Future studies on paired primary and metastatic tumors with comprehensive treatment information through intensive multi-region sampling and single cell sequencing may provide more solutions for these processes.Nevertheless,large sample,multicenter clinical data are still needed to confirm our findings,and morefunctional experiments are needed to explain the driving ability of key genes and find better targets for clinic.Part 1: Changes of mutations in colorectal cancer with pulmonary metastasis and its clinical enlightenmentObjective: To detect the expression and mutation of specific genes in primary and lung metastases of colon cancer,and to determine whether there is abnormity of the key gene.Methods: To construct tissue microarray,to detected the expression of Ki67 and PRR11 in primary and lung metastases of colon cancerby immunohistochemistry and to detect the mutation between Braf V600 E and Kras and Nras by Arms.Results:13 tissue chips containing primary and lung metastases of colon cancer was constructed.The expression rate of Ki67 in the primary lesion was 92.3%(12/13),the average expression intensity was 13.69,the expression rate of Ki67 in lung metastases was92.3%(12/13),and the average expression intensity was 12.4.There was 5 cases that the expression intensity in the primary foci was higher than that in the metastatic foci,1 case was the same,and 7 below.The expression rate of PRR11 in primary colorectal cancer was46.2%(6/13)and the average expression intensity was 7.69.The expression rate of PRR11 in lung cancer was 69.2%(9/13)and the average expression intensity was 18.08.There was 1 case that the expression intensity of primary tumor in colon cancer was higher than that in metastatic foci,5 cases were the same,and 7 cases below.Only one of the primary lesions and lung metastases had BRAF V600 E mutations in the detectable ten pairs.2cases of colorectal cancer in the presence of Kras codon12 mutation,1 case of codon61 mutation,the 3 cases of lung metastases also contain similar mutations.However,Kras codon12 mutations occurred in one lung metastasis and Kras codon61 mutations occurred in the other case,but these two patients did not contain mutations in the primary lesion.Conclusions: The differences in protein expression(Ki67 and PRR11)and gene mutation(Kras)between lung metastases and primary intestinal foci suggest a genetic variation between colorectal cancer and lung metastases.This variation suggests new revelation for clinical guidance.For patients with colorectal cancer who have undergone lung metastases,it is best to re-examine these metastatic tumor cells if they are able to obtain lung metastases.If there is a mutation,they can not be treated with the C225.Part 2:The change rule of the gene-mutation profiles in the process of lung metastasis of colorectal cancerObjective: To perform a whole exome sequencing of primary tumor and multiple metastatic tumor cells from one patient who has experienced two time of consecutive lung metastases of colorectal cancer,with a view to obtaining the gene-mutation profiles in the lung metastasis of colorectal cancer.Methods: The adjacent non-tumor mucosa,primary tumor,primary lung metastases and second lung metastases were obtained.The tumor cells were extracted and the whole exon was sequenced by Hiseq platform and analyzed by bioinformatics.Results: There were 183 missense mutations in the T/N(primary lesion/normal tissue),10 of In Del;813 missense mutations in M1/N(first lung metastatic/normal tissue),83 of In Del;M2/N(second lung metastases/normal tissue)missense mutation in 12049,In Del1381.These figures indicate an increase in the number of gene mutations as the number of shifts increases.Functional analysis and pathway analysis also showed that the tumor cell mutations in the second lung metastases were mainly involved in the invasion or metastasis of related molecules or pathways,suggesting that the tumor cells in the second metastases were more invasive.Conclusion: There are significant heterogeneity of gene phenotype before and after tumor metastasis.The data show that the number of mutations and mutation sites in tumor cells are exponentially increasing as the number of shifts increases.The evolutionary phenomena of tumor cells provides theoretical support for the clinical guidance and prevention of drug resistance in cancer patientsPart 3: MTA3 and the role of mutant MTA3 in colorectal cancerObjective:The results of sequencing showed that there was a mutation of MTA3 in second lung metastases.This study was to determine whether this mutation might be the cause of multiple functions of MTA3.Methods: the wild-type and mutant plasmids of MTA3 were designed and constructed to transfect colorectal cancer cells for observing the effect of tumor cell function(proliferation and invasion).Immunohistochemistry was used to detect the expression of MTA3 in lung metastases and primary intestinal foci.Results: The expression intensity of MTA3 in normal tissue(N),primary tumor(PT),lymph node metastasis(LN)and lung metastasis(LM)were:N=7.3±10.2,PT: 53.8±71.6,LM: 5.4±10.7.Statistical analysis showed that the expression intensity in PT was significantly higher than that in normal tissues,but the expression in metastases was significantly lower than that in primary tumors.The effect of wild type MTA3 and mutant MTA3 on the proliferation of HGC27 cells was not significant.Wild type MTA3 promoted invasion of intestinal cancer cells,while mutant MTA3 reduced its ability to attack.Conclusion: MTA3 is highly expressed in primary colorectal cancer,but with tumor metastasis,its expression disappears and may be related to gene mutation inactivation.Wild type MTA3 promotes migration of colorectal cancer cells,but the ability to mutate after mutations suggests that deletion mutations or point mutations are potential factors leading to functional diversity of MTA3.