The Comparison Of The Grade Of Concordance In Terms Of K-ras Status Between Primaries And Related Liver Metastases In Colorectal Cancer

1. BACKGROUND & OBJECTIVESColorectal cancer (CRC) is the third most common cancer worldwide, with over a million new cases diagnosed each year. In the United States alone, an estimated 154,000 new cases will be diagnosed and over 56,000 deaths are expected from the disease every year, and CRC is the second most common cause of cancer mortality. In China, the incidence rate of CRC is the fourth among all of the cancers with an increasing rate of 4.2% per year and the population is much younger than before. Compared with that in 20th century 70s, the incidence rate increased 31.95% in cities and 8.51% in countries in 20th century 90s. In 2006 the mortality rate of CRC in china is the fifth among all of the cancers. As reported, the liver is the most frequent site of haematogenous metastatic spread of CRC. Approximately 20-25% of patients with CRC have liver metastases when diagnosed and 60% patients will develop hepatic lesions during their course of their illness. The recurrence rate is about 50% after the curative resection and approximate 45%-71% patients died from CRC had liver metastases. Although surgical resection,local ablation and radiosurgery are the common used therapies, medical therapy is the important method when the lesions cannot be removed or the hepatic insufficiency existed.Now chemotherapy is the major therapy in advanced CRC. In the past 40 years, Fluorouracil(5-FU) is the major drug in the therapy of CRC and the patient survival prolongs with Leucovorin(CF) plus 5-FU continued infusion. In 20th century 90s, the new cytotoxic drug Oxaliplatin and Irinotecan(CPT-11) improve the short-term effect and survival of CRC. In recent years the molecular targeted drug Bevacizumab,Cetuximab(C225) and Panitumumab make the new therapy of CRC.Epidermal growth factor receptor (EGFR) is a kind of tyrosine kinase activity transmembrane receptor, now found that the receptor family includes four subtypes: EGFR (HER1/ErbB1), HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4). More than 10 kinds of ligands can selectively combine with the EGFR monomer. Ligand-receptor binding has caused the role of receptor oligomerization, activated receptor tyrosine kinase, and then makes the receptor cells in parts of the tyrosine residues cis-phosphorylation. Receptor phosphorylation after the conformation change to form a point of parking spaces to work with a SH2 (Src-homology 2) domain and phosphorylated tyrosine binding domain of the protein, thereby triggering including mitogen-activated protein kinase (mitogen -activated protein kinase, MAPK) pathway and phosphatidylinositol -3 kinase (phosphoinositide 3-kinase, PI3K) pathway of signal transduction cascade. Then it plays a very important role in of organ formation and tumor growth in the process of cell proliferation, inhibiting apoptosis, angiogenesis and metastases.Epidermal growth factor receptor (EGFR) in CRC is over-expressed and it is an ideal cancer therapy target. EGFR over-expression is closely linked to the cell malignant transformation, tumor cells invasion, metastasis, tumor angiogenesis and apoptosis resistance. Cetuximab (C225) is a recombinant human-mouse chimeric IgGl monoclonal antibody, its affinity of EGFR on the cell membrane is 5-10 times to the endogenous ligand. Combined with EGFR, it can block endogenous ligand binding with EGFR, cut off the signal transduction and block the phosphorylation of tyrosine kinase, thereby inhibiting cell cycle progression,inducing apoptosis of tumor cells and reducing the matrix metalloproteinase and vascular endothelial growth factor production. It can also cause receptor dimerization, internalization and down-regulation to inhibit tumor invasion and metastasis. In addition to inhibiting EGFR, Cetuximab can also activate the host immune response by antibody-dependent cellular cytotoxicity (ADCC) to kill tumor cells. A number of recent studies show that in metastatic CRC, the efficacy and the clinical benefit of K-ras wild-type patients receiving C225 and combination with chemotherapy are significantly better than those of K-ras mutant group, strongly suggesting that K-ras status is related with efficacy of C225 and it is an independent predictor of C225 efficacy. Only K-ras wild-type patients are recommended to recieve the EGFR inhibitors treatment, such as C225 and Panitumumab(including single drug or in combination with chemotherapy). Therefore, K-ras gene detection was written in 2009 edition of "U.S. National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines for colorectal cancer." That is, K-ras gene status should be detected in patients with metastatic CRC.At present the view that the incidence of CRC is the body's internal factors (genetic susceptibility to CRC) and exogenous (dietary and environmental factors) together results. A large number of experiments prove the occurrence of CRC is related with multiple oncogene activation or inactivation of tumor suppressor genes. Ras gene mutation is thought to play a catalytic role to carcinogenesis and is an early event. Ras gene was first discovered in Kirston and Hayvey rat sarcoma and with the transduction of the ras gene the mouse fibroblast cells could turn to malignant transformation. So it determined the status of its oncogene. Ras gene family is made up of three cancer-related genes in humans, K-ras, H-ras and N-ras, the sequences of which are highly conserved. Ras gene mapping to human chromosome No.12, contains four coding exons and a 5'non-coding exon that together code the ras protein containing 189 amino acids, and because of its relative molecular mass 21×103, also known as p21 protein. The most common way of K-ras gene activation is the point mutations which are observed mainly at, codon 12, or 13 in exon 1 and sometimes at codon 61 in exon 2. The wild-type of K-ras gene at codon 12 is glycine, while the mutant type often are cysteine, valine or arginine. These mutations result in amino acid changes, resulting in carcinogenic activity p21 protein. The guanosine triphosphatase (GTPase) activity decreased when the K-ras gene abnormalities occurred, then p21GTP with a solid combination cannot be hydrolyzed by GTPase and has been in active, continuing to stimulate cell growth, development, proliferation and causing cell malignant, so CRC may occur. Furthermore K-ras gene activation can also be observed by amplification or over-expression, but with a lower frequency, which are majority found in animal experiments. Algar et al found that the over-expression of K-ras gene significantly reduced the anti-tumor effect of natural killer cells (NK cells) and might be the features of metastasis and disease progression in CRC. The study concluded that K-ras gene mutations occurred early during the tumor formation and it has given a great tendency to the possibility of infiltration and metastasis of tumor cells.The mutation rate of K-ras gene in CRC in Europe and the United States is about 30% -68%, and in Taiwan and Japan the K-ras mutation rate is 26.5% -36.9%, lower than that in Europe and the United States. Oliveira etc have demonstrated that the frequency of K-ras mutations in lymph node metastases is higher than that in the related primary CRCs. Molinari analyzed K-ras mutational status in a small sample of 30 consecutive patients with CRC with synchronous or metachronous metastasis. The same mutational pattern between primaries and corresponding metastases was observed in 26 cases, with a total grade of concordance of 86.6%.Daniele evaluated the grade of concordance in terms of K-ras status between primaries and related metastases in 99 cases with CRC and found the concordance rate was 96.0%. Fotios analyzed K-ras mutational status between primaries and related metastases in 43 cases of CRC and found the concordance rate was 95.0%. However, can the K-ras mutational status in primaries predict that of related liver metastases in CRC? Above are some reports abroad to answer this question and there were some different conclusions. Furthermore there is little domestic-related information. This study evaluated the grade of concordance in terms of K-ras status between primaries and related liver metastases in 75 cases with CRC and provided a new basis of selection of molecular-targeted drugs.2. METHODS75 cases of CRC diagnosed in the First People's Hospital of Foshan from January 1,2003 to May 30,2008 were enrolled in this study. These cases were all received the synchronous or metachronous surgical resection of colorectal cancer and related liver metastasis and the pathological specimens were obtained. Enrolled in this study, all of the patients were not received any molecular target drug treatment. All of the specimens were conventional formalin-fixed, paraffin-embedded, and cut into 10μm thick sections to be placed on slides, then the tissue DNA were extracted from primary tumors and related liver metastases respectively by the application of QIAamp DNA FFPE Tissue Kit Organization Box. The extracted DNA was amplified to get K-ras gene by conventional PCR. Then PCR products were purified by 2% agarose gel electrophoresis and sequenced through DNA sequencing instrument. We mainly detected K-ras codon 12 and 13 mutations in primary tumors and related liver metastases. Data results were stated by rates and analyzed by pairedχ2 test (McNemar test) with SPSS 13.0 software pack, with test level a=0.05.3. RESULTS3.1 Clinical data75 cases of CRC diagnosed in the First People's Hospital of Foshan from January 1,2003 to May 30,2008 were enrolled. These cases were all received the synchronous or metachronous surgical resection of colorectal cancer and related liver metastasis and the pathological specimens were obtained.75 patients were composed of 43 males (57.3%) and 32 females (42.7%); median age at diagnosis,56 years (range,31-74 years).64 cases (85.3%) received synchronous surgical resection and 11 cases(14.7%) received metachronous resection. There were 55 cases (73.3%) of colon cancer with liver metastasis and 20 cases (26.7%) of rectal cancer with liver metastases.26(34.7%) were affected by right colon,29(38.6%) by left colon and 20(26.7%) by rectal cancer. Lymph node metastases were found in 57 cases (76.0%) and 18 cases (24.0%) with none. Pathological types:5 cases (6.7%) of poorly differentiated adenocarcinoma,67 cases (89.3%) of differentiated adenocarcinoma and mucinous adenocarcinoma in 3 cases (57.3%).3.2 ResultsThree pairs were excluded from the analysis because of the low quality of DNA extraction. And here is the analysis of 72 cases.3.2.1 K-ras mutational status in primary tumorsK-ras gene mutations were found in 24 cases (33.3%) in primary colorectal cancer tissues, of which 18 cases (75.0%) with colon cancer,6 cases (25.0%) with rectal cancer. Specific mutation types:G12C in 3 cases (12.5%), G12D in 10 cases (41.7%), G12V in 9 cases (37.5%) and G13D in 2 cases (8.3%).3.2.2 K-ras mutational status in liver metastasesK-ras gene mutations were found in 23 cases (31.9%) in liver metastases tissues. Specific mutation types:G12C in 4 cases (17.4%), G12D in 11 cases (47.8%), G12V in 7cases (30.5%) and G13D in 1 cases (4.3%).3.2.3 The grade of concordance in terms of K-ras status between primaries and related liver metastasesAmong 24 cases of K-ras mutation in primary tumors,21 cases (87.5%)of K-ras mutation were found in related liver metastases. And 46 cases were K-ras wild-type both in primaries and related liver metastases. The rate of concordance was 93.1%, with no significant difference (P=1.00) by McNemar test. And the value of kappa was 0.842, it means that the degree of matching between the two was high. Discordance was observed in 5(6.9%) patients. In 2 patients K-ras was wild-type in the primary tumor and mutated in the liver metastases. In 3 patients K-ras was mutated in primary tumor and wild-type in the metastatic site.4. CONCLUSION4.1 In this study, the mutation rate of K-ras gene in primary tissues was 33.3% and similar with that of Asia. And the major mutation type of codon 12 was G12D(GGT→GAT)and G12V(GGT→GTT) which was similar with the reports above.4.2 In this study, the rate of concordance in terms of K-ras status between primaries and related liver metastases was 93.1%. Therefore, we believe that to Chinese people, in metastatic colorectal cancer, K-ras mutational status between primaries and related liver metastases was concordant. K-ras status of the primary tumor can guide the clinical use of drugs and the metastases have not to be taken re-biopsy, or if the specimens of the primary tumor can not be got, we can take the pathological specimens from the metastatic site to test K-ras gene status. It will reduce duplication of medical operations, reduce complications, save time and decrease medical costs.4.3 Of course, to obtain more accurate information, it needs for researches with more samples, detection of related protein expression and analysis of clinical follow-up cases. We provide this initial research data as a reliable reference to further researches.