| Background and PurposeMalignant tumor has become a common disease which threaten the human health and lead human to death. According to’World cancer report 2014’released by the world health organization (WHO), the number of new cancer case will take on a trend of dramatic increase each year in the world, which was about 14 million in 2012 and to 2025 will be as high as 19 million; about 3.07 million new cancer cases and about 2.2 million cases of cancer death occurred in China in 2012, respectively accounted for 21.9% and 26.8% of total global cases, ranking first in the world. The prevention and treatment of tumor has now become the world’s largest public health problem.With the rapid development of tumor molecular biology, tumor gene plays a more and more important role in malignancy screening, diagnosis, treatment, prognostic evaluation, therapy monitoring, and so on. The occurrence and development of malignant tumors began in tumor gene point mutation, and then to cause cell dividing indefinitely, until cell deterioration and migration. Screening of genes associated tumor of healthy people on a regular basis, especially of people at high risk of cancer, is advantageous for early discovery, early diagnosis, early treatment and to win more survival opportunities for patients with cancer. With a rich accumulation of clinical experience of tumor diagnosis and treatment, the killing effect of chemotherapy drugs on tumor cells have been found to significantly associate with specific gene expression and gene polymorphisms. Genetic diagnosis can help to select the best treatment for cancer patients to improve the therapeutic effect and the quality of survival of them. In recent years, molecular targeted therapy has achieved great effect in clinic which provides a new effective model for cancer therapies. Molecular targeted drug therapy mainly uses small molecules, polypeptide, monoclonal antibodies to target specific molecules that closely associated with cancer onset, progression and prognosis, thus inhibiting cell growth factor receptor activation, signal transduction pathway and angiogenesis, intervening tumor cell biology behavior, and ultimately achieving the purpose of suppressing tumor development. Molecular targeted drugs with little side-effect, are highly selective for killing cancer cells without killing or only very little damaging normal cells, can effectively improve the patients life quality and treatment effect. Malignant tumors have high heterogeneity at the molecular level, and results from many mutually coordinated genes and genotypes, process steps. Therefore, patients should be clear whether there is a related drug target before using targeted drug therapy, and on this to guide the individual targeted therapy and to improve medication safety and efficacy for the malignant tumor patients.Compared to other current common genetic diagnosis methods, Next Generation Sequencing(NGS) has gradually from the laboratory research to clinical application with many superiorities in term of high-throughput, low-cost, rapid speed simple operation, and screening multiple mutations in multiple genes simultaneously by its single platform. As one of Next Generation Sequencing, the technology of Ion Torrent sequencing is based on the semiconductor chip technology and the natural principle of base pairing, uses ion sensor to capture the local change of the pH value caused by H+ that released from DNA synthesis and bases extension, and interprets bases in real time by instantly converting chemical signals into digital signals, thus acquires the sequence of each DNA fragment. Rather than other sequencing technologies, Ion Torrent sequencing breaks through the limitations of the traditional technology that relies on fluorescent tags and optical imaging theory to collect the data. This makes it has incomparable advantages on its equipment costs, speed of sequencing and sequencing cost.This research adopts the technology of Ion Torrent sequencing to detect 22 genes of patients with malignant tumor, namely EGFR, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, MET, ALK, KRAS, NRAS, BRAF, STK11, MAP2K1, PTEN, PIK3CA, AKT1, DDR2, TP53, CTNNB1 and SMAD4, FBXW7, NOTCH1, and validate the results of some gene locus of EGFR, KRAS, BRAF by Sanger sequencing and ADx-ARMS, in order to evaluate the accuracy and clinical applicability of the technology of Ion Torrent sequencing for cancer gene detection.Method1. The research object collection 110 FFPE specimens with neoplastic tissues from patients with malignant tumor and 96 DNA samples that were extracted from malignant tumor patients and had been detected EGFR, KRAS, or BRAF gene by ADx-ARMS according to the requirement of clinic were collected, totaling 206 cases in this study.2. The DNA extraction and the preliminary quantitation The DNA of 110 FFPE specimens were extracted by QIAamp DNA FFPE Tissue Kit of QIAGEN. The DNA concentration of 206 cases were measured by Qubit (?) 3.0.3. According to the standard process of AmpliseqTM olon and Lung Cancer panel of Life Technology, to construct DNA library, prepare DNA template and enrich of positive template, finally sequence on Ion Proton System.4. To analyze the data of the Ion Torrent sequencing.5. To detect three genes of EGFR, KRAS, BRAF in 206 cases by Sanger sequencing, and then to analyze and report the results of 39 kinds of common mutant genotypes covered by the detection and compare with the results of Ion Torrent sequencing.6. To compare and analyze the results of EGFR, KRAS, BRAF of 96 samples detected by ADx-ARMS and Ion Torrent sequencing.Result1. In this study, a total of 206 patients with malignant tumor were enrolled. 131 (65.6%) males and 75 (36.4%) females, between 23-90 years old, the average age was 63+12 years old. There were 114 cases younger than 65 years old accounting for about 53.3%,92 cases older than 65 years old, accounting for about 46.7%. Colon cancer was the most frequent type, a total of 104 case (50.5%), lung cancer (72 cases) accounting for about 34.9% and malignant melanoma (30 cases) accounting for about 14.6%.126 DNA samples (61.2%) extracted from FFPE sample,69 (33.5%) from frozen tissue,7 (3.4%) from pleural fluid and 4(1.9%) from biopsy tissue.2.22 cancer genes were detected by Ion Torrent sequencing technology in 206 cases of malignant tumor samples, the analysis results were as follows:(1) The mean number of sequencing mapped reads was 1592934±302945.3, and the minimum was 482469; the mean number of sequencing mean depth was 15753X±3082.638X, and the minimum was 4826X; the mean sequencing uniformity was 97.1%±2.30%, and the minimum was 84.14%; the minimum allelic frequency of mutation detected was 1.6%. There were statistically significant differences about sequencing mapped reads and mean depth between the colon cancer, lung cancer and malignant melanoma groups (P<0.01), but not statistically significant differences betweent the FFPE, frozen tissue, pleural fluid, biopsy tissue groups (P> 0.05); while sequencing uniformity was opposite on above two points. (2) The gene mutation rates in the top six of 22 cancer genes were EGFR (100%), FGFR3 (100%), TP53 (85.4%), ERBB4 (67.0%), DDR2 (50.5%), KRAS (31.6%); while the mutation of FGFR2 or MAP2K1 were not detected. There were statistically significant differences only about the mutation rates of KRAS and BRAF between the colon cancer, lung cancer and malignant melanoma groups. The mutation rate of KRAS in colon cancer was higher than in others(P< 0.01), and the mutation rate of BRAF was higher in malignant melanoma(P< 0.01). (3) The number of simultaneous mutant genes detected in one cancer was five on average, and was two at least. (4) Except for MAP2K1 and FGFR2 mutation undetected, the other twenty genes were detected with at least one kind or at most sixty-nine different kinds of mutant genotypes. The most kinds of mutant genotypes was TP53, had 69 kinds; the second is the EGFR, had 19; and PTEN, KRAS and SMAD4, FBXW7, PIK3CA, all has more than 10 kinds of. And This part of above genes all have one genotype of them with higher mutant rate than others, between 21.4% to 100%. While the number of cases with ERBB2, AKT1 or NOTCH 1 mutation were consistent with their number of genotypes, namely one case with one differrent genotypes. (5) The way of gene mutation was mainly monotype mutation. MET, STK11, BRAF, FBXW7, ALK, NRAS, CTNNB1, ERBB2, FGFR1, AKT1, NOTCH1, these eleven genes were all monotype mutation detected in one case. SMAD4, PIK3CA, ERBB4, DDR2, KRAS, FGFR3, these six genes were detected with one or two mutant genotypes in one case and the monotype mutation rates of them were between 92.3%～92.3%. While TP53, EGFR with one to four mutant genotypes and PTEN with one, two, or six mutant genotypes in one case, the monotype mutation rates of them were 54.6%,41.5% and 85.7% in turn.3. EGFR, KRAS, or BRAF gene were detected by Sanger sequencing in 206 cases, and the analysis results of 39 kinds of common mutant genotypes of them were as follows:(1) There were 96 cases that EGFR, KRAS, or BRAF mutated, accounting for 46.6%,114 cases with wild-type EGFR, KRAS, or BRAF gene, accounting for 54.6%. (2) 96 cases that EGFR, KRAS, or BRAF mutated were all single-gene mutations. There were 33 cases that EGFR mutated, accounting for 16.0%,55 cases (26.7%) that KRAS mutated, and 8 cases (3.9%) that BRAF mutated. (3) 33 cases that EGFR mutated were all lung cancer samples. There were 2 cases in 33 cases that exon 18 mutated, accounting for 6.1%,8 cases (24.2%) that exon 19 mutated,1 case (3.0%) that exon 20 mutated,20 cases (60.6%) that exon 21 mutated and even all was L858R mutated, and 2 cases (6.1%) that exon 20 and 21 double-mutated. (4) 55 cases that KRAS mutated contained 51 cases (92.7%) of colon cancer samples, mainly for Gly12Asp, Gly13Asp, Gly12Val mutation and each 2 (3.6% each) cases of Lung cancer, malignant melanoma samples. (5) 8 cases that BRAF V600E mutated contained 5 cases (62.5%) of colon cancer samples and 3 cases (37.5%) of malignant melanoma samples.4. The testing results of the Ion torrent sequencing technology compared with the corresponding results of Sanger sequencing, and the result were as follows:There were 101 cases of EGFR, KRAS or BRAF mutation, accounting for 49.0%, and 105 cases (51.0%) with wild-type EGFR, KRAS, or BRAF gene.5 cases in 206 cases had inconsistent test results of these two methods, containing 3 case of EGFR mutations, each 1 case of KRAS and BRAF mutations. Ion torrent sequencing technology had a sensitivity of 100%, specificity of 95.5%, the coarse consistency of 97.6%, KAPAA value of 0.951 (P< 0.01). Through the analysis of the Ion torrent sequencing dates about gene mutations of 101 cases, we found that the allelic frequency of mutation were all over 5% in 96 cases with consistent test results of these two methods, and all below 5% in 5 cases with inconsistent test results.5. Comparing with the testing results of ADx-ARMS and the Ion Torrent sequencing in 96 cases, the results of ADx-ARMS were 67 cases with EGFR, KRAS or BRAF mutation, accounting for 69.8%, and 29 cases with wild-type EGFR, KRAS, or BRAF gene, accounting for 30.2%; the results of the Ion Torrent sequencing were 60 (62.5%) cases with EGFR, KRAS or BRAF mutation and 36 cases (37.5%) with wild-type EGFR, KRAS, or BRAF gene. There were 7 cases with inconsistent test results and the positive coincidence rate of these two methods was 89.6%, the negative coincidence rate was 100%, the coarse consistency was 92.7%, KAPPA value was 0.838 (P< 0.01).Conclusion:This research using the technology of Ion torrent sequencing, successfully tested the 22 cancer genes of 206 cases of patients with malignant tumor, and through the validation test of some mutations of EGFR, KRAS, BRAF gene by Sanger sequencing and ADx-ARMS, to confirmed that the technology of Ion torrent sequencing has high specificity and sensitivity, suitable for clinical application on a large scale in early screening and individualized diagnosis and treatment of malignant tumors. |
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