High Resolution Melting Analysis Application And Technology Evaluation In Malignant Tumors With EGFR Activated Mutation

BACKGROUNDS:With the deciphering of the human genome,conducting of genome-wide association studies in many diseases and learning the molecular mechanism of many diseases more in-depth,it is revealed gradually that the effect of genetic variants on disease occurrence,development,drug sensitivity and prognosis.Targeted therapies,based on specific gene variation,opened a new era of disease treatment,especially cancer therapy.Carcinogenesis is a multi-steps process involving multi-genes mutation.The driver or activated mutation is various among different tumors,even among different patients with the same tumor.It is possible that the different modality of treatment is used for the same tumor and the same treatment modality for the different tumor,while not the same standardized treatment for the same tumor like traditional therapy.Therefore,it is necessary to profile tumor molecular characters and provide a specific molecular portrait of each individual in order to aid clinicians in improving diagnosis and tailoring the best therapy to each patient.EGFR tyrosine kinase inhibitor(EGFR-TKI)is one of the most widely used molecular targeted drugs.It has been used in the treatment of non-small lung cancer(NSCLC)with EGFR mutation in exon 19 and 21 with good response.Although there is high proportion of NSCLC patients with EGFR mutation in China,about half of the patients can't be predicted the response to EGFR-TKI.Moreover some of the patients who have good response to it would obtain resistance after treatment with it for a while.It would be helpful to resolve the problems by profiling the molecular characters related to EGFR mutation in NSCLC and then finding new molecular markers.It is valuable to detect if other tumors can be treated with EGFR-TKI.EGFR mutation has been found in head and neck neoplasm,gastric cancer and colorectal cancer,etc.and the patients with EGFR mutation also had good response to EGFR-TKI.Glioma is the most common malignant tumor of central nervous system.The rampant proliferation and recurrence are its obvious biological characters.Despite the available of many traditional treatment options,the prognosis is often dismal.It is still unclear if EGFR-TKI can be used to treat glioma,though the activation EGFR was often found in glioma,including EGFR mutation in extracellular domain(point mutation and deletion in exon 2 to 7),amplification and over expression.Gene variance detection is the basis of molecular targeted therapy.Compared to germline mutation detection with blood samples of genetic disease,gene variance detection is harder with tumor samples.The proportion of variant DNA is unknown,because normal cells will be mixed unavoidably in the tumor samples.In addition,unknown mutation is often found in tumor samples.It is key to select a gene variance detection method which is sensitive enough and can be used to detect the unknown mutations.High resolution melting analysis(HRMA)is a new gene variance detection technique,which was first introduced in 2003.Because HRMA is a simple,high sensitivity,high speed and low contamination risk method,it is rapidly adapted used into most real-time PCR instruments as well as widely used in research and clinical diagnostics for detecting DNA variants,either known or unknown.Detection of gene variance by HRMA depends on subtle melting curve differences,including shape changes and melting temperature(Tm)shifts.The accuracy and detection sensitivity were affected by chemical factors from PCR,amplicon features such as fragment lengths,gene variance types and sequences,and hardware and software from the instruments.The effect of chemical factors and amplicon features can be controlled by the design of the assay,but the instrument factor is often hard to avoid.There are large differences among HRMA instruments,including sample format,melting rates,detection mode,data density,acquisition mode and software.It is not clear if the differences would affect gene variance detection accuracy.Therefore,it is necessary to select the proper instrument to detect the gene variance by the metric of quality control built depending on comparison the genotyping accuracy among 9 instruments.It would be also helpful to identify those factors that really matter for continued improvement of HRMA.The fast detection speed is often expected when a method used in clinical molecular diagnostics.HRMA may have fast detection speed,but many real-time PCR instrument with HRMA slow the melting rate down to obtain adequate resolution.Because it was thought for a long time that increasing the melting rate means sacrificing the detection resolution.The effect of melting rate on genotyping accuracy is controversial.It is very important to explore the problem systematically in order to make HRMA used in clinical gene variance detection better.METHODS AND RESULTS:1.Expression of the mismatch repair gene hMLH1 is enhanced in NSCLC with EGFR mutationsThe expression of MMR proteins hMSH2 and hMLH1,and the proliferation markers PCNA and Ki67 were measured by immunohistochemistry in 181 NSCLCs.EGFR and KRAS mutations were identified by high resolution melting analysis.In 181 NSCLCs,the frequencies of EGFR and KRAS mutation were 36.5%and 5.5%;the frequencies of hMSH2?hMLH1?PCNA and Ki67 positive expression were 59.6%,71.3%,88.4%and 61.9%,respectively.Stronger hMLH1 expression correlated to a higher frequency of EGFR mutations in exon 19 and 21(p<0.0005).Overexpression of hMLHl and the adenocarcinoma subtype were both independent factors that related to EGFR mutations in NSCLCs(p=0.013 and p<0.0005).The expression of hMLHl,hMSH2 and PCNA increased,while Ki67 expression significantly decreased(p=0.030)in NSCLCs with EGFR mutations.2.BRAF overexpression inducing the rampant proliferation in glioma independent on EGFR activationEGFR,KRAS,BRAF,PI3K,phospho-EGFR and Ki67 expressions were detected with immunohistochemistry in 82 glioma specimens.The positive expression of phospho-EGFR was used as the marker of EGFR activation,or vice versa.In 82 glioma,the frequencies of phospho-EGFR?EGFR?KRAS?BRAF?PI3K and Ki67 positive expression were 59.8%?75.6%?63.8%?72.0%?68.1%and 69.5%.The expression of EGFR positively correlated with patients' age.There were no significant differences in clinicopathological characteristics between gliomas with and without EGFR activation.EGFR overexpression was significantly related to its activation.In gliomas with EGFR activation,overexpressions of EGFR,BRAF and PI3K were each significantly related to proliferation respectively.However,in gliomas without EGFR activation,only BRAF overexpression was significantly related to proliferation of tumors cells.3.The feasibility of HRMA to detect EGFR,KRAS mutations in pleural effusion samples of NSCLC patientsThe mutations of KRAS gene in exon 2 and EGFR gene in exon 19,21 were detected in 36 pleural effusion samples with HRMA.The difference of EGFR and KRAS mutation frequencies between pleural effusion samples and cancer tissue samples from patients with NSCLC were analyzed with Chi square.There were 2 cases(5.6%)with KRAS mutations and 10 cases(27.8%)with EGFR mutations found in 36 pleural effusion samples with HRMA.The frequencies of KRAS and EGFR mutation,detected in 181 NSCLC tissues before,were 5.5%and 36.5%,respectively.There was no significant difference in either KRAS or EGFR between pleural effusion samples and cancer tissue samples(p=1.000 and p=0.318).4.Genotyping accuracy in nine HRMA instrumentsThree genotype of the single nucleotide variance(c.3405-29A>T)of CPS 1 was amplified by PCR in the presence of LCGreen Plus in four PCR product lengths.After blinding and genotype randomization,samples were melted in 9 instruments(10 instrument configurations)under conditions recommended by the manufacturer.For each configuration and PCR product length,32 to 96 samples(depending on batch size)were analyzed with both commercial and custom software.The accuracy of heterozygote detection and homozygote were assessed.Overall,the heterozygote accuracy was 99.7%(n=2141),while homozygote accuracy was 70.3%(n=4441).Instruments with single sample detection as opposed to full plate imaging better distinguished homozygotes(78.1%and 61.8%,respectively,p<0.0005).PCR products of 51,100,272 and 547 bp had accuracies of 72.2%,83.1%,59.8%and 65.9%,respectively(p<0.0005).Slower melting rates did not improve accuracy.The index,Log(?Tm/Tm SD),was a good predictor of an assay's ability to distinguish homozygotes(AUC=0.863,p<0.0005).5.The effect of melting rate to genotyping accuracyThree genotype of the single nucleotide variance(c.3405-29A>T)of CPS1 was amplified by PCR in the presence of LCGreen Plus in four PCR product lengths.All samples in triplication were melted in HR-1 with the ramp rate 0.01?0.02?0.04?0.08?0.16?0.32?0.64?/s,separately.The corrected coefficient was calculated with the difference between the instrument temperature and sample temperature at each ramp rate.All raw data were corrected with the coefficient before analysis.When the ramp rate increased,melting curves of all samples translated to the right(high temperature).Heterozygotes(<300 bp)and homozygotes(51 and 100 bp)were easier to detected with the ramp rate increasing,while the homozygote(272 bp)was easier to be distinguished with the ramp rate decreasing.For 547 bp gragment,heterozygote was the most difficult to detect in the middle ramp rate(0.8?/s),it was easier with the ramp rate increasing or decreasing.On the contrary,547 bp homozygotes were the easiest to be distinguished in the middle ramp rate,it was more difficult with the ramp rate increasing or decreasing.CONCLUSIONS:Overexpression of hMLHl could be a new molecular marker to predict the response to EGFR-TKIs in NSCLCs.BRAF overexpression would be an independent factor inducing rampant proliferation in gliomas independent on EGFR activation;the molecular target drug with multi-targets of EGFR,BRAF,PI3K could be more effective in treating glioma than EGFR-TKI.HRMA detects heterozygotes with excellent accuracy,but homozygote accuracy is dependent on instrumentation,PCR product size as well as melting temperature difference,and variation within homozygotes.Fast ramp rate is helpful to increase the sensitivity of genotyping of shrot amplicons,either heterozygote or homozygote.HRMA is a simple,sensitive,low contamination risk,high speed method of gene variance(both known and unknown)detection.It is suitable for gene variance detection in clinic,especially for tumor samples including pleural effusion,which is mainly with heterozygous mutations.It is possible to make the detection more fast and sensitive by increasing the ramp rate when the amplicon is not too long.