Detection Of NRAS/KRAS Mutations In Acute Myeloid Leukemia By Drop-off Droplet Digital PCR And Its Clinical Application

Purpose:1.The purpose of this study is to establish a drop-off droplet digital PCR method to detect RAS gene mutations in acute myeloid leukemia(AML),the performance test of this method was confirmed by using wild-type and mutant plasmids or DNA samples of clinical patients,including its sensitivity,specificity,repeatability,and linearity.The limit of blank and the minimum detection limit of the method in clinical samples were established.2.The established drop-off droplet digital PCR method was applied to detect bone marrow blood DNA of clinical samples of newly diagnosed AML patients.The results were standardized with Sanger sequencing and Next-generation sequencing(NGS).3.The established drop-off droplet digital PCR method was applied to dynamically monitor the change in mutant frequency of RAS mutation and to evaluate the relationship between the change in mutant frequency and clinical efficacy.Research methods:1.Extract bone marrow DNA samples from patients who have already been confirmed to have this mutation,and design sequencing PCR primers and amplification primers which cover the hot mutation region of NRAS G12/13,NRAS Q61 or KRAS G12/13 by comparing the conserved sequence of RAS gene.Wild-type NRAS/KRAS plasmids and mutant NRAS/KRAS plasmids were constructed by using designed primers and isolated clinical samples.2.Two wild-type probes were designed to cover the hot spot mutation regions of NRAS G12/13,NRAS Q61 or KRAS G12/13 and relatively conservative regions outside the hot spot mutation.Using wild-type and mutant plasmids or clinical samples as templates,the specificity of amplified primers and probes respectively were tested by gradient PCR and Quantitative Real-time PCR(Q-PCR),to optimize the reaction system,and select the appropriate annealing range.3.The established drop-off droplet digital PCR method was used to adjust the density of primers and probes and select an annealing temperature that could significantly distinguish the positive and negative droplets,by using wild-type and mutant clinical samples as templates,and then verify the performance of the established reaction system,establish the detection limit,and evaluate the repeatability of this detection method.4.A new drop-off droplet digital PCR method was used to screen consecutive newly diagnosed clinical samples,and the results were compared with the previous Sanger sequencing results and then NGS was used for further verification.5.Monitor the mutant frequency in patients with RAS gene mutations and evaluate whether the frequency changes of gene mutations are related to clinical efficacy.6.The clinical characteristic of 140 newly AML patients were classified according to whether existed RAS gene mutations.The indicators were analyzed,and the statistically significant indicators were analyzed in depth.Results:1.In this research,a drop-off droplet digital PCR method was established to detect the hot spot mutations of NRAS G12/13.This method had high specificity and sensitivity,by which the NRAS G12/13 mutation could be well detected in clinical samples when the mutant frequency exceeded 0.158%,and the repeatability and linearity were good(R~2=0.999).The blank limit of detecting clinical samples was 5.4 copies/microliter and the minimum detection limit was 15.84copies/microliter.2.In this research,a drop-off droplet digital PCR method was established to detect the hot spot mutations of NRAS Q61.This method has strong specificity and high sensitivity,by which the NRAS Q61 mutation could be well detected in clinical samples when the mutant frequency exceeded 0.05%,and good repeatability and linear relationship(R~2=0.9991).The blank limit of detecting clinical samples was 1.51 copies/microliter,and the minimum detection limit was 5.12copies/microliter.3.In this research,a drop-off droplet digital PCR method was established to detect the hot spot mutations of KRAS G12/13.This method had high specificity and sensitivity,by which the KRAS G12/13 mutation could be well detected in clinical samples when the mutant frequency exceeded 0.16%,and the repeatability and linearity were good(R~2=0.9988).The blank limit of detecting clinical samples was 5.04 copies/microliter and the minimum detection limit was 16.47copies/microliter.4.Among 140 newly diagnosed AML patients,NRAS G12/13 mutations were detected in 28clinical samples(20%)by drop-off droplet digital PCR,and the mutation frequency was 0.26%-52.85%(median mutation frequency was 2.14%).However,only 7 clinical samples(5%)of the previous Sanger sequencing were positive.To further verification,the rest 21 clinical samples with negative results of Sanger sequencing were tested by NGS.The results showed that 14 clinical samples were positive among 21clinical samples.For these 14 samples,the mutation frequency detected by drop-off droplet digital PCR ranged from 0.26%to 9.64%(median mutation frequency was 2.82%),and NGS detected mutant frequencies ranged from 1.03%to 9.5%(median mutation frequency was 1.46%)for these 14 samples.The drop-off ddPCR mutation frequency was 0.53%-1.5%(median mutation frequency was 1.1%).The correlation analysis between the mutation frequencies detected by next generation sequencing and drop-off ddPCR,including 2 cases of NRAS G12/13 mutation detected by Sanger sequencing and confirmed by NGS,showed a good correlation between the mutation frequencies detected by the two methods(R~2=0.9158).5.Among 140 newly diagnosed AML patients,KRAS G12/13mutations were detected in 9clinical samples(6.4%)by drop-off droplet digital PCR,and the mutant frequency was 1.56%-32.75%(median mutation frequency was 9.6%).The previous Sanger sequencing method only detected 4 clinical samples(2.86%).To verify the reliability of the results,9 samples with KRAS G12/13 mutations detected by drop-off droplet digital PCR were all subjected to KRAS G12/13high-throughput sequencing again.The results of NGS showed that KRAS G12/13 mutations were exactly existed in these 9 patients,and the mutant frequency detected by drop-off droplet digital PCR was well correlated with that detected by NGS(R~2=0.9697).6、During the dynamic monitor of patients with NRAS G12/13 mutations,this study found that the frequency of NRAS G12/13 mutations had a significant upward trend before or at the time of disease relapse while there was a significant downward trend when the disease achieved complete remission(CR).Conclusion:A drop-off droplet digital PCR method was established to detect common hot spot mutations of RAS gene in AML patients.The established drop-off droplet digital PCR method was highly sensitive to identify single gene mutations compared with Sanger sequencing and NGS.This method can be used to screen the common hot spot mutation of RAS gene in newly diagnosed AML patients and can dynamically and quantitatively monitor the mutation frequency of RAS gene during clinical treatment,to predict the prognosis and monitor the disease of AML patients alone or in combination with other gene mutations,to achieve the purpose of clinical individualized precision treatment.