Development Of A Drop-off Droplet Digital PCR Method For C-KIT Gene Mutation Detection In Acute Myeloid Leukemia And Its Clinical Application

Objective:Acute myeloid leukemia(AML)is a hematological malignancy characterized by clonal expansion of myeloid progenitor cells in bone marrow and peripheral blood,with high heterogeneity.C-KIT mutations often occur in AML patients with t(8;21),inv(16),or t(16;16).AML patients with C-KIT mutations often have poor prognosis and a high risk of recurrence after complete remission.The Chinese AML guideline classifies these patients into subgroups with moderate prognosis.The European Leukemia Network Working Group recommends to combine C-KIT mutations with other markers for measurable residual disease(MRD)monitoring.The purpose of this study is to develop a method for quantitative detection of mutations at D816 and N822 sites of C-KIT gene based on drop-off droplet digital PCR(dd PCR)technology,complete methodological evaluation,and assess the application value of this method in prognosis evaluation,recurrence monitoring and treatment guidance of AML.Methods:(1)Design a pair of specific primers and three probes for the exon 17 of the C-KIT gene,two of which are located on the D816 and N822 mutation hotspots,and the other wild-type probe is located outside the two mutation hotspots mentioned above;(2)To establish and optimize the drop-off dd PCR method for detecting D816 and N822 mutation of C-KIT gene,and assess the specificity,sensitivity,repeatability and linearity of the method;(3)Use the established method to test bone marrow samples from 140 newly diagnosed AML patients who have previously Sanger sequenced,and validate the results with nextgeneration sequencing(NGS)to compare the detection efficiency of C-KIT gene mutation using three methods;(4)Collect and organize various clinical parameters of the study subjects,divide them into mutation group and wild group based on dd PCR results,and analyze whether there are statistical differences in various clinical parameters between the two groups,Simultaneously analyze whether there are statistical differences in clinical parameters between the C-KIT mutation group and the wild group in patients with core binding factor acute myeloid leukemia(CBF-AML);(5)Six patients with C-KIT mutation were monitored for MRD using the drop-off dd PCR method.Results:(1)The limit of blank(LOB)of C-KIT gene D816 mutation detected by drop-off dd PCR is 4.26copies/μl.The limit of detection(LOD)is 12.98 copies/μl.Its sensitivity is 0.13%,the repeatability and linear relationship were good when the concentration was above 5copies/μl.(2)The LOB of C-KIT gene N822 mutation by drop-off dd PCR is 1.62 copies/μl,and the LOD is 10.12 copies/μl.Its sensitivity is 0.10%,the repeatability and linear relationship were good when the concentration was above 5 copies/μl.(3)Sanger sequencing and drop off dd PCR were used to detect C-KIT gene mutation in 140 newly diagnosed AML patients.Among them,Sanger sequencing revealed only two(1.43%)positive cases,while drop-off dd PCR revealed twenty-one(15.00%)positive cases.Among them,dd PCR identified nineteen(13.58%)KIT-D816 mutation with the mutation burden ranged from 0.18% to 48.85%(median 0.66%),seven(5.00%)KIT-N822 mutation with the mutation burden ranging from 0.29% to 7.41%(median 1.42%),and five patients detected D816 and N822 double mutation.Subsequently,the results were further confirmed by NGS.NGS revealed eight(5.71%)positive cases with variant allele frequency(VAF)ranging from 1.26%～37.70%(median 22.25%).Using the results detected by NGS as the gold standard,the specificity and sensitivity of the drop-off dd PCR method for detecting C-KIT gene D816 and N822 mutations were 100%.(4)According to the results of dd PCR,140 newly diagnosed AML patients were divided into the mutated group(n=21)and the wild-type group(n=119).The clinical parameters between the two groups were analyzed and compared for any differences.The results showed that there was a statistically significant difference(P=0.001)in chromosome karyotype and risk level grouping between the two groups,C-KIT mutations are mainly found in patients with t(8;21)chromosomal translocation(7/12,58%).However,in terms of gender,age,WBC,Hb,Plt,blast cell counts,whether complete remission has been achieved,FAB classification and common gene mutations in AML were not statistically significant(P>0.05).Further,we analyzed whether there were differences in clinical parameters between the two groups in thirteen patients with CBF-AML,including 7 cases of C-KIT mutation and six cases of wild type.The results showed that there was no significant difference between the two groups in WBC,Hb,Plt,blast cell counts,and whether they reached CR(P>0.05).(5)The established drop-off dd PCR method was used to dynamically monitor the mutation burden after chemotherapy in six patients with C-KIT mutations.The results showed that the mutation burden of C-KIT gene was in accordance with the tendency of complete remission or recurrence in clinical practice.Conclusions:(1)Based on the drop-off dd PCR technology,this study developed a method to detect the KITD816 and KIT-N822 mutations,which has good methodological performance.Compared with Sanger sequencing and NGS,dd PCR has a higher mutation detection rate and a better ability to detect low-frequency mutations;(2)The dd PCR is anticipated to be used for monitoring measurable residual disease and guiding individualized treatment following remission in positive patients.