Cytogenetic Analysis And Clinical Significance Of AML And MDS Patients With DMs

Objective:The purpose of the research is a preliminary exploration of the genetic characteristics and molecular structure of double minute chromosomes(DMs)in patients with acute myeloid leukemia(AML)and myelodysplastic syndrome(MDS),providing research direction and foundations for future indepth study into the mechanism of the DMs.Methods:This research uses the retrospective analysis method to present cytogenetic features of 2726 patients with AML and 1741 patients diagnosed with MDS in the Chinese People’s Liberation Army General Hospital starting from January 2011 to June 2021,among which patients with acute myeloid leukemia with myelodysplasia-related changes(AML-MRC)and MDS-defining cytogenetic abnormalities(namely AML-MRC-C group)and patients with MDS and high-risk cytogenetic abnormalities(namely MDS-PC group)were chosen for testing the presence of DMs and the significant genetic markers associated with prognosis.All participants underwent the chromosome analysis and the Fluorescence in situ hybridization(FISH)test in which two genes(MYC and KMT2A)will be probed to present the molecular structure of DMs;the chromosome analysis and FISH test were conducted to detect TP53 deletion,and Sanger sequencing was used to detect TP53 mutation,and all patients were followed up for survival status.Results:The research revealed that a total of 42 patients with AML and MDS had clonal DMs.0.99%of patients with AML and 0.86%of patients with MDS harbor DMs.And 22.5%(20/89)of patients in AML-MRC-C group and 24.5%(13/53)of patients in MDS-PC group were tested positive in DMs,which showed significant differences compared with the entire cohort(p=0.00).Besides,patients with AML-MRC-C and patients with MDS-PC were divided into two groups based on the presence or absence of DMs:DMs-positive group&control group.The median patient age among patients diagnosed with AML-MRC-C(n=20)was higher in DMs than that of the AML-MRC-C control group(n=69)[66(39-90)years vs.55.5(20-91)years,p=0.009].Among AML-MRC-C patients,the positive rates of genetic markers of poor prognosis indicator in the DMspositive group were higher than those in the control group(p<0.05),for example,complex karyotypes(CK)(100%vs.60.9%,p=0.013),monosomal karyotype(MK)(90%37.7%,p<0.001),TP53 deletion(70.6%30.6%,p=0.004),TP53 mutation(76.5%25.9%,p<0.001),TP53 double hit(66.7%11.1%,P<0.001),overall survival(OS)(2.7 months vs.12 months,p=0.013).DMspositive patients with AML-MRC-C and TP53 deletion(n=12)had a worse prognosis than those without TP53 deletion(n=5)(2 months vs.18 months,p=0.047)and a significantly shorter OS than the control group(n=15)(2 months vs.8.3 months,p=0.006).DMs-positive patients with AML-MRC-C and with TP53 mutation(n=13)had a worse prognosis than those without TP53 mutation(n=4)(median overall survival 2.7 months vs.18 months,p=0.0032).COX multivariate survival analysis showed that DMs and 5q-/-5 are independent risk factors of OS in patients with AML-MRC-C.The median patient age among patients with MDS-PC(n=13)was higher than that of the MDS-PC control group(n=40)[68.7(48-89)years vs.59.1(3286)years,p=0.046].Among patients with MDS-PC,there were statistically differences(p<0.05)between DMs-positive group and control group in terms of 5q-/-5(84.6%40%,p=0.005)and TP53 deletion(83.3%17.9%,p=0.007).And there was no significant difference in the positive rate of other genetic markers of poor prognosis indicator when compared to the control group(p>0.05),for example,complex karyotype(100%90%,p=0.561),monosomal karyotype(76.9%vs.60%,p=0.44),TP53 mutation(63.6%29.6%,p=0.114),TP53 double hit(33.3%vs.10%,p=0.457),and shorter overall survival(OS)(7 months vs.18 months,p=0.037).COX multivariate survival analysis showed that among patients with MDS-PC,age,5q-/-5,7q-/-7,and TP53 deletion are independent risk factors that have a strong bearing on patients’ overall survival,and DMs were not an independent risk factor of OS in this group of patients.Of a total of 27 AML patients with positive DMs(20 patients with AMLMRC-C and 7 non-AML-MRC-C patients),24 patients had enough samples for the FISH test and the results showed that the positive rate of the MYC gene in the AML group(n=27)was 37.5%(9/24),and the positive momentum of KMT2A was 12.5%(3/24).Of a total of 15 MDS patients with positive DMs(13 MDSPC patients and 2 non-MDS-PC patients),13 patients had enough samples for FISH detection,the results showed that the positive rate of the MYC gene in the MDS group(n=15)was 7.6%(1/13),KMT2A positivity was not detected.Of 10 patients with amplified MYC genes(9 in the AML group and 1 in the MDS group),8 patients performed the TP53 deletion test,and the positive rate was 50%(4/8)and 9 patients performed the TP53 mutation test,and the positive rate was 44.4%(4/9).The survival time of 3 patients with MYC amplification and TP53 mutation was short,one month,two months,and four months respectively;the OS of 5 patients without TP53 mutation was more than 12 months.All three patients with positive KMT2A showed TP53 double-hit,and the OS was short(1 month,five months,and 2.7 months respectively).Of MYC-positive AML patients,88.9%(8/9)of the cases showed a significant amplification of the MYC signal.The amplified signals were found clustered inside and outside the nucleus,and they were also observed on the ring chromosomes and homogeneously stained regions.Conclusion:DMs are rare in patients with AML and MDS and more common in patients with AML-MRC-C and MDS-PC.The AML-MRC-C patients with positive DMs are associated with older age and the main cytogenetic characteristics of DMs-positive AML-MRC-C and DMs-positive MDS-PC are CK and MK.The genetic factors of adverse prognoses,such as 5q-/-5,TP53 deletion,TP53 mutation,and TP53 double hit,presented a high prevalence,indicating the instability of the genome and poor prognosis.The AML-MRC-C DMs patients with TP53 deletion and TP53 mutation had a worse prognosis.Multivariate analysis also suggested that DMs and 5q-/-5 were independent risk factors for OS in AML-MRC-C patients.MDS-PC patients with DMs also tend to be old-age individuals with CK and MK as the main cytogenetic features,and a greater incidence of 5q-/-5 and TP53 deletion,which all suggest a poor prognosis.Multivariate analysis of MDS-PC patients showed that age,5q-/-5,7q-/-7,and TP53 deletion were independent risk factors for OS,and the presence of DMs was not an independent risk factor.Extensive amplification of the proto-oncogene MYC is common in AML patients with DMs.The AML patients with DMs and MYC amplification showed heterogeneity survival,and those with TP53 mutations had a worse prognosis.In addition,patients with KMT2A amplification on DMs all showed TP53 double hits and a very short OS.DMs are associated with micronuclei,ring chromosomes,and regions of homogeneously stained regions.