Identification And Clinical Application Of Novel Molecular Markers In Myelodysplastic Syndromes

Part1 Development and validation of a novel prognostic risk classification(IPSS-Rm)in MDS Objective:To retrospectively analyse the clinical and biological characteristics of 2215 patients with myelodysplastic syndromes in our center from 1995 to 2016.And to reveal the role of next gene sequencing for prediction of outcome of MDS patients.Methods:1.From 1995 to 2016,a total of 2215 MDS patients who diagnosed at our center were enrolled in this study and had available data were included.The diagnosis and classification of MDS were based on the 2008 World Health Organization(WHO)criteria and the latest 2016 WHO criteria.2.We then analyzed the impact of mutations on the outcome of 169 patients with MDS through next generation sequencing.And the newly-established prognostic model of incorporating molecular data into IPSS-R was established by statistical analysis.Results:1.Of the 2215 MDS patients,there were 1304 males and 911 females,and the median age was 55 years(6-93 years old).According to the IPSS-R stratification:32patients(1.9%)had very low risk MDS;405(24.3%),low;596(35.7%),intermediate;385(23.1%),high;and 252(15.1%),very high risk MDS.42.5%(941/2215)patients had clonal chromosome abnormalities,in which 271(12.2%)patients with complexed karyotype.And the main types of chromosomal aberrations were unbalance abnormality,especially in trisomy or monosomy.The most common abnormity was+8.Other aberrations in frequent order was del(5q),-7/del(7q),del(20q)and so on.2.According to the WHO(2016)criterion:99 patients with MDS-RS-SLD,52 patients with MDS-RS-MLD,253 patient with MDS-SLD,620 patient with MDS-MLD,28 patients with 5q-syndrome,421 patients with MDS-EB-1,458 patients with MDS-EB-2,284 patients with MDS-U.3.130/169 patients(76.9%)harbored at least one mutation(median,1 per patient;range,0-6).The mutation frequencies were:U2AF1(25.4%),ASXL1(15.4%),TP53(10.1%),RUNX1(10.1%),DNMT3A(9.5%),BCOR/BCORL1(7.7%),TET2(7.7%),KRAS/NRAS(7.7%)and so on.In multivariate analysis,only age,IPSS-R score,TP53,KRAS/NRAS and GATA2 had independent prognostic significance(P<0.05).A risk model was developed incorporating the weighted coefficients of these variables:TP53×1+KRAS/NRAS×1+GATA2×2+age×0.04+IPSS-R×0.2.And the new model had a better C-index(0.73)compared with the IPSS-R(0.68).Conclusion:1.In our study,the MDS patients showed the unique clinical and biological features.We found that the characteristics of cytogenetics have significant differences from western MDS patients.The most common abnormity was+8.Other aberrations in frequent order was del(5q),-7/del(7q),del(20q),and so on.2.Our new model serves as a proof of concept that the incorporation of molecular data into the IPSS-R may enhance its ability to predict outcome in MDS patients.Part2 Clinical and laboratory characteristics of myelodysplastic syndromes patients with monosomal karyotype Objective:To investigate the clinical and genetic characteristics of the monomer karyotype myelodysplastic syndromes(Monosomal karyotype myelodysplastic syndromes,MK~+MDS).Methods:The cytogenetics of MDS patients diagnosed in our center were retrospectively analyzed,and the patients with monosomal karyotype were selected.Meanwhile,the patients without monosomal karyotype were considered as a control group to describe the clinical and laboratory characteristics of MK~+MDS patients.Results:MK was observed in 8.1%patients(168/2080),and monosomies of chromosome 5/7were the most frequent types of MK.We further found that MK was significantly associated with elderly patients,higher bone marrow blasts and relatively poor cytogenetics.In addition,MDS patients with MK(n=59)had poor survival than those without MK(n=491)in total cohort(P<0.001),and there was significant difference in the OS between the patients with MK(n=56)and without MK(n=53)in the relatively poor cytogenetics group(P=0.0025).Incorporation of MK into IPSS-R could further stratify MDS patients into different prognostic groups(P<0.001).Interestingly,monosomies of chromosome 5/7 rather than MK were significantly related to shorter OS(HR=2.709,P<0.001)by multivariate analysis.Conclusion:8.1%MDS patients were presented with MK,and the incidence of MK increased with the number of cytogenetic abnormalities.Monosomies of chromosome 5/7 were the most frequent MK as well as an independent poor risk factor for OS in Chinese MDS patients.Part3 The clinical and biological studies of MDS patents with der(1;7)(q10;p10)Objective:To analyse systematically the clinical and laboratorial characteristics of MDS patients with der(1;7)(q10;p10)who diagnosed in our center and to reveal the unique features of MDS patients with der(1;7)(q10;p10),improving the study on cytogenetics and molecular characteristics.Methods:1.We screened patients with der(1;7)(q10;p10)from the chromosome database of our center,and retrospectively analyzed the clinical and prognostic characteristics.2.We performed NGS on the target DNA with a panel of 390 genes in a cohort of 22 MDS patients with der(1;7)(q10;p10)and 32 MDS patients with-7/del(7q).3.The influence of RUNX1-muts on protein function was predicted by structural modeling.The sub-location of RUNX1-wt and its mutants in Hela transient cell lines was observed by immunofluorescence.4.We then performed the gene expression array in 7der(1;7)(q10;p10)and 6-7/del(7q)samples to find a possible pathogenetic gene with the RUNX1 mutation.Results:1.A strong male predominance was showed in 70 patients with der(1;7)(q10;p10)(84.3%,59/70).The median age was 56 years(range,16-82 years).Based on review of the patient records,these patients were further classified as follows:50(71.4%)MDS,15(21.4%)AML,3(4.3%)MM,1 CML,and 1 HES.The frequency of der(1;7)(q10;p10)in MDS was 2.6%(50/1,934)in our center.Compared to-7/del(7q)cases,der(1;7)(q10;p10)MDS cases showed lower blast counts and higher hemoglobin concentrations.According to the IPSS-R,der(1;7)(q10;p10)cases were more likely to be relatively low risk groups.In addition,MDS patients with der(1;7)(q10;p10)showed better outcome:the 2-year OS was50%compared to 15%in the MDS patients with-7/del(7q)(P=0.018).Karyotypic analysis showed that the der(1;7)(q10;p10)appeared as the sole aberration in 60%cases,and accompanied only by a limited number and variety of additional abnormalities,mostly trisomy 8 and/or loss of 20 q.2.We performed NGS on the target DNA with a panel of 390 genes in a cohort of 22 MDS patients with der(1;7)(q10;p10)and 32 MDS patients with-7/del(7q).Mutations were detected in 53 of the 54(98%)patients.9 of the 54(16.7%)patients carried one and14/54(25.9%)cases harbored two(n=14),three(n=11)or at least four(n=19)mutations(Figure 1A).In the der(1;7)(q10;p10)cohort,the most frequently mutated genes were RUNX1(9/22,40.9%),ASXL1(5/22,22.9%),EZH2(4/22,18.2%),and DNMT3A(4/22,18.2%),while TP53(9/32,28.1%),ASXL1(9/32,28.1%),SETBP1(7/32,21.9%),and TET2(6/32,18.8%)were the most frequently mutated genes in the-7/del(7q)cohort.Notably,we observed high frequency of RUNX1 mutations in der(1;7)(q10;p10)patients compared to-7/del(7q)group(40.9%vs.12.5%,P=0.016).3.We performed structural modeling to understand the possible biological effect of RUNX1 mutations,and found these mutations significantly affected the DNA or protein binding ability of RUNX1 and might participate in the pathogenesis of MDS patients with der(1;7)(q10;p10).By indirect immunofluorescence staining,wild-type RUNX1 protein,as well as N136 K,was found to localize in the nucleus.The S141X、R201X和R204X protein was found to localize not only in the nucleus,but also in the cytoplasm.4.The gene expression profiling was significantly different between der(1;7)(q10;p10)and-7/7q-patients by cluster analysis.There were 151 dysregulated genes located on chromosome 1q and the majority of 1q associated genes were reported to be directly or indirectly involved in the disease development and pathogenesis.For instance,over-expression of NUF2 could cause defective spindles,misaligned chromosomes,and activated spindle assembly checkpoint,and thus inhibited chromosome segregation and metaphase-anaphase transition.The NUF2 gene mRNA level was significantly higher in der(1;7)(q10;p10)patients compared to-7/del(7q)(P=0.0005).Conclusion:1.We observed that MDS patients with der(1;7)(q10;p10)present male predominance and have a better outcome than the-7/del(7q)group.Karyotypic analysis showed that the der(1;7)(q10;p10)appeared as the sole aberration in 60%cases,and accompanied only by a limited number and variety of additional abnormalities,mostly trisomy 8 and/or loss of 20 q.2.We demonstrate for the first time,to our knowledge,that der(1;7)(q10;p10)is associated with a high frequency of mutations in RUNX1(40.9%).Cellular localization revealed RUNX1-S141 X,R201X,R204 X can lead to the abnormal localization of protein encoded by the gene.