Dimitriou, Marios

Hellstrom-Lindberg, Eva

Jansson, Monika

Karimi, Mohsen

Kere, Juha

Lehmann, Soren

Matsson, Hans

Nilsson, Christer

Unneberg, Per

Haematologica

PubMed

Helsingin yliopiston digitaalinen arkisto

High-throughput mutational screening adds clinically important information in myelodysplasticsyndromes and secondary or therapy-related acutemyeloid leukemiaRecurrent mutations are implicated in the prognosis ofmyeloid malignancies and are likely to be incorporated intothe next WHO classification. We evaluated a poolingapproach for cost-effective targeted sequencing in combi-nation with a clinically accredited method for validatingpoint mutations, Sequenom®. We found the pooling strate-gy adequate for detecting clonal mutations, but not sensi-tive enough to confidently detect subclonal mutations. Ourdata indicate that targeted sequencing adds prognosticinformation to existing risk scores in myelodysplastic syn-dromes (MDS), mixed myeloproliferative/MDS neoplasms(MDS-MPN) and acute myeloid leukemia (AML). More than one-third of patients with MDS have higher-risk disease with poor overall survival and a high rate ofprogression to AML.1 Several clinical trial programs, there-fore, include higher-risk MDS in current protocols forAML.2,3 Moreover, based on clinical and cytogenetic char-acteristics, a biological similarity between higher-risk MDSand poor prognosis AML has been suggested. Poor-riskcytogenetic abnormalities encompassing complete or par-tial loss of chromosome 5, 7, and 17, and complex kary-otype are found in both diseases; but outside this group,the cytogenetic pattern differs markedly between MDS andAML.4 FLT3, NPM1, CEBPA and KIT mutations are inte-grated in the current WHO classification for AML, but arerarely mutated in MDS, or secondary or therapy-relatedAML.5 Consecutive adult patients were enrolled in the MDSand AML registers at the Karolinska University Hospitaland diagnostic bone marrow (BM) samples werebiobanked. The MDS cohort (n=100) consisted of patientswith MDS and MDS-MPN (n=100) and the AML cohort(n=92) of patients with AML evolving from MDS (MDS-AML) or MPN (MPN-AML), therapy-related AML (tAML),and AML with MDS like cytogenetics (OnlineSupplementary Appendix and Online Supplementary Table S1).First-line treatment for both cohorts followed current ther-apeutic guidelines developed by the EuropeanLeukemiaNet project.6,7 Eighteen patients (7 MDS and 11AML) underwent allogeneic stem cell transplantation.DNA samples from 20 healthy subjects were used as con-trols. The study was approved by the local ethics commit-tee and samples were obtained after informed consent.Categorical data were compared using Fisher’s exact test,survival was estimated using Kaplan-Meier method andcompared using the log rank test, and Cox proportionalhazards model was used for uni- and multivariable analy-ses. Variables included in the model for MDS were muta-tions found to be significant in the univariable analyses(Online Supplementary Table S2) together with age, sex andIPSS-R classification. In the corresponding model for AML,IPSS-R was replaced by the cytogenetic risk group. Two-sided P values with a significance level of 0.05 were used inall analyses.8Halogenomics target amplification technology wasapplied to amplify all exons of 22 selected recurrentlymutated genes9,10 from BM mononuclear cell DNA (OnlineSupplementary Appendix). To analyze samples in a high-throughput and cost effective way we used a poolingapproach with 10 samples per pool. A total of 22 poolswere defined by 6 bp barcoding and samples weresequenced in 2 Illumina Hiseq2000 sequencing systemusing a 100 bp paired end protocol. Sequencing data werefiltered based on the barcodes of each pool and aligned tothe human genome reference hg19 with BWA version0.5.9-r16.11Only variants covered by 4 or more reads wereconsidered for variant calling, and reads with more than 4mismatches were disregarded. Finally, variants were anno-tated with ANNOVAR version 2011-11-28.12 Non-synony-mous variants in protein coding regions (232 in total) wereselected and analyzed by Sequenom® technology. In addi-tion, we analyzed hotspot mutations in three splicing factorgenes, SF3B1, SRSF2 and U2AF1. The median sequencing coverage in each pool was 4658and we detected 261 variations with functional effects inhaematologica 2015; 100:e223LETTERS TO THE EDITORFigure 1. Distribution of mutations and cytogenetic aberrations in 100 patient samples with myelodysplastic syndromes (MDS) and 92samples with acute myeloid leukemia (AML). Each column represents one patient. The colors represent different subgroups of the dis-eases. *Denotes patients with MDS who subsequently progressed to AML and AML with a morphologically identified preceding MDSphase. ¤Denotes patients with normal karyotype and no mutation. aberr.: aberration; MK: monosomal karyotype; TSG: tumor suppressorgene. protein coding regions of which 41 variations already werereported as germ-line polymorphisms and were excludedfrom further analysis. Of the 232 variations analyzed bySequenom®, 85 were identified as real mutations and wereincluded in further analysis. As we noticed a TP53 andASXL1 mutational frequency lower than expected inpatients with del(5q) deletions and tAML, we performedre-sequencing of TP53 in 47 patients (all 20 del(5q) MDS,18 other MDS and 9 AML samples). We discovered twoadditional TP53 mutations and three additional ASXL1mutations. In 3 of these samples, the allele burden was20% or less. Adding these mutations to the statisticalmodel did not change outcome data, which remainedbased on the whole cohort.Sixty-one percent of MDS and 50% of AML patients hadmutations in at least one gene, and 22% and 15%, respec-tively, had mutations in more than one gene. In the MDScohort, the most frequently mutated genes were SF3B1,TET2, SRSF2 and IDH2 (Figure 1), confirming the results oftwo recent large studies of MDS.9,10 In the AML cohort,TET2, SRSF2, U2AF1 and IDH2 were the most frequentlymutated genes (Figure 1 and Online Supplementary S3).SF3B1 mutations dominated in RARS/RCMD-RS, whilemutations in epigenetic regulators dominated in otherMDS subtypes. As expected, patients with 5q- syndromehad the lowest frequency of gene mutations, 21%. TET2mutations were observed more frequently in CMML thanin MDS (60% vs. 9%; P=0.001) (Figures 1 and 3A), withSRSF2 being the second most common mutation.13Within the AML cohort, mutational frequencies variedsignificantly between different subgroups, which indicatesthat these patients are more heterogeneous than previouslythought (Figure 1). MDS-AML shared features with higherrisk MDS with mutations predominantly in epigenetic andsplicing genes, and with a minor proportion of mutations insignaling genes and oncogenes. By contrast, AML withMDS-like cytogenetics showed lower frequency of splicefactor and epigenetic mutations (Figure 1). The mutationalpattern of MPN-AML differed from the other AML by ahigher incidence of signaling and oncogenic mutations.Interestingly, 87% of patients with tAML showed cytoge-netic abnormalities, while the mutational rate was relative-ly low (33%), indicating that chromosomal aberrationsconstitute the main driver of disease in these patients.However, albeit only 13% of tAML carried del(5q), anunderestimation of TP53 mutations cannot be excluded. Inorder to discover mutations associated with transformationfrom MDS to AML in MDS, we grouped MDS patientswith subsequent progression to AML with AML patientswith morphologically identified preceding MDS and com-pared them with MDS without AML progression (Figure2). The only gene that was positively associated with pro-gression was U2AF1 (3 of 11 MDS with AML progressionand 5 of 28 MDS-AML, compared to 4 of 89 MDS withouttransformation; P=0.008). No other mutation was signifi-cantly associated with transformation and our data pointtowards a distinct prognostic role of U2AF1. We also con-firm the previously described negative correlation betweenSF3B1 mutation and risk of AML progression (20 of 89MDS without transformation versus 1 of 34 with AMLtransformation; P=0.004).9,10We assessed the additive prognostic value of recurrentmutations (Figure 3). As SF3B1mutations alone were asso-ciated with a favorable prognosis both in the univariableanalysis and in previous studies,14 patients were grouped asno mutations or SF3B1 mutation only versus any mutationexcept SF3B1. Furthermore, MDS patients were groupedaccording to the Revised International Prognostic ScoreSystem (IPSS-R) in IPSS-R very low, low and intermediaterisk versus high and very high risk. Overall survival (OS)was better in patients with no or SF3B1mutations than inpatients with other mutations, and the difference was morepronounced in the low/intermediate risk group (P<0.001)(Figure 3A). Similar results were obtained when OS wasreplaced by progression-free survival (P<0.001) (Figure 3B).This strongly supports the additional prognostic value ofmutational screening in addition to IPSS-R in MDS. Acute myeloid leukemia patients were grouped based oncytogenetic risk assessment15 in low risk + intermediate risk(n=40) versus high risk (n=52) and then further divided intomutated and non-mutated patients. No AML patient hadSF3B1mutation only. As illustrated in Figure 3C, the muta-tional status did not contribute to OS in high-risk AMLpatients. However, the OS of low + intermediate riskpatients with mutations was lower than that of patientswithout mutations, indicating that gene mutations per semay influence disease pathogenesis (Figure 3C).Finally, a Cox proportional hazards model was used toanalyze OS in the MDS cohort (Online Supplementary TableS2). In univariable analysis, SF3B1 significantly correlatedwith better, and SRSF2, IDH2, U2AF1 and RUNX1 withworse survival. After adjusting for sex, age and IPSS-R, onlySF3B1 and U2AF1 remained independent prognostic fac-tors [mutated vs. non-mutated (HR 0.34, 95%CI: 0.14-0.82)and (HR 2.78, 95%CI: 1.07-7.25)]. In the correspondingmodel for AML, TP53 and NRAS showed a significant cor-relation with worse survival, even after adjusting for sex,age and cytogenetic risk, although the number of patientshaematologica 2015; 100:e224LETTERS TO THE EDITORFigure 2. Proportion of patients with acertain gene mutation that did or didnot progress from myelodysplasticsyndromes to acute myeloidleukemia. Blue bars consist of MDSwith subsequent progression to AMLand AML with an identified precedingdiagnosis of MDS (n=39). Red barsconsist of all cases from themyelodysplastic syndromes cohortwithout a known progression to acutemyeloid leukemia (n=89). Median fol-low up was 57.3 months (9.7–113,SD±27.0) for MDS and 88.5 months(27.6–190, SD ±40.5) for acutemyeloid leukemia patients. were very few entailing wide confidence intervals.In conclusion, mutational screening by targeted sequenc-ing provides important clinical information for MDS andAML and will develop to become a prognostic tool in cen-ters managing these disorders. Targeted sequencing willconstitute a cornerstone in patient management during thenext years. While the pooling approach is adequate fordetecting clonal mutations, important subclonal mutations,such as TP53 and ASXL1 may be missed by this strategy.Sequenom® methodology, or equivalent quick genotypingtests, may prove to be a rapid and cost-effective method todetect hotspot mutations.Mohsen Karimi,1 Christer Nilsson,1 Marios Dimitriou,1Monika Jansson,1 Hans Matsson,2 Per Unneberg,3Sören Lehmann,1 Juha Kere,2,4 and Eva Hellström-Lindberg11Center for Hematology and Regenerative Medicine, Department ofMedicine Huddinge, Karolinska Institutet, Sweden; 2Department ofBiosciences and Nutrition and Center for Innovative (CIMED),Karolinska Institutet, Sweden; 3Science for Life Laboratory, Departmentof Biochemistry and Biophysics, Stockholm, Sweden; 4MolecularNeurology Research Program, University of Helsinki and FolkhälsanInstitute of Genetics, Helsinki, FinlandAcknowledgments: we would like to thank SciLifeLab and MAFcore facilities at Karolinska Institutet for their generous help with high-throughput sequencing and mutation analysis services.Funding: this study was supported by research grants from theSwedish Research Council, Cancerfonden and Karolinska Institutet toEHL, SL and JK. Correspondence: Eva.Hellstrom-Lindberg@ki.sedoi:10.3324/haematol.2014.118034Key words: myelodysplastic syndromes, acute myeloid leukemia,secondary, therapy-related, high-throughput mutational screening.Information on authorship, contributions, and financial & other disclo-sures was provided by the authors and is available with the online versionof this article at www.haematologica.org.References1. Greenberg PL, Tuechler H, Schanz J, et al. Revised international prog-nostic scoring system for myelodysplastic syndromes. Blood. 2012;120(12):2454-2465.2. Garcia-Manero G, Tambaro FP, Bekele NB, et al. Phase II trial ofvorinostat with idarubicin and cytarabine for patients with newlydiagnosed acute myelogenous leukemia or myelodysplastic syn-drome. J Clin Oncol. 2012 ;30(18):2204-2210.3. Lim ZY, Ingram W, Brand R, et al. Impact of pretransplant comorbidi-ties on alemtuzumab-based reduced-intensity conditioning allogene-ic hematopoietic SCT for patients with high-risk myelodysplasticsyndrome and AML. Bone Marrow Transplant. 2010;45(4):633-639.4. Lubbert M, Muller-Tidow C, Hofmann WK, Koeffler HP. Advancesin the treatment of acute myeloid leukemia: from chromosomalaberrations to biologically targeted therapy. J Cell Biochem. 2008;104(6):2059-2070.5. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification ofTumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon,France: International Agency for Research on Cancer, 2008.6. Döhner H, Estey EH, Amadori S, et al. Diagnosis and managementof acute myeloid leukemia in adults: recommendations from an inter-national expert panel, on behalf of the European LeukemiaNet.Blood. 2010;115(3):453-474. 7. Malcovati L, Hellström-Lindberg E, Bowen D, et al. Diagnosis andtreatment of primary myelodysplastic syndromes in adults: recom-mendations from the European LeukemiaNet. Blood. 2013;122(17):2943-2964.8. R Core Team. R: A language and environment for statistical comput-ing. 3.0.3 ed. Vienna, Austria: R Foundation for StatisticalComputing, 2014.9. Haferlach T, Nagata Y, Grossmann V, et al. Landscape of geneticlesions in 944 patients with myelodysplastic syndromes. Leukemia.2014;28(2):241-247.10. Papaemmanuil E, Gerstung M, Malcovati L, et al. Clinical and biolog-ical implications of driver mutations in myelodysplastic syndromes.Blood. 2013;122(22):3616-3627; quiz 99.11. Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26(5):589-595.12. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation ofgenetic variants from high-throughput sequencing data. NucleicAcids Res. 2010;38(16):e164. 13. Meggendorfer M, Roller A, Haferlach T, et al. SRSF2 mutations in275 cases with chronic myelomonocytic leukemia (CMML). Blood.2012;120(15):3080-3088.14. Papaemmanuil E, Cazzola M, Boultwood J, et al. Somatic SF3B1mutation in myelodysplasia with ring sideroblasts. N Engl J Med.2011;365(15):1384-1395.15. Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytoge-netic classification in acute myeloid leukemia: determination of prog-nostic significance of rare recurring chromosomal abnormalitiesamong 5876 younger adult patients treated in the United KingdomMedical Research Council trials. Blood. 2010;116(3):354-365.haematologica 2015; 100:e225LETTERS TO THE EDITORFigure 3. Survival according to risk classification combined withmutational status. (A) Survival in IPSS-R very low+intermediaterisk MDS versus high+very high risk myelodysplastic syndromes(MDS) with or without the presence of any mutation other thanSF3B1. (B) Acute myeloid leukemia (AML)-free survival in IPSS-Rvery low+intermediate risk myelodysplastic syndromes (MDS) ver-sus high+very high risk myelodysplastic syndromes (MDS) with orwithout the presence of any mutation other than SF3B1. (C)Survival in low+intermediate cytogenetic risk acute myeloidleukemia (AML) versus high cytogenetic risk acute myeloidleukemia (AML) with or without the presence of any mutation.  ABC% SurvivingYearsYearsYears% Surviving% Surviving1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 10100806040200100806040200100806040200

High-throughput mutational screening adds clinically important information in myelodysplastic syndromes and secondary or therapy-related acute myeloid leukemia

http://dx.doi.org/10.3324/haematol.2014.118034

High-throughput mutational screening adds clinically important information in myelodysplastic syndromes and secondary or therapy-related acute myeloid leukemia

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