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Monitoring of Residual Disease by Next-Generation Deep-Sequencing of RUNX1 Mutations Can Identify Acute Myeloid Leukemia Patients with Resistant Disease

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Monitoring of Residual Disease by Next-Generation Deep-Sequencing of RUNX1 Mutations Can Identify Acute Myeloid Leukemia Patients with Resistant Disease Leukemia (2014) 28, 129–137 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE Monitoring of residual disease by next-generation deep-sequencing of RUNX1 mutations can identify acute myeloid leukemia patients with resistant disease A Kohlmann, N Nadarajah, T Alpermann, V Grossmann, S Schindela, F Dicker, A Roller, W Kern, C Haferlach, S Schnittger and T Haferlach We studied the utility and clinical relevance of RUNX1 (runt-related transcription factor 1) mutations and their application as residual disease detection markers using next-generation deep-sequencing. Mutation screening was prospectively performed in 814 acute myeloid leukemia patients. At diagnosis, 211/814 (25.9%) patients harbored mutations with a median clone size of 39% (range: 2–96%). Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed. In 47/57 (82.5%) cases the same alterations detected at diagnosis were present at relapse, whereas in 1/57 (1.8%) cases the mutation from the diagnostic sample was no longer detectable. Discrepancies were observed in 9/57 (15.8%) cases, also including the occurrence of novel RUNX1 mutations not restricted to those regions affected at diagnosis. Moreover, in 103 patients the prognostic impact of residual levels of RUNX1 mutations during complete remission was studied. Separation of patients according to median residual mutation burden into ‘good responders’ and ‘poor responders’ (median: 3.61%; range: 0.03–48.0%) resulted in significant differences of both event-free (median 21.0 vs 5.7 months, Po0.001) and overall survival (OS; median 56.9 vs 32.0 months, P ¼ 0.002). In conclusion, deep-sequencing revealed that RUNX1 mutations qualify as patient-specific markers for individualized disease monitoring. The measurement of mutation load may refine the assignment into distinct risk categories and treatment strategies. Leukemia (2014) 28, 129–137; doi:10.1038/leu.2013.239 Keywords: RUNX1; mutation analysis; next-generation sequencing INTRODUCTION example, PML–RARA, RUNX1–RUNX1T1 and CBFB–MYH11,isan 9 Alterations of RUNX1 (runt-related transcription factor 1) constitute established concept. In addition, also some molecular mutations, disease-defining aberrations in acute myeloid leukemia (AML).1 for example, NPM1 mutations, have been shown to be useful 10 Mechanistically, deregulations occur either through balanced targets for quantitative PCR-based MRD detection. However, the translocations, deletions, amplifications or molecular mutations. number of recurrently altered genes, which are potential targets 4,5,11,12 In particular, RUNX1 mutations were proposed as clinically useful for MRD assessment, as illustrated by mutations in RUNX1, 13,14 15–17 18,19 biomarkers to follow disease progression from myelodysplastic TP53, TET2, or ASXL1, is constantly increasing. syndromes to secondary AML,2 as well as to monitor minimal Moreover, it is expected that translating the wealth of know- residual disease (MRD).3 Moreover, RUNX1 mutations were ledge that is so rapidly being generated by whole-genome and demonstrated to be frequent in de novo AML with non-complex whole-exome sequencing studies into actionable gene panels will 20–22 karyotype and conferred an unfavorable prognosis,4 explained by have a profound impact on the molecular landscape in AML. an association with resistance to chemotherapy.5 This can already be exemplified by pivotal discoveries such as 23 24,25 26 The importance of detecting small subclones in hematological IDH1, DNMT3A, BCOR or cohesin complex (STAG2, SMC3, 27 malignancies has increased. This has become highly relevant RAD21 and SMC1A) gene mutations. As such, there is a great either in a setting of assessing MRD or identifying molecular unmet need for unbiased standardized objective methodologies mutations with prognostic or predictive relevance to direct to provide information on molecular alterations not only per se but treatment strategies.6 Indeed, applications of MRD monitoring also at a level of sensitivity and throughput necessary for have been proposed to include early assessment of response to diagnostics processes.28 therapy to improve risk stratification and to guide post-remission In principle, it seems that RUNX1 mutations qualify as a marker therapy. Further, post-treatment monitoring would be suited to for MRD detection, as RUNX1 is frequently mutated in AML. Overall detect impending relapse and guide pre-emptive therapy frequencies of RUNX1 mutations were reported to range from strategies.7 Particularly in AML, risk-adapted strategies can be 5.6 to 32.7% of AML patients, depending on the age group of established based on MRD data on an individual basis for each the patients and chromosomal aberrations.4,5,11,29 Therefore, here patient.8 we investigated for the first time whether amplicon Today, PCR techniques and multiparameter flow cytometry deep-sequencing has the potential to become such a have become standard methods to investigate MRD in AML.8 In standardized routine diagnostic methodology, enabling particular, the monitoring of MRD kinetics using quantitative PCR unprecedented levels of sequencing sensitivity and allowing an assays for the detection of leukemia-specific fusion genes, for individualized monitoring of disease progression and treatment MLL Munich Leukemia Laboratory, Munich, Germany. Correspondence: Dr A Kohlmann, MLL Munich Leukemia Laboratory, Max-Lebsche-Platz 31, 81377 Munich, Germany. E-mail: [email protected] Received 31 July 2013; accepted 12 August 2013; accepted article preview online 20 August 2013; advance online publication, 17 September 2013 Residual disease in AML using NGS A Kohlmann et al 130 efficacy. However, in contrast to other candidate genes involved in Data analysis and detection of variants (ref. 10) (ref. 23) myeloid malignancies, such as NPM1 or IDH1 ,no Image and data processings were performed using default amplicon mutational hotspots are present in RUNX1. As such, offering pipeline settings of the GS FLX Sequencer Instrument software version 2.3 patient-specific quantitative real-time PCR assays to detect the or higher (Roche Applied Science). Subsequent sequence alignment and broad spectrum of mutations would require the need to apply variant detection was performed using default settings of the GS Amplicon individualized assays, which is not feasible on a per-patient basis Variant Analyzer software version 2.5.3 (Roche Applied Science). The results during routine diagnostics procedures. were further processed and visualized in R/Bioconductor using the package R453Plus1Toolbox (version 1.0.1) and the Sequence Pilot software Thus far, next-generation sequencing (NGS) has demonstrated version 3.4.0 (JSI Medical Systems, Kippenheim, Germany).34 For the its potential as a diagnostic platform and amplicon deep- detection of variants, filters were set to display sequence variants occurring sequencing assays have been envisioned to be applied in 41% of bidirectional reads per amplicon in at least one patient. The cut- 28,30–32 clinically. In the scenario of RUNX1 mutation analyses, off was set after sequencing a control cohort of 10 healthy individuals as seven amplicons are sufficient to test the complete coding region previously reported.35 Depending on the actual type of the mutation, that and to offer unprecedented levels of sensitivity in sequencing of is, single-nucleotide changes versus more complicated variants, higher 35 genomic alterations.33 Here we studied the use of amplicon deep- levels of sensitivity can be reached. Single-nucleotide polymorphisms sequencing in a routine diagnostics environment to characterize according to dbSNP build 131 or higher and alterations within introns with the spectrum of RUNX1 mutations in 814 cases with AML and to the exception of splice-site mutations, which were both validated on cDNA level for their effect on splicing, were not scored.36 assess the stability of RUNX1 mutations in 57 paired samples from An additional functional interpretation of the variants detected in RUNX1 diagnosis and relapse. Moreover, this deep-sequencing assay was performed using MutationTaster (www.mutationtaster.org),37 was applied to monitor the composition and mutation load PolyPhen (http://genetics.bwh.harvard.edu/pph2/index.shtml)38 and SIFT kinetics of RUNX1 mutations, following in detail 103 patients (http://sift.jcvi.org/) algorithms.39 during their course of disease, and the prognostic impact is presented allowing to identify AML patients responding poorly to Serial NGS analyses using deep-sequencing chemotherapy. For longitudinal studies, two additional cohorts were investigated. The overlap of cases from these cohorts with the cohort of 814 prospectively collected cases is shown in Supplementary Figure S1. A first cohort included 57 known RUNX1-mutated AML cases of whom MATERIALS AND METHODS samples were also available for a serial deep-sequencing study at relapse Patient cohorts stage. This cohort included 114 specimens in total, collected from We here report on prospective deep-sequencing molecular analyses in 814 November 2005 to May 2012. The median sampling interval from untreated AML cases at diagnosis, including 645 de novo AML, 109 diagnosis to relapse for these cases was 360 days (range: 56–1578 days). secondary AML and 60 therapy-related AML cases. The median age of the Of note, nine cases had previously been reported.4 patients was 69.6 years (range: 0.4–93.1 years), including 375 female and In a second independent cohort of 103
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