Prognostic Implications and Molecular Associations of NADH Dehydrogenase Subunit 4 (ND4) Mutations in Acute Myeloid Leukemia

Prognostic implications and molecular associations of NADH dehydrogenase subunit 4 (ND4) mutations in acute myeloid leukemia

F Damm1, T Bunke1, F Thol1, B Markus1, K Wagner1,GGo¨hring2, B Schlegelberger2, G Heil1,3, CWM Reuter1,KPu¨llmann1, RF Schlenk4,KDo¨hner4, M Heuser1, J Krauter1,HDo¨hner4, A Ganser1 and MA Morgan1

1Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; 2Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany; 3Department of Internal Medicine V, Klinikum Lu¨denscheid, Lu¨denscheid, Germany and 4Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany

To study the prevalence and prognostic importance of muta- novel somatic mutations affecting other genes, such as IDH1/ tions in NADH dehydrogenase subunit 4 (ND4), a mitochondrial IDH2 or DNMT3A, suggesting that multiple genetic aberrations encoded transmembrane component of the electron transport contribute to leukemic clone evolution and expansion.4–6 Using chain respiratory Complex I, 452 AML patients were examined for ND4 mutations by direct sequencing. The prognostic impact the same approach, mutations in the mitochondrial NADH of ND4 mutations was evaluated in the context of other clinical dehydrogenase subunit 4 (ND4) were described in 3 out of prognostic markers and genetic risk factors. In all, 29 of 452 93 AML patients.6 patients (6.4%) had either somatic (n ¼ 12) or germline (n ¼ 17) Mitochondria contribute to several key components of ND4 mutations predicted to affect translation. Somatic muta- cellular physiology, including ATP production, reactive oxygen tions were more likely to be heteroplasmic (Po0.001), to species generation, maintenance of intracellular Ca2 þ homeo- occur in predicted transmembrane domains (Po0.001) and 7,8 were predicted to have damaging effects upon translation stasis and regulation of cell death pathways. Thus, it is not (Po0.001). Patients with somatically acquired ND4 mutations surprising that mitochondrial dysfunction has been linked to had significantly longer relapse-free survival (P ¼ 0.017) and human degenerative diseases and cancers, including leuke- overall survival (OS) (P ¼ 0.021) than ND4wildtype patients. Multi- mia.9–12 Disrupted electron transport chain function due to variate analysis also demonstrated a tendency for increased mtDNA mutations can cause increased production of reactive survival in patients with somatic ND4 mutations (RFS: hazard oxygen species, which may promote transformation by generat- ratio (HR) 0.25, confidence interval (CI) 0.06–1.01, P ¼ 0.052; OS: mutated ing oncogenic mutations and/or by stimulating cell proliferation HR 0.29, CI 0.74–1.20, P ¼ 0.089). Somatic ND4 patients 8 had a higher prevalence of concomitant DNMT3A mutations through activation of mitogenic signal transduction pathways. (P ¼ 0.023) and a higher percentage of the NPM1/FLT3-ITD low- Furthermore, mtDNA mutations have been reported in bone risk genotype (P ¼ 0.021). Germline affected cases showed marrow failure syndromes such as Pearson’s syndrome,13 and in higher BAALC (P ¼ 0.036) and MLL5 (P ¼ 0.051) expression de novo and secondary AML.6,14,15 Many of these mutations levels. Further studies are warranted to validate the favorable involve mitochondrial genes encoding components of respi- prognostic influence of acquired ND4 mutations in AML. Leukemia (2012) 26, 289–295; doi:10.1038/leu.2011.200; ratory Complex I of the electron transport chain. Complex I published online 9 August 2011 is located within the mitochondrial inner membrane and is Keywords: AML; mutation; ND4; DNMT3A; prognostication the initiating step in the electron transport chain of mitochondrial oxidative phosphorylation. Mutations contributing to impaired Complex I function can result in altered NADH/ NAD þ levels and deregulated citric acid cycle activity. ND4, one of the seven Complex I subunits encoded by mtDNA, is Introduction predicted to be important for proton translocation across the membrane based on its homology to the bacterial proton- 16 Acute myeloid leukemia (AML) is a heterogeneous disease translocating NADH-quinone oxidoreductase subunit NuoM. characterized by different cytogenetic aberrations, acquired As the prevalence and prognostic importance of ND4 muta- mutations and impaired gene expression. Cytogenetic and tions in AML are unknown, we screened diagnostic samples molecular analyses provide the most important prognostic from 452 intensively treated AML patients for ND4 mutations. information at diagnosis.1,2 Recurrent mutations in several The impact of ND4 mutations on patient outcome and the genes have been described in AML, including CEBPA, FLT3, prognostic value of ND4 mutations were evaluated in the KIT, MLL, NPM1, NRAS, RUNX1 and WT1. Some of these context of other prognostic molecular markers. genetic alterations have been associated with treatment outcome in cytogenetically normal AML, and serve as a basis for 3 molecularly guided risk assessment and treatment stratification. Patients and methods Results from whole genome sequencing strategies identified Diagnostic bone marrow or peripheral blood samples were Correspondence: Dr MA Morgan, Department of Hematology, analyzed from 452 adult patients (aged 17–60 years) with Hemostasis, Oncology, and Stem Cell Transplantation, Hannover de novo (n ¼ 402) or secondary AML (AML with previous history Medical School, Carl-Neuberg Str. 1, 30625 Hannover, D-30625, of MDS (n ¼ 34); therapy-related AML (n ¼ 16)) with French– Germany. E-mail: [email protected] American–British classification M0–M2, or M4–M7, who were Received 16 April 2011; revised 28 June 2011; accepted 5 July 2011; entered into the multicenter treatment trials AMLSG 0704 published online 9 August 2011 (ClinicalTrials Identifier NCT00151242, n ¼ 105), AML SHG ND4 mutations in acute myeloid leukemia F Damm et al 290 0199 (ClinicalTrials Identifier NCT00209833, June 1999 to Statistical analysis September 2004, n ¼ 249) or AML SHG 0295 (February 1995 CR, overall survival (OS) and relapse-free survival (RFS) were to May 1999, n ¼ 98), and for whom DNA was available. defined following recommended criteria.32 Primary analysis was All patients received intensive induction and consolidation performed on OS. Sensitivity analyses were performed on CR therapy. Detailed treatment protocols have been reported and RFS, and results are displayed for exploratory purposes. previously.3,17,18 To analyze the incidence of ND4 mutations Median follow-up time for survival was calculated according to in healthy controls, peripheral blood samples from 124 healthy the method of Korn.33 OS end points, measured from the date of blood donors (age 18–60 years) were obtained from the Institute entry into one of the prospective studies, were death (failure) of Transfusion Medicine, Hannover Medical School. Written and alive at last follow-up (censored). RFS end points, measured informed consent was obtained according to the Declaration of from date of documented CR, were relapse (failure), death in CR Helsinki, and the study was approved by the institutional review (failure) and alive in CR at last follow-up (censored). Pair-wise board of Hannover Medical School. comparisons of variables for exploratory purposes were performed using the Mann–Whitney U test for continuous variables Cytogenetic and molecular analysis and two-sided Fisher’s exact tests for categorical variables. The Pretreatment samples from all patients were studied centrally Kaplan–Meier method and log-rank test were used to estimate by G- or R-banding analysis. Chromosomal abnormalities were the distribution of OS and RFS, and to compare differences described according to the International System for Human between survival curves, respectively. Mutations/polymor- Cytogenetic Nomenclature.19 Other genes were assessed for phisms in the analyzed genes were used as categorical variables. frequently occurring mutations as previously described (that is, To assess the impact of gene expression of MLL5, MN1, BAALC, FLT3-ITD,3 NPM1,3 NRAS,3 IDH1,20 IDH221 and DNMT3A22). ERG or WT1 on patient outcome, expression values were dicho- In the subgroup of cytogenetically normal AML, additional tomized (only for MLL5 quartiles were used29) at the median mutation analyses were performed for CEBPA23 and WT1.24 expression value and used as categorical variables (median BAALC,25 ERG,26 MN1,27,28 MLL529 and WT124 expression normalized copy number, gene transcripts per ABL transcripts).31 levels were quantified as previously described using cDNA from For multivariate analysis, a Cox proportional hazards model the KG1A cell line (BAALC, ERG and MLL5) or plasmids (MN130 was constructed for OS and RFS. Variables considered for model and WT124) to construct a standard curve.31 inclusion were age, WBC count, hemoglobin concentration, platelet count, sex, European LeukemiaNet (ELN) risk group Analysis of ND4 mutations (adverse vs intermediate I vs intermediate II vs favorable),34 Mononuclear cells were prepared as previously reported.20,24 de novo vs secondary AML, and ND4 mutation status. In Genomic and mitochondrial DNA were extracted from samples all multivariable models no variable selection was performed and using the All Prep DNA/RNA Kit (Qiagen, Hilden, Germany) full models were presented. To provide quantitative information on according to the manufacturer’s recommendations. Complete the relevance of results, 95% confidence intervals (CIs) of odds coverage of the mitochondrial ND4 gene sequence was ratios and hazard ratios (HR) were computed. accomplished using three overlapping PCR. Primer sequences were 1F 50-GCCAATATTGTGCCTATTGC-30 and 1R 50-TTCTT GGGCAGTGAGAGTGA-30 (680 bp amplicon); 2F 50-TGAACG Results CAGGCACATACTTC-30 and 2R 50-GGGGGTAAGGCGAGG TTAG-30 (685 bp amplicon); 3F 50-GCCTAGCAAACTCAAAC ND4 mutations were analyzed in pretreatment samples from TACGA-30 and 3R 50-GGGGCATGAGTTAGCAGTTC-30 (499 bp 452 newly diagnosed AML patients of all cytogenetic subgroups amplicon). Samples were amplified using the following PCR from multicenter treatment trials AMLSG 0704 (n ¼ 105), AML conditions: 95 1C (10 min); 35 cycles of 95 1C (1 min), 56 1C SHG 0199 (n ¼ 249) and AML SHG 0295 (n ¼ 98; Supplemen- (1 min) and 72 1C (2 min). Purified PCR fragments were directly tary Table S1). Clinical characteristics, including outcome, of sequenced using forward primers (PCR1 þ 2) or primer 50-GT these 452 patients were representative of all patients entered in CGCATCATAATCCTCTCTC-30 (PCR3) and compared with the these trials (Supplementary Material). Ensembl ND4 coding sequence (transcript: MT-ND4-201 Genetic alterations resulting in amino-acid substitutions were ENST00000361381). Nucleotide and amino-acid residue found in 34 of 452 AML samples (7.5%). The codon most numbering was done according to MITOMAP:mtDNA Somatic recurrently affected was the homoplasmic I165T substitution, Mutations (4 October 2010 updated version) (http://www. which was observed in 6 cases (1.3%). This alteration was found mitomap.org/MITOMAP/MutationsSomatic). Mutations were to be a germline event in all cases and was also detected in 2 out confirmed in independent experiments. Heteroplasmic sequence of 124 healthy controls (1.6%). Therefore, the I165T substitution alterations were defined by detection of both wild-type and most likely represents a polymorphism and was classified as mutant mtDNA, whereas in cases of homoplasmic mutations only such for all further analyses in our study. Twenty-seven patients mutant ND4 mtDNA was detected. Genetic alterations resulting had single-point mutations resulting in amino-acid substitutions, in predicted amino-acid substitutions and not listed in the SNP one patient had two separate missense point mutations and databank were classified as mutations, unless they were also another patient had a heteroplasmic one base pair deletion detected in at least one of the 124 healthy controls, in which case predicted to result in a truncated protein. The 30 detected aberrations were classified as polymorphisms. The one-nucleo- mutations occurred throughout the ND4 coding region, with 14 tide deletion in patient UPN12 was confirmed by cloning the PCR cases (47%) affecting predicted transmembrane domains amplicon using the pGEM-T vector system following the (TMDs) (Figure 1). In total, 18 of the 30 (60%) ND4 mutations manufacturer’s protocol (Promega, Madison, WI, USA). Somatic were homoplasmic point mutations. The V6I, the F50L and the or germline status of ND4 mutations was established by evalu- A404T substitutions were each observed in two patients, while ating matched buccal swabs, with follow-up samples obtained all other mutations were only detected in single cases. To define when patients were in complete remission (CR) or CD3 þ the somatic or germline origin of mutations, leukemia samples CD45brightCD34À T cells purified from diagnostic leukemia were compared with non-tumoral DNA. Constitutional DNA samples by flow cytometry. was obtained from buccal swabs (n ¼ 3), follow-up material

Leukemia ND4 mutations in acute myeloid leukemia F Damm et al 291

somatic germline

100 200 300 400 459

Figure 1 Amino acids affected by the 30 ND4 mutations detected in 29 of 452 AML patients. The 11 predicted mitochondrial transmembrane regions are depicted by crosshatches and are in accordance with data from the Protein Knowledgebase (UniProtKB) (http://www.uniprot.org/ uniprot/P03905).

Figure 2 Examples demonstrating sorting strategy and analysis of (a) a heteroplasmic somatic ND4 mutation and sequencing analysis of (b) a homoplasmic germline ND4 mutation.

while patients were in CR (n ¼ 7), or from CD3 þ T cells (Supplementary Table S1). However, ND4mutated patients tended (n ¼ 23). Three cases in which mutation origin was determined to be younger (P ¼ 0.059), with a median age of 39 vs 47 years based on examination of CR material were confirmed by CD3 þ for ND4wildtype patients. ND4 mutations did not correlate with T-cell analysis. These analyses demonstrated 12 AML patients any specific cytogenetic subgroup, but they tended to occur had somatic and 17 AML patients had germline ND4 mutations more often in patients with cytogenetic aberrations classified as (Figure 2). Detailed information on the 29 mutated AML patients ‘others’ (P ¼ 0.069). is given in Table 1. Comparison of somatic with germline ND4mutated patients To determine the incidence of ND4 alterations in the general revealed these groups to be similar with respect to age, sex, population, we used the same sequencing strategy to evaluate ECOG performance status, hemoglobin levels, WBC count, type 124 healthy blood donor samples. The I165T alteration of AML (de novo vs secondary vs therapy-related AML) or blast mentioned above was the only alteration detected in both the count (Supplementary Table S3). However, concomitant NPM1 AML cohort and the healthy controls. In addition to known mutations were detected more often in patients with acquired synonymous single-nucleotide polymorphisms (SNPs), 59 novel ND4 mutations vs germline cases (42 vs 12%, P ¼ 0.092). This SNPs were identified in the 452 AML patients and the 124 difference translated into a significantly higher prevalence in the healthy blood donors. The affected codons are listed in NPM1mut/FLT3-ITD low-risk group (33 vs 0%, P ¼ 0.021) and Supplementary Table S2. also by trend in the favorable ELN-classification group (P ¼ 0.067). Furthermore, mutations in DNMT3A were more often observed in somatic ND4mutated than in germline-affected Correlation of ND4 mutations with clinical and patients (50 vs 7%, P ¼ 0.023). In contrast, patients with biological characteristics germline ND4 mutations showed a trend for lower platelet ND4mutated and ND4wildtype patients were similar with respect counts (P ¼ 0.097) and higher expression levels of BAALC to sex, ECOG performance status, cytogenetic risk group, (P ¼ 0.036) and MLL5 (P ¼ 0.051). BAALC transcript values did hemoglobin levels, WBC count, platelet count, type of AML not differ between ND4wildtype and acquired ND4mutated patients (de novo vs secondary vs therapy-related AML) or blast count (P ¼ 0.521).

Leukemia 292 Leukemia

Table 1 ND4 mutations from the total AML cohort

UPN Nucleotide position Origin Protein Polyphen prediction Age Sex WBC Cytogenetics Other mutations Survival status change change (score rank) ( Â 109/ml)

1 T10941C (heteroplasmic) Somatic L61Pa Probably damaging (0.906) 55 M 7.16 Normal FLT3-ITD, DNMT3A Deceased 2 C11151T (heteroplasmic) Germline A131V Benign (0.009) 23 F 3.2 t(8;21)(q22;q22) None Alive ND4 G12009A (heteroplasmic) Somatic G417D Probably damaging (0.990) uain nauemeodleukemia myeloid acute in mutations 3 G11226C (heteroplasmic) Somatic G156Aa Probably damaging (0.984) 46 F 61.3 Normal NPM1, DNMT3A Alive 4 G11330C (homoplasmic) Somatic A191P Possibly damaging (0.296) 60 F 4.0 Normal NPM1, IDH1 Alive 5 T11366C (heteroplasmic) Somatic F203La Possibly damaging (0.692) 31 F 52.6 Normal FLT3-ITD, IDH1, DNMT3A Alive 6 G11484A (heteroplasmic) Somatic G242Da Probably damaging (0.996) 49 M 26.0 inv(16)(p13q22) NRAS Deceased 7 A11535G (heteroplasmic) Somatic Y259Ca Probably damaging (0.996) 38 F 25.1 Normal NPM1, DNMT3A Alive 8 T11568C (heteroplasmic) Somatic I270Ta Possibly damaging (0.834) 48 F 1.7 Normal NPM1, DNMT3A Alive 9 G11742A (heteroplasmic) Somatic C328Ya Probably damaging (0.953) 31 M 8.6 t(8;21)(q22;q22) None Alive 10 T11973C (heteroplasmic) Somatic L405Pa Probably damaging (0.952) 32 F 45.2 del(11)(q23) None Alive Damm F 11 T12090C (heteroplasmic) Somatic I444T Benign (0.028) 48 M 25.1 Normal CEBPA Alive 12 delA11032–1103840,42 Somatic Truncateda 31 M 19.1 Normal FLT3-ITD, NPM1, CEBPA Alive

(heteroplasmic) al et 13 G10775A (homoplasmic) Germline V6I Benign (0.000) 35 F 117.8 +8,inv(16)(p13q22) NRAS, KIT, FLT3-ITD Deceased 14 G10775A (homoplasmic) Germline V6I Benign (0.000) 59 M 122.2 ÀY NRAS, FLT3-ITD, NPM1 Deceased 15 A10899G (homoplasmic) Germline N47S Benign (0.000) 25 F 13.6 i(21)(q10) NRAS Deceased 16 T10907C (homoplasmic) Germline F50L Benign (0.000) 28 M 137.2 t(9;22)(q34;q11) None Deceased 17 T10907C (homoplasmic) Germline F50L Benign (0.000) 28 M 18.8 t(6;11)(q27;q23) None Deceased 18 C10920T (homoplasmic) Germline P54L Benign (0.000) 38 F 6.8 ÀX,t(8;21)(q22;q22) FLT3-ITD Alive 19 G11016A (homoplasmic) Germline S86N Benign (0.000) 39 M 3.7 +8 FLT3-ITD Alive 20 G11150A (homoplasmic) Germline A131T Benign (0.001) 31 F 140.0 del(9)(q21q33) None Alive 21 T11204C (homoplasmic) Germline F149La Possibly damaging (0.692) 44 F 34.1 Normal FLT3-ITD, WT1, IDH2 Deceased 22 A11930G (homoplasmic) Germline I391Va Benign (0.000) 44 M 40.0 Normal NRAS, CEBPA, WT1 Alive 23 A11337G (homoplasmic) Germline N193S Benign (0.000) 55 F 10.7 Normal CEBPA, DNMT3A Deceased 24 G11447A (homoplasmic) Germline V230Ma Benign (0.000) 60 M 9.5 +1,der(1;16)(q10;p10) None Deceased 25 G11969A (homoplasmic) Germline A404Ta Benign (0.000) 48 F 176.6 Normal NRAS Deceased 26 G11969A (homoplasmic) Germline A404Ta Benign (0.000) 48 M 3.8 Normal CEBPA, IDH1 Deceased 27 T11984C (homoplasmic) Germline Y409Ha Probably damaging (0.952) 33 F 170.0 Normal NRAS Alive 28 C12000T (homoplasmic) Germline T414M Probably damaging (0.993) 36 F 0.9 Normal IDH2 Deceased 29 C12063T (homoplasmic) Germline T435I Benign (0.000) 46 M 69.0 Normal FLT3-ITD, NPM1, IDH2 Deceased Abbreviations: AML, acute myeloid leukemia; ND4, NADH dehydrogenase subunit 4; UPN, unique patient number. aDenotes that mutation is within or affects a predicted transmembrane domain; ND4 mutations and nucleotide/amino-acid residue numbering were based on Ensembl transcript: MT-ND4-201 (ENST00000361381) and MITOMAP:mtDNA Somatic Mutations (October 4, 2010 updated version) (http://www.mitomap.org/MITOMAP/MutationsSomatic); ND4 mutations were determined to be somatic or germline based on cell sorting experiments (somatic when mutation was only present in leukemic blasts and absent in T-cell populations or germline when mutation was present in both leukemic blasts and T-cell populations), analysis of follow-up material obtained during CR (mutation was somatic when not detected in CR sample or germline when detected in CR sample) and/or by buccal swab analysis (mutation was called somatic when not detected in buccal swab sample or germline when detected in buccal swab sample); predictions and scores for damaging missense mutations were calculated using the PolyPhen-2 software tool (http://genetics.bwh.harvard.edu/pph2/). ND4 mutations in acute myeloid leukemia F Damm et al 293 Influence of ND4 mutations on treatment response and evidence linking altered metabolic function to leukemo- survival genesis35 led us to investigate the prevalence and prognostic The median follow-up time for patients was 4.4 years. In all, 333 value of ND4 mutations in a large cohort of AML patients ND4wildtype patients (79%) achieved a CR compared with 23 (n ¼ 452). ND4 mutations were detected with a prevalence of ND4mutated patients (79%; P ¼ 0.94). In univariate analysis, 6.4% and were associated by trend with younger age ND4mutated patients had similar 5-year RFS (50% (n ¼ 23) vs (P ¼ 0.059), but were not associated with other baseline 41% (n ¼ 333); P ¼ 0.377) and 5-year OS (38 vs 43%; P ¼ 0.506) parameters or molecular aberrations. compared with ND4wildtype patients (Figure 3). Subgroup analyses Analogously to the discovery of recurrent mutations in IDH according to the NPM1/FLT3 mutation status, cytogenetically genes, which led to renewed efforts to decipher the role of normal AML or cytogenetic risk groups34 revealed no significant altered metabolic processes in cancer,36 our results demonstrate differences for RFS or OS between ND4mutated and ND4wildtype ND4 mutations as an additional example of genetic alteration of patients (data not shown). Next, the prognostic impact of somatic essential components of the citric acid cycle. ND4 is a part of (n ¼ 12)orgermline(n ¼ 17) ND4 mutations was investigated. CR respiratory Complex I, which generates the cofactor NAD þ after induction therapy was achieved in 79% of ND4wildtype,92% required for several steps of the citric acid cycle. NAD þ also of somatic ND4mutated and 71% of germline ND4mutated patients serves as the substrate for NAD kinase to produce NADP þ .37 (P ¼ 0.392). Univariate analysis revealed that somatic ND4mutated IDH1 and IDH2 use the cofactor NADP þ , while IDH3 uses patients had significantly longer RFS and OS compared with NAD þ to convert isocitrate to a-ketoglutarate in the citric acid ND4wildtype patients (5-year RFS 81% vs 41%; P ¼ 0.025; 5-year cycle. Cells expressing mutated IDH1/2 exhibited decreased OS 83% vs 43%; P ¼ 0.029) and showed significantly longer RFS a-ketoglutarate production and increased generation of the and OS compared with patients with germline ND4 mutations oncometabolite 2-hydroxyglutarate, which elicited genome- (RFS: P ¼ 0.003; OS: P ¼ 0.002, Figure 3). Germline ND4mutated wide alteration of histone and DNA methylation patterns.38,39 patients showed similar RFS (P ¼ 0.134) and OS (P ¼ 0.225) Thus, mutations which lead to decreased Complex I activity and compared with ND4wildtype patients. subsequently decreased NAD þ generation may also result in Multivariate analysis revealed a tendency for improved altered a-ketoglutarate production and epigenetic modulation survival in patients with the somatic ND4 mutation status similar to that observed in cells containing mutated IDH1/2. (RFS: HR 0.25, 95% CI 0.06–1.01, P ¼ 0.052; OS: HR 0.29, 95% Supporting this hypothesis, others have shown that cells CI 0.74–1.20, P ¼ 0.089) when considered together with age, harboring one of the ND4 mutations we observed, the somatic ELN risk category and WBC count (Table 2). 1-base-pair deletion resulting in early translation termination (UPN12, Table 1), had markedly reduced Complex I activity.40 Furthermore, the 11 predicted TMDs of ND4 may be important Discussion for mitochondrial proton transport and 47% of the mutations we detected are within these domains (Figure 1). The S407P ND4 Recent descriptions of mutations in the mitochondrial ND4 gene mutation discovered by others in an AML sample6 is predicted in leukemia patient samples6 coupled with accumulating to reside in the same ND4 transmembrane domain as the four

Figure 3 (a) RFS and (b) OS of 452 AML patients according to ND4 mutation status (log-rank test). (c) RFS and (d) OS of 452 AML patients according to ND4 mutation status (log-rank test).

Leukemia ND4 mutations in acute myeloid leukemia F Damm et al 294 Table 2 Univariate and multivariate analysis for relapse-free and overall survival including all 452 analyzed AML patients

Endpoint Variables in the model Univariate analysis Multivariate analysis

HR 95% CI P HR 95% CI P

RFS ND4 mutation status: somatic vs all others 0.23 0.06–0.92 0.017 0.25 0.06–1.01 0.052 Age: above vs below median 1.35 1.02–1.79 0.035 1.26 0.94–1.67 0.119 ELN risk group: adverse vs int-II vs int-I vs favorable risk 1.51 1.30–1.74 o0.001 1.49 1.29–1.72 o0.001 WBC count: below vs above median 1.40 1.05–1.86 0.021 1.45 1.09–1.94 0.011 OS ND4 mutation status: somatic vs all others 0.23 0.06–0.94 0.021 0.29 0.74–1.20 0.089 Age: above vs below median 1.73 1.33–2.25 o0.001 1.64 1.25–2.13 o0.001 ELN risk group: adverse vs int-II vs int-I vs favorable risk 1.65 1.45–1.87 o0.001 1.60 1.42–1.82 o0.001 WBC count: below vs above median 1.26 0.98–1.64 0.074 1.31 1.01–1.69 0.045 Abbreviations: AML, acute myeloid leukemia; CI, conﬁdence interval; HR, hazard ratio; ND4, NADH dehydrogenase subunit 4; OS, overall survival; RFS, relapse-free survival.

additional mutations (I391V, A404T, L405P, Y409H) that we but only 3/17 (18%) germline mutations were predicted to be identified. However, ND4 mutation site (for example, inside vs damaging and to cause important functional consequences outside TMD) did not influence ND4mutated patient outcome in at the protein level (Po0.001; Table 1). To demonstrate that our study. In contrast, univariate analysis demonstrated that mutated ND4 mRNAs are expressed and not subjected to somatic ND4mutated patients enjoyed significantly longer RFS RNA-mediated decay, we sequenced cDNA from somatic and OS when compared with either germline ND4mutated and germline-mutated patients, and observed the same hetero- patients or ND4wildtype patients. Multivariate analysis also plasmic or homoplasmic status that was detected on the demonstrated a tendency for increased survival of somatic mitochondrial DNA level for all analyzed samples (data not ND4mutated patients (RFS: HR 0.25, 95% CI 0.06–1.01, shown). Additionally, we investigated samples collected while P ¼ 0.052; OS: HR 0.29, 95% CI 0.74–1.20, P ¼ 0.089). patients were in CR to analyze MRD levels, which demonstrated Our study raises the interesting point that germline or somatic loss of ND4 mutations in 6/6 patients tested who presented with mutations of the same gene can have drastically different somatic mutations. impacts on patient outcome. Although somatic ND4mutated In summary, our observations support the hypothesis of patients more often had the favorable NPM1mut/FLT3-ITDneg leukemogenesis in which cooperation between altered epige- genotype (P ¼ 0.021) and the ELN-favorable risk group status netic and metabolic pathways has an important role. Our data (P ¼ 0.067), restricting survival analysis to these subgroups still suggest that germline and somatic ND4 mutations have different demonstrated a trend for superior RFS and OS for somatic effects on leukemia cell biology, such as contribution to ND4mutated patients (Supplementary Figure S1 shows analysis for leukemia maintenance or sensitization of leukemia cells to cytogenetically normal AML). Our patients with acquired ND4 therapy. The significant impact of acquired ND4 mutations on mutations received allogeneic stem cell transplantation more patient outcome may become important for improved risk often than patients with wild-type ND4 or germline ND4 stratification and risk-guided therapy. It will be of interest to mutations (55 vs 30 vs 29%, respectively; P ¼ 0.264). Although determine which ND4 mutations affect mitochondrial function, not statistically significant, this may have contributed to the ultimately endowing leukemia cells with altered survival and/or superior survival we observed for patients with acquired ND4 proliferative capacities. mutations. Of note, patients with acquired ND4 mutations more often had concomitant DNMT3A mutations and lower MLL5 expression levels, which were described to have a negative Conflict of interest influence on patient outcome.5,22,29 Although our analysis may allow identification of DNMT3A mutated patients with favor- The authors declare no conflict of interest. able outcome, three of these patients were also classified as favorable risk and two as Intermediate-I risk using ELN criteria. The observation of unfavorable molecular markers (for example, Acknowledgements DNMT3A mutations) in patients classified as ELN-favorable risk likely reflects the fact that AML results from the cooperation of We thank all patients and physicians for provision of study several factors, and emphasizes the importance of discovering material; Kerstin Go¨rlich, Elvira Lux, Sylvia Horter, Diana how interactions of these factors contribute to AML. The obser- Dudacy, Irina Scha¨fer, Susanne Wolf and Uwe Borchert for their vation of concomitant IDH1 or IDH2 mutations in some ND4 support in sample and data acquisition; Dr Sarvari Velaga for mutated patients may be of interest, but further functional helping in the preparation of Figure 2; Dr Thomas Winkler and analysis is required in order to understand the relevance of this Dr Olivier Bernard for critically reading the manuscript. We finding. Beyond the different concomitant aberrations detected acknowledge the assistance of the Cell-Sorting-Core-Facility of the in somatic and germline ND4-mutated AML patients, we Hannover Medical School supported in part by Braukmann- observed that 9/12 (75%) somatic but only 6/17 (35%) germ- Wittenberg-Herz-Stiftung and Deutsche Forschungsgemeinschaft. line ND4 mutations occurred in predicted TMD (Po0.001). This study was supported by the Hannelore-Munke Fellowship, Additionally, 11/12 (91.6%) somatic ND4 mutations were grants HILF 2010 and no. 109686 by the Deutsche Krebshilfe heteroplasmic mutations and 16/17 (94%) germline cases were awarded to FD; grant no. M 47.1 from the HW & J Hector Stiftung; homoplasmic mutations (Po0.001). Using the PolyPhen-2 and grants no. 01GI0378 (Kompetenznetz ‘Akute und chronische (http://genetics.bwh.harvard.edu/pph2/) software tool to predict Leuka¨mien’) and 01KG0605 from the Bundesministerium fu¨r damaging missense mutations,41 10/11 (91%) somatic mutations, Bildung und Forschung.

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