To study the prevalence and prognostic importance of mutations in NADH dehydrogenase subunit 4 (ND4), a mitochondrial encoded transmembrane component of the electron transport chain respiratory Complex I, 452 AML patients were examined for ND4 mutations by direct sequencing. The prognostic impact of ND4 mutations was evaluated in the context of other clinical prognostic markers and genetic risk factors. In all, 29 of 452 patients (6.4%) had either somatic (n=12) or germline (n=17) ND4 mutations predicted to affect translation. Somatic mutations were more likely to be heteroplasmic (P<0.001), to occur in predicted transmembrane domains (P<0.001) and were predicted to have damaging effects upon translation (P<0.001). Patients with somatically acquired ND4 mutations had significantly longer relapse-free survival (P=0.017) and overall survival (OS) (P=0.021) than ND4wildtype patients. Multivariate analysis also demonstrated a tendency for increased survival in patients with somatic ND4 mutations (RFS: hazard ratio (HR) 0.25, confidence interval (CI) 0.06–1.01, P=0.052; OS: HR 0.29, CI 0.74–1.20, P=0.089). Somatic ND4mutated patients had a higher prevalence of concomitant DNMT3A mutations (P=0.023) and a higher percentage of the NPM1/FLT3-ITD low-risk genotype (P=0.021). Germline affected cases showed higher BAALC (P=0.036) and MLL5 (P=0.051) expression levels. Further studies are warranted to validate the favorable prognostic influence of acquired ND4 mutations in AML.
Acute myeloid leukemia (AML) is a heterogeneous disease characterized by different cytogenetic aberrations, acquired mutations and impaired gene expression. Cytogenetic and molecular analyses provide the most important prognostic information at diagnosis.1, 2 Recurrent mutations in several genes have been described in AML, including CEBPA, FLT3, KIT, MLL, NPM1, NRAS, RUNX1 and WT1. Some of these genetic alterations have been associated with treatment outcome in cytogenetically normal AML, and serve as a basis for molecularly guided risk assessment and treatment stratification.3 Results from whole genome sequencing strategies identified novel somatic mutations affecting other genes, such as IDH1/IDH2 or DNMT3A, suggesting that multiple genetic aberrations contribute to leukemic clone evolution and expansion.4, 5, 6 Using the same approach, mutations in the mitochondrial NADH dehydrogenase subunit 4 (ND4) were described in 3 out of 93 AML patients.6
Mitochondria contribute to several key components of cellular physiology, including ATP production, reactive oxygen species generation, maintenance of intracellular Ca2+ homeostasis and regulation of cell death pathways.7, 8 Thus, it is not surprising that mitochondrial dysfunction has been linked to human degenerative diseases and cancers, including leukemia.9, 10, 11, 12 Disrupted electron transport chain function due to mtDNA mutations can cause increased production of reactive oxygen species, which may promote transformation by generating oncogenic mutations and/or by stimulating cell proliferation through activation of mitogenic signal transduction pathways.8 Furthermore, mtDNA mutations have been reported in bone marrow failure syndromes such as Pearson's syndrome,13 and in de novo and secondary AML.6, 14, 15 Many of these mutations involve mitochondrial genes encoding components of respiratory Complex I of the electron transport chain. Complex I is located within the mitochondrial inner membrane and is 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+ levels and deregulated citric acid cycle activity. ND4, one of the seven Complex I subunits encoded by mtDNA, is predicted to be important for proton translocation across the membrane based on its homology to the bacterial proton-translocating NADH-quinone oxidoreductase subunit NuoM.16
As the prevalence and prognostic importance of ND4 mutations in AML are unknown, we screened diagnostic samples from 452 intensively treated AML patients for ND4 mutations. The impact of ND4 mutations on patient outcome and the prognostic value of ND4 mutations were evaluated in the context of other prognostic molecular markers.
Patients and methods
Diagnostic bone marrow or peripheral blood samples were analyzed from 452 adult patients (aged 17–60 years) with de novo (n=402) or secondary AML (AML with previous history of MDS (n=34); therapy-related AML (n=16)) with French–American–British classification M0–M2, or M4–M7, who were entered into the multicenter treatment trials AMLSG 0704 (ClinicalTrials Identifier NCT00151242, n=105), AML SHG 0199 (ClinicalTrials Identifier NCT00209833, June 1999 to September 2004, n=249) or AML SHG 0295 (February 1995 to May 1999, n=98), and for whom DNA was available. All patients received intensive induction and consolidation therapy. Detailed treatment protocols have been reported previously.3, 17, 18 To analyze the incidence of ND4 mutations in healthy controls, peripheral blood samples from 124 healthy blood donors (age 18–60 years) were obtained from the Institute of Transfusion Medicine, Hannover Medical School. Written informed consent was obtained according to the Declaration of Helsinki, and the study was approved by the institutional review board of Hannover Medical School.
Cytogenetic and molecular analysis
Pretreatment samples from all patients were studied centrally by G- or R-banding analysis. Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature.19 Other genes were assessed for frequently occurring mutations as previously described (that is, FLT3-ITD,3 NPM1,3 NRAS,3 IDH1,20 IDH221 and DNMT3A22). In the subgroup of cytogenetically normal AML, additional mutation analyses were performed for CEBPA23 and WT1.24 BAALC,25 ERG,26 MN1,27, 28 MLL529 and WT124 expression levels were quantified as previously described using cDNA from the KG1A cell line (BAALC, ERG and MLL5) or plasmids (MN130 and WT124) to construct a standard curve.31
Analysis of ND4 mutations
Mononuclear cells were prepared as previously reported.20, 24 Genomic and mitochondrial DNA were extracted from samples using the All Prep DNA/RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Complete coverage of the mitochondrial ND4 gene sequence was accomplished using three overlapping PCR. Primer sequences were 1F 5′-IndexTermGCCAATATTGTGCCTATTGC-3′ and 1R 5′-IndexTermTTCTTGGGCAGTGAGAGTGA-3′ (680 bp amplicon); 2F 5′-IndexTermTGAACGCAGGCACATACTTC-3′ and 2R 5′-IndexTermGGGGGTAAGGCGAGGTTAG-3′ (685 bp amplicon); 3F 5′-IndexTermGCCTAGCAAACTCAAACTACGA-3′ and 3R 5′-IndexTermGGGGCATGAGTTAGCAGTTC-3′ (499 bp amplicon). Samples were amplified using the following PCR conditions: 95 °C (10 min); 35 cycles of 95 °C (1 min), 56 °C (1 min) and 72 °C (2 min). Purified PCR fragments were directly sequenced using forward primers (PCR1+2) or primer 5′-IndexTermGTCGCATCATAATCCTCTCTC-3′ (PCR3) and compared with the Ensembl ND4 coding sequence (transcript: MT-ND4-201 ENST00000361381). Nucleotide and amino-acid residue numbering was done according to MITOMAP:mtDNA Somatic Mutations (4 October 2010 updated version) (http://www.mitomap.org/MITOMAP/MutationsSomatic). Mutations were confirmed in independent experiments. Heteroplasmic sequence alterations were defined by detection of both wild-type and mutant mtDNA, whereas in cases of homoplasmic mutations only mutant ND4 mtDNA was detected. Genetic alterations resulting in predicted amino-acid substitutions and not listed in the SNP databank were classified as mutations, unless they were also detected in at least one of the 124 healthy controls, in which case aberrations were classified as polymorphisms. The one-nucleotide deletion in patient UPN12 was confirmed by cloning the PCR amplicon using the pGEM-T vector system following the manufacturer's protocol (Promega, Madison, WI, USA). Somatic or germline status of ND4 mutations was established by evaluating matched buccal swabs, with follow-up samples obtained when patients were in complete remission (CR) or CD3+CD45brightCD34− T cells purified from diagnostic leukemia samples by flow cytometry.
CR, overall survival (OS) and relapse-free survival (RFS) were defined following recommended criteria.32 Primary analysis was performed on OS. Sensitivity analyses were performed on CR and RFS, and results are displayed for exploratory purposes. Median follow-up time for survival was calculated according to the method of Korn.33 OS end points, measured from the date of entry into one of the prospective studies, were death (failure) and alive at last follow-up (censored). RFS end points, measured from date of documented CR, were relapse (failure), death in CR (failure) and alive in CR at last follow-up (censored). Pair-wise comparisons of variables for exploratory purposes were performed using the Mann–Whitney U test for continuous variables and two-sided Fisher's exact tests for categorical variables. The Kaplan–Meier method and log-rank test were used to estimate the distribution of OS and RFS, and to compare differences between survival curves, respectively. Mutations/polymorphisms in the analyzed genes were used as categorical variables. To assess the impact of gene expression of MLL5, MN1, BAALC, ERG or WT1 on patient outcome, expression values were dichotomized (only for MLL5 quartiles were used29) at the median expression value and used as categorical variables (median normalized copy number, gene transcripts per ABL transcripts).31
For multivariate analysis, a Cox proportional hazards model was constructed for OS and RFS. Variables considered for model inclusion were age, WBC count, hemoglobin concentration, platelet count, sex, European LeukemiaNet (ELN) risk group (adverse vs intermediate I vs intermediate II vs favorable),34 de novo vs secondary AML, and ND4 mutation status. In all multivariable models no variable selection was performed and full models were presented. To provide quantitative information on the relevance of results, 95% confidence intervals (CIs) of odds ratios and hazard ratios (HR) were computed.
ND4 mutations were analyzed in pretreatment samples from 452 newly diagnosed AML patients of all cytogenetic subgroups from multicenter treatment trials AMLSG 0704 (n=105), AML SHG 0199 (n=249) and AML SHG 0295 (n=98; Supplementary Table S1). Clinical characteristics, including outcome, of these 452 patients were representative of all patients entered in these trials (Supplementary Material).
Genetic alterations resulting in amino-acid substitutions were found in 34 of 452 AML samples (7.5%). The codon most recurrently affected was the homoplasmic I165T substitution, which was observed in 6 cases (1.3%). This alteration was found to be a germline event in all cases and was also detected in 2 out of 124 healthy controls (1.6%). Therefore, the I165T substitution most likely represents a polymorphism and was classified as such for all further analyses in our study. Twenty-seven patients had single-point mutations resulting in amino-acid substitutions, one patient had two separate missense point mutations and another patient had a heteroplasmic one base pair deletion predicted to result in a truncated protein. The 30 detected mutations occurred throughout the ND4 coding region, with 14 cases (47%) affecting predicted transmembrane domains (TMDs) (Figure 1). In total, 18 of the 30 (60%) ND4 mutations were homoplasmic point mutations. The V6I, the F50L and the A404T substitutions were each observed in two patients, while all other mutations were only detected in single cases. To define the somatic or germline origin of mutations, leukemia samples were compared with non-tumoral DNA. Constitutional DNA was obtained from buccal swabs (n=3), follow-up material while patients were in CR (n=7), or from CD3+ T cells (n=23). Three cases in which mutation origin was determined based on examination of CR material were confirmed by CD3+ T-cell analysis. These analyses demonstrated 12 AML patients had somatic and 17 AML patients had germline ND4 mutations (Figure 2). Detailed information on the 29 mutated AML patients is given in Table 1.
To determine the incidence of ND4 alterations in the general population, we used the same sequencing strategy to evaluate 124 healthy blood donor samples. The I165T alteration mentioned above was the only alteration detected in both the AML cohort and the healthy controls. In addition to known synonymous single-nucleotide polymorphisms (SNPs), 59 novel SNPs were identified in the 452 AML patients and the 124 healthy blood donors. The affected codons are listed in Supplementary Table S2.
Correlation of ND4 mutations with clinical and biological characteristics
ND4mutated and ND4wildtype patients were similar with respect to sex, ECOG performance status, cytogenetic risk group, hemoglobin levels, WBC count, platelet count, type of AML (de novo vs secondary vs therapy-related AML) or blast count (Supplementary Table S1). However, ND4mutated patients tended to be younger (P=0.059), with a median age of 39 vs 47 years for ND4wildtype patients. ND4 mutations did not correlate with any specific cytogenetic subgroup, but they tended to occur more often in patients with cytogenetic aberrations classified as ‘others’ (P=0.069).
Comparison of somatic with germline ND4mutated patients revealed these groups to be similar with respect to age, sex, ECOG performance status, hemoglobin levels, WBC count, type of AML (de novo vs secondary vs therapy-related AML) or blast count (Supplementary Table S3). However, concomitant NPM1 mutations were detected more often in patients with acquired ND4 mutations vs germline cases (42 vs 12%, P=0.092). This difference translated into a significantly higher prevalence in the NPM1mut/FLT3-ITD low-risk group (33 vs 0%, P=0.021) and also by trend in the favorable ELN-classification group (P=0.067). Furthermore, mutations in DNMT3A were more often observed in somatic ND4mutated than in germline-affected patients (50 vs 7%, P=0.023). In contrast, patients with germline ND4 mutations showed a trend for lower platelet counts (P=0.097) and higher expression levels of BAALC (P=0.036) and MLL5 (P=0.051). BAALC transcript values did not differ between ND4wildtype and acquired ND4mutated patients (P=0.521).
Influence of ND4 mutations on treatment response and survival
The median follow-up time for patients was 4.4 years. In all, 333 ND4wildtype patients (79%) achieved a CR compared with 23 ND4mutated patients (79%; P=0.94). In univariate analysis, ND4mutated patients had similar 5-year RFS (50% (n=23) vs 41% (n=333); P=0.377) and 5-year OS (38 vs 43%; P=0.506) compared with ND4wildtype patients (Figure 3). Subgroup analyses according to the NPM1/FLT3 mutation status, cytogenetically normal AML or cytogenetic risk groups34 revealed no significant differences for RFS or OS between ND4mutated and ND4wildtype patients (data not shown). Next, the prognostic impact of somatic (n=12) or germline (n=17) ND4 mutations was investigated. CR after induction therapy was achieved in 79% of ND4wildtype, 92% of somatic ND4mutated and 71% of germline ND4mutated patients (P=0.392). Univariate analysis revealed that somatic ND4mutated patients had significantly longer RFS and OS compared with ND4wildtype patients (5-year RFS 81% vs 41%; P=0.025; 5-year OS 83% vs 43%; P=0.029) and showed significantly longer RFS and OS compared with patients with germline ND4 mutations (RFS: P=0.003; OS: P=0.002, Figure 3). Germline ND4mutated patients showed similar RFS (P=0.134) and OS (P=0.225) compared with ND4wildtype patients.
Multivariate analysis revealed a tendency for improved survival in patients with the somatic ND4 mutation status (RFS: HR 0.25, 95% CI 0.06–1.01, P=0.052; OS: HR 0.29, 95% CI 0.74–1.20, P=0.089) when considered together with age, ELN risk category and WBC count (Table 2).
Recent descriptions of mutations in the mitochondrial ND4 gene in leukemia patient samples6 coupled with accumulating evidence linking altered metabolic function to leukemogenesis35 led us to investigate the prevalence and prognostic value of ND4 mutations in a large cohort of AML patients (n=452). ND4 mutations were detected with a prevalence of 6.4% and were associated by trend with younger age (P=0.059), but were not associated with other baseline parameters or molecular aberrations.
Analogously to the discovery of recurrent mutations in IDH genes, which led to renewed efforts to decipher the role of altered metabolic processes in cancer,36 our results demonstrate ND4 mutations as an additional example of genetic alteration of essential components of the citric acid cycle. ND4 is a part of respiratory Complex I, which generates the cofactor NAD+ required for several steps of the citric acid cycle. NAD+ also serves as the substrate for NAD kinase to produce NADP+.37 IDH1 and IDH2 use the cofactor NADP+, while IDH3 uses NAD+ to convert isocitrate to α-ketoglutarate in the citric acid cycle. Cells expressing mutated IDH1/2 exhibited decreased α-ketoglutarate production and increased generation of the oncometabolite 2-hydroxyglutarate, which elicited genome-wide alteration of histone and DNA methylation patterns.38, 39 Thus, mutations which lead to decreased Complex I activity and subsequently decreased NAD+ generation may also result in altered α-ketoglutarate production and epigenetic modulation similar to that observed in cells containing mutated IDH1/2. Supporting this hypothesis, others have shown that cells harboring one of the ND4 mutations we observed, the somatic 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 for mitochondrial proton transport and 47% of the mutations we detected are within these domains (Figure 1). The S407P ND4 mutation discovered by others in an AML sample6 is predicted to reside in the same ND4 transmembrane domain as the four additional mutations (I391V, A404T, L405P, Y409H) that we identified. However, ND4 mutation site (for example, inside vs outside TMD) did not influence ND4mutated patient outcome in our study. In contrast, univariate analysis demonstrated that somatic ND4mutated patients enjoyed significantly longer RFS and OS when compared with either germline ND4mutated patients or ND4wildtype patients. Multivariate analysis also demonstrated a tendency for increased survival of somatic ND4mutated patients (RFS: HR 0.25, 95% CI 0.06–1.01, P=0.052; OS: HR 0.29, 95% CI 0.74–1.20, P=0.089).
Our study raises the interesting point that germline or somatic mutations of the same gene can have drastically different impacts on patient outcome. Although somatic ND4mutated patients more often had the favorable NPM1mut/FLT3-ITDneg genotype (P=0.021) and the ELN-favorable risk group status (P=0.067), restricting survival analysis to these subgroups still demonstrated a trend for superior RFS and OS for somatic ND4mutated patients (Supplementary Figure S1 shows analysis for cytogenetically normal AML). Our patients with acquired ND4 mutations received allogeneic stem cell transplantation more often than patients with wild-type ND4 or germline ND4 mutations (55 vs 30 vs 29%, respectively; P=0.264). Although not statistically significant, this may have contributed to the superior survival we observed for patients with acquired ND4 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 influence on patient outcome.5, 22, 29 Although our analysis may allow identification of DNMT3A mutated patients with favorable 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, DNMT3A mutations) in patients classified as ELN-favorable risk likely reflects the fact that AML results from the cooperation of several factors, and emphasizes the importance of discovering how interactions of these factors contribute to AML. The observation of concomitant IDH1 or IDH2 mutations in some ND4 mutated patients may be of interest, but further functional analysis is required in order to understand the relevance of this finding. Beyond the different concomitant aberrations detected in somatic and germline ND4-mutated AML patients, we observed that 9/12 (75%) somatic but only 6/17 (35%) germline ND4 mutations occurred in predicted TMD (P<0.001). Additionally, 11/12 (91.6%) somatic ND4 mutations were heteroplasmic mutations and 16/17 (94%) germline cases were homoplasmic mutations (P<0.001). Using the PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) software tool to predict damaging missense mutations,41 10/11 (91%) somatic mutations, but only 3/17 (18%) germline mutations were predicted to be damaging and to cause important functional consequences at the protein level (P<0.001; Table 1). To demonstrate that mutated ND4 mRNAs are expressed and not subjected to RNA-mediated decay, we sequenced cDNA from somatic and germline-mutated patients, and observed the same heteroplasmic or homoplasmic status that was detected on the mitochondrial DNA level for all analyzed samples (data not shown). Additionally, we investigated samples collected while patients were in CR to analyze MRD levels, which demonstrated loss of ND4 mutations in 6/6 patients tested who presented with somatic mutations.
In summary, our observations support the hypothesis of leukemogenesis in which cooperation between altered epigenetic and metabolic pathways has an important role. Our data suggest that germline and somatic ND4 mutations have different effects on leukemia cell biology, such as contribution to leukemia maintenance or sensitization of leukemia cells to therapy. The significant impact of acquired ND4 mutations on patient outcome may become important for improved risk stratification and risk-guided therapy. It will be of interest to determine which ND4 mutations affect mitochondrial function, ultimately endowing leukemia cells with altered survival and/or proliferative capacities.
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We thank all patients and physicians for provision of study material; Kerstin Görlich, Elvira Lux, Sylvia Horter, Diana Dudacy, Irina Schäfer, Susanne Wolf and Uwe Borchert for their support in sample and data acquisition; Dr Sarvari Velaga for helping in the preparation of Figure 2; Dr Thomas Winkler and Dr Olivier Bernard for critically reading the manuscript. We acknowledge the assistance of the Cell-Sorting-Core-Facility of the Hannover Medical School supported in part by Braukmann-Wittenberg-Herz-Stiftung and Deutsche Forschungsgemeinschaft. This study was supported by the Hannelore-Munke Fellowship, grants HILF 2010 and no. 109686 by the Deutsche Krebshilfe awarded to FD; grant no. M 47.1 from the HW & J Hector Stiftung; and grants no. 01GI0378 (Kompetenznetz ‘Akute und chronische Leukämien’) and 01KG0605 from the Bundesministerium für Bildung und Forschung.
The authors declare no conflict of interest.
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Damm, F., Bunke, T., Thol, F. et al. Prognostic implications and molecular associations of NADH dehydrogenase subunit 4 (ND4) mutations in acute myeloid leukemia. Leukemia 26, 289–295 (2012) doi:10.1038/leu.2011.200
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