Decreased survival in normal karyotype AML
with single-nucleotide polymorphisms in genes
encoding the AraC metabolizing enzymes
cytidine deaminase and 5'-nucleotidase
Ingrid Jakobsen Falk, Anna Fyrberg, Esbjörn Paul, Hareth Nahi, Monica Hermanson, Richard Rosenquist, Martin Höglund, Lars Palmqvist, Dick Stockelberg, Yuan Wei,
Henrik Gréen and Kourosh Lotfi
Linköping University Post Print
N.B.: When citing this work, cite the original article.
Original Publication:
Ingrid Jakobsen Falk, Anna Fyrberg, Esbjörn Paul, Hareth Nahi, Monica Hermanson, Richard Rosenquist, Martin Höglund, Lars Palmqvist, Dick Stockelberg, Yuan Wei, Henrik Gréen and Kourosh Lotfi, Decreased survival in normal karyotype AML with single-nucleotide polymorphisms in genes encoding the AraC metabolizing enzymes cytidine deaminase and 5'-nucleotidase, 2013, American Journal of Hematology, (88), 12, 1001-1006.
http://dx.doi.org/10.1002/ajh.23549 Copyright: Wiley
http://eu.wiley.com/WileyCDA/
Postprint available at: Linköping University Electronic Press http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-98699
Decreased Survival in Normal Karyotype AML with Single Nucleotide
Polymorphisms in Genes Encoding the AraC Metabolizing Enzymes
Cytidine Deaminase and 5ˈ-Nucleotidase
Running title: Influence of CDA, cN-II, and DCK SNPs in AML
Ingrid Jakobsen Falk, M.Sc 1*, Anna Fyrberg, Ph.D 2*, Esbjörn Paul, M.D 3, Hareth Nahi, M.D,
Ph.D 3, Monica Hermanson, Ph.D 4, Richard Rosenquist, M.D, Ph.D 4, Martin Höglund, M.D,
Ph.D5, Lars Palmqvist, M.D, Ph.D 6, Dick Stockelberg, M.D, Ph.D 7, Yuan Wei, M.D, Ph.D 7,
Henrik Gréen, Ph.D 1, 8, 9**, and Kourosh Lotfi, MD, Ph.D 1, 10**
1Clinical Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping
University, Linköping, Sweden; 2Centre for Biomedical Resources, Faculty of Health Sciences, Linköping
University, Linköping Sweden; 3Division of Hematology, Department of Medicine, Karolinska Institutet,
Huddinge, Stockholm, Sweden; 4Department of Immunology, Genetics and Pathology, Rudbeck Laboratory,
Uppsala University, Uppsala, Sweden; 5Division of Hematology, Department of Medical Sciences, Uppsala
University, Uppsala, Sweden; 6Department of Clinical Chemistry and Transfusion Medicine, Institute of
Biomedicine, University of Gothenburg, Gothenburg, Sweden; 7Section for hematology and coagulation,
Department of internal medicine, Sahlgrenska University Hospital, Gothenburg, Sweden; 8Science for Life
Laboratory, KTH Royal Institute of Technology, School of Biotechnology, Solna, Sweden; 9Department of
Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden;
10Department of Hematology, Linköping University Hospital, Linköping, Sweden. *IJF and AF share first authorship
**HG and KL share last authorship.
Key words: AML, cytidine deaminase, single nucleotide polymorphism, cytarabine
Corresponding author: Ingrid Jakobsen Falk, Clinical Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Linköping, Sweden. Phone: +46 10 1032029; Fax: +46 13 10 41 95. E-mail: ingrid.jakobsen.falk@liu.se
Word counts: Abstract: 227 words. Manuscript text (excluding references): 3813.
Abstract
Purpose: De novo acute myeloid leukemia with normal karyotype (NK-AML) comprises a large group of patients with no common cytogenetic alterations and with a large variation in treatment response. Single nucleotide polymorphisms (SNPs) in genes related to the metabolism of the nucleoside analogue AraC, the backbone in AML treatment, might affect drug sensitivity and treatment outcome. Therefore, SNPs may serve as prognostic biomarkers aiding clinicians in individualized treatment decisions, with the aim of improving patient outcomes. Experimental design: We analyzed polymorphisms in genes encoding cytidine deaminase (CDA 79A>C rs2072671 and -451C>T rs532545), 5´-nucleotidase (cN-II 7A>G rs10883841), and deoxycytidine kinase (DCK 3´UTR 948T>C rs4643786) in 205 de novo NK-AML patients. Results: In FLT3-ITD-positive patients, the CDA 79C/C and -451T/T genotypes were associated with shorter overall survival compared to other genotypes (5 vs. 24 months, p <0.001, and 5 vs. 23 months, p = 0.015, respectively) and this was most pronounced in FLT3-ITD-positive/NPM1-positive patients. We observed altered in vitro sensitivity to topoisomerase inhibitory drugs, but not to nucleoside analogues, and a decrease in global DNA methylation in cells carrying both CDA variant alleles. A shorter survival was also observed for the cN-II variant allele, but only in FLT3-ITD-negative patients (25 vs. 31 months, p = 0.075). Conclusion: Our results indicate that polymorphisms in genes related to nucleoside analog drug metabolism may serve as prognostic markers in de novo NK-AML.
Introduction
Acute myeloid leukemia (AML) is characterized by failed differentiation and uncontrolled proliferation of hematopoietic myeloid progenitor cells. With modern treatments, the majority of patients with AML obtain a complete remission (CR), but more than two-thirds of adult AML patients relapse and the 5-year survival rate is only 30–40%; less than 15% in patients above the age of 60 [1]. The most common treatment for AML currently in use is a combination of the nucleoside analog cytosine arabinoside (AraC) and an anthracycline such as daunorubicin or idarubicin, followed by consolidation chemotherapy and/or stem cell transplantation.
Cytogenetic characterization of AML at diagnosis is crucial for treatment strategies and prognosis. Based on cytogenetic aberrations, patients can be stratified into high, intermediate, and low-risk patient groups [2, 3]. However, almost half of the AML patients do not show any cytogenetic aberrations and are referred to as normal karyotype AML (NK-AML) [2]. Patients (<70 years) in this group have a 5-year overall survival (OS) of 35–40%, but this intermediate risk group is very heterogeneous with respect to treatment response and risk of relapse [4].
A number of mutations are known to predict response and outcome in the NK-AML group [5, 6]. The fms-like tyrosine kinase 3 gene (FLT3) is mutated and constitutively active in about 30% of the AML patients, the majority of these mutations being internal tandem duplications (ITD) in the coding region for the juxtamembrane loop of this enzyme [7, 8]. The FLT3-ITD mutation in NK-AML is associated with higher leukocyte and blast counts, increased relapse rates and decreased OS [9, 10].
On the other hand, mutation in the nucleolar protein nucleophosmin, encoded by the NPM1 gene, is a favorable prognostic factor for achieving CR [11]. This gene is mutated in
approximately 50–60% of NK-AML patients. However, this mutation provides no protective benefit when it occurs together with the ITD of FLT3, which is the case in approximately 40% of patients with NPM1 mutations[5]. As with NPM1 mutations, CCAAT/enhancer binding protein alpha (CEBPA) gene mutations, found in around 10% of all AML patients, are associated with a favorable prognosis in de novo NK-AML but only in the absence of FLT3-ITD [12].
One reason for the poor treatment outcome in AML could be deregulated expression of AraC-metabolizing enzymes. AraC is activated intracellularly by the enzyme deoxycytidine kinase (DCK), which converts itto the triphosphate, Ara-CTP, and poor treatment outcome and chemotherapy resistance might be due to decreased expression or activity of this enzyme [13]. There are also several enzymes responsible for the inactivation of AraC, including cytidine deaminase (CDA) and 5´-nucleotidases, and altered activity of these enzymes can lead to decreased sensitivity to AraC [14, 15]. The balance of these enzyme activities determines the cellular concentration of active Ara-CTP, which is a metabolite that varies widely in patients with AML [16]. The ratio of deoxycytidine to cytosolic 5´-nucleotidase II (cN-II) expression has been shown to correlate with intracellular production of Ara-CTP in primary AML cells [17] and is a predictor of survival in AML patients treated with AraC [18].
Single nucleotide polymorphisms (SNPs) can significantly alter the expression and activity of these drug-metabolizing enzymes [19-23]. Provided confirmation in clinical trials, SNPs may serve as biomarkers guiding the clinician in the choice and dosing of drugs according to the drug-metabolizing capacity of the patient. Hopefully, this will lead to less drug resistance, reduce the toxic side effects of treatment, and increase survival rates.
In this study we investigated the potential of polymorphisms in the DCK, CDA, and cN-II genes to act as markers of drug sensitivity and prognosis in 205 de novo NK-AML patients. Our findings indicate that CDA and cN-II polymorphisms may have potential as biomarkers for risk stratification in NK-AML patients.
Methods
Patients
The study was approved by the local Ethics Committees and conducted in compliance with the Helsinki declaration. All patients provided informed consent to be included in this study. Bone marrow sampling was performed at diagnosis and before the initiation of treatment in 205 Swedish patients diagnosed with de novo NK-AML between 1988 and 2009. All patients received chemotherapy treatment with curative intent, and all patients except 2 were given induction treatment regimens including AraC. From 2004, patients have been treated
according to nationwide AML treatment guidelines
(http://www.sfhem.se/Filarkiv/Nationella-riktlinjer, accessed 2013-05-13). Thus, the majority of the patients received induction treatments including daunorubicin 60mg/m2 q.d. for three days combined with AraC as 1000mg/m2 b.i.d. in 2h i.v infusions for 5 days. Before 2004, regional guidelines most commonly included AraC doses of 200 mg/m2 as 24 h i.v infusions for 7 days combined with either daunorubicin or idarubicin [24]. In addition to chemotherapy, fifty-seven patients were consolidated with allogeneic hematopoietic stem cell transplants (allo-SCT). Chemotherapy response, defined as morphological CR or non-complete remission (no CR), was evaluated according to the International Working Group criteria, and patients were followed up for up to 4 years. Median censoring time was 10 months (0.03-48 months); patients treated by allo-SCT were censored at the time of transplantation. Without censoring at time of transplantation, median censoring time was 17
months (0.03-48 months). Table I (Supplemental online material) summarizes patient characteristics.
Reference group
The HapMap-CEU reference population were used
(http://www.ncbi.nlm.nih.gov/SNP/snp_viewTable.cgi?pop=1409 accessed 2013-02-08) for comparisons between the NK-AML patients and the general population with regard to genotype distributions.
SNP genotyping
The two CDA SNPs, 79A>C (rs2072671) and -451C>T (rs532545), and the cN-II SNP 7A>G (rs10883841) were evaluated using TaqMan SNP Genotyping Assays according to the manufacturer´s instructions with the exception that we used a total reaction volume of 10 L. The DCK SNP 3´UTR 948T>C (rs4643786) was detected using PCR followed by pyrosequencing. DNA was isolated from patients with the QIAamp® DNA mini-kit (Qiagen, Sweden). Amplification of DCK covering the SNP region was performed using the HotStarTaq master mixture (VWR International, Sweden) with primers and MgCl2 at final
concentrations of 0.4 mM and 1.5 mM, respectively,in a total volume of 25 µL. The PCR primers were forward AAC CCA AGT TTT TAA TCG and reverse biotin-CCA CTT TGC AAC TTT TAA TA. The SNP was analyzed on a PSQ96MA instrument (Qiagen, Sweden) according to the manufacturer’s instructions and as previously described [25]. In brief, the amplified and biotinylated PCR product was isolated with a Vacuum Prep Workstation (Qiagen, Sweden). The sequencing primer (Invitrogen, Pisley, UK) AAT GAA TCT TAT GCA AAA CT was annealed to the single-stranded DNA template for 2 min at 80°C. The plate was then transferred to the pyrosequencer instrument and sequencing, by incorporation
of dispensed nucleotides, was performed with the following dispensing order: CTG ACT CTA G.
NPM1 and FLT3-ITD analysis
Insertion mutations in exon 12 of the NPM1 gene and the ITDs in the FLT3 gene were detected by PCR as described previously [26, 27] using 10 ng of genomic DNA. The PCR products were separated by capillary electrophoresis in an ABI 3130 XL genetic analyzer (Applied Biosystems, Foster City, CA, USA) and fragment sizing was performed using the GeneMapper 4.0 software (Applied Biosystems).
In vitro cytotoxicity
Leukemic cells from a subgroup of the NK-AML patients (n = 56) were isolated using Lymphoprep (Axis-Shield PoC, Oslo, Norway). To mimic intracellular drug concentration during AML treatment the cells were incubated for 96 h with AraC 0.5 µM, for 1 h with daunorubicin 0.2 µM, etoposide 20 µM. or mitoxantrone 0.1 µM, with all samples in duplicate and including drug free controls [28, 29]. After culturing during 96 h, the cell viability was determined by measuring the intracellular ATP concentrations using a bioluminescence assay as previously described [30]. The half maximal inhibitory concentrations expressed as IC50 values were calculated by comparing the ATP
concentrations to those in the drug free controls.
DNA methylation assay
Global DNA methylation was detected using the colorimetric version of the MethylFlash Methylated DNA Quantification Kit (Epigentek, Farmingdale, NY) according to the manufacturer´s instructions. DNA samples from 82 NK-AML patients were included, and all samples were measured in duplicate.
Statistics
Fisher´s exact test was used to make comparisons between genotypes and allelic distributions and between CR and no CR. Kruskal Wallis Test and Chi2 or Fisher´s exact test were used to investigate differences between genotype groups in terms of mean age, distribution of FLT3-ITD/NPM1 mutation and treatment regime (chemotherapy vs. chemotherapy followed by allo-SCT) (Data on distributions across groups are available in Supplemental data A). Kaplan-Meier analysis was applied together with the log-rank test to determine the significance of differences in survival times. These tests were performed with both the entire material and with the data stratified by FLT3 and NPM1 status. Survival analysis was performed with and without censoring patients treated by allo-SCT at the time of transplantation in the survival analysis. Significant findings were also further investigated by multivariable Cox regression analysis using the forced entry method. Due to the wide diagnostic period for the material (1988–2009), the analyses were also performed with the inclusion of year of diagnosis as a possible confounder, to indirectly adjust for minor changes in treatment protocols over the years. When comparing groups in terms of differences in sensitivity to the in vitro drug panel, a two-sided non-equal variance Student’s t-test was used. The significance level was defined as P = 0.05. All analyses were performed using IBM SPSS Statistics software version 20 (IBM, Armonk, NY, USA).
Results
No differences in genotype frequencies between NK-AML patients and controls
All 205 patients with de novo NK-AML were successfully genotyped for SNPs in CDA (79A>C and -451C>T) and DCK (3´UTR 948T>C). cN-II (7A>G) genotyping was successful for all but 3 patients. Data on FLT3-ITD and NPM1 mutations were available for 203 patients. No significant differences in genotype distributions between patients and the
reference population were found (Table II, supplemental online material). The distribution of the SNPs were in accordance with the Hardy-Weinberg equation except for the DCK 3´UTR 948T>C SNP where the C/C genotype is absent in the European population. FLT3-ITD and NPM1 mutations were found at the expected frequencies. The distributions of patient characteristics did not differ significantly between the different CDA or cN-II genotypes. Heterozygous DCK T/C patients were significantly younger than DCK T/T patients, mean age at diagnosis 48 and 60 years, respectively (p=0.007).
No significant effects of the SNPs on treatment response
There were no significant differences between genotypes in terms of treatment response for the CDA or cN-II SNPs. For the DCK 3´UTR 948T>C SNP, all 16 patients with the T/C genotype achieved CR compared to 152 out of 185 of the homozygous T/T patients, but this difference was not statistically significant (p = 0.08, Table III, supplemental online material). Notably, the heterozygous T/C patients were also more frequently transplanted than T/T patients (50% and 26%, respectively, p=0.047).
Impact on survival of the CDA and cN-II SNPs
The SNPs and the FLT3 and NPM1 mutations were analyzed for any correlations with progression free survival (PFS) or OS.
No significant differences in OS were seen for any of the SNPs when analyzing the entire material. However, when the patients were stratified according to FLT3 status, a significant effect on OS was observed both for CDA 79A>C and -451C>T SNPs in the FLT3-ITD-positive patients. This was the case with as well as without censoring patients at time of allo-SCT, although some survival differences between genotypes were indicated in the unstratified material for CDA 79A>C when censoring at time of allo-SCT was not applied (p=0.067; for survival data without censoring at time of allo-SCT, see Supplemental data
A). Patients that were FLT3-ITD-positive and homozygous C/C or T/T had a worse outcome compared to FLT3-ITD-positive patients with other genotypes (5 vs. 24 months, p <0.001, for CDA 79A>C (Figure 1A) and 5 vs. 23 months, p = 0.015, forCDA -451C>T (Figure 1B)). Similar patterns were seen for PFS (2 vs. 18 months, p<0.001, for CDA 79A>C; and 4 vs. 17 months, p=0.09, for CDA -451C>T). Stratification based on combined FLT3/NPM1 status showed that the effect of the CDA SNPs on OS was most pronounced in patients who were both FLT3-ITD-positive and NPM1-positive (4 vs. 28 months for homozygous CDA 79 C/C vs. other genotypes, p <0.001, and 4 vs. 27 months for CDA -451 T/T vs. other genotypes, p = 0.001).
For the cN-II 7A>G SNP, patients carrying at least one variant allele appeared to have a shorter OS compared to cN-II A/A patients, but only in FLT3-ITD-negative patients and not statistically significant (25 vs. 31 months, p = 0.075, Figure 1C). Inclusion of both FLT3 and NPM1 status in the Kaplan Meier analysis did not increase significance. (p = 0.13–0.20, data not shown).
As expected, there was a significant difference in survival between FLT3-ITD-positive and FLT3-ITD-negative patients, and negative patients showed significantly longer PFS and OS than positive patients (mean PFS 28 vs. 17 months, p = 0.004; mean OS 29 vs. 22 months, p = 0.055; for survival data without censoring at time of allo-SCT, see Supplemental data A). Patients with the FLT3-ITD-negative/NPM1-positive genotype had a better prognosis compared to FLT3-ITD-positive/NPM1-negative patients (mean PFS 28 vs. 11 months, p = 0.007; mean OS 32 vs. 16 months, p = 0.02).
Multivariable Cox regression analysis confirms the influence of CDA SNPs on survival
The poor prognosis for the patients with the homozygous CDA 79C/C or -451T/T genotypes, as indicated by the log rank test, was further investigated in a multivariable analysis using
Cox regression. OS without censoring patients at time of allo-SCT was analyzed, taking age, gender, treatment (chemotherapy alone or chemotherapy followed by allo-SCT) and FLT3/NPM1 genotype group into account. The homozygous CDA 79C/C or -451T/T genotypes remained indicative as markers of decreased survival together with higher age and the FLT3-ITD-positive/NPM1-negative or FLT3-ITD-positive/NPM1-positive genotype (Table IV). Adjusting for year of diagnosis further increased the significance for the CDA SNPs. Additional analyses were performed including an interaction variable between CDA genotype and treatment, but there was no significant interaction (data not shown). Results from a cox regression comparing groups based on FLT3 showed that the influence of CDA genotype differed between FLT3 subgroups, as indicated by the Kaplan Meier analysis (grouped data presented in Supplemental data A).
Effect of CDA SNPs on in vitro cytotoxicity
The in vitro sensitivity of leukemic cells from 56 patients was tested against several chemotherapeutic drugs commonly used to treat AML. The CDA -451C>T SNP affected the sensitivity to mitoxantrone and etoposide, and cells from homozygous T/T patients were significantly more sensitive than those from heterozygous C/T or homozygous C/C individuals (p = 0.002 for mitoxantrone and p = 0.006 for etoposide, data not shown). The CDA 79A>C SNP also showed a higher sensitivity to mitoxantrone (p = 0.003) and etoposide (p = 0.019) in the homozygous C/C compared to the homozygous A/A patients and a borderline significance (p = 0.051) for daunorubicin. No significant differences between genotypes in terms of sensitivity could be observed for AraC. There were no differences in mean age, frequencies of FLT3-ITD/NPM1 mutations, or SNP genotype distributions between the patients in the in vitro test and the entire material (data not shown). Survival data for the in vitro-population showed the same direction as reported above for the entire material, but the sample size was too small for a stratified analysis (data not shown).
Differences in global methylation status between different CDA genotypes
Global DNA methylation was examined in duplicate from 82 patient DNA samples using the MethylFlash Methylated DNA Quantification Kit and the positive and negative controls provided in the kit. There was a significantly higher degree of DNA methylation in patients homozygous for both CDA 79A>C and -451C>T (A/A + C/C) compared to patients heterozygous in both positions (p = 0.018, Figure 2). There was a further decrease in global DNA methylation in the homozygous C/C + T/T group, but this was not statistically significant.
Discussion
NK-AML patients display a wide variation in treatment response and survival, and the analysis of FLT3-ITD, NPM1, and CEBPA mutations have become important clinical markers. There is still a need, however, for additional tools to classify subgroups among these intermediate-risk patients as a way of individualizing treatment. This is especially evident in patients lacking the above-mentioned mutations and patients with ambiguous mutational status. DCK, CDA and cN-II are all enzymes with important functions in the metabolism of nucleoside analog drugs, and polymorphisms in their genes might influence gene expression, enzyme function, and the subsequent treatment.
Our results showed that the CDA 79C/C and -451T/T genotypes decrease OS in FLT3-ITD-positive patients with NK-AML. The CDA polymorphisms also affected the in vitro sensitivity to topoisomerase inhibitor drugs, but not to AraC. This differential drug sensitivity might be explained by differences found in the degree of global DNA methylation between CDA variants.
The impact of the CDA polymorphisms on OS seemed to be most pronounced in FLT3-ITD-positive/NPM1-positive patients. Because stratification according to FLT3/NPM1 status
results in small groups, these results must be interpreted with care. However, CDA genotype remained in the multivariable analysis as a significant factor for OS. In addition, the FLT3-ITD-positive/NPM1-positive patients with homozygous 79C/C or -451T/T genotype did not differ from the other patients regarding specific treatment regime or age, which excluded these factors as possible confounders.
The T allele of the CDA -451C>T SNP has previously been shown to result in altered enzyme expression and activity [19, 31]. A study by Mahlknecht et al. reported a higher incidence of AraC-related grade III and grade IV liver toxicity in patients with the CDA 79A>C variant allele and a shorter survival time in CDA -451T/T AML patients [22]. This study is in accordance with our results; however, no toxicity data were available on our patients. The CDA 79C/C variant has been reported to result in lower CDA allelic expression [19], decreased enzyme activity [32], poor outcome in patients with non-small cell lung cancer [33], and reduced in vitro AraC deamination [34], while other studies report a higher CDA activity in the 79C variants [23]. We could not investigate CDA phenotypes in our patients, which would have provided further explanations to the survival differences seen between the CDA genotypes. However, despite differences in explanatory hypotheses, most studies describe a worse outcome in homozygous 79C/C patients, which is in accordance with our findings. The impact of SNPs affecting CDA activity may also depend on drug dosage. In patients receiving lower dose AraC, a decrease in CDA activity might result in improved treatment response, while in higher dose regimens it could increase the risk of toxicities.
Surprisingly, none of the SNPs affected the sensitivity to AraC in vitro. Instead, the CDA variants were associated with an increased sensitivity to topoisomerase inhibitors. There may be methodological explanations to the lack of any detectable difference in AraC sensitivity. AraC acts on cells mainly in the S-phase, and in this short term incubation assay, cells are mainly kept alive without extensive cell division. To achieve significant cytotoxic effects in
vitro, AraC concentrations higher than those achieved in vivo are required, which in turn may overcome any differential enzyme activity between variants. Influence of CDA polymorphisms on AraC sensitivity should therefore not be excluded based on our in vitro results. Why the effect of topoisomerase inhibitors would be enhanced in the CDA variants remains a question. Cytidine deamination was first discovered as a mechanism of RNA editing [35], allowing for modification of mRNA transcripts in order to alter the amount of gene product without directly affecting the genes. The activation-induced cytidine deaminase has been shown to reduce the expression of topoisomerase by suppressing its mRNA through deamination [36]. We speculate, therefore, that an altered CDA activity may lead to altered expression of topoisomerase enzymes in the cells and to a potentiation of drug effect. Unfortunately, we did not have sufficient amounts of RNA to measure the expression of topoisomerase mRNA.
Another possible mechanism explaining the differential drug sensitivity could be that CDA might affect the degree of DNA methylation, and thereby the amount of DNA available to these drugs. Pharmacological inhibition of CDA reduces the degree of DNA methylation [37, 38] and we speculated, therefore, that individuals carrying the CDA variant alleles may not only have reduced CDA activity, but also overall reductions in DNA methylation. We found significant differences in global methylation between cells from patients homozygous for both CDA SNPs (79A/A and -451C/C) and patients heterozygous in both positions (p = 0.018). The group with the alternative homozygous genotype (79C/C together with -451T/T) was limited in number but appeared visually to be less methylated than the other two groups. We speculate, therefore, that CDA may somehow take part in the DNA methylation process and thereby affect topoisomerase inhibitor sensitivity.
The cN-II 7A>G SNP did not show any correlation to response, outcome, or cytotoxicity in the entire material, although some differences in mRNA expression have been shown
previously between cN-II genetic variants [19]. Some survival differences were seen in FLT3-ITD-negative patients, where the cN-II G allele seemed to be associated with shorter OS, but this did not hold in the multivariable analysis.
It was interesting that patients heterozygous for the DCK 3´UTR 948C>T SNP all achieved initial CR. Lamba (2007) showed that polymorphisms in the 3´UTR region of DCK could affect mRNA expression and the levels of Ara-CTP in AML blasts [39]. This may influence the response to AraC treatment, but the differences indicated here may also be related to the younger age of our T/C patients.
In summary, the clinical relevance of our findings on the cN-II and DCK SNPs is unclear, and warrants further investigation.
Conclusions
Our results show that polymorphisms of CDA, and possibly cN-II, have potential as prognostic markers of survival in NK-AML. The CDA results are of particular interest because NK-AML patients with simultaneous FLT3-ITD/NPM1 mutations comprise a patient group with few or ambiguous genetic markers for use in risk stratification. Providing confirmation in clinical trials, our results indicate that CDA SNP analysis may be a useful tool for making decisions regarding individual treatment regimes such as dose adjustments or allocation to early bone marrow transplantation.
Acknowledgements/Grant support
This work was supported by grants from the Swedish Cancer Foundation, the County Council of Östergötland, AFA Insurance, Stockholm Cancer Society, Karolinska Institutet, and the Swedish Research Council. We thank Dr. Roza Chaireti, Department of Hematology,
Linköping University Hospital, and Christer Paul, Division of Hematology, Karolinska Institutet, Huddinge, for valuable input and help with clinical data. The authors would also like to thank Sofia Bengtzén at Karolinska University Hospital, Huddinge, for technical support, and Karl Wahlin, Division of Statistics, Department of Computer and Information Science, Linköping University, Linköping, for statistical consultation.
Author contributions: IJF and AF: Research, data compilation, statistical analysis, manuscript writing; EP and MHe: Research, data collection; RR: Data analysis; HN, MHo, LP, DS, and YW: Patient material and data collection; HG: Research, study design, data and statistical analysis; KL: Study design, patient material, and data collection. All authors contributed with critical revision of the manuscript.
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Figure 1. Differences in overall survival (OS) depending on CDA and cN-II genotypes. In FLT3-ITD-positive patients, homozygous CDA 79C/C and CDA -451T/T genotypes were significantly associated with a shorter OS. (A) The mean OS was 5 vs. 24 months for 79C/C vs. other genotypes, p<0.001. (B) The mean OS was 5 vs. 23 months for -451T/T vs. other genotypes, p = 0.015. In FLT3-ITD-negative patients, a shorter survival was seen in
individuals carrying at least one cN-II 7A>G variant allele. (C) The mean OS was 25 vs. 31 months for 7A/A vs. other genotypes, p = 0.075.
Figure 2. CDA SNPs and global DNA methylation. Global DNA methylation in relation to the CDA 79A>C and -451C>T genotypes was evaluated in 82 patient DNA samples. There was a significant increase in DNA methylation in patients harboring the A/A + C/C genotype compared to the heterozygotes (p = 0.018).
Supplemental data A
Supplemental data A contains Kaplan Meier survival curves with data for FLT3 and CDA SNPs 79A>C and -451C>T, without censoring at time of bone marrow transplantation (Figure 1-3).
Distributions of patient characteristics in the entire material and in CDA, cN-II and DCK genotype subgroups are presented in Table I. Subgroup comparisons (independent samples Mann-Whitney U test and Chi2 or Fisher’s exact test) revealed no significant differences in terms of mean age or distributions of gender, FLT3-ITD, NPM1 mutation, treatment (chemotherapy or chemotherapy followed by allo-SCT), or treatment response (CR/non-CR) across CDA or cN-II genotypes. DCK T/C patients were significantly younger than T/T patients, and were more frequently transplanted.
Figure and table legends
Figure 1. Differences in overall survival (OS) depending on CDA 79A>C genotype. (A) No significant
survival differences between 79A>C genotypes were seen in the unstratified material (mean OS was 24 vs. 29 months for 79C/C vs. other genotypes, p=0.067). (B) In FLT3-ITD-positive patients,
homozygous CDA 79C/C genotype was significantly associated with a shorter OS (mean OS was 5 vs. 26 months for 79C/C vs. other genotypes, p<0.001).
Figure 2. Differences in overall survival (OS) depending on CDA -451C>T genotype. (A) No
significant differences between -451C>T genotypes were seen in the unstratified material (mean OS was 25 vs. 29 months for -451T/T vs. other genotypes, p=0.125). (B) In FLT3-ITD-positive patients, homozygous CDA -451T/T genotype was significantly associated with a shorter OS (mean OS was 5 vs. 25 months for -451T/T vs. other genotypes, p=0.002).
Figure 3. Differences in overall survival (OS) and progression free survival (PFS) between FLT3-wt and FLT3-ITD patients. (A) Patients with FLT3-ITD have a significantly shorter OS than patients with
FLT3-wt. Mean OS was 24 vs. 31 months for FLT3-ITD vs. FLT3-wt, p=0.02. (B) FLT3-ITD patients also have a significantly shorter PFS. Mean PFS was 17 vs. 28 months for FLT3-ITD vs. FLT3-wt, p=0.004.
Table I. Patient characteristics in the entire material (n=205) and in CDA, cN-II and DCK genotype
subgroups. Comparisons between groups in terms of mean age using the Independent samples Mann-Whitney U test, and Chi2 or Fisher’s exact test for comparing distributions of categorical variables.
Table II. Cox regression comparing groups based on FLT3; including the covariates age, gender,
treatment (chemotherapy alone or chemotherapy followed by allo-SCT), NPM1, and CDA genotype. Adjusted for year of diagnosis.
26
Table I.
Characteristic All patients (n=205) 79A/A or A/C (n=179) 79C/C (n=26) p -451C/C or C/T (n=184) -451T/T (n=21) p cN-II A/A (n=138) cN-II A/G or G/G (n=64) p DCK T/T (n=189) DCK T/C (n=16) p Mean age (range) 59 (18–85) 58 (18-84) 62 (27-78) 0.12 58 (18-84) 62 (37-78) 0.14 58 (18-84) 60 (18-82) 0.38 60 (18-84) 48 (23-73) 0.007
Gender: Male Female 96 109 83 96 13 13 0.83 86 98 10 11 1.0 64 74 31 33 0.88 89 100 7 9 1.0 FLT3: FLT3-ITD negative FLT3-ITD positive Missing information 136 67 2 118 60 1 18 7 1 0.66 121 62 1 15 5 1 0.62 87 49 2 47 17 0.20 126 61 2 10 6 0.78 NPM1: NPM1 wildtype NPM1 mutation Missing information 111 92 2 98 80 1 13 12 1 0.83 101 82 1 10 10 1 0.81 74 62 2 35 29 1.0 103 84 2 8 8 0.80 Treatment: Chemotherapy Chemotherapy+allo-SCT 148 57 126 53 22 4 0.16 131 53 17 4 0.45 97 41 48 16 0.51 140 49 8 8 0.05 Treatment response: CR Non-CR Not evaluated 168 33 4 149 27 3 6 19 1 0.26 153 28 3 15 5 1 0.34 116 20 2 50 12 2 0.53 152 33 4 16 0 0.08
27 Table II. FLT3-ITD- FLT3-ITD+ Covariates HR 95% CI p HR 95% CI p Age 1.026 1.001-1.052 0.044 0.993 0.954-1.034 0.734 Gender 1.535 0.887-2.658 0.126 0.636 0.297-1.363 0.245 NPM1-mutation 0.736 0.413-1.313 0.299 0.467 0.224-0.973 0.042 Treatmenta 0.425 0.165-1.096 0.077 0.306 0.105-0.886 0.029 CDA 79C/C genotypeb 1.455 0.671-3.154 0.342 6.697 2.405-18.650 <0.001 Age 1.027 1.002-1.054 0.035 0.992 0.954-1.031 0.684 Gender 1.630 0.933-2.849 0.086 0.632 0.299-1.337 0.230 NPM1-mutation 0.701 0.390-1.259 0.234 0.451 0.217-0.935 0.032 Treatmenta 0.437 0.169-1.130 0.088 0.272 0.094-0.787 0.016 CDA -451T/T genotypec 2.110 0.906-4.915 0.083 7.424 2.294-24.029 0.001 Age 1.027 1.002-1.054 0.035 0.992 0.954-1.031 0.684 Gender 1.630 0.933-2.849 0.086 0.632 0.299-1.337 0.230 NPM1-mutation 0.701 0.390-1.259 0.234 0.451 0.217-0.935 0.032 Treatmenta 0.437 0.169-1.130 0.088 0.272 0.094-0.787 0.016
Combined CDA 79C/C and -451T/T genotyped 2.110 0.906-4.915 0.083 7.424 2.294-24.029 0.001
aChemotherapy followed by allo-SCT compared to chemotherapy alone; bcompared to CDA 79A/A+A/C; ccompared to CDA
28 Supplemental online material
Table I. NK-AML patient characteristics.
AML patient characteristics Total N =205
Gender
Male 96 (47%)
Female 109 (53%)
Age at diagnosis, mean (range) 59 (18–85)
FLT3 status FLT3 wild type 136 (66%) FLT3 mutated 67 (33%) Missing information 2 (1%) NPM1 status NPM1 wild type 111 (54%) NPM1 mutated 92 (45%) Missing information 2 (1%) Treatmenta Dnr + AraC 121 (59%) Ida + AraC 38 (18.5%) Ida + AraC + CdA 12 (6%)
AraC 11 (5%)
Dnr + AraC + 6-TG 8 (4%) Mitox + AraC + Eto 6 (3%) Ida + AraC + Eto 3 (1.5%) Mitox + AraC 3 (1.5%) FaraA + AraC + G-CSF 1 (0.5%) Dnr + mylotarg 1 (0.5%) Ida 1 (0.5%) Treatment responseb CR 168 (82%) Non-CR 33 (16%) Not evaluated 4 (2%)
aDnr = Daunorubicine; AraC = Cytarabine; 6-TG = 6-thioguanine; Ida = Idarubicine; Cda = Cladribine; Eto =
etoposide; FaraA = Fludarabine; G-CSF = Granulocyte-colony stimulating factor; Mitox = Mitoxantrone; mylotarg= Gemtuzumab ozogamicin. bCR = complete remission
29
Table II. Polymorphisms analysed and their genotype frequencies. P-values indicate differences between patient
genotype and the expected genotype distribution based on the HapMap-CEU reference. The cN-II genotype was missing in 3 patients.
SNP Position Genotype N Freq. (%) Ref seq. (%) p CDA rs2072671 79A>C A/A 84 41 44 0.80 A/C 95 46.3 45 C/C 26 12.7 11 CDA rs532545 -451C>T C/C 83 40.5 43 0.81 C/T 101 49.3 48 T/T 21 10.2 9 cN-II rs10883841 7A>G A/A 138 68.3 76 0.19 A/G 60 29.7 23 G/G 4 2 1 DCK
rs4643786 3´UTR 948C>T C/C 0 Not present in Europeans 0.40
T/C 189 92.2 90 T/T 16 7.8 10
30
Table III. Genotypes in relation to treatment response. No significant association was seen between complete
remission (CR) and the CDA, cN-II, or DCK variants, although all DCK heterozygotes achieved CR. CR was not evaluable in 4 (2%) patients.
Gene Genotype Non-CR CR p
FLT3 Wild type 24 109 0.31
ITD 8 58
NPM1 Wild type 22 87 0.12
Mutated 10 80
CDA 79A>C A/A 16 66 0.19
A/C 11 83
C/C 6 19
CDA -451C>T C/C 17 64 0.10
C/T 11 89
T/T 5 15
cN-II 7A>G A/A 20 116 0.46
A/G 12 47
G/G 0 3
DCK 3´UTR 948C>T T/C 0 16 0.08
31
Table IV. Cox regression analysis of OS (not censoring patients at time of allo-SCT). The analyses were performed separately for CDA 79A>C, -451C/T, and the
combination of CDA 79A>C and -451C>T. Results are presented with and without adjustment for year of diagnosis.
Adjusted for year of diagnosis
Covariates HR 95% CI p HR 95% CI p Age 1.020 1.000-1.041 0.045 1.017 0.966-1.037 0.109 Gender 1.075 0.705-1.638 0.737 1.140 0.747-1.742 0.543 FLT3-ITD-positive/NPM1-negativea 3.289 1.645-6.576 0.001 3.014 1.506-6.032 0.002 FLT3-ITD-positive/NPM1-positivea 1.920 1.002-3.678 0.049 1.978 1.035-3.781 0.039 FLT3-ITD-negative/NPM1-negativea 1.365 0.766-2.430 0.291 1.378 0.771-2.445 0.282 Treatmentb 0.451 0.239-0.851 0.014 0.409 0.213-0.786 0.007 CDA 79C/C genotypec 1.880 1.047-3.375 0.035 2.043 1.134-3.682 0.017 Age 1.021 1.001-1.042 0.038 1.018 0.977-1.038 0.091 Gender 1.107 0.727-1.687 0.634 1.173 0.769-1.790 0.459 FLT3-ITD-positive/NPM1-negativea 3.388 1.686-6.807 0.001 3.103 1.542-6.242 0.001 FLT3-ITD-positive/NPM1-positivea 1.968 1.023-3.789 0.043 3.031 1.058-3.898 0.033 FLT3-ITD-negative/NPM1-negativea 1.421 0.795-2.541 0.236 1.438 0.803-2.576 0.222 Treatmentb 0.446 0.236-0.841 0.013 0.407 0.212-0.780 0.007 CDA -451T/T genotyped 2.124 1.107-4.074 0.023 2.313 1.200-4.457 0.012 Age 1.021 1.001-1.042 0.038 1.018 0.977-1.038 0.091 Gender 1.107 0.727-1.687 0.634 1.173 0.769-1.790 0.459 FLT3-ITD-positive/NPM1-negativea 3.388 1.686-6.807 0.001 3.103 1.542-6.242 0.001 FLT3-ITD-positive/NPM1-positivea 1.968 1.023-3.789 0.043 3.031 1.058-3.898 0.033 FLT3-ITD-negative/NPM1-negativea 1.421 0.795-2.541 0.236 1.438 0.803-2.576 0.222 Treatmentb 0.446 0.236-0.841 0.013 0.407 0.212-0.780 0.007
Combined CDA 79C/C and -451T/T genotypee 2.124 1.107-4.074 0.023 2.313 1.200-4.457 0.012 aCompared to FLT3 ITD-negative/NPM1-positive; bchemotherapy+allo-SCT compared to chemotherapy alone; ccompared to 79A/A + A/C; dcompared to -451C/C + C/T; ecompared to 79A/A or A/C and -451C/C or T/T. Bold indicates significant p-values.
