OGG1 is a novel prognostic indicator in acute myeloid ... - Nature

Oncogene (2010) 29, 2005–2012

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SHORT COMMUNICATION

OGG1 is a novel prognostic indicator in acute myeloid leukaemia K Liddiard, R Hills, AK Burnett, RL Darley and A Tonks Department of Haematology, School of Medicine, Cardiff University, Cardiff, UK

OGG1 (8-oxoguanine DNA glycosylase) constitutes a key component of the DNA base excision repair pathway, catalysing the removal of 8-oxoguanine nucleotides from DNA, thereby suppressing mutagenesis and cell death. We found that OGG1 expression was significantly downregulated by the RUNX1-ETO fusion protein product of the t(8;21) chromosome translocation in normal haematopoietic progenitor cells and in patients with acute myeloid leukaemia (AML). Further examination of OGG1 expression in 174 AML trial patients using Affymetrix microarrays showed that the prevalence rate of OGG1 expression was 33% and correlated strongly with adverse cytogenetics. OGG1-expressing patients had a worse relapse-free survival and overall survival and an increased risk of relapse at 5-years of follow-up. There remained a trend towards increased relapse rate among OGG1-expressing patients, even after adjusting for other known risk factors in comprehensive stratified analyses. We also determined a trend for OGG1 expression to have a more adverse impact on disease outcome in the context of the FLT3-ITD mutation. This study highlights OGG1 as a valuable prognostic marker that could be used to sub-stratify AML patients to predict those likely to fail conventional chemotherapies but those likely to benefit from novel therapeutic approaches that modulate DNA repair activity. Oncogene (2010) 29, 2005–2012; doi:10.1038/onc.2009.462; published online 21 December 2009 Keywords: OGG1; acute myeloid leukaemia; prognosis; DNA repair

Expression of the DNA repair gene, OGG1, is downregulated by RUNX1-ETO in normal haematopoietic progenitor cells and AML patients One of the most common molecular abnormalities (B12% frequency) in acute myeloid leukaemia (AML) is the t(8;21)(q22;q22) translocation (Peterson and Zhang, 2004) that produces the RUNX1-ETO (also known as AML1-ETO or RUNX1-RUNX1T1) fusion protein (Miyoshi et al., 1991; Erickson et al., 1992). This Correspondence: Dr A Tonks, Department of Haematology, School of Medicine, Cardiff University, Cardiff, CF14 4XN, UK. E-mail: [email protected] Received 7 August 2009; revised 6 November 2009; accepted 12 November 2009; published online 21 December 2009

translocation disrupts wild-type RUNX1 transcription factor signalling with significant impact on myeloid differentiation and function (Yergeau et al., 1997; Kitabayashi et al., 1998). RUNX1 signalling is also perturbed by the related AML chromosomal aberration, inv(16)(p13q22), which fuses the RUNX protein-binding partner, core binding factor (CBF)b, with myosin-11 (Liu et al., 1993). De novo diagnosed CBF leukaemias (t(8;21) and inv(16)) are generally associated with good patient prognosis in terms of complete remission (CR), relapse risk (R) and overall survival (OS) compared with other subtypes (Keating et al., 1988; Grimwade et al., 1998). Hence, although CBF fusion proteins are leukaemogenic, they are not linked to aggressive cellular transformation and patients remain responsive to therapy. To identify genes dysregulated by the RUNX1-ETO fusion protein that could contribute to the characteristic t(8;21) FAB-M2 AML disease pathology (Oshimura et al., 1982), we previously carried out Affymetrix microarray gene expression profiling using transgenic haematopoietic progenitor cells overexpressing RUNX1-ETO as a single molecular abnormality (Tonks et al., 2007). Importantly, we found expression of the DNA repair enzyme, OGG1 (8-oxoguanine glycosylase), to be significantly downregulated in RUNX1-ETOoverexpressing progenitor cells (twofold downregulated; Po0.05). OGG1 is a critical component of the base excision repair pathway required for the removal of oxidized guanine nucleotides (8-oxoguanine) from both nuclear and mitochondrial DNA exposed to reactive oxygen species (ROS) (Van Der Kemp et al., 1996; Nishioka et al., 1999). Indeed, OGG1 expression and activity have previously been found to be increased in response to increased ROS levels (Ishchenko et al., 2003) and oxidative DNA damage (Jankowska et al., 2008). The excision of 8-oxoguanine residues by OGG1 protects against aberrant adenine–cytosine and guanine–thymine conversions (Sunaga et al., 2001) that can lead to heritable mutagenesis, particularly in nonproliferative cells in which lesions accumulate without dilution by cell division (Klungland et al., 1999). OGG1 expression therefore preserves genomic integrity, and Ogg1-deficient mice experience enhanced incidences of mutations and tumours (Klungland et al., 1999; Osterod et al., 2001; de Souza-Pinto et al., 2001). Furthermore, in humans, a polymorphic variant (S326C) with reduced repair activity has been associated with increased risk of several cancers (Weiss et al., 2005). To validate the downregulation of OGG1 expression by RUNX1-ETO, we performed quantitative reverse

Significance of OGG1 in AML K Liddiard et al

2006

transcriptase PCR (qRT–PCR) to measure OGG1 mRNA expression in three independent sets of CD34 þ RUNX1-ETO-overexpressing haematopoietic progenitor cells and matched controls (vector alone) generated by retroviral transduction. Levels of transduction efficiency were B70%, as determined by flow cytometric analysis of surrogate green fluorescent protein (GFP)

expression from the same retroviral vector, using mocktransduced control cells to set threshold autofluorescence (Figure 1a). In accordance with previous reports (Alcalay et al., 2003; Krejci et al., 2008), RUNX1-ETO expression resulted in a greater than seven-fold reduction in OGG1 mRNA levels compared with control cells when measured by qRT-PCR (Figure 1b) using

RUNX1-ETO

GFP control 239

222

Events

296 Events

OGG1 FOLD expression over GFP control

2

62% GFP+

148 74 0 100

101

102 103 GFP

104

179

72% GFP+

120 60 0 100

101

102 103 GFP

* 2

* *

1

*

0

-2 -4 -6 -8 -10 †

-12 GFP control

RUNX1-ETO

3

* M2 (–)

t(8;21)

Patient subgroup

Normalized OGG1 microarray expression

Normalized OGG1 microarray expression

3

104

0

* 2

*

* *

1

*

0

* M2 (–)

M4 (–)

inv(16)

Patient subgroup

Figure 1 Regulation of 8-oxoguanine DNA glycosylase (OGG1) expression by RUNX1-ETO in human CD34 þ haematopoietic progenitor cells and acute myeloid leukaemia (AML) patients. (a) Normal human CD34 þ haematopoietic progenitor cells were purified from human cord blood using the MiniMACS CD34 þ cell isolation kit (Miltenyi Biotec, Bisley, UK). Ectopic expression of RUNX1-ETO and green fluorescent protein (GFP) was achieved using the PINCO retroviral vector (Grignani et al., 1998). Retroviral transduction was carried out over 2 sequential days of culture as described previously (Tonks et al., 2007). After transduction, total RNA was harvested for expression analyses on day 3 of culture after verification of transduction efficiency by flow cytometric detection of the GFP surrogate marker (filled histograms). GFP þ threshold was defined by autofluorescence (open histograms) of mocktransduced cells. Typical percentages of viable GFP þ (transduced) cells are shown for each culture. (b) Quantitative reverse transcriptase–PCR (qRT–PCR) analysis of OGG1 expression in control and RUNX1-ETO-transduced cells. Transduced haematopoietic progenitor cell total RNA was purified and DNase-treated using the Qiagen RNeasy mini kit before 1 mg was reverse transcribed using random hexamers. Reactions for qRT–PCR were prepared using LightCycler FastStart DNA SYBR Green I master mix (Roche, Hertfordshire, UK), 500 nM forward and reverse pan-OGG1 or ABL primers, 3 mM magnesium and 50 ng each of cDNA. A 40-cycle qRT-PCR was performed on the LightCycler 2.0 (Roche) with a melting temperature of 60 1C. Normalized expression values were determined by crossing-point analysis of OGG1 signal compared with the ABL housekeeping gene (Beillard et al., 2003) control in duplicate samples. Fold change was calculated using the 2DDCt method (Livak and Schmittgen, 2001). Expression of OGG1 is 7.3-fold reduced in RUNX1-ETO-expressing cells compared with GFP control alone. Data represent mean plus 1 s.d. (n ¼ 3). Statistical significance was calculated by paired t-test. wPo0.05. (c) Reduced expression of OGG1 is associated with t(8;21) and (d) inv(16) favourable cytogenetics compared with other M2 and M4 patients. Patient and healthy donor-derived blast cell total RNA was extracted by lysis in Trizol. RNA quantity, quality and purity were assessed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Stockport, UK). The preparation and processing of labelled and fragmented cRNA targets, as well as Affymetrix HGU133A GeneChip hybridization and scanning, was carried out according to the manufacturer’s protocol (http://www.affymetrix.com). Affymetrix MicroArray Suite version 5 (MAS 5.0) and robust multi-chip average (RMA) software were used to translate the scanned images into expression analysis files. Unique MAS 5.0 and RMA signal values for each gene together with a ‘Detection P-value’ (associated with the MAS 5.0 analysis), indicating whether a transcript was reliably detected (present) or not detected (absent) were exported into Genespring GXv10.0 (Agilent Technologies, Palo Alto, CA, USA). Target intensity values from each array underwent baseline transformation to the median of all samples. OGG1 expression analysis was based on signal intensities and detection values for Affymetrix probe set 205760_s_at. M2() patients without a t(8;21) or inv(16); M4() patients without an inv(16); *data outliers. Horizontal bars represent median values. OGG1 expression was 7.5-fold downregulated (Po0.05) in t(8;21) patients compared with M2 patients; 7.7-fold downregulated (Po0.05) in inv(16) patients compared with M2 patients and 1.5-fold downregulated (not significant) in inv(16) patients compared with M4 patients. Statistical significance was determined by one-sided analysis of variance (ANOVA) using Tukey’s honestly significant differences. Oncogene


2007

primers designed to hybridize to both type I and type II isoforms (Nishioka et al., 1999) of OGG1 (pan-OGG1 primers, Supplementary Figure S1c; see Supplementary information). To determine whether the inhibition of OGG1 expression observed in normal haematopoietic progenitor cells expressing RUNX1-ETO is also seen in AML patients with the t(8;21) chromosomal translocation, we examined an Affymetrix gene expression database containing data obtained from 174 individual AML patients entered into MRC/NCRI AML trials 10–15 (1988–2005), representing the full range of FAB phenotypic groups (Bennett et al., 1985) except FABM3 patients, who were excluded owing to disparate treatment regime and prognosis. Importantly, patients included in this expression database experienced no difference in survival rates from trial patients excluded from the database (P ¼ 0.5) when examined by univariate statistical analysis. Expression data were normalized using robust multi-chip average software and exported into Genespring GXv10 to compare OGG1 signal values (probe set 205760_s_at) for 12 t(8;21) AML patients (FAB-M2) with those of 27 other FABM2 patients lacking this molecular abnormality (Figure 1c). We determined a greater than seven-fold reduction in median OGG1 gene expression in t(8;21) patients compared with other FAB-M2 patients, corroborating initial microarray analyses (Tonks et al., 2007) performed with RUNX1-ETO overexpressing CD34 þ cells. This suggests a similar extent of OGG1 gene expression regulation by RUNX1-ETO in the context of both normal and leukaemic haematopoietic cells, supporting evidence that the suppression of OGG1 gene expression is directly mediated by the RUNX1-ETO fusion protein (Krejci et al., 2008). To further evaluate the hypothesis that OGG1 is dysregulated by aberrant CBF signalling, we also examined OGG1 expression levels in AML patients with the inv(16) chromosomal rearrangement that produces an aberrant fusion protein containing the CBFb subunit (Liu et al., 1993). OGG1 robust multi-chip average signal values from inv(16) patients were compared with other FAB-M2 and FAB-M4 patient values, as our Affymetrix microarray database contained inv(16) patients with both these AML disease phenotypes. We found that median OGG1 expression was reduced by more than seven-fold in inv(16) patients compared with other FAB-M2 patients (Figure 1d). However, median OGG1 expression was only 1.5-fold downregulated in inv(16) patients compared with other FAB-M4 patients (not statistically significant). These results indicate that OGG1 expression is downregulated in association with different CBF abnormalities that interfere with RUNX1 signalling, but not significantly within the context of FAB-M4 disease. These data support the identification by Ross et al. (2004) of OGG1 as a component of a unique gene expression signature that can be used in the diagnosis of CBF leukaemias. To validate the microarray data, we performed correlative qRT–PCR analysis. RNA samples obtained from 19 AML patients (selected at random from those

previously subjected to microarray analysis) were reverse transcribed and used in qRT–PCR experiments with OGG1 type I or II isoform-specific (Nishioka et al., 1999) or pan-OGG1 primers. We found that the magnitude of OGG1 expression change was analogous regardless of which OGG1 primers were used (data not shown), suggesting that nuclear- and mitochondriallocalized isoforms of OGG1 are similarly regulated by the RUNX1-ETO fusion protein. Type II isoformspecific primers were used in subsequent analyses as these amplify an equivalent pool of transcripts to the Affymetrix 205760_s_at probe set. OGG1 gene expression measured by Affymetrix gene microarray profiling (robust multi-chip average-normalized) and qRT–PCR showed a high degree of correlation using Pearson’s correlation coefficient (R ¼ 0.631, Po0.01), validating both methods of OGG1 signal detection (Supplementary Figure S1a). To extend this validation, we performed an additional MAS5.0 summarization of the Affymetrix gene expression data to generate ‘present’, ‘absent’ or ‘marginal’ calls as an evaluation of whether OGG1 transcripts were reliably detected in each patient sample. Using this method, we determined that the prevalence of OGG1 expression (OGG1present) is only 33% among our sample of AML patients (58 of 174 patients). Seven OGG1present and nine OGG1absent AML samples were subsequently selected for measurement of OGG1 expression by qRT–PCR. As shown in Supplementary Figure S1b, OGG1present samples were confirmed as having higher OGG1 expression levels than OGG1absent samples, hence corroborating the robust multi-chip average-normalized data. Reduced OGG1 expression in t(8;21) and inv(16) patients may contribute to leukaemogenesis in these patients by reducing the capacity for DNA repair and increasing the likelihood of cooperating mutations arising. Indeed, both RUNX1-ETO (Krejci et al., 2008) and reduced Ogg1 (Klungland et al., 1999) expression lead to increased mutation rates, although each is insufficient to promote oncogenic transformation (Higuchi et al., 2002; Xie et al., 2004). The combination of both molecular abnormalities might then facilitate conversion of pre-leukaemic clones to full-blown leukaemias over time. Importantly, RUNX1-ETO expression results in both increased p21 expression (Peterson et al., 2007) and p53 activation (Krejci et al., 2008), which promote cell-cycle arrest and growth inhibition characteristic of these cells (Tonks et al., 2004). The RUNX1-ETO-mediated reduction in OGG1 expression may also contribute to dysregulated p21 expression, as a polymorphic variant (R229Q) with reduced DNA repair capacity identified in the KG-1 leukaemic cell line (Hyun et al., 2000) is associated with an upregulation of p21 in response to ROS (Hyun et al., 2006). We have also measured a two-fold increase in p21 expression in CD34 þ RUNX1-ETO overexpressing haematopoietic progenitor cells by microarray analysis (data not shown), but, notably, this is not retained among t(8;21) AML patients. It has recently been proposed that the activation of p21 by RUNX1-ETO actually facilitates leukaemic transformation through an arrest in Oncogene


2008

cell-cycle progression that permits DNA repair and subsequent maintenance of the leukaemic stem cell pool, rather than apoptosis of damaged cells (Viale et al., 2009). Alternatively, in the context of reduced OGG1 expression, this p21-conferred resistance to apoptosis may facilitate the accumulation of secondary genetic insults, such as loss of p21 (Peterson et al., 2007) or p53 (Krejci et al., 2008), which result in leukaemic progression. Thus, the effects of RUNX1-ETO may be mediated through its impact on DNA repair pathways and cell-cycle regulators, and may contribute to leukaemic transformation and the pathogenesis of AML in t(8;21) patients. Patients with CBF leukaemias are considered to have favourable prognosis compared with other de novo diagnosed AML subtypes (Keating et al., 1988; Grimwade et al., 1998). Thus, the reduced expression of OGG1 in these patients may conversely contribute to their ‘good risk’ status after diagnosis and clinical treatment. A reduction in DNA repair capacity can increase cellular sensitivity to the chemotherapeutics and radiation used to treat patients (Kinsella, 2009) so that low expression levels of repair pathway components can be predictive of better patient survival in chemotherapy (Husain et al., 1998; Shirota et al., 2001). This is exemplified by the KG-1 polymorphic OGG1 cell line that shows increased sensitivity to the chemotherapeutic, menadione, resulting in cell death (Hill and Evans, 2007). Recently, Krejci et al. (2008) showed that OGG1 downregulation by RUNX1-ETO was linked to enhanced cellular sensitivity to DNA-damaging agents, reduced ability to repair DNA damage and increased cell death. Thus, the favourable prognosis of t(8;21) AML patients may be directly linked to reduced OGG1 expression resulting in improved responsiveness to therapy even within the context of acquired ‘secondhit’ genetic aberrations. As t(8;21) patients with therapyinduced AML do not enjoy the same favourable prognosis as de novo diagnosed cases (Quesnel et al., 1993; Gustafson et al., 2009), downregulation of OGG1 expression may only be associated with favourable prognosis in clones that have not evolved alternative mechanisms of chemoresistance. Alternatively, OGG1 may not be downregulated at all in therapy-induced AML patients, which would merit further investigation. Furthermore, it would be of therapeutic and diagnostic value to assess the expression and activation of p21 and p53 within these different patient subsets to elucidate the impact of this gene axis on patient prognosis. Significance of OGG1 expression for patient prognosis As both upregulated (Saebo et al., 2006) and downregulated (Klungland et al., 1999) OGG1 expression or function is associated with experimental and clinical malignancy, we sought to determine the relevance of dysregulated OGG1 expression for AML patient prognosis using our database of 174 intensively treated AML patients. To facilitate coherent statistical analysis, we used MAS5.0 summarized data in association with clinical Oncogene

follow-up information for the same AML patients (Table 1). Statistical analysis of patient survival data (median follow-up 5.25 years, range 7 months to 12.6 years) showed that OGG1 gene expression (OGG1present) is strongly associated (P ¼ 0.0001) with adverse cytogenetics linked to poor prognosis (abnormalities of chromosomes 5, 7 and 3q and complex karyotypes consisting of X5 different abnormalities). Of the 20 patients with adverse cytogenetics, 12 (60%) were classified as OGG1present. Overall, 21% of all OGG1present patients had adverse cytogenetics compared with only 7% of OGG1absent patients. Importantly, the OGG1 gene is located on the human chromosome 3p25.3, and therefore would not be expected to be directly affected by the gross karyotypic changes used to determine the adverse cytogenetics group. To determine whether the correlation of OGG1 expression with adverse cytogenetics had any impact on patient prognosis, we examined survival statistics for OGG1present versus OGG1absent patients. We found that patients with OGG1present experienced a significant reduction in both relapse-free survival (RFS) and overall survival (OS) and an increased risk of relapse (R) (Figure 2). A decrease in RFS over 5 years from CR of 39 to 21% was observed (P ¼ 0.007), resulting in a similar reduction in OS from 39 to 22% over a 5-year period from trial entry (P ¼ 0.01). Conversely, the rate of relapse over 5 years was found to increase from 53 to 75% (P ¼ 0.005) in patients expressing OGG1. Hazard ratio and confidence interval estimates for these observations are: RFS: 1.9(1.18, 3.05) P ¼ 0.008, OS: 1.79(1.19, 2.7) P ¼ 0.005, R: 2.16(1.29, 3.6) P ¼ 0.003. There was also a trend towards a reduction in complete remission (CR) for OGG1present patients, but this did not reach significance (OR 1.6(0.74, 3.48), P ¼ 0.2). Use of OGG1 as an independent prognostic marker in AML We next addressed the question of whether OGG1 expression has a significant impact on individual patient parameters such as age, sex, diagnosis (de novo, secondary or myelodysplastic syndrome disease), performance status, white blood cell count and induction therapy by performing stratified statistical analyses (Figure 3a and Supplementary Figures S2 and S3). These analyses revealed that OGG1 expression had a significantly adverse effect on prognosis (RFS, OS and R, but not CR) in association with these known risk factors and in particular with the 50–59 age bracket, the male gender, de novo disease and high white blood cell count. When adjusting for all these factors except induction therapy (which had no effect on survival outcomes, data not shown), there remained a trend towards increased risk of relapse in OGG1present patients, although this did not reach statistical significance (P ¼ 0.05). The point estimates and confidence intervals for these stratified analyses were RFS: 1.51(0.93, 2.48) P ¼ 0.1, OS: 1.45(0.95, 2.23) P ¼ 0.08, R: 1.69(0.99, 2.87) P ¼ 0.05 and CR: 1.04(0.44, 2.46) P ¼ 0.9 (Figure 3a).


2009 Relapse-free survival by OGG1

Parameters of patients included in study

Overall AML10 AML11 AML12 AML14 AML15 Age (years) 0–14 15–29 30–39 40–49 50–59 60–69 70 þ Median (range)

174 1 5 28 51 89

Absent 116 1 3 20 31 61

Present 58 0 2 8 20 28

0.8

0 23 22 28 42 39 20 53.5 (15–77)

0 16 15 18 30 26 11 53 (15–77)

87 87

57 59

30 28

0.7

160 11 3

109 6 1

51 5 2

0.3

32 71 32 39

22 44 26 24

10 27 6 15

1.0*

33.6 (1.3–316)

35.3 (1.3–316.0)

Performance status WHO 0 WHO 1 WHO 2 WHO 3 WHO 4

101 61 8 3 1

70 39 4 2 1

31 22 4 1 0

0.5*

Cytogenetics Favourable Intermediate Adverse Unknown

32 100 20 22

29 63 8 16

3 37 12 6

0.0001*

Diagnosis De novo Secondary MDS WBC o10 10–49.9 50–99.9 100 þ Missing Median (range)

Absent Present

NO. NO. Events Patients Obs. Exp. 95 58 69.0 Absent 43 34 23.0 Present

75

2P = 0.01

50 39% 25

21%

0.5* 0 0

1

2

3

4

5

Years from CR Overall survival by OGG1 100

Absent Present

NO. NO. Events Patients Obs. Exp.

75

Absent

116

71

84.3

Present

58

45

31.7

2P = 0.007

50 39% 25

22%

0 0

30.7 (2.2–306.0)

1

2 3 Years from entry

4

5

Relapse by OGG1 100

% Relapsing

Sex Female Male

0 7 7 10 12 13 9 54 (17–74)

100

P-value % Alive in CR

Total

% Alive

Table 1

Absent Present

75

75% 53%

50 NO. NO. Patients Obs.

25

Events Exp.

Absent

95

48

59.3

Present

43

31

19.7

2P = 0.005

0 0

1

2

3

4

5

Years from CR

FAB type M0 M1 M2 M4 M5 M6 M7 Bilineage RAEB-t Other/unknown

11 36 46 35 24 2 2 2 2 14

3 24 37 31 6 1 2 1 2 9

8 12 9 4 18 1 0 1 0 5

o0.0001

Induction treatment ADE DA/DAT FLAG-Ida MAE MAC

29 96 37 9 3

18 62 30 4 2

11 34 7 5 1

0.2

Abbreviations: AML, acute myeloid leukaemia; MDS, myelodysplastic syndrome; WBC, white blood cell; WHO; World Health Organization. Stratified patient parameters relating to all 174 intensively treated AML trials patients included in statistical analyses. ‘Present’ and ‘Absent’ refer to the reliable or lack of reliable MAS5.0 signal detection for Affymetrix probe set 205760_s_at. Statistical significance (P-value) of correlates with OGG1 expression is given in the far right column. * Denotes the use of the Mantel–Haenszel test for trend, all other tests use w2.

Figure 2 Expression of OGG1 is associated with poor prognosis. Kaplan–Meier plots of (a) relapse-free survival, (b) overall survival and (c) relapse rate in AML patients expressing OGG1 (present) or not (absent). CR; complete remission. OGG1 expression analysis was based on MAS5.0 normalized detection values for Affymetrix probe set 205760_s_at. Statistical comparisons were performed using SAS version 9.1.3 software (SAS Institute Inc., Buckinghamshire, UK). Comparisons of survival data were performed using log-rank tests or Cox regression; significance was set at Po0.05.

We extended these analyses to determine whether the impact of OGG1 expression was also influenced by patient FLT3 (Fms-like tyrosine-3) mutation status, as the FLT3-ITD (internal tandem duplication) variant is a frequently occurring (B18% prevalence) and significant risk factor among AML patients (Rombouts et al., 2000) that has also been linked to increased intracellular ROS levels (Sallmyr et al., 2008). We found that both FLT3-wild type and FLT3-ITD patients had worse prognosis when also expressing OGG1 (reductions in OS from 38 to 22% and 29 to 10% in OGG1absent versus Oncogene


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Figure 3 Summary of stratified analyses of prognostic effects of OGG1 expression (present versus absent). (a) Relapse-free survival (RFS), overall survival (OS), relapse (R) and complete remission (CR) data for 174 intensively treated AML patients were stratified by the individual known risk factors denoted in the table (detailed in full in Supplementary Figure S3) or (b) by FLT3 mutation status. Point estimates and 95% confidence intervals (in parentheses) are listed with associated significance values included below. An OR o1 indicates a beneficial prognostic impact for OGG1 present. The overall adjusted multivariate analyses are entered in the bottom row. OGG1 expression analysis was based on detection values for Affymetrix probe set 205760_s_at. Statistical comparisons were performed using SAS version 9.1.3 software (SAS Institute Inc.). Comparisons of survival data were performed using log-rank tests or Cox regression; comparisons of demographics were by the Mantel–Haenszel test or the Wilcoxon rank-sum test; significance was set at Po0.05.

OGG1present patients, respectively, data not shown), but that this did not achieve statistical significance because of the small numbers of known FLT3-ITD patients in this study (34 of 174 patients, 20%). Adjusting for FLT3 mutation status further reduced the statistical significance of OGG1 as an independent prognostic marker. The point estimates and confidence intervals for these stratified analyses are RFS: 1.38(0.80,2.41) P ¼ 0.2, OS: 1.32(0.83, 2.11) P ¼ 0.2, R: 1.52(0.83, 2.80) P ¼ 0.17 and CR: 0.96(0.38, 2.45) P ¼ 0.9 (Figure 3b). Previous research (Bullinger et al., 2007) has shown that, even within the generally favourable prognostic groups of CBF leukaemia patients, subsets of patients with significantly worse prognosis can be identified and that these patients have higher levels of DNA repair enzymes, including BRCA1, RAD51 and CHEK2. Importantly, we found that the presence of OGG1 expression strongly correlates with poor prognosis among our cohort of AML trial patients. This is in keeping with findings showing an upregulation of OGG1 in colorectal cancer (Saebo et al., 2006) and in a subset of patients with the myelodysplastic syndrome (Jankowska et al., 2008). The increased expression of OGG1 may result in a more globalized destabilization of cellular DNA repair pathways that provokes genomic instability (Frosina, 2000), as well as a reduction in sensitivity to chemotherapeutic agents that contributes to the poor prognostic outcome of these patients. The regulation of OGG1 expression and activity is closely linked to intracellular ROS levels, which promote the accumulation of 8-oxoguanine DNA modifications (Floyd, 1990). Recent evidence indicates Oncogene

that cells are particularly sensitive to imbalances in DNA repair pathways when mitochondrial-specific expression is targeted, as mitochondria are the sites of ROS production and associated oxidative stress (Fishel et al., 2003; Radyuk et al., 2006). It is noted that expression of the KG-1 OGG1 polymorphic variant with reduced DNA repair capacity results in more cell death in response to oxidative damage when specifically targeted to the mitochondria, rather than the nucleus (Chatterjee et al., 2006). By qRT–PCR expression analysis, we found that type I and type II isoforms of OGG1 were analogously regulated by the transgenic expression of RUNX1-ETO. This suggests that both mitochondrial- and nuclear-localized OGG1 enzymes (Nishioka et al., 1999) may be similarly affected by the t(8;21) translocation, with consequences for genome integrity in both organelles (de Souza-Pinto et al., 2001; Radyuk et al., 2006). Significantly, we also found that OGG1 expression associated with the FLT3-ITD mutation in patients with poor prognosis. The FLT3-ITD mutation has been shown to effect genomic instability and increased DNA damage through enhanced intracellular ROS generation (Sallmyr et al., 2008). Thus, OGG1 expression in these patients may function as a marker of cellular exposure to oxidative stress, as well as contributing to the incidence of additional mutations, such as loss of p21 and p53, that promote more aggressive disease. In summary, this research represents the first large-scale investigation of OGG1 expression and ensuing prognostic significance for AML patients. We have shown that OGG1 expression is specifically


2011

downregulated in good-risk CBF leukaemia patients with subverted RUNX1 signalling. OGG1 expression significantly correlates with adverse cytogenetics, resulting in reduced survival and increased incidence of relapse among OGG1present patients. Although this research has not characterized the nature of the association of OGG1 expression with adverse cytogenetics, it is likely that dysregulated expression promotes genomic instability and the occurrence of additional mutations that facilitate oncogenic transformation or clonal survival (Frosina, 2000). Additional studies with larger groups of patients are required to determine whether OGG1 expression could serve as a valuable independent prognostic marker for AML patients or whether this knowledge can be used to sub-stratify patients within different risk categories for therapeutic benefit. OGG1 expression may be a useful indicator of patients for whom standard chemotherapy will be inadequate to effect disease remission. These patients may benefit from novel approaches based on rational targeting of DNA

repair pathways to deplete the leukaemic stem cell pool (Viale et al., 2009).

Conflict of interest The authors declare no conflicts of interest.

Acknowledgements This study was supported by the Wales Office of Research and Development (Welsh Assembly Government) and the Medical Research Council UK. Dr A Tonks is also supported by Leukaemia Research UK. We thank Amanda Gilkes and Megan Musson (Cardiff University) for their technical assistance in processing microarray samples. We also thank Professor Ken Mills (Queen’s University, Belfast) for help in microarray analyses. We acknowledge the MRC for access to patient sample material enrolled in the NCRI clinical trials.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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