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Prognostic value of TP53 gene mutations in myelodysplastic syndromes and acute myeloid leukemia treated with azacitidine

Leukemia Research

Abstract

TP53 mutations are found in 5 to 10% of MDS and AML, where they are generally associated with complex karyotype and an overall poor prognosis. However, the impact of TP53 mutations in MDS treated with Azacitidine (AZA) remains unclear. We analyzed TP53 mutations in 62 patients with high risk MDS or AML treated with AZA. A TP53 mutation was found in 23 patients (37.1%), associated with complex karyotype in 18 (78.3%) of them. TP53 mutations had no significant impact on response or complete response to AZA (p = 0.60 and p = 0.26, respectively). By univariate analysis, OS was negatively influenced by the presence of TP53 mutation (median OS 12.4 months versus 23.7 months, p < 10−4), abnormal cytogenetics (median OS 14.4 months vs 33 months, p = 0.02) complex cytogenetics (median OS 12.7 months versus 23.7 months, p= 0.0005), and a diagnosis of AML (median 14.5 months vs 21.2 months for MDS or CMML, p = 0.02). By multivariate analysis, only TP53 mutational status (HR 2.89 (95% confidence interval 1.38-6.04; p = 0.005) retained statistical significance for OS. Results were similar when the analysis was restricted to MDS and CMML patients, excluding AML (HR = 2.46 (95% confidence interval: 1.1- 6.4); p = 0.04)).

Thus, TP53 mutations strongly correlated with poorer survival in higher risk MDS and AML treated with AZA.

Keywords: myelodysplastic syndromes, TP53 mutation, Azacitidine, acute myeloblastic leukemia, prognostic factor.

1. Introduction

Azacitidine (AZA), a hypomethylating agent, can improve survival in higher risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with 20-30% marrow blasts [1] and has become a reference first line treatment in those disorders. Prognostic factors of response and survival with AZA in high risk MDS and low blast AML are not completely known. No clear correlation between baseline gene methylation patterns (and/or their modification with treatment) and response and/or survival after hypomethylating agents has been identified [2] . However, conventional parameters, including higher marrow blast percentage and unfavorable karyotype[3] and [4] or higher revised International prognostic scoring system (IPSS) [5] are associated to poorer response rates and/or survival. Acquired mutations of a few genes, including the TET2 gene [6] IDH1 and 2 and DNMT3A [7] gene have been also correlated with better or poorer response, but in relatively small series.

TP53 gene mutation is seen in 5 to 10% of MDS and AML [8], [9], [10], [11], [12], and [13]. With the exception of low risk MDS with isolated del 5q, where they are observed in 20 to 25% of the cases [14] TP53 gene mutation are generally associated with complex karyotype, generally resulting in del (17p) (ie with loss of the remaining TP53 allele)[15], [16], and [17]. Irrespective of treatment administered, TP53 mutations have been clearly associated with poor overall outcome in MDS and AML, independently [11], [12], and [13] or not [18] of complex karyotype. TP53 mutations have more specifically been associated with resistance to chemotherapy, including anthracycline-Aracytine (AraC) combinations and low-dose cytarabine in AML and MDS [18] . In patients treated with AZA, a recent study found no difference in response between mutated and wild type TP53 patients, and survival based on TP 53 status was not reported [13] .

We assessed here the impact of TP53 mutations on response and survival after AZA treatment in MDS and AML, and found TP53 mutations to be the only prognostic factor for survival in multivariate analysis.

2. Patients And Methods

2.1. Patients and treatment

We analyzed 62 patients with MDS, AML and chronic myelomonocytic leukemia (CMML) treated with AZA, generally according to the approved schedule (75 mg/m2/day, 7 days every 4 weeks), and response to treatment was evaluated after 4-6 cycles of treatment (unless progression occurred before), according to IWG 2006 response criteria. Responders were to continue treatment until progression. All patients signed a consent form for analysis of TP53 mutation by FASAY and sequencing. This study was approved by the Institutional Review Board of the Groupe Francophone des Myélodysplasies (GFM).

3. Methods

TP53 status was determined on RNA by the functional analysis of separated alleles in yeast (FASAY) assay which evaluates the transactivation activity of p53 on a p53-responsive promoter stably integrated in the yeast genome, as described by Flaman et al. (19). Briefly, RNA was reverse transcripted in cDNA using Random Hexamer and Superscript II. p53 transcripts were amplified by polymerase chain reaction (PCR) (exon 4 to 10) and cotransfected with the Gap repair plasmid in yeast. In this assay, yeast colonies transformed with wild-type or mutated TP53 sequences appear as white and large or red and small, respectively. p53 was considered non functional when more than 10% of the yeast colonies were red. Analysis of the split version of the test was performed to confirm this result and to localize the defect in the 5′ or 3′ part of the gene. The detection limit of this technique is around 10%, including in our hands [19] and [20].

When p53 was considered non functional with FASAY, mutant yeast colonies were analyzed to identify the genetic defect by Sanger sequencing. The TP53 mutation was also confirmed by sequencing on patient's marrow cell DNA by Next-Generation Sequencing (NGS) assay using pyrosequencing (GS Junior System, Roche with the IRON II plate design), whose sensitivity is in our hands of 1 to 2% [21] .

In a few cases with difficulties in interpreting the FASAY test (i.e when the split version did not confirm global result), analysis by NGS was also performed.

3.1. Statistical analysis

Patient outcomes were compared according to TP53 gene mutational status. Baseline characteristics and response rates according to IWG 2006 criteria were compared by nonparametric tests (Fisher's exact test for qualitative variables, Kruskal-Wallis test for quantitative variables). Censored endpoints were estimated by the non-parametric Kaplan-Meier method, and compared by the log-rank test. Survival was measured from the onset of hypomethylating agent therapy. Type I error was fixed at the 5% level. Univariate and multivariate analyses were performed with log-rank tests and proportional hazard Cox models, respectively, the latter including all variables with P < 05 in univariate analysis. Statistical analysis was performed on StataSE 10.1 (StataCorp, College Station, Texas, USA).

4. Results

Between March 2007 and July 2011, 62 patients from one center (Hôpital Avicenne- Paris 13 University) were analyzed. Forty four patients had MDS, 18 had AML with 20 to 30% marrow blasts (Refractory anemia with excess of blasts in transformation (RAEB-T) according to FAB classification, and 4 had CMML, AZA being also approved for treatment of the last 2 subsets in the EU. IPSS was high or intermediate 2 (int 2) in 54 patients, low or int 1 in 6 patients and could not be assessed in 2 patients. The 6 patients with low or int 1 IPSS received AZA treatment for different reasons: 3 had isolated 5q deletion but had progressed or lost response after Lenalidomide, 2 had unfavourable karyotype (one monosomy 7 and one complex karyotype with del 5q), 1 had low risk MDS without response to EPO and was included in a trial with AZA treatment

4.1. Baseline patient characteristics and response to treatment

Baseline characteristics of the 62 patients are included in Table 1 . Median age was 71 (range 42-90). According to WHO classification, 44 patients had MDS: 5q syndrome (n = 2); RCMD (n = 7); RAEB1 (n = 10); RAEB2 (n = 21); CMML (n = 4)) and 18 had AML 20-30%/RAEB-T.

Table 1 Baseline characteristics of the patients according to TP53 mutational status.

  All TP53 mutated TP53 WT P value
N 62 23 39  
Age median(Range) 71(42-90) 71(47-81) 70(42-90) 0.65
Sex
Male 38 12 26 0.29
Female 24 11 13  
WHO diagnosis        
RCMD 7 2 5 1
5q syndrome 2 1 1 1
RAEB1 10 4 6 1
RAEB2 21 7 14 0.78
AML (20-30% marrow blasts) 18 9 9 0.25
CMML
  4 0 4 0.29
marrow blast count
0-14% 35 11 24 0.43
> = 15% 27 12 15  
Cytogenetics (n = 60)
Normal 12 0 12 0.002
Complex 30 18 12 0.001
IPSS
Low 1 1 0 0.46
Int-1 5 1 4 0.05
Int-2 24 5 19 0.03
High 30 16 14 -
Failure 2 0 2  
medianN° of cycles of azacydine 8(1-36) 7(1-23) 8(1-36) 0.38
IWG 2006 response (n = 62)
CR
Overall response 20 (32%) 5 (22%) 15 (38%) 0.26
  30 (48%) 10 (44%) 20 (51%) 0.6
OS (median) (in months) 15.6 m 12.4 m 23.7 m <10-4 (Log Rank)
         
         
         
         
         
         

Karyotype was normal in 12 patients, had 1 abnormality in 11 patients, 2 abnormalities in 7 patients and was a failure in 2 patients. Thirty patients had complex karyotype, including del 5q or monosomy 5 in 29 cases, monosomy 7 or del 7q in 19 cases, and both chromosome 5 and 7 deletion in 13 cases.

The median number of cycles of AZA administered was 8 (range 1-36). Response to AZA was observed in 30 (48%) of the patients, including 20 CR (32%), 3 PR (5%), 2 marrow CR (3%), and 5 (8%) stable disease with hematological improvement (HI). Median overall survival from onset of AZA was 15.6 months.

4.2. TP 53 mutations and their prognostic value for AZA treatment

Twenty three (37.1%) patients had a TP53 mutation detected by FASAY technique, and always confirmed by NGS (n = 18) or Sanger method (n = 5). No false positive with FASAY technique was observed. Details of mutations observed are shown in Table 2 . Eighteen of the 23 patients (78.3%) with TP53 mutation had a complex karyotype. In the 5 patients with TP53 mutation without complex karyotype, 2 had isolated del 5q, 1 had monosomy 5, two had del 17p (isolated in 1 patient, and associated with del 20q in 1 patient). Nine (75%) of the 12 patients with chromosome 17 rearrangement leading to del 17p had a TP53 mutation.

Table 2 Description of TP 53 gene mutations.

Patient TP53 mutation Karyotype
1 Intron 9 insertion 46,XX,i(17)(q10) [22]
2 R282 W 46,XY,del(5)(q13q33) [2] /46,sl,add(4)(p16) [2] /47,sl,+8 [2] /46,XY [16]
  C238Y  
3 R273H 45,XY,del(5)(q13q33),der(17;20)(p13;q1?) [3] /44,sl,-16 [8] /44,sl,-18 [3] /43,sl,-16,-18 [3] /45,sl,der(4)t(4;17)(q35;p10) [2]
4 A161D 44-45,XX,-5,-7,dic(9;16)(p10;q10),der(12)t(5;12;5), dup(19)(q12q13)der(19)t(7;19)(?q31;q13),+mar[cp7]/
  S241 C 43-44,sl, der(11)t(8;11)(p12;?q22),-18 [7]
5 R280G 44,XY,dic(5;17)(q11;p11),-7 [17] /88,sl,x2 [4]
6 C238F 45,XY,del(3)(p13),del(5)(q13q33),-7,der(12)t(7;?;12)(q11;?;p12-13),-21,+mar [8] /44,sl-Y [8] /46,XY [3]
  R273H  
7 C176Y 45,X-Y,del(5)(q13q33),+11,del(17)(p11),-18 [4] /69-71,XXY,-2,-3,+4,del(5)(q13q33),+del(5)(q13q33) [5] ,-7 [4] ,+8 [5] ,+11,+11 [3] ,+13 [6] ,-16 [5] ,del(17)(p11)[cp8]/46,XY [2]
8 H179R 46,XX,del(5)(q13q33),der(12)t(12;17)(q24;q21),del(16)(q22),der(17)t(16;17)(q22;?q21),del(18)(q22) [1] /46,sl,add(8)(p11) [8] /47,sl,+8 [7] /46,XX [2]
9 G245D 46,XY,del(20)(q12) [3] /47,sl,+21 [5] /47,sdl1,del(17)(p11) [14] /46,XY [1]
10 R248G 43-44,XY,add(1)(q23),add(1)(q11),del(5)(q21q32),-7,del(12)(p12),-13,add(13)(p11),-16,add(18)(p11),+/- mar[cp23]
11 R337S 46,XY,-3,der(5)t(3;5)(q21;q31),+8 [10] /45,sl,t(4;16)(q21;?p13),add(6)(p25),-14 [5]
12 Early stop codon (codon53) 47,XX,+21 [1] /47,XX,der(4)ins(4;17)(q11;?p11p13)del(4)(q13),der(5)t(5;21)(q10;q11),der(7)t(4;7;11)(q?;q21;q14),der(11)t(4;7;11)(q?;q21;q14),r(17)(?p11q25),+tas (21)(qter;qter) [4] /48,sl,+min [5] /46,XX [11]
13 I162 N 45,XY,-5 [6] /46,XY [3]
14 L257R 43-45,XX,del(5)(q13q31),-7,+8 [7] ,-16 [4] ,-17 [7] ,-19 [7] [cp15]/46,XY [9]
15 V272L 47,XY,del(5)(q13q33),-18,+19,+mar [5] /46,XY [11]
  I195F  
16 R273H 45,XX,t(3;12)(p13;p11),del(5)(q13q33),der(9;14)(q10;q10),del(17)(p11) [6] /46,sl,+8 [5] /44,sl,-7 [7] /46,XX [1]
17 E224X 46,XX,add(3)(q13),del(5)(q14q34),ins(7,?)(p21,?),del(20)(q11q13) [2] /44,idem,del(6)(p22),-18,-21 [15] /46,XX [3]
  (STOP codon)  
18 P177H 44-45,XX,del(4)(q21),del(6)(q14),-7,-13,-20 [3] ,+2mar [cp4]/46,XX [1]
19 R248Q 46,XX,del(5)(q13q33) [7] /47,XX,+min [4] /46,XX [9]
20 C176P 46,XX,del(5)(q13q34) [1] /46,sl,del(7)(q22) [2] /47,sl,+8,der(9)t(1;9)(p22;q34) [11] /46,XX [3]
  G245S  
21 R282 W 46,XY,del(5)(q21q34) [16] /46,sl,-6,add(14)(q32),+mar [10] /45,sdl1,-7,+mar2 [4]
22 R175H 46,XY,del(5)(q15q33) [7] /46,XY [12]
23 R158L 45XY,del5(q13q33), der(16;17)(p10;q10) [11] /44,sl,-Y [5]

Overall Response rate to AZA was 10/23 (44%) in patients with TP 53 mutation, and 20/39(51%) in wild type cases (p= 0.60). The CR rate was 5/23 (22%) in patients with TP 53 mutation, and 15/39(38%) in wild type cases (p= 0.26). On the other hand, OS was negatively influenced by the presence of TP53 mutation (median OS 12.4 months versus 23.7 months in non mutated cases, p < 10−4, figure 1 A), abnormal cytogenetics (median OS 14.4 months vs 33 months in patients with normal karyotype, p = 0.02), complex cytogenetics (median OS 12.7 months versus 23.7 months in patients without complex karyotype, p= 0.0005), a diagnosis of AML with 20-30% marrow blasts (median 14.5 months vs 21.2 months in MDS or CMML, p = 0.02, figure 1 B). Prognostic factors for OS are reported in Table 3 .

gr1

Figure 1 Overall survival according to TP53 mutational status in patients treated with Azacitidine. (A) All patients. (B) in patients with MDS according to WHO classification.

Table 3 Prognostic factors for overall survival.

  All patients (n = 62)
  Univariate analysis Multivariate analysis
  Median OS (months) p HR [95% CI] p
Gender        
Female 17.5 0.28    
Male 14.1      
WHO        
MDS/CMML 21.2 m 0.02 1.36[0.62- 3.0] 0.44
AML 14.5      
marrow blasts        
<15%        
>15% 23.7 0.05 1.41[0.61- 3.25] 0.40
  15      
Cytogenetics        
Normal 33 m vs 14.4 0.02 0.56[0.19-1.63] 0.29
Complex 12.7 vs 23.7 0.0005 1.37[0.60-3.16] 0.44
TP53 mutation 12.4 m vs 23.7 <10-4 2.89[1.38-6.04] 0.005

By multivariate analysis, only TP53 mutational status (HR 2.89 (95% confidence interval 1.38-6.04; p = 0.005) retained statistical significance for OS. Results were similar when the analysis was restricted to MDS and CMML patients, excluding AML (HR = 2.46 (95% confidence interval:1.1- 6.4); p = 0.04)).

5. Discussion

In this work, we showed presence of TP53 mutation to constitute the only independent prognostic parameter of poorer survival in MDS, AML of the elderly with 20-30% blasts considered unfit for intensive chemotherapy and advanced CMML patients treated with AZA. Those patients fulfilled criteria for AZA treatment in the European Union, except for a few patients with lower risk MDS, with anemia resistant to EPO, or with del 5q and resistant to lenalidomide, known to have poor prognosis [22], [23], and [24].

The FASAY method used in this work [19] is a functional method that can detect TP53 mutations through their loss of p53 transcriptional activity in a yeast system. By comparison with methods detecting TP53 mutations through p53 protein stabilisation, FASAY has the advantage of also detecting mutations leading to decreased or absent of p53 protein (particularly stop codon mutations). Moreover, detecting p53 accumulation, by immunohistochemical methods, does not necessarily reflects a mutated TP53 gene and can lead to misinterpretations. While the sensitivity of the FASAY technique is only 10%, this restriction may not be a major issue in high risk MDS and AML with TP53 mutations where, particularly due to loss of the TP 53 wild type allele (through del 17p) in most cases, the proportion of the mutated allele is generally high[8] and [9]. In addition we found, in a preliminary work in MDS samples analyzed with both FASAY and NGS methods, that only one of 47 patients with negative FASAY had a TP 53 mutation detected by NGS (manuscript in preparation). In that patient, with low-risk MDS and del 5q, the mutated clone size was very small (3%). Finally the FASAY test, in the present study, was not associated with false positive results in any patient

The impact of TP53 mutation on response to azacitidine has not so far been well established. In our work, TP53 gene mutations were not significantly associated with response, although there was a trend for lower CR rate in mutated cases. Recently, Kulasekararaj et al. [13] also found no influence of TP53 mutations on response to AZA, although responses were short in mutated cases. In their paper the correlation between TP53 mutation and survival with AZA was not reported. We found TP53 gene mutations to be associated with significantly shorter OS, other poor prognostic factors of shorter OS being presence of a complex karyotype and of greater than 20% marrow blasts. The 2 last factors confirmed our previous prognostic analysis in MDS/low blast count AML treated with AZA [4] .

In multivariate analysis for OS, only TP53 mutation persisted as prognostic factor, while cytogenetics and marrow blast percentage lost their prognostic value.

TP53 mutations, especially when associated with loss of the remaining TP53 allele through 17 p deletion, are associated to resistance to chemotherapy in many cancers including AML and MDS, in particular because they appear to reduce chemotherapy induced cell apoptosis [18] . How TP53 mutation and/or inactivation may influence response to hypomethylating agents is currently unknown. Biological studies have shown that mutant TP53 alters DNA methylation, leading to gene activation or silencing. When re-introducing wild-type p53 in those experiments, expression of these genes could be improved and when combined to azacitidine, normal expression of these genes could be restored[25] and [26]. Another study showed that colon tumor cell lines expressing wild-type p53 were more sensitive to azacitidine than those expressing mutant p53 [27] . Consequently, response to azacitidine could be worse in patients with mutant p53. Finally, the mechanism of action of azacitidine, even at the relatively low doses used in MDS, appears to also include apoptosis induction[28] and [29], which could be abrogated by p53 inactivation.

Thus, overall, in the present work, TP53 mutations had no impact on response to AZA in patients with high risk MDS, but they strongly correlated with a poorer OS and appeared as the strongest prognostic factor.

Acknowledgement

CB, MS and MJM recruited the patients and collected clinical data; CB, AR and JLC performed the laboratory work for this study; LA performed the statistical analysis; PF, CP, HDT and JLC co-ordinated the research; CB, LA, PF and JLC wrote the paper. The authors report no potential conflicts of interest.

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Footnotes

a Department of Hematology, Assistance Publique- Hôpitaux de Paris (AP-HP), Hôpital Saint Louis, Université Paris 7, France

b Laboratoire d’Hématologie, Centre de Biologie-Pathologie, CHRU de Lille, France

c Laboratoire d’Hématologie, Assistance Publique- Hôpitaux de Paris (AP-HP), Hôpital Avicenne, Bobigny, France

d Laboratory for molecular biology and cytogenetics, Institut Paoli-Calmettes, Marseille, France

e Univ Paris Diderot, Sorbonne Paris Cité, CNRS UMR7212/INSERM U944, Paris, France

f AP-HP, Hosp Saint-Louis, Laboratory of Biochemistry, Paris, France

lowast Corresponding author.