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| ORIGINAL ARTICLE |
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| Year : 2012 | Volume
: 37
| Issue : 4 | Page : 252-256 |
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Role of P53 deletion in patients with acute myeloid leukaemia
Mona A. Ismail1, Tamer M. Ahmed2, Iman Z. Ahmed2
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Ain Shams University, Cairo, Egypt
| Date of Submission | 02-Jul-2012 |
| Date of Acceptance | 14-Jul-2012 |
| Date of Web Publication | 21-Jun-2014 |
Correspondence Address:
Mona A. Ismail
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.7123/01.EJH.0000419282.79533.ff
Background
Loss of the P53 tumour suppressor gene located on the short arm of chromosome 17 (17p13.1) is frequently associated with aggressive disease courses and drug resistance. The prognostic association of P53 deletion has been confirmed for chronic myeloid leukaemia, but not for acute myeloid leukaemia (AML).
Aim of work
The aim of the current study was to investigate the prognostic value of P53 deletion in de-novo patients with AML and to correlate this with known prognostic parameters as well as clinical outcome.
Patients and methods
This study was conducted on 91 patients with de-novo AML meeting WHO criteria for AML. The fluorescence in-situ hybridization technique was applied for detection of P53 deletion at the time of diagnosis. Follow-up studies were carried out to evaluate therapeutic response and clinical outcome.
Results
Loss of P53 was detected in eight out of 91 (8.8%) patients with AML. Among them seven (87.5%) had complex aberrations. A significant difference was detected between P53 deletion positive and negative patients as regards age and total leukocyte count. A highly significant association was detected between P53 deletion and incomplete remission. Interestingly, the coexistence of P53 deletion with old age and high total leukocyte count exhibited a negative impact on the outcome in patients.
Conclusion
P53 deletion can be considered as an indicator of poor therapeutic response in AML patients. Developing new drugs targeting the P53 pathway could be a way to improve treatment of AML. Keywords: acute myeloid leukaemia, fluorescence in-situ hybridization, P53 deletion
How to cite this article:
Ismail MA, Ahmed TM, Ahmed IZ. Role of P53 deletion in patients with acute myeloid leukaemia. Egypt J Haematol 2012;37:252-6 |
| Introduction | |  |
The overall long-term disease-free survival in acute myeloid leukaemia (AML) rarely exceeds 50%, even in younger patients who can receive the most intensive treatment 1. To improve the favourable prognostic rate of AML, morphologic, immunologic, cytogenetic and molecular biological classifications are used. Cytogenetic abnormalities are important prognostic factors in AML and provide a basis for current risk scores. Examination of the P53 gene is not included routinely in AML diagnostics, partly because P53 deletions and mutations are infrequent features. On the basis of the action of new therapeutics on P53 related proteins, the question of whetherprotein P53 analysis will provide important information for the therapy of AML arises 2.
The P53 tumour suppressor gene located on the short arm of chromosome 17 (band 17p13.1) possesses a range of biological activities that may contribute to its role in tumour suppression, including its ability to trigger various cell cycle checkpoints, apoptosis, autophagy, differentiation and cellular senescence 3,4. Moreover, loss of P53 fuels genomic instability, thus further facilitating tumour evolution. P53 is best known for its action in response to cellular stress, whereby it elicits one or more of the above biological responses to limit proliferation. Activation of P53 has two main outcomes: cell cycle arrest to allow for DNA repair and induction of apoptosis 5. Inactivation of P53 is usually caused by loss of one P53 allele and a point mutation in the remaining allele. Deletion of P53 plays an important role in neoplastic transformation in solid tumours and it has also been reported in haematological malignancies in association with progression of the disease 6.
In view of these data, the present study was aimed to assess the prognostic value of P53 deletion in de-novo patients with AML and to correlate this with known prognostic parameters as well as clinical outcomes.
| Subjects and methods | | |
The current study included 91 patients with de-novo AML. They were subclassified into two groups according to their age: children (31 patients) and adults (60 patients). The age in the children group ranged from 1.5 to 8.5 years with a mean value of 4.6±2.1 years; there were 18 male patients and 13 female patients with an M/F ratio of 1.4 : 1. The age in the adult group ranged from 21 to 78 years with a mean value of 47.2±23.2 years; there were 37 male and 23 female patients with an M/F ratio of 1.6 : 1. All patients were diagnosed and followed up at the Ain Shams University Hospitals, Pediatric Clinics and Clinical Hematology/Oncology Unit between December 2007 and December 2011.
All patients were subjected to thorough history and clinical examination, complete blood picture (using LH750 Coulter; Beckman, California, USA), bone marrow aspiration with examination of Leishman-stained blood smears and fluorescence in-situ hybridization (FISH) using the TRITC-labelled locus specific identifier probe 17p13 (P53) for detection of P53 deletion.
Fluorescence in-situ hybridization analysis
Principle
The target DNA sequence and the TRITC-labelled locus specific identifier probe 17p13 (P53) were codenaturated and hybridized, followed by examination using fluorescence microscopy for detection of P53 deletion 7.
Interpretation
Results were interpreted by scanning most of the metaphases and interphases using chromoscan (CytoVision 2.7; Santa Clara, California, USA) to detect the target abnormalities. The case was considered negative if two red signals were detected, positive for heterozygous P53 deletion if one red signal was detected and positive for homozygous P53 deletion if no red signal was detected at all.
All patients were followed up over a study period of 40 months. Patients were reported as responders (R) and nonresponders (NR) at day 14 (for children) and day 28 (for adults) of chemotherapy, respectively. One year disease-free survival (DFS) and overall survival (OS) were calculated using the Kaplan–Meier method. Complete remission (CR) was defined as the presence of morphologically normal bone marrow and at least 1.5×109/l granulocytes and 100×109/l platelets in blood 8.
Statistical analysis
IBM SPSS statistics (V. 20.0; IBM Corp., New York, USA, 2011) were used for data analysis. Data were expressed as median percentiles for quantitative nonparametric measures and as both number and percentage for categorized data.
The following tests were performed:
- The χ2-test to study the association between two variables or to compare two independent groups as regards the categorized data. The probability of error at 0.05 was considered significant, whereas that at 0.01 and 0.001 was considered highly significant.
- Calculated relative risk assessments (relative risk ratio) that measure how many times the risk was present among diseased individuals as compared with nondiseased ones. They were calculated as absolute figures and as a standard error of estimate (95P).
- Survival analysis including survival estimates, confidence intervals, Kaplan–Meier survival curves and overall comparison using the log-rank test.
| Results | | |
The results of this study are summarized in [Table 1], [Table 2] and [Table 3] and [Figure 1], [Figure 2] and [Figure 3]. | Table 1: Association between P53 deletion and standard prognostic factors
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 | Table 2: Association between standard prognostic factors in relation to therapeutic response
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 | Table 3: Association between P53 deletion and survival of patients with acute myeloid leukaemia
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 | Figure 1: Interphase fluorescence in-situ hybridization technique showing a positive P53 deletion denoted by the presence of one red signal.
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 | Figure 2: Interphase fluorescence in-situ hybridization technique showing negative P53 deletion denoted by the presence of two red signals.
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 | Figure 3: Kaplan–Meier curve illustrating overall survival (OS) and disease-free survival (DFS) for all patients distributed according to P53 deletion. Cum survival, cumulative survival.
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Interphase FISH analysis of the 91 samples was successfully carried out using the LSI 17p13 (P53) probe, revealing positive results for heterozygous P53 deletion in 8/91 (8.8%; [Figure 1]) samples, 4/31 (12.9%) in the childhood AML group and 4/60 (6.6%) in the adult AML group, and negative results in 83/91 (91.2%) samples [Figure 2]. No positive samples for homozygous deletions were detected.
A significant difference was detected between P53 positive and negative patients as regards total leukocyte count (TLC) and age, where a highly significant association was detected between P53 deletion and TLC at least 50×109/l (87.5%; P-value<0.001). In addition, further subdivision of adults into young adults (<60 years) and elders (≥60 years) revealed that elders show a highly significant association with P53 deletion and a poor response to chemotherapy [Table 1],whereas a nonsignificant association was found with other standard prognostic factors.
Furthermore, studying the association between other cytogenetic aberration patterns and P53 deletion revealed a significant association in which 7/8 (87.5%) patients with P53 deletion had a complex aberrant karyotype and only 5/83 (6%) patients negative for P53 deletion were associated with complex karyotyping [Table 1].
Patients were followed up after the first induction to detect their response to therapy. Out of the 91 patients, 55(60.4%) were responders (achieved CR), whereas 36/91 (39.6%) were nonresponders (had incomplete remission). With regard to the relationship between the standard prognostic factors and therapeutic response, a significant association was observed between poor therapeutic response and age greater than 60 years, TLC at least 50×109/l and complex karyotyping [Table 2].
In this study, a highly significant association was detected between P53 deletion and the poor response of patients to chemotherapy (P-value=0.000). All patients with P53 deletion were nonresponders to chemotherapy, whereas 55/83 (66.3%) patients without deletion responded to chemotherapy [Table 2].
There was a highly significant association between P53 and clinical outcome, in which OS and DFS were higher for P53 negative patients when compared with those in the positive group (P=0.001 and 0.002; [Table 3]).
Patients were followed up over a period of 40 months to evaluate their outcome. Using Kaplan–Meier curves, all patients with P53 deletion were estimated to have an OS less than 23 months and DFS less than 18 months. In contrast, 35/83 (42.2%) and 46/83 (62.2%) patients negative for P53 deletion had OS and DFS greater than 18 months and greater than 23 months, respectively (P-value=0.002; [Table 3] ; [Figure 3]).
Multivariate analysis of patient outcomes and other parameters in the studied patients revealed that P53, age and presenting TLC were most significantly associated with CR (data not shown).
| Discussion | | |
Although the outcome in patients with AML has improved significantly, the majority of patients still die from their disease, mainly as a result of primary or acquired drug resistance 9. The evaluation of the prognostic impact of many clinical and genetic features has established the basis for risk adapted treatment approaches in patients with AML 10.
P53 is a sequence-specific transcription factor that can halt progression through the cell cycle or initiate apoptosis on activation by genomic stimuli 11. Inactivation of P53 plays an important role in neoplastic transformation in solid tumours and it has been reported in haematological malignancies in association with progression of the disease 12,13.
In the current study, a deletion of 17p13 was detected by the FISH technique in 8/91 (8.8%) patients with de-novo AML; a similar incidence was detected previously in about 9% of AML patients 14. However, Byrd and colleagues 8,15 reported a lower incidence of 5% in a large study that included 1213 AML patients. In a previous study on patients with myelodysplastic syndromes and AML, the tumour suppressor effect of the P53 gene was lost through mutation of the remaining P53 allele in 16 out of 17 patients examined (94%) 16,17. The variation in the P53 deletion frequency may be because of differences in FISH efficiency or intrinsic differences in the different patient populations examined.
Patients were subdivided according to age (<60 and ≥60 years); a significant difference was found between the two groups as regards P53 deletion, where 50% of the patients with P53 deletion were at least 60 years of age. In addition, a positive association was found between P53 deletion and TLC, in which 87.5% of patients showed a TLC of at least 50×109/l. This was consistent with other studies 18 that found a significant association between P53 deletion and both parameters and also found that TLC, age, complex aberrations and P53 deletion were strong factors for poor OS.
With regard to the cytogenetic results, a statistically significant difference was found between P53 deletion and complex karyotyping, where 7/8 (87.5%) patients with P53 deletion were associated with complex karyotyping. Similar results were found by other authors 19–22, who observed a positive association between P53 deletion and established high risk aberrations (del 5q, −5, −7).
The loss of 17p in AML patients is often accompanied by a P53 mutation, resulting in a loss of heterozygosity 23,24. Several studies 25,26 reported P53 deletions with a wild-type configuration of the remaining allele. Data from experiments on mice revealed that loss of one P53 allele could be sufficient for tumourigenesis 27. Furthermore, overexpression of genes inhibiting P53 (MDM2) and promoting degradation of P53 can be considered.
Moreover, an interesting investigation by Sankar et al. 28 revealed only 3/17 patients presenting P53 inactivation on FISH, in contrast to 14 patients with locus 17p13.3 deleted. Thus, other tumour suppressor genes on the short arm of chromosome 17 may be involved in the development of leukaemia.
The current study revealed a significant association between P53 deletion and the response to therapy, in which all the patients with P53 deletion had failed to achieve remission. These data were consistent with those reported by other researchers who found that the P53 complex is an essential factor as regards response to chemotherapy 29 and that the samples with hemizygous P53 deletion were more resistant to all drugs when compared with samples with a normal karyotype. This strongly suggests that normal P53 function is important for chemotherapy-induced apoptosis in AML 30.
In addition, on comparing cells with chromosome 17 abnormalities with samples having complex karyotypes, a significantly greater resistance to almost all the tested drugs was reported among patients with chromosome 17 aberrations. Furthermore, on comparing chromosome 17 abnormalities with other unfavourable karyotypes including monosomy 7 and/or 7q deletion, a more pronounced in-vitro drug resistance was found among patients with chromosome 17 aberrations 30.
Patients were followed up to detect their clinical outcome. A significant association was detected between P53 deletion and clinical outcome, in which patients with P53 deletion had DFS less than 18 months and OS less than 23 months. This was in agreement with the study by Seifert et al. 18 who revealed P53 deletion as an independent negative prognostic factor for DFS, relapsed risk and OS. Nahi et al. 30 found that patients with chromosome 17 abnormalities had a very poor rate of OS, and all patients died within 1 year of diagnosis. In addition, on comparing this group with patients with normal karyotypes, a significantly inferior OS was observed 30.
To assess the predictive value of P53 deletion relative to other pretreatment prognostic factors, a multivariate analysis was performed. This analysis demonstrated that P53, age and presenting TLC were most significantly associated with predicting attainment of CR. These analyses confirm the importance of cytogenetics as a prognostic tool for predicting CR and OS.
This was in agreement with other studies that revealed that patients with a single P53 deletion or with only one additional aberration had a median OS of only 5 months. In multivariate analysis, a single P53 deletion was a strong and independent negative prognostic factor for DFS and OS 30–32.
| Conclusion | | |
Our data derived from patients with de-novo AML and prolonged follow-up show that P53 deletion at diagnosis is predictive of treatment and clinical outcome. Because of poor outcome, it is necessary to provide an alternative therapy strategy, other than conventional treatment strategies including allogeneic stem cell transplantation, to patients with P53 deletion. However, further large prospective studies are necessary to confirm the risk of P53 deletion with and without complex aberrant karyotypes.[32]
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
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