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Mutations With Loss of Heterozygosity of p53 Are Common in Therapy-Related Myelodysplasia and Acute Myeloid Leukemia After Exposure to Alkylating Agents and Significantly Associated With Deletion or Loss of 5q, a Complex Karyotype, and a Poor Prognosis

From the Section of Hematology and Oncology, Cytogenetic Laboratory, and Department of Clinical Genetics, The Juliane Marie Center, Rigshospitalet, Copenhagen, Denmark.

Address reprint request to Debes H. Christiansen, MS, Section of Hematology/Oncology, Department of Clinical Genetics, Section 4052, The Juliane Marie Center, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen Ø, Denmark; email: Debes{at}rh.dk

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To study mutations and loss of heterozygosity (LOH) of p53 in therapy-related myelodysplasia (t-MDS) and acute myeloid leukemia (t-AML).

PATIENTS AND METHODS: Fifty-two unselected patients with t-MDS and 25 patients with t-AML were studied by polymerase chain reaction (PCR)–single-strand conformational polymorphism (SSCP) at the DNA level and by reverse transcriptase (RT)-PCR–SSCP at the mRNA level, and cases with aberrant SSCP patterns were sequenced.

RESULTS: Somatically acquired mutations of p53 were observed in 21 of 77 cases of t-MDS or t-AML, and 19 of these 21 patients had received alkylating agents. Single-base substitutions at A:T pairs were more common in t-MDS and t-AML, whereas single-base substitutions at G:C pairs are most common in MDS and AML de novo and in solid tumors. Six patients demonstrated a cytogenetic loss of 17p13, and these six and an additional nine patients with p53 mutations demonstrated LOH of p53 at the DNA or mRNA level. This suggests a cytogenetic loss of the normal p53 allele in these nine cases combined with duplication of the homologous chromosome 17 carrying the mutated p53 allele. Mutations of p53 were significantly associated with deletion or loss of 5q (P < .0001) and a complex karyotype (P = .0001), but surprisingly were not associated with deletion or loss of 7q (P = .73), and were infrequent in patients with balanced chromosome translocations (P = .03). Mutations of p53 were more common in older patients (P = .036) and were associated with an extremely poor prognosis (P = .014), apparently restricted to the 15 cases with LOH of p53 ( P = .046).

CONCLUSION: Mutations with loss of function of p53 are significantly associated with deletion or loss of 5q in t-MDS and t-AML after previous treatment with alkylating agents and are associated with genetic instability.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
MUTATIONS OF the p53 tumor-suppressor gene located on chromosome band 17p13 are important in the multistep process of malignant transformation. Whereas germline p53 mutations as observed in Li-Fraumeni syndrome are very rare, somatically acquired p53 mutations are observed in more than 50% of patients with solid tumors^1,2 and have been related to tumor progression and a poor prognosis.³ DNA damage of several types activates the p53 protein, resulting in a p53-dependent cell cycle arrest at the G₁ and G₂ cell cycle checkpoints, allowing time for DNA repair.^4,5 If the DNA is not repaired, the p53-dependent apoptotic pathway is activated.^4,5 Loss of p53 function may result in increased frequency of mutations,⁶ chromosomal abnormalities,⁷ gene amplification,^8,9 deregulation of centrosome replication with abnormal chromosome segregation,¹⁰ and disruption of the mitotic spindle checkpoint control.¹¹ Thus, p53 is of major importance for genetic stability and genomic integrity of the cell, and a normal p53 function prevents malignant transformation.^5,12

Characteristic spectra of p53 mutations have been observed in hepatocellular carcinoma associated with dietary aflatoxin B1 intake, in skin cancer associated with sunlight exposure, and in lung cancer associated with cigarette smoking.^1,13 In myelodysplasia (MDS) and acute myeloid leukemia (AML) de novo single-base substitutions at G:C pairs predominate, and the mutational spectrum does not show a unique pattern if compared with other hematologic malignancies or solid tumors.^13,14 In patients with MDS and AML de novo, mutations of p53 have been observed in less than 10%, often associated with loss of chromosome band 17p13,^15,16 a complex karyotype, resistance to chemotherapy, and a short survival.¹⁷ Interestingly, many cases with deletion of 17p and mutation of p53 have been reported also to present loss or deletion of the long arm of chromosomes 5 and 7,^16-21 and a cooperation between loss of p53 function and loss of a putative tumor suppressor gene at 5q has recently been suggested.^19,22

By comparison, the mutational spectrum of p53 in therapy-related MDS (t-MDS) and AML (t-AML) is possibly of greater interest, because most of these cases are directly induced by previous chemotherapy with alkylating agents, platinum derivatives, or topoisomerase II inhibitors.²³ These agents each have their unique cellular mechanism of action, and they are mutagenic, clastogenic, and carcinogenic.^24,25

We examined 77 unselected patients with t-MDS or t-AML for mutation of p53 at the DNA level by polymerase chain reaction (PCR)–single-strand conformational polymorphism (SSCP) and at the mRNA level by reverse transcriptase (RT)-PCR–SSCP. Cases with aberrant SSCP patterns were sequenced to confirm and classify the mutations. The mutational spectrum of p53 was related to type of previous treatment and to the mutational spectrum of MDS and AML de novo.² Furthermore, we evaluated loss of heterozygosity (LOH) of p53 by conventional cytogenetics, by fluorescence in situ hybridization (FISH), and by SSCP at the DNA and mRNA levels. Finally, we related the results to other cytogenetic findings and to clinical parameters of all 77 patients, in order to define more precisely the significance of p53 mutations in t-MDS and t-AML

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells Studied
Bone marrow cells or leukemic blasts from peripheral blood of 52 unselected patients presenting with t-MDS and 25 patients presenting with overt t-AML were obtained at diagnosis and stored in liquid nitrogen until the present study. The studies were performed on mononuclear cells obtained by Ficoll-Hypaque fractionation for the first 62 patients, whereas unfractionated white cells were studied in the last 15 patients. The clinical and cytogenetic characteristics of the first 71 of these 77 patients diagnosed and treated at our institution have been described in detail previously as cases 58, 64, 73, 78, 79 to 84, 86 to 89, 91, 93 to 95, 97, 98, 100, 103 to 106, 108, 110 to 112, 114 to 119, 122 to 124, 129, 132, 135 to 139, 142, 143, 145, 150, 151, 154 to 156, 159, 160, 162 to 170, 172, and 175 to 180.^26-28

Cytogenetic and FISH Analysis
Cytogenetic studies were performed on aspirates from the iliac crest using standard trypsin-Giemsa banding technique after a 1- to 2-day culture of the bone marrow samples. FISH analysis was performed in all cases with mutation of p53 or a cytogenetic loss or deletion of 17p if bone marrow material was available. A p53-specific probe was hybridized to interphase or metaphase cells using standard procedures (Vysis, Downers Grove, IL). Furthermore, a painting probe for whole chromosome 17 (Vysis) was applied to metaphases in selected cases. FISH signals were visualized by a Zeiss Axioskop epifluorescence microscope (Oberkochen, Germany), and images were captured by the Quips Smart Capture FISH imaging software (Vysis). For each patient, 200 interphase cells and/or 25 metaphase cells were analyzed if material was available. In the interphase analyses, deletion of p53 was diagnosed if only one fluorescence signal was present in at least 10% of the analyzed nuclei. This cutoff level had been determined in studies of cells from normal controls.

PCR Amplification of cDNA and Genomic DNA
Total RNA and genomic DNA was extracted from cryopreserved cells with the TRIzol reagent (Gibco Life Technologies, Grand Island, NY). First-strand cDNA synthesis of 1 µg of total RNA was performed with the SuperScript II RNase H^- Reverse Transcriptase kit (Gibco).

For each patient, 0.5 µL of the cDNA solution (RT-PCR) and 25 ng of genomic DNA (PCR) were amplified by PCR in separate tubes in a total volume of 25 µL containing 1x PCR buffer II (50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3), 1.5 mmol/L MgCl₂, 0.1 mmol/L dNTP (Pharmacia Biotech, Uppsala, Sweden), 10 µmol/L of each primer (DNA Technology, Aarhus, Denmark), and 0.75 units of AmpliTaq polymerase (Perkin-Elmer, Foster City, CA). Nested RT-PCR was performed, in the first round with the forward/reverse primers: 5'-CAGACTGCCTTCCGGGTCAC-3'/5'-GGAGGCTGTCAGTGGGGAAC-3' and in the second round with the forward/reverse primers: 5'-TCTTGCATTCTGGGACAGCC-3'/5'-AACATCTCGAAGCGCTCACG-3', which generated a band of 685 bp encompassing exons 5 to 9. Before SSCP analysis, the RT-PCR fragments were digested with Sau96I (New England BioLabs, Hitchin, England) to generate three fragments of 182, 227, and 276 bp. PCR of exons 2 through 10 of the p53 gene was performed using the following intronic, forward/reverse primers: 5'-TGCCTTCCGGGTCACTGCC-3'/5'-AGCCCTCCAGGTCCCAGCC-3' for exons 2 and 3, 5'-ATCTACAGTCCCCCTTGCCG-3'/5'-GCAACTGACCGTGCAAGTCA-3' for exon 4, 5'-GCTGCCGTGTTCCAGTTG-3'/5'-ACCAGCCCTGTCGTCTCTC-3' for exon 5, 5'-CCAGGCCTCTGATTCCTCAC-3'/5'-GCCCCCCTACTGCTCACC-3' for exon 6, 5'-GCCACAGGTCTCCCCAAG-3'/5'- TGTGCAGGGTGGCAAGTG-3' for exon 7, 5'-TGCCTCTTGCTTCTCTTTTCC-3'/5'-GGCATAACTGCACCCTTGG-3' for exon 8, 5'-GCGGTGGAGGAGACCAAG-3'/5'-GCTACAACCAGGAGCCATTG -3' for exon 9, and 5'-CCCCCTCCTCTGTTGCTGC-3'/5'-GGCAGGGGAGTAGGGCCAG-3' for exon 10. RT-PCR and PCR conditions were as follows: touchdown for 10 cycles including denaturation at 94°C for 30 seconds, annealing at 70°C for 30 seconds (which was decreased by 1°C in each cycle), and extension at 72°C for 30 seconds, followed by 30 cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes.

Identification of p53 Mutations
To identify p53 mutations, SSCP analysis was performed on digested RT-PCR fragments (RT-PCR–SSCP) and PCR fragments (PCR-SSCP). The fragments were loaded on PhastGel homogeneous 20% gels (Pharmacia Biotech) and run with PhastGel native buffer strips (Pharmacia Biotech) on the PhastSystem (Pharmacia Biotech) according to Separation Technique File no. 131 (Pharmacia Biotech). Each sample was run at 20°C, 12°C, and 4°C to maximize chances of detecting mutations. After electrophoresis, gels were silver-stained to visualize the SSCP bands according to the PhastSystem Development Technique File no. 210 (Pharmacia Biotech). All PCR fragments with abnormal SSCP patterns were confirmed by a second independent amplification and SSCP, and subsequently subjected to direct DNA sequencing. Before sequencing, PCR fragments were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). PCR fragments were sequenced in both directions using the aforementioned primers with the ABI Prism Dye Terminator Cycle Sequencing kit (Perkin-Elmer) and analyzed on an ABI Prism 377 Genetic Analyzer (Perkin-Elmer).

LOH Analysis
PCR-SSCP and RT-PCR–SSCP were applied to analyze LOH at the DNA and mRNA levels in the cases with p53 mutations. Cases with total absence of (or only faintly visible) SSCP bands, representing the wild-type p53 allele, were considered to present LOH. These results were confirmed by the direct DNA sequencing of PCR or RT-PCR fragments performed to verify the mutations.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Frequency and Spectrum of p53 Mutations
Aberrantly migrating SSCP bands, confirmed by sequencing to represent mutated p53 alleles, were observed in 21 (27%) of 77 patients with t-MDS or t-AML ( Table 1). The mutations were located within exons 5 to 8 of the p53 gene in 19 patients and in the conserved splice sites in intron 8 in two patients (cases 111 and N). Single-base substitutions were observed in 14 patients, small deletions in three patients (cases 80, 123, and 151), a single-base insertion in one patient (case 166), and a tandem mutation in one patient (case 100). The single-base substitutions and the tandem mutation resulted in single–amino acid substitutions in these 15 patients, whereas the splice site mutations, the deletions, and the insertion in the other six resulted in frameshifts and protein truncation.

View larger version (87K): [in this window] [in a new window]   Fig 1. Representative SSCP bands of exon 6 of p53 demonstrating variation in LOH at DNA and mRNA levels. Lanes 1 and 5 are normal controls. Lane 2 (case 58) demonstrates heterozygosity for p53 at DNA and mRNA level, lane 3 (case 115) demonstrates LOH at DNA and mRNA level, and lane 4 (case 155) demonstrates LOH at the mRNA level only.

Mutations of p53 and Chromosome Aberrations
The relationship between mutations of p53 and the most important recurrent chromosome abnormalities in 77 patients with t-MDS and t-AML is shown in Tables 1 and 5. All eight patients with a normal karyotype presented a wild-type p53. A complex karyotype with three or more chromosome aberrations was significantly associated with mutations of p53 (P = .0001). Mutations of p53 were also significantly associated with deletion or loss of 5q or monosomy of chromosome 5 (15 of 21 v 4 of 56, P < .0001) ( Table 5). On the other hand, the most common cytogenetic abnormality in t-MDS and t-AML, deletion or loss of 7q or monosomy of chromosome 7, was not associated with p53 mutations (11 of 21 v 25 of 56, P = .73). Furthermore, patients with balanced chromosome aberrations rarely presented p53 mutations (P = .03). p53 mutations aside, significant associations were observed between the presence of a complex karyotype and deletion or loss of 5q and 17p (P < .0001 and P = .005, respectively), whereas a similar association was not observed for deletion or loss of 7q (P = .56).

View larger version (14K): [in this window] [in a new window]   Fig 2. (A) Survival of 21 patients with t-MDS or t-AML and with p53 mutations as compared with 56 patients without mutations of p53 (Kaplan-Meyer estimate). (B) Survival of 15 patients with p53 mutations and LOH of p53 as compared with six patients with p53 mutations and heterozygosity of the gene.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Mutations of p53 were observed in 27% of our patients with t-MDS or t-AML as compared with only 7% in previous studies in MDS or AML de novo.^29-40 Our results thus confirm the findings in two smaller series of adult patients with t-MDS and t-AML^21,41 and in a study of t-MDS or t-AML with chromosome 17p deletions²⁰ in which p53 mutations likewise were common. In another study of pediatric cases of t-MDS and t-AML,⁴² p53 mutations were rare. In the latter study, however, most patients had previously been treated with DNA topoisomerase II inhibitors without alkylating agents, and the leukemias predominantly presented balanced chromosome translocations. In our study, p53 mutations were significantly less common in younger patients (P = .036) and likewise rare in the subgroup of patients with t-AML and balanced translocations (P = .03). Mutations of p53 were observed more frequently in patients treated without radiotherapy (P = .019) (Table 6) and were apparently related to previous therapy with alkylating agents, as 19 of 21 patients with mutations of p53 had previously received either a classic alkylating agent or cisplatin. Only seven patients had received DNA topoisomerase II inhibitors, in all cases combined with either an alkylating agent or cisplatin.

The mutational spectrum of p53 in t-MDS and t-AML has not been discussed previously. In the present study the spectrum was demonstrated, as far as base substitutions in general are concerned, to differ significantly from that of MDS and AML de novo (Table 2). Our results so far support experimental studies analyzing the preferential sites for substitution in DNA and the mutational spectrum for different alkylating agents. Only two of 21 patients with p53 mutations presented A:T-to-C:G transversions (cases 98 and 172) and both were previously treated with the aromatic alkylating agent chlorambucil. This drug has been shown preferentially to substitute at adenine N³ in the minor groove of the DNA and to induce transversions at A:T pairs.^43-45 Single-base substitutions at G:C pairs were observed in five patients previously treated with mechlorethamine, lomustine, or cisplatin. These drugs have been shown preferentially to substitute at guanine O⁶ in the major groove of DNA and to induce transitions at G:C pairs.^46-48 Two patients previously treated with cisplatin and without other alkylating agents (cases 178 and N) were the only two to present single-base substitutions at a 5'-AGG-3' sequence (underlined in Table 1) in the nontranscribed strand. This abnormality has been demonstrated to be common in an in vitro study of cisplatin-induced single-base substitutions.⁴⁸ As far as cyclophosphamide is concerned, our results suggest that this drug, like the aromatic alkylating agents, preferentially could induce single-base substitutions at A:T pairs. If confirmed, these results are of importance as a possibly new example of a significant association between previous treatment with a specific leukemogenic drug and subsequent development of t-MDS and t-AML with characteristic genetic abnormalities.

In many types of tumor, including MDS and AML de novo, mutations of p53 have often been associated with a cytogenetic loss of the other nonmutated p53 allele. This was also the case in the present study, and supplementary FISH added only sparse information to conventional cytogenetics, as microdeletion of the p53 gene was not observed in any of our patients. Unlike most other studies, and because cytogenetics may not reveal all cases with LOH of p53, we also examined our cases with p53 mutations for LOH at the DNA level by PCR-SSCP and at the mRNA level by RT-PCR–SSCP. If positive, we performed direct sequencing of the PCR and RT-PCR fragments. By means of all these methods, our study demonstrated LOH of p53 in 15 of the 21 patients with p53 mutations, with major variations according to the technique used (Table 4). The fact that three of the six cases with cytogenetic loss of band 17p13 and one FISH signal for p53 in all cells showed heterozygosity for p53 at the DNA level most likely relates to a mixture of leukemic and nonleukemic hematopoietic precursor cells. At the mRNA level, however, no wild-type p53 mRNA was detected in any of the six patients with cytogenetic loss of the p53 gene. As previously discussed in solid tumors⁴⁹ and de novo leukemias,⁵⁰ this probably reflects a very low level of p53 mRNA in normal hematopoietic cells, as compared with a high level in leukemic cells.

By analogy with the experience from other recessive tumor-suppressor genes such as the Rb-1 gene⁵¹ and the WT1 gene,^52,53 the LOH of p53 at the DNA level in the four patients with apparently normal chromosomes 17 and two FISH signals for p53 may be explained by a cytogenetic loss of the wild-type p53 allele combined with a chromosomal reduplication including the mutated p53 allele. Previous studies of patients with Li-Fraumeni syndrome support such a mechanism for p53.⁵⁴ Finally, the lack of wild-type p53 mRNA in an additional five patients with cytogenetically normal chromosomes 17 and heterozygosity of p53 at the DNA level most likely also reflects LOH attributable to cytogenetic deletion and duplication of the p53 gene. In conclusion, our results indicate that in t-MDS and t-AML a substantial number of patients with mutations of p53 present LOH of the gene despite cytogenetically normal chromosomes 17. These cases can best be detected at the mRNA level, which probably circumvents the problem of contamination with normal hematopoietic cells. The observed difference in survival between the 15 patients with p53 mutations and LOH of the gene, and the six patients heterozygous for the mutated p53 gene (Fig 2B), although only significant in a one-sided test, supports the clinical importance of LOH of p53 as determined in the present study. Likewise, it is noteworthy that two of three patients with p53 mutations and only up to two chromosome aberrations did not show LOH of the gene (cases 58 and 178).

From a clinical point of view, mutations of p53 are important because, in patients with LOH, the survival is so short that therapy other than the traditional combination of an anthracycline with cytarabine should be considered. In younger patients with an HLA-matched sibling donor, immediate allogenic bone marrow transplantation is an option,⁵⁵ without first attempting to bring the patient into complete remission. Also, high-dose chemotherapy and total-body irradiation followed by tandem autologous stem cell transplantation must be considered, and for the future, mutations of p53 may allow new types of gene-based therapy. The present study confirms that t-MDS and t-AML still represent an important model for studies of the etiology and pathogenesis of MDS and AML, with alternative genetic pathways. The reason for the close association between mutations of p53 and deletion or loss of 5q previously also observed in solid tumors^56-58 remains to be determined.

	ACKNOWLEDGMENTS

 
Supported by grants from the Danish Cancer Society, HS forskningspulje 1997, and Anders Hasselbalchs Foundation.

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TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted June 2, 2000; accepted November 16, 2000.

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