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Clinical Cancer Research Vol. 8, 973-985, May 2002
© 2002 American Association for Cancer Research

Advances in Brief

p53 Mutations in Leukemia and Myelodysplastic Syndrome after Ovarian Cancer1

Debra G. B. Leonard, Lois B. Travis, Kathakali Addya, Graca M. Dores, Eric J. Holowaty, Kjell Bergfeldt, David Malkin, Betsy A. Kohler, Charles F. Lynch, Tom Wiklund, Marilyn Stovall, Per Hall, Eero Pukkala, Diana J. Slater and Carolyn A. Felix2

Department of Pathology and Laboratory Medicine [D. G. B. L.], Molecular Diagnostic Core Facility, University of Pennsylvania Cancer Center [D. G. B. L., K. A.], Division of Oncology, The Children’s Hospital of Philadelphia [D. J. S., C. A. F.], and Department of Pediatrics [C. A. F.], University of Pennsylvania School of Medicine, Philadelphia, PA 19104; Division of Cancer Epidemiology and Genetics [L. B. T., G. M. D.] and Division of Cancer Prevention [G. M. D.], National Cancer Institute, Bethesda, MD 20892; Cancer Care Ontario, Toronto, Ontario, M5G 2L7 Canada [E. J. H.]; Karolinska University Hospital, Karolinska Institute, SE–177 77 Stockholm, Sweden [K. B., P. H.]; Division of Hematology/Oncology, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, MG5 1X8 Canada [D. M.]; New Jersey Department of Health and Senior Services, Trenton, New Jersey 08625 [B. A. K.]; Department of Epidemiology, University of Iowa, Iowa City, Iowa 52242 [C. F. L.]; Helsinki University Central Hospital, FIN–00290 Helsinki, Finland [T. W.]; The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 [M. S.]; and Finnish Cancer Registry, The Institute for Statistical and Epidemiological Cancer Research, FIN–00170 Helsinki, Finland [E. P.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Patients and Methods
 Results
 Discussion
 REFERENCES
 
Purpose: Although p53 mutations occur in alkylating agent-related leukemias, their frequency and spectrum in leukemias after ovarian cancer have not been addressed. The purpose of this study was to examine p53 mutations in leukemias after ovarian cancer, for which treatment with platinum analogues was widely used.

Experimental Design: Adequate leukemic or dysplastic cells were available in 17 of 82 cases of leukemia or myelodysplastic syndrome that occurred in a multicenter, population-based cohort of 23,170 women with ovarian cancer. Eleven of the 17 received platinum compounds and other alkylating agents with or without DNA topoisomerase II inhibitors and/or radiation. Six received other alkylating agents, in one case, with radiation. Genomic DNA was extracted and p53 exons 5, 6, 7, and 8 were amplified by PCR. Mutations and loss of heterozygosity were analyzed on the WAVE instrument (Transgenomic) followed by selected analysis by sequencing.

Results: Eleven p53 mutations involving all four exons studied and one polymorphism were identified. Genomic DNA analyses were consistent with loss of heterozygosity for four of the mutations. The 11 mutations occurred in 9 cases, such that 6 of 11 leukemias after platinum-based regimens (55%) and 3 of 6 leukemias after other treatments (50%) contained p53 mutations. Two leukemias that occurred after treatment with platinum analogues contained two mutations. Among eight mutations in leukemias after treatment with platinum analogues, there were four G-to-A transitions and one G-to-C transversion.

Conclusions: p53 mutations are common in leukemia and myelodysplastic syndrome after multiagent therapy for ovarian cancer. The propensity for G-to-A transitions may reflect specific DNA damage in leukemias after treatment with platinum analogues.


   Introduction
Top
 ABSTRACT
 Introduction
Patients and Methods
 Results
 Discussion
 REFERENCES
 
Leukemias comprise a small fraction of all second cancers, although they represent the major carcinogenic complication of chemotherapy (reviewed in Ref. 1 ). Since the first observations of alkylating agent-related leukemia and MDS3 (2) , leukemia has become increasingly common after effective chemotherapy (reviewed in Ref. 3 ). Chemotherapy is implicated in AML of several morphological subtypes, MDS, ALL, and even chronic myelogenous leukemia (reviewed in Ref. 1 ). Therapeutic radiation also has been linked to an increased risk of leukemia (4 , 5) .

Two broad classes of cytotoxic drugs are associated with leukemia: alkylating agents and DNA topoisomerase II inhibitors (1 , 3 , 6) . Germ-line mutations in the conserved region of the p53 gene, a critical element in the cellular DNA damage response pathway, have been associated with leukemias after alkylating-agent treatment (7, 8, 9) , suggesting that certain individuals are genetically predisposed. Complex numerical and structural karyotypic abnormalities in alkylating-agent-related leukemias associated with germ-line p53 mutations, typically including loss of chromosomes 5, 7, and 17p, suggest genomic instability, which accompanies loss of wild-type p53 (7, 8, 9) . In a recent study, p53 mutations were observed in the leukemic cells in 21 of 77 unselected cases (27%) of treatment-related AML and treatment-related MDS (10) . Alkylating-agent exposure was documented in 19 of the 21 cases with p53 mutations, indicating that p53 mutations are common molecular alterations in AML and MDS after alkylating-agent treatment (10) . In contrast, in DNA topoisomerase II inhibitor-related leukemias, p53 mutations generally are absent, suggesting that the p53 component of the DNA damage response pathway is intact (7) .

The classic alkylating agents used as anticancer drugs include nitrogen mustard and its analogues (11 , 12) . These agents form covalent bonds with bases in the DNA and can cause interstrand cross-links. Resultant O-6-methylguanine residues are carcinogenic (11 , 12) . Although not a classic alkylating agent, cisplatin, which has been in clinical use since 1969, forms intrastrand N-7-alkyl adducts on adjacent deoxyguanosines or deoxyguanosine and deoxyadenosine (11 , 13 , 14) . Monoadducts and interstrand cross-links can also be formed by cisplatin (11 , 13 , 14) . The platinum analogues cisplatin and carboplatin typically are used in combination with other leukemogenic drugs. In the early 1990s, it was suggested that cisplatin administered with DNA topoisomerase II inhibitors could be leukemogenic (13 , 15) . AML with the t(8;21) translocation was observed after osteosarcoma treatment with cisplatin and the DNA topoisomerase II inhibitor doxorubicin (15) . Cases of AML with translocations involving chromosome band 11q23 or 21q21, recurrent translocations in leukemias related to DNA topoisomerase II inhibitor, were reported after treatment of germ-cell tumors with cisplatin and etoposide (16, 17, 18) . Characteristic of alkylating-agent-related leukemias, other leukemias that occurred after regimens including platinum analogues contained monosomies of chromosomes 5 and 7 (8 , 9 , 19) .

Although platinum analogues are instrumental in ovarian-cancer treatment (20) , they are linked with significantly increased, dose-dependent risks of leukemia (21) . In a large, case-control study of women with ovarian cancer who received cisplatin- or carboplatin-containing regimens, a 4-fold risk of leukemia was observed (trend for cumulative platinum dose, P < 0.001; Ref. 21 ). A similar, highly significant dose-response relationship was found between the cumulative cisplatin dose used in testicular cancer treatment and leukemia risk (22) . However, few studies have described the molecular features of leukemias after successful treatment of ovarian cancer, and data on specific p53 mutations that incorporate cumulative doses of cytotoxic drugs and radiation are sparse. The goal of the present study was to determine the frequency and spectrum of mutations in conserved exons of the p53 gene in cases of leukemia after ovarian-cancer treatment.


   Patients and Methods
Top
 ABSTRACT
 Introduction
 Patients and Methods
Results
 Discussion
 REFERENCES
 
Study Population.
All available tissue samples were requested from five of seven population-based cancer registries that participated previously in an international case-control investigation of leukemia after ovarian cancer (21) . The case-control investigation was conducted within a population-based cohort that included 28,971 women with ovarian cancer in which 96 cases of leukemia were observed (21) . Of the 96, 82 were reported to the five cancer registries participating in the present study (Ontario, Sweden, New Jersey, Iowa, Finland) and occurred within the cohort of 23,170 women with ovarian cancer in these registries. Tissue samples were obtained from 27 (33%) of the 82 cases. There were sufficient archived bone marrow aspirate specimens or, in one case, peripheral blood with evidence of leukemia or MDS for p53 mutation analysis in 17 cases. Ten cases in which materials were available were excluded because of insufficient amount or poor quality of DNA. Molecular studies are difficult on tissues preserved in fixatives other than formalin or alcohol. Bone marrow core biopsies are unusable because decalcification is incompatible with molecular studies. Therefore, several available tissue samples could not be used for the present study. Data on specific cytogenetic aberrations were not available because the diagnoses of leukemia and MDS in the population-based cohort occurred in many different institutions, which were often local hospitals where cytogenetic analyses were not performed routinely.

DNA Preparation.
Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue blocks of bone marrow aspirates. 15–20-µm thick sections were first cut from a control blank block and then from the blocks containing tissue. The sections were deparaffinized with xylenes followed by 100% ethanol. Deparaffinized sections were incubated at 55°C for 1–3 h in 200 µL of a solution containing 288 µg proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, IN) and x1 PCR buffer (PE Biosystems, Foster City, CA). To deactivate the proteinase K, the mixture was incubated at 100°C for 10 min, followed by microcentrifugation at 13,500g for 10 min. The supernatant containing the DNA was removed to a clean, 1.5-ml microcentrifuge tube and used directly for PCR.

For genomic DNA extraction from bone marrow or peripheral blood smears on glass slides, 50 to 100 µL of Cell Lysis Solution (Gentra Systems, Inc., Minneapolis, MN) containing one-tenth volume of 14.4 mg/ml proteinase K solution (Boehringer Mannheim Biochemicals) were added directly to the slides, causing solubilization and lysis of the cells. The solution was transferred to a 1.5-ml microcentrifuge tube and incubated at 55°C for 1–3 h. One-third volume of Protein Precipitation Solution (Gentra Systems) was added and the mixture was vortex-mixed and microcentrifuged at 13,500g for 10 min. The supernatant was transferred to a clean 1.5-ml microcentrifuge tube. After the addition of an equal volume of isopropanol, the mixture was incubated at -20°C from 20 min to overnight and microcentrifuged at 13,500g for 20 min. The supernatant was removed, and the DNA pellet was washed with 70% ethanol, dried, and, depending on the pellet size, resuspended in 30–100 µL of 10 mM Tris/0.1 mM EDTA solution for use in PCR.

PCR Amplification of p53 Exons 5–8.
Exons 5, 6, 7, and 8 of the p53 gene and flanking intronic sequences were amplified in separate PCRs. The 50-µl reaction mixtures contained 1 µl of DNA, sense and antisense primers at 0.2 µM each, 0.2 mM each dNTP, 1.25 units of AmpliTaq Gold DNA polymerase (Perkin-Elmer; Foster City, CA), 1.5 mM MgCl2, 50 mM KCl, and 10 mM Tris-HCl, pH 8.4. A master mixture of all reagents except AmpliTaq Gold DNA polymerase and DNA was made in advance and stored at -20°C for up to 2 months. Similar primers have been used previously (23) . The sense and antisense primers for the p53 exons were as follows: p53 exon 5, 5'-TTCCTCTTCCTACAGTACTC-3' and 5'-GCAACCAGCCCTGTCGTCTC-3'; p53 exon 6, 5'-ACCATGAGCGCTGCTCAGAT-3' and 5'-AGTTGCAAACCAGACGTCAG-3' p53 exon 7, 5'-GTGTTGTCTCCTAGGTTCGC-3' and 5'-CAAGTGGCTCCTGACCTGGA-3'; and p53 exon 8, 5'-CCTATCCTGAGTAGTGGTAA-3' and 5'-TGAATCTGAGGCATAACTGC-3'. Underlined nucleotides in the exon 6 and 7 primers are mismatches with the p53 genomic sequence. Expected PCR-product sizes were 238, 236, 139, and 212 bp for p53 exons 5, 6, 7, and 8, respectively. Amplification was performed in a Perkin-Elmer 9600 thermal cycler (Emeryville, CA). The three-step PCR consisted of an initial cycle at 95°C for 11 min, 55°C for 30 s, and 72°C for 1 min, followed by 44 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, a final extension at 72°C for 3 min and a hold at 4°C. Samples that did not amplify with 1.25 units of AmpliTaq Gold were amplified again with 2.5 units of AmpliTaq Gold and an additional 10 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s for a total of 55 cycles. For amplification of exon 7, 2.5 units of AmpliTaq Gold were used and the cycling included 94°C for 9 min followed by 45 cycles of 94°C for 75 s, 58°C for 75 s, and 72°C for 30 s (increase elongation time by 1 s/cycle) followed by 72°C for 10 min and a hold at 4°C.

Genomic DNA samples with known p53 mutations in exons 5, 6, 7, and 8 were used as positive controls (24, 25, 26) . The positive control DNA for exon 5 was from ALL cells with a TGC-to-TTC transversion at codon 141 (24) . The positive control DNA for exon 6 was from ALL cells with a 2-bp (TA) deletion at codons 214–215 (25) . The positive control DNA for exon 7 was from ALL cells with a TTT-to-TGT transversion at codon 270 (25) . The positive control DNA for exon 8 was from the RD cell line with a CGG-to-TGG transition at codon 248 (26) . DNA from peripheral blood mononuclear cells of a normal subject was used as a negative control.

WAVE Detection System for p53 Mutations.
The presence of a mutation was detected with the WAVE System (Transgenomic, Omaha, NE). The method is based on ion-pair, reverse-phase high-performance liquid chromatography and temperature-modulated heteroduplex analysis. Sequence variations create mismatched heteroduplexes during reannealing of PCR products containing wild-type and mutant sequences. The difference in melting temperature between heteroduplexes and homoduplexes allows for their separation by ion-pair, reverse-phase high-performance liquid chromatography and identification of the presence of mutations. PCR products with mutant sequences were detected with the protocol specified by the manufacturer. First, 5 µL of the PCR product were run under nondenaturing conditions to determine DNA concentrations. PCR products then were mixed 1:1 by concentration with PCR products from negative control DNA to assure detection of mutant sequences in the presence of LOH. The mixtures were denatured at 95°C for 5 min followed by a 45-min ramp to 25°C and incubation at 25°C for 30 s. The denatured, reannealed samples were run on the WAVE instrument with a melting temperature of 63°C and the regular gradient for p53 exon 5, 62°C and the regular gradient for p53 exon 6, 61°C and a gradient shifted by -3° for p53 exon 7, and 63°C and the regular gradient for p53 exon 8. WAVEMaker software was used to calculate the regular gradient for each PCR product on the basis of the length and melting temperature of each DNA fragment.

Sequence Confirmation of Mutations.
The specific sequence alteration in each PCR product with a mutant sequence or polymorphism detected by WAVE analysis was identified by direct sequencing. The PCR product was directly sequenced first. If the mutation was not identified by direct sequencing, the heteroduplex peak from WAVE analysis containing 50% mutant DNA and 50% normal DNA was collected and the collected peak was sequenced. The eluted peak containing the heteroduplex DNA was collected in a fraction collector according to the WAVE System protocol. Mutations in collected peaks were confirmed by direct sequencing in both directions with the Big Dye Terminator Kit (PE Applied Biosystems) and the Prism ABI 310 instrument (PE Applied Biosystems).

A base substitution in the sequence and absence or near absence of the normal sequence was interpreted as evidence of LOH, although the unlikely presence of the same mutation in both p53 alleles was not excluded. Detection of a heterozygous pattern by direct sequencing could have been attributable to either heterozygosity in the leukemic cells or the presence of nonleukemic cells in the specimen. Therefore, in cases with substantial nonleukemic cells, as in MDS, LOH in the leukemic cells could not be excluded by the heterozygous pattern.

Analysis of Sensitivity.
The sensitivity of mutation detection by WAVE analysis for the primers and conditions used was evaluated by serial dilution of DNA samples with known p53 mutations into normal DNA.

Statistical Analyses.
Data analyses were performed by Wilcoxon’s rank-sum test. All statistical tests were two-sided and P < 0.05 was considered statistically significant.


   Results
Top
 ABSTRACT
 Introduction
 Patients and Methods
 Results
Discussion
 REFERENCES
 
The p53 gene was examined in secondary leukemia or secondary MDS specimens from 17 women with primary ovarian cancer. Clinicopathological data including demographic features, all therapy for ovarian cancer, latency and type of leukemia are summarized in Table 1Citation . Cisplatin and/or carboplatin were administered in 11 cases (patients 1, 2, 3, 5, 6, 7, 8, 10, 12, 14, 17) in combination with other alkylating agents in four cases, or in combination with both other alkylating agents and DNA topoisomerase II inhibitors in the other seven. Patient 3 received both cisplatin and carboplatin. Table 1Citation shows the cumulative platinum analogue doses expressed in total milligrams in accordance with the underlying case-control study from which these patients were derived (21) . The median cumulative cisplatin dose administered to the nine patients who received cisplatin (patients 1, 2, 3, 5, 6, 8, 10, 12, 14) was 710 mg (range, 422–910 mg). The median cumulative carboplatin dose administered to the three patients who received carboplatin (patients 3, 7, 17) was 2660 mg (range, 1710–3550 mg), and the median cumulative carboplatin dose expressed as a cisplatin-equivalent dose (27) was 665.0 mg (range 427.5–887.5 mg). In six patients, therapy included alkylating agents but not platinum analogues (patients 4, 9, 11, 13, 15, 16). Three patients also received radiation (patients 5, 8, 11), and the radiation dose to total active bone marrow is shown in Table 1Citation .


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  		Table 1 Clinicopathological features and secondary leukemias in patients with primary ovarian cancer

 
Eight of the 17 patients presented with AML, one with chronic myelogenous leukemia and eight with MDS. Latencies between the diagnosis of ovarian cancer and the diagnosis of leukemia or MDS ranged from 1.3 to 7.8 years (median, 4.0 years). Sixteen of the 17 patients are deceased. The median duration of survival from time of diagnosis of secondary leukemia or secondary MDS to death or end of study was 0.2 year (range, <0.1 to 2.5 years).

Eleven p53 mutations and one known p53 exon 6 polymorphism were detected in 9 of the 17 cases studied (Table 2)Citation . Serial-dilution experiments showed that the presence of 3.25% mutant allele in a wild-type background was detectable by WAVE analysis with the primers and conditions used for p53 exons 5, 6, 7, and 8, indicating that somatic-mutation detection was feasible in specimens containing a significant proportion of nonleukemic cells.


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  		Table 2 p53 mutations in cases of leukemia and MDS after ovarian-cancer treatment

 
Three mutations involved exon 5. The codon 141 TGC-to-TAC transition in the AML of patient 3 was associated with LOH (Table 2Citation ; Fig. 1Citation ) and would result in a Cys-to-Tyr substitution. The French-American-British M2 AML of patient 5 contained both an exon 5 codon 143 GTG-to-ATG transition that would result in a Val-to-Met substitution and an exon 8 codon 274 GTT-to-GCT transition that would change Val to Ala; LOH was not detected at either of these codons. The codon 134 TTT-to-CTT transition in the AML of patient 16 would result in a Phe-to-Leu change and was not associated with LOH.



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  		Fig. 1. Detection of p53 exon 5 mutation in AML of patient 3 using the WAVE system (Transgenomic). A, p53 exon 5 PCR product from AML DNA of patient 3 was mixed with an equal amount of wild-type exon 5 PCR product and analyzed on the WAVE instrument (top panel). Arrows indicate heteroduplex peaks in the AML specimen (top panel) and in the positive control (middle panel). Under the partially denaturing temperature conditions of the assay, heteroduplexes are denatured and eluted from the column before homoduplexes. There was no heteroduplex peak in the negative control (bottom panel). B, direct sequencing of p53 exon 5 PCR product confirmed a codon 141 TGC-to-TAC transition with associated LOH in AML of patient 3 (top panel). The bottom panel shows the normal sequence.

 
One mutation and one polymorphism were identified in exon 6. In patient 10 with RARS, analysis of DNA samples from a paraffin-embedded bone marrow aspirate and from a bone marrow smear, both from the time of MDS diagnosis, indicated a codon 220 TAT-to-TGT transition that would result in a Tyr-to-Cys substitution. LOH was detected in one of the specimens (bone marrow smear slide) containing both normal and dysplastic cells (Table 2Citation ; Fig. 2Citation ). In the RAEB of patient 14, WAVE positivity identified an exon 6 sequence alteration. Direct sequencing was consistent with the known CGA-to-CGG polymorphism involving codon 213 (28) , although a new, silent mutation was not excluded because DNA from normal tissue from the patient was not available for comparison. In the RAEB of the same patient, there also was an exon 7 codon 236 TAC-to-TGC transition that would result in a Tyr-to-Cys substitution with suggested LOH (Table 2)Citation .



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  		Fig. 2. Detection of p53 exon 6 mutation in RARS of patient 10. A, p53 exon 6 PCR product from RARS DNA of patient 10 was mixed with an equal amount of wild-type exon 6 PCR product and analyzed on the WAVE instrument (top panel). Arrows indicate heteroduplex peaks in the RARS specimen (top panel) and in the positive control (middle panel). There was no heteroduplex peak in the negative control (bottom panel). B, direct sequencing indicated that the p53 exon 6 mutation in the RARS of patient 10 was a codon 220 TAT-to-TGT transition with LOH. Normal sequence is shown in the bottom panel.

 
In addition to the p53 codon 236 mutation in the RAEB of patient 14, there were three other mutations involving exon 7. The RAEB of patient 1 contained both an exon 7 codon 248 CGG-to-CCG transversion that would change Arg to Pro (Table 2Citation ; Fig. 3, A and BCitation ) and an exon 8 codon 273 CGT-to-CAT transition that would change Arg to His (Table 2Citation ; Fig. 4, A and BCitation ). LOH was suggested at codon 248, but not at codon 273. Alternatively, if both p53 alleles were present in the affected cells, the same exon 7 mutation may have been present on both alleles (see "Discussion"). The AML of patient 15 contained the same codon 248 CGG-to-CCG transversion with suggested LOH (Table 2)Citation . The AML of patient 4 contained a silent codon 236 TAC-to-TAT transition without LOH (Table 2)Citation .



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  		Fig. 3. Detection of p53 exon 7 mutation in RAEB of patient 1 by WAVE analysis. A, p53 exon 7 PCR product from RAEB DNA of patient 1 was mixed with an equal amount of wild-type exon 7 PCR product and analyzed on the WAVE instrument (top panel). Arrows indicate heteroduplex peaks in the RAEB specimen (top panel) and in the positive control (middle panel). A heteroduplex peak is absent in the negative control (bottom panel). B, direct sequencing of the exon 7 PCR product from the RAEB DNA of patient 1 revealed a codon 248 CGG-to-CCG transversion with LOH (top panel). The bottom panel shows the normal sequence.

 


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  		Fig. 4. Detection of p53 exon 8 mutation in RAEB of patient 1 by WAVE analysis. A, p53 exon 8 PCR product from RAEB DNA of patient 1 was mixed with an equal amount of wild-type exon 8 PCR product and analyzed on the WAVE instrument (top panel). Arrows indicate heteroduplex peaks in the RAEB specimen (top panel) and in the positive control (middle panel). Heteroduplex peak is absent in the negative control (bottom panel). B, LOH was not detected with the p53 exon 8 codon 273 CGT-to-CAT transition in the RAEB of patient 1 as indicated by the normal G peak in addition to the mutant A peak in the sequence. The normal sequence is shown in the bottom panel. Apparent LOH in exon 7, but not in exon 8, may indicate the presence of the exon 7 mutation on both alleles (compare with Fig. 3Citation ).

 
One other p53 exon 8 mutation was identified in addition to the codon 274 mutation in the AML of patient 5 and the codon 273 mutation in the RAEB of patient 1. The MDS of patient 12 contained a codon 275 TGT-to-TAT transition that was associated with LOH and would change Cys to Tyr (Table 2)Citation .

In two of the 17 leukemias (patients 15, 16), not all four exons were examined either because there was no amplification or because there was insufficient DNA for additional PCR analyses.

Specific genotoxic exposures relative to the observed p53 mutations are summarized in Table 3Citation . Six of 11 leukemias after platinum-based therapy contained p53 mutations; in two of these six cases (patients 1, 5), there were two mutations. In the leukemias of patients exposed to any platinum analogue, the mutations were predominantly transitions; seven transitions and only one transversion were observed. Four of these seven transitions were G-to-A substitutions.


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  		Table 3 Exposures and p53 mutations in cases of leukemia and MDS after ovarian cancer treatment

 
The median ages at primary cancer diagnosis in cases with and without p53 mutations of 67.0 years (range, 45–72 years) and 59.0 years (range, 35 to 76 years), respectively, were not significantly different (rank-sum test, P > 0.05). In patients whose leukemias harbored p53 mutations, the median latency from ovarian-cancer diagnosis to leukemia diagnosis was 3.2 years (range, 1.3 to 7.7 years), compared with 4.4 years (range, 1.3 to 7.8 years) in cases without p53 mutations (rank-sum test, P > 0.05).


   Discussion
Top
 ABSTRACT
 Introduction
 Patients and Methods
 Results
 Discussion
REFERENCES
 
We investigated the frequency of p53 mutations in all available cases of leukemia and MDS after primary ovarian cancer within five of seven population-based cancer registries participating in an epidemiological study of treatment-related leukemia (21) . The percentages of cases in which material was available (33%) and in which the material was adequate (21%) is comparable with proportions collected for other molecular studies undertaken in these or similar population-based cancer registries (29 , 30) . Because the retrospective availability and adequacy of archived materials is unlikely related to the occurrence of selected molecular alterations, but rather reflective of hospital protocol and storage practices, the results should not be biased.

Eleven confirmed mutations and one known polymorphism occurred in 9 of 17 cases, indicating that p53 mutations are common in this setting. Previous therapy included a platinum analogue in six of the nine cases with mutations and other alkylating agents without a platinum analogue in the other three. p53 mutations were present in 6 of 11 leukemias after platinum-based regimens. In two of these six cases, there were two mutations; in one case, there was a polymorphism as well as a mutation.

We and others previously observed associations between germ-line p53 mutations and leukemias after alkylating-agent treatment (7, 8, 9) . The types of malignancies with which germ-line p53 mutations are associated generally portend the types of malignancies where somatic p53 mutations will be found. Indeed, Christiansen et al. (10) recently demonstrated that p53 mutations are common in leukemias after alkylating-agent treatment. Of 21 secondary leukemias with p53 mutations, 17 occurred after exposure to a classic alkylating agent, and two occurred after exposure to cisplatin (10) . The purpose of the present study was to examine p53 mutations in leukemias after primary ovarian cancer, for which platinum analogues and other alkylating agents have been widely used. The results demonstrate prevalent p53 mutations in leukemias after treatment with platinum analogues, as well as after other treatments. The high frequency of p53 mutations in leukemia and MDS after ovarian-cancer treatment is in contrast to lower reported frequencies of p53 mutations in cases of primary MDS (31 , 32) . Sugimoto et al. (32) detected only three p53 mutations in 50 cases of primary MDS or MDS-derived leukemia. Jonveaux et al. (31) identified five p53 mutations among 135 cases of primary and 16 cases of secondary MDS.

The trend toward transition-type p53 mutations, especially G-to-A transitions, in the leukemias after platinum analogues is noteworthy (Table 3)Citation . Although this observation may suggest that platinum analogues cause specific damage to the DNA, we remain circumspect given the low number of cases and the use of combination chemotherapy. Others have suggested that cisplatin preferentially induces mutations at G residues (10 , 33) . Consistent with these observations (10 , 33) , five of eight mutations in leukemias after treatment with platinum analogues in the present study (63%) involved G residues, including four G-to-A transitions and one G-to-C transversion. The other three mutations in leukemias after treatment with platinum analogues in the present study included two A-to-G transitions and one T-to-C transition. It has also been observed that cisplatin- induced mutations in the aprt gene in Chinese hamster ovary cells preferentially occurred at or proximal to 5'-AGG-3' and 5'-GAG-3' sequences (34) . Seven of the eight p53 mutations in leukemias after treatment with platinum analogues in the present study were proximal to the 5'-AGG-3' or 5'-GAG-3' sequences in either the sense or antisense strand of the DNA (Fig. 5)Citation . The two p53 mutations described by Christiansen et al. (10) in leukemias after treatment with cisplatin were G-to-A transitions at 5'-AGG-3' sequences. This mutation spectrum appears to be distinct from the preferred mutations at A:T base pairs suggested by Christiansen et al. (10) in leukemias after various cyclophosphamide-, cyclophosphamide-busulfan-, or chlorambucil-based treatments. Taken together with the study by Christiansen et al. (10) , these results may suggest that the types of p53 mutations in treatment-related leukemias are the result of specific drug exposures; however, the significance of the sequence specificity with respect to the formation of specific platinum adducts and their processing remains to be determined.



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  		Fig. 5. 5'-AGG-3' and 5'-GAG-3' sequences (34) proximal to identified p53 mutations in leukemias after treatment with platinum analogues (brackets). Codons containing p53 mutations are as indicated. Arrows show specific single base-pair changes in the coding strands. Mutations were verified by sequencing the noncoding strand (not shown).

 
No archival germ-line tissues were available for analysis of constitutional DNA samples in this retrospective study, and the somatic versus germ-line origin of the observed p53 mutations could not be assessed. It is likely, however, that they were of somatic origin. The ages of the women at primary cancer diagnosis generally were older than that of patients with the Li-Fraumeni syndrome; ovarian cancer also does not occur in classic Li-Fraumeni families with germ-line p53 mutations (35 , 36) . In the leukemias in this study, the codon 248 CGG-to-CCG transversion that changes Arg to Pro, comprised 2 of the 11 mutations. Germ-line codon 248 mutations are prevalent in the Li-Fraumeni syndrome, but typically these are CGG-to-TGG (Arg-to-Trp) or CGG-to-CAG (Arg-to-Gln) transitions (35 , 36) . The codon 220 TAT-to-TGT transition observed in the leukemia of patient 10, however, has been reported in Li-Fraumeni syndrome (36) .

Different types of p53 mutations also were observed in other cases of leukemia after multimodality, alkylating-agent-containing regimens for sarcomas where the mutations were of germ-line origin (7, 8, 9 , 37) . The mutations in these cases included a 2-bp TA deletion at p53 codon 209 (7) , a CGA-to-TGA nonsense mutation at codon 306 (37) , and 1- and 4-bp deletions, suggesting replication errors in regions of mononucleotide C repeats at codons 250 and 299–301 (8 , 9) . In contrast, in the present study, the mutations were all single-bp changes and either missense or, in one case, silent. The somatic versus germ-line origin of the frequent p53 mutations in leukemias after primary ovarian cancer warrants future investigation including correlation with chemotherapy and radiotherapy exposures.

The sequence changes and resultant amino-acid substitutions in p53 associated with 70 cases of ovarian cancer have been studied previously (38) . The five most frequent p53 mutation sites in ovarian cancer were codons 273, 248, 282, 245, and 175; CGT-to-CAT transitions at codon 273 leading to Arg-to-His substitutions were most common (38) . This same codon 273 transition represented only 1 of the 11 mutations in the leukemias in this series. The p53 codon 248 mutations observed in ovarian cancer (38) differed from the p53 codon 248 CGG-to-CCG transversion detected in two leukemias in the present study.

The results of genomic DNA analysis were consistent with LOH for 4 of the 11 identified mutations. The RAEB of patient 1 contained both exon 7 codon 248, and exon 8 codon 273 mutations, with LOH suggested for the exon 7 but not the exon 8 mutation. If both p53 alleles were present, as suggested by the analysis of exon 8, the same exon 7 mutation may have been present on both alleles. Chromosomal reduplication of the allele containing the exon 7 mutation, followed by mutation of exon 8 on one allele, may be one explanation for this finding. Alternatively, mixtures of leukemic and normal cells in the dysplastic marrow may have confounded LOH detection for the exon 8 mutation, although this seems less likely because the amplification of exon 7 and exon 8 should have been affected equally by the presence of the normal cells. Similar explanations may apply to the RAEB of patient 14, in which there was an exon 7 codon 236 mutation with suggested LOH, but heterozygosity at codon 213 in exon 6. It has been proposed that RNA-based studies may be more sensitive than genomic-DNA approaches for LOH detection because mutant p53 mRNA is generally more stable and present at high levels compared with very low-level p53 mRNA expression in normal hematopoietic cells (10) . RNA was unavailable for comparison in this study.

Cooperating mutations affecting proliferation and differentiation are believed essential to the pathogenesis of AML (39) . The predicted tumor suppressor genes at critical regions of chromosomes 5q and 7q where deletions are observed in many alkylating-agent-related leukemias are yet to be identified (39) . Specific, well-characterized molecular fusions involving MLL, and other balanced translocations, are considered primary alterations in DNA topoisomerase II inhibitor-related cases (reviewed in Refs. 40 , 41 ). The nature of the mutations cooperating with these hallmark alterations, including p53 mutations, are of major interest. RAS mutations are features of alkylating agent-related but not DNA topoisomerase II inhibitor-related cases (42 , 43) . Germ-line mutations in the NF1 tumor suppressor gene, the product of which is a GTPase protein that accelerates GTP hydrolysis on Ras proteins (44) , predispose to MDS with monosomy 7 after alkylating-agent treatment (45 , 46) . p53 mutations are also features of alkylating agent-related cases (7 , 10) , but not of cases after DNA topoisomerase II inhibitors (7) as described above. Segmental jumping translocations are chromosomal abnormalities in which multiple copies of various oncogenes are dispersed throughout the genome and extrachromosomally (37 , 47) . Gene amplification potential accompanies loss of wild-type p53 (48) , and p53 mutations and MLL segmental jumping translocations are strongly correlated and occur after alkylating-agent treatment (49) . The p53 mutations may be of germ-line origin (37) . Noteworthy is the recent finding that internal tandem duplications in the FLT3 tyrosine kinase, which confer a myeloproliferative phenotype (50) and are common in de novo AML (39) , are infrequent in treatment-related cases (51) . Besides mutations in oncogenes and tumor suppressor genes, the observation of microsatellite instability has suggested that a mutator phenotype coupled with the relevant exposures may be of importance (52) . In addition, genetic polymorphisms affecting the CYP3A4, NQO1, and GST drug-metabolizing enzymes confer susceptibility (53, 54, 55) .

The WAVE system (Transgenomic; Ref. 56 ) provides a new approach for p53-mutation screening. Mutations and LOH can be analyzed with this automated scanning method, which resolves hetero- and homoduplexes using temperature-modulated liquid chromatography column separation (56) . The method is gel-free (56) and, therefore, more rapid than single-strand conformation polymorphism analysis (7 , 24, 25, 26 , 37 , 57, 58, 59, 60, 61) , constant denaturant gel electrophoresis (62) , or conformation-sensitive gel electrophoresis (53 , 63) . The sensitivity of the WAVE system for detection of mutations in other genes has been reported to approach 100% (64 , 65) . The WAVE system (56) proved a practical approach for rapid analysis of unknown p53 mutations for this study.

A high frequency of p53 mutations was observed in leukemia and MDS after primary ovarian-cancer treatment with regimens containing platinum analogues as well as other regimens. The acquisition of p53 mutations in the setting of platinum-based chemotherapy has been reported in other tumor systems including an osteosarcoma cell line (66) . Additional studies have suggested that resistance to chemotherapy and {gamma}-radiation may exist in part because of the presence of mutant p53 or deleted p53 function (67, 68, 69) . Both cisplatin and etoposide, as well as {gamma}-radiation, induce wild-type p53 to activate downstream signals that initiate cell-cycle arrest and/or apoptosis. In the presence of mutant p53, these biological endpoints are not achieved and the cells become resistant (67, 68, 69) . Experimental systems to investigate the relevance of p53 mutations and alterations in activation of the downstream targets of p53 to the emergence of the leukemic clone in treatment-related leukemias should also be developed.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants CA80175, CA77683, and CA85469 (all to C. A. F.), and NO1-CP-51034 (to C. F. L.) and a Leukemia and Lymphoma Society Scholar Award (to C. A. F.). Back

2 To whom requests for reprints should be addressed, at Division of Oncology, Leonard and Madlyn Abramson Pediatric Research Building, Room 902B, The Children’s Hospital of Philadelphia, 3516 Civic Center Boulevard, Philadelphia, PA 19104-4318. Phone: (215) 590-2831; Fax (215) 590-3770; E-mail: felix{at}email.chop.edu Back

3 The abbreviations used are: MDS, myelodysplastic syndrome; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; LOH, loss of heterozygosity; RARS, refractory anemia with ringed sideroblasts; RAEB, refractory anemia with excess blasts. Back

Received 12/19/01; revised 2/18/02; accepted 2/20/02.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Patients and Methods
 Results
 Discussion
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J. Breton, F. Sichel, A. Abbas, J. Marnay, D. Arsene, and M. Lechevrel
Simultaneous use of DGGE and DHPLC to screen TP53 mutations in cancers of the esophagus and cardia from a European high incidence area (Lower Normandy, France)
Mutagenesis, May 1, 2003; 18(3): 299 - 306.
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