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Exon 5 mutations in the p53 gene in relapsed childhood acute lymphoblastic leukemia
Leukemia Research Vol. 21, No. 8, pp. 721-729. 1997. 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 014552126/97 $17.00 + 0.00
Pergamon PII: SO145-2126(97)000386
EXON 5 MUTATIONS IN THE P53 GENE IN RELAPSED CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA Orit Blau, Smadar Avigad, Batia Stark, Yona Kodman, Drorit Luria, Ian J. Cohen and Rina Zaizov Cancer Molecular Genetics, Felsenstein Medical Research Center, Department of Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Beilinson Medical Campus,Petha-Tikva, SackIer Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel (Received 30 December 1996.Accepted 28 February 1997) Abstract-Thirty seven children with relapsed acute lymphoblastic leukemia (ALL), 25 Blineage and 12 T-lineage, were analyzed for p53 alterations at different stages of the disease. Loss of heterozygosity (LOH) was detected in the relapse phase in three patients. ~53 mutations were identified by single strand conformation polymorphism (SSCP) and sequencing analyzes in seven of the 37 ALL patients (19%); three B-lineage (12%) and four T-lineage (33%). Most of the mutations were identified in the relapse phase. In two exceptional cases, one of the mutations was indicated as a germ line and the other was already present at diagnosis. No ~53 mutation was identified in any of the other 20 available bone marrow samples obtained at diagnosis. No correlation between the ~53 status and clinical outcome could be determined. The majority of the mutations (four out of seven, 57%) were clustered at exon 5. Our data implicate that p53 exon 5 is a frequent site of mutations in relapsed childhood ALL. 0 1997 Elsevier Science Ltd Key words: relapse,
childhood,
ALL, ~53, mutations.
Introduction
in MDS, AML and CML patients having 17p monosomy [2,4,6,9, lo]. In lymphoid malignancies, aberrations of the ~53 gene have been reported in B-cell chronic leukemia (B-CLL), especially in the stage of progression known as Richter’s transformation (40%) [ll], in Burl&t’s lymphoma, in its leukemic counterpart B-cell ALL (2040%) [ll-151 and in the transition from lymphoma to acute T-cell leukemia in adults (ATL) (30-50%) [16-181. ~53 mutations in MDS, AML and CLL are known to be poor prognostic factors [19]. In precursor B-cell ALL, mutations of the ~53 gene are relatively rare, ca 3%. Only 11 ~53 mutations were identified among 355 B-lineage ALL patients in five different reports [ 11, 13, 14, 20,211. In the distinct molecular subgroups of B-lineage ALL, different incidences of ~53 mutations have been observed. In Philadelphia chromosome (Ph’)-positive ALL, the ~53 mutations appear more often (17%) in cases accompanied by loss of the distal part or the whole chromosome 17 [22,23]. In ALL patients with translocation t(l:19), the ~53 mutations are infrequent at diagnosis but associated with disease progression and poor clinical outcome [24]. High incidence of ~53 mutation has been reported in cases of B-cell ALL with chromosomal breakpoints at 8q24 (45%) and also with chromosomal breakpoints at 1lq23 (21%) [25]. Only
Acute lymphoblastic leukemia (ALL) is one of the most common childhood neoplasms (30%). The ~53 tumor suppressor gene has been implicated in the pathogenesis of a wide variety (ca 85%) of human cancers, being inactivated by rearrangement, deletion and/or point mutations [ 11. Tbe majority of the p53 mutations cluster in exons 5-8, the four ‘hot spot’ regions of the gene. Alterations of the ~53 gene have been found in nonrandom distribution among the different subtypes of hematological neoplasms. In the myeloid malignancies, a relatively low incidence of structural abnormalities and point mutations has been observed especially, in myelodysplastic syndrome (MDS) (5-10%) [2-4] and in acute myeloid leukemia (AML) (10%) [5-71. A significantly bigher incidence of ~53 mutations was found during the disease course of chronic myeloid leukemia (CML) to blast crisis (20-30%) [8,9, lo] and Abbreviations: ALL, acute lymphoblastic leukemia; LOH, loss of heterozygosity; SSCP, solid phase conformation polymorphism; RFLP, restriction fragment length polymorphism; VNTR, variable number of tandem repeats. Correspondence to: Smadar Avigad, Ph.D., Department Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah-Tikva 49202, Israel (Tel.: 972-39376792;
972-3-9393042). 721
122
0. Blau er al.
Table 1. ~53 Gene involvement in childhood relapsedALL patients Patient No. 1
Lineage
Diseasestage*
T
A.D. Rl R2 R3 RM
LOH
Mutation
Exon
Codon
Nucleotide substitution
Amino acid
132 132 132 -
GGC+GAC GGC+GAC GGC+GAC -
Lys+Arg Lys+Arg Lys+Arg -
275 275
TGT+TAT TGT-tTAT
Cys-rTyr Cys+Tyr
-I-
+
+ -
+ -
5 5 5 -
A.D. Rl
-
+ +
8 8
Rl R2
-
+
-
-
5
175
CGC-XAC
Arg-+His
Rl
-
-
7
248
CGG-+CAG
Arg+Gln
R2
-
+
5
175
CGC-CAC
Arg+His
Rl RM
-
+ +
5 5
154 154
GGC+GAC GGC+GAC
Gly+Asp Gly+Asp
Rl RM
+ -
+ -
7 -
248 -
CGG+CCG -
Arg+Pro -
R2 R4
+
-
-
-
-
-
-
-
+
A.D., at diagnosis; RM, remission; Rl, 1st BM relapse; R2, 2nd BM relapse; R3, 3rd BM relapse; R4, 4th BM relapse; +, harboring a mutation or LOH; -, no mutation nor LOH *: available PBL/BM samplesfor the study.
one ~53 mutation was found in six series of 116 newly diagnosed T-lineage ALL cases [ 11,13, 21, 26-281, although it seems to be a frequent feature in relapsed Tcell ALL (25%) [21,27,28] and in T-cell ALL cell lines (60%) [29]. Since leukemia is considered a component tumor of the Li-Fraumeni syndrome [30], germ line ~53
Patient
mutations might be responsible for the hereditary susceptibility of familial leukemia. To gain insight into the mechanism of leukemogenesis, we studied the involvement of p53 tumor suppressor gene in the initiation and progression of childhood ALL. Thirty-seven relapsed ALL patients
no. 1
Fig. 1. LOH analysis using VNTR in i&on 1 of the ~53 gene. All samples represent different stages of the disease in patient No. 1. LOH was identified in R2 and R3 samples. In R2, residuals of the wild type allele are also seen. A.D., at diagnosis; RM, remission; Rl, 1st BM relapse; R2, 2nd BM relapse; R3, 3rd BM relapse.
123
Exon 5 mutations in the ~53 gene
Fig. 2. SSCP analysis of p53 geneexon 5. Abnormal mobility shifts are detectedin patient No. 1 (in Rl and R3; R2-not shown), in patient No. 3 (only in R2) and in all samples of patients Nos 5 and 6. N, normal control; A.D., at diagnosis; RM, remission; R, single
BM relapse; Rl, 1st BM relapse; R2, 2nd BM relapse; R3, 3rd BM relapse.
were evaluated at different stages of disease by loss of heterozygosity (LOH), single strand conformation polymorphism (SSCP) and sequencing analyzes. Seven missense mutations and three cases of LOH were identified during progression of the disease. Four of the mutations (57%) were detected in exon 5, one of them was indicated as a germ line mutation.
Materials and Methods
to our center during the last 10 years. Thirteen of the Blineage patients and eight of the T-lineage patients were studied at both diagnosis and relapse; the remainder were obtained at subsequent relapses only. In 20 cases (17 B-lineage and 3 T-lineage) bone marrow (BM) cells from the remission period were also available for analysis.
DNA extraction
Patients The study included a group of 37 children with relapsed ALL, 25 B-lineage and 12 T-lineage, referred
High molecular weight genomic DNA was extracted from BM cells or peripheral blood leukocytes (PBL) using standard methods [31].
Patient no. 1 A. D.
23.9.88
RI 1.7.90
RM
R2 15.10.90
19.8.90
Rs 30.7.91
II-I-
\
;: z 2
0
T
T T T T
T T T G
G T A
T A G
p 2 5
1
q Arg A
C Exon 5 - codon
132
Fig. 3. Sequencing analysis of p53 exon 5 in patient No. 1. Mutation in codon 132 was identified in Rl, R2 and R3. A.D., at diagnosis; RM, remission; Rl, 1st BM relapse; R2, 2nd BM relapse; R3, 3rd BM relapse.
724
0. Blau et al. Patient no. 3 RI
R2
18.10.85
20.1.88
-II
G
A
T
C
G
A
T
C
Exon 5-codon
Fig. 4. Sequencing analysis of ~53 exon 5 in patient No. 3. Mutation in codon 175 was identified only in m sample. A.D., at diagnosis; Rh4, remission; Rl, 1st BM relapse; R2, 2nd BM relapse.
LOH analysis
LOH analysis was performed comparing BMIPBL samples of the same patient at least in two different stages of the clinical course of the disease, at diagnosis, remission and subsequent relapses. PCR amplification was carried out using primers for the following three polymorphic loci in the ~1.53gene: variable number of tandem repeats (VNTR)-intron 1 [32]; restriction fragment length polymorphism (RFLP)-exon 4 [33]; and RFLP-intron 6 [34] (custom made by Operon Tech, Inc., Alameda, CA). The digested PCR products of exon 4 and intron 6 were electrophoresed on 3% agarose gel and the VNTR analysis of intron 1 was subjected to 18% polyacrylamide gel. All gels were stained by etbidium bromide. SSCP analysis
SSCP analysis, based on the method of Orita et al. [35], was performed for the detection of ~53 mutations in exons 5-8 of the ~53 gene, using primers as described before [36]. An aliquot of each PCR amplified product was mixed with 0.1% sodium dodecyl sulfate (SDS), 10 mM ethylenediamine-tetra-acetic acid (EDTA) and gel-loading solution (95% formamide, 20 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol), boiled for 2 mm, cooled immediately on ice and loaded on MDE (Mutation Detection Enhancement) gel (FMC BioProduct, Rockland, ME, U.S.A.). The samples were electrophoresed at 3 W for 14-16 h at room temperature and/or 4°C and silver stained. Solid phase sequencing
Samples exhibiting
analysis
SSCP mobility
shifts were sub-
jected to sequencing. The appropriate PCR product, labeled with biotin, was purified with centricon 30, denaturated to single strand PCR using Dynabeads (Dynal) covered with streptavidin and sequenced using the sequenase version 2.0 protocol (United States Biochemical Corp., Cleveland, OH, U.S.A.). Samples that were identified for LOH were also subjected to sequencing analysis. Each sequence was performed by two new independent PCR products and the identified mutations were reconfirmed by sequencing the antistrand. Results LOH analysis
Twenty-four B-lineage patients and 10 T-lineage patients were available for LOH analysis (three patients had only one sample). Three of them (12%) displayed LOH of the ~53 locus during progression of the disease, two B-lineage (Nos 6 and 7) (out of 18 informatives) and one T-lineage (No. 1) (out of eight informatives) (Table 1). Fig. 1 demonstrates LOH analysis using VNTR marker in intron 1 of the ~53 gene. LOH was identified in the second and the third BM relapse samples of the Tlineage patient. The second BM relapse sample contained only 60% leukemic blasts; therefore, residual wild type allele was also present. SSCP analysis
All 37 patients (149 samples) were screened for mutations in exons 5-8 of the ~53 gene using SSCP analysis. Nine electrophoretic mobility shifts were observed in five B-lineage and four T-lineage patients.
125
Exon 5 mutations in the ~53 gene Family
of Patient
no. 6
w ,i.
T ”
C”G
A
T *-I(
C”G
A
T
C’
C
‘A
s 01 I C G G
GUY
Exon 5-codon
Fig. 5. Sequencing analysis of ~53 exon 5 in patient No. 6. Mutation in codon 154 was identified in R, RM and PBL of patient’s father. (sequencing analysis of mouth epithelial cells is not shown). C, normal control; A.D., at diagnosis; RM, remission; R, single BM relapse; F. father.
Four of the shifts were identified in exon 5, two in exon 7, one in exon 8 and two in exon 6. The SSCPmobility shifts in exon 5 are shown in Fig. 2. Sequencing analysis
Sequencinganalysis confirmed sevenmissensemutations (Table l), and two silent mutations. The two silent mutations were detectedin exon 6 codon 213, which has been previously shown to be polymorphic [37]. Four of the seven missensemutations were identified in exon 5. Fig. 3 demonstratesa ~53 mutation in exon 5, codon 132, in a T-lineage patient (No. 1). The mutation was not detected at diagnosis but was identified in all the three relapses. In the sample of the first BM relapse, the presenceof the wild type allele indicates a heterozygous mutation. In the sampleof the secondBM relapse,LOH was identified, but the sample contained only 60% leukemic blasts. Therefore, our findings of mutant and wild type alleles indicate the presenceof a mixture of leukemic cells harboring a hemizygous mutation with normal marrow cells. The hemizygous mutation was clear in the third BM relapsein the leukemic clone from pleural fluid. The same mutation in codon 175 exon 5 was identified in the relapse phase of two different patients, one T-lineage (No. 3) and one B-lineage (No. 5). Fig. 4 demonstrates that the codon 175 mutation developed only in the secondBM relapse of patient No.
3. An additional ~53 mutation in exon 5 was identified in codon 154 in the relapse phase of a B-lineage patient (No. 6) (Fig. 5). This mutation was also present in BM samples from two different remissions which have occurred before the patient had allogeneic BM transplantation. Thus, in order to determine if it is a germ line mutation we analyzed normal tissue (mouth epithelial cells) from the patient and PBL from his father. The mutation was identified in both tissues (Fig. 5). The other three n-&sense mutations were identified two in exon 7, codon 248 (patients Nos 4 and 7) and one in exon 8, codon 275 (patient No. 2). In patient No. 2 the mutation was already identified at diagnosis. Clinical outcome and ~53 involvement
The clinical profile and outcome of all 37 ALL patients were studied in correlation with the ~53 status in the leukemic cells. Table 2 summarizesthe clinical characteristics and the outcome of the three B-lineage and the four T-lineage ALL patients harboring ~53 mutation. No significant difference in outcome was detectedbetween patients with and without ~53 alterations. Discussion In this study we analyzed 37 relapsedchildhood ALL
7+
6
5
4
3”
2+
1
M
54 81
(HLADR+g:9+CD Common
(HLADR+g:9+CD 10”) Pre-B ALL (HLADR+CD19+CDlO+CD20+)
10’)
M
97
M
M
M
194 181
F
M
133
66
Sex
T-ALL (CD7+CD2+CD4+CD3+) Pre T-ALL (CD7’) T-ALL (CD7+CD2+CD8+) T-ALL (CD7+CD3+) Common
Phenotype
Age (months)
Jewish
Moslem
Moslem
Jewish
Moslem
Jewish
Jewish
Ethnic origin
2
4
4.2
50
10
47
14
4
100
4
30
0
90
43
Ll
Ll/L2
Ll
-
-
L2
LlrL2
White blood cell count Blast in (109L-‘) PBL (%) FAB
-
3
3
0
2
4
4
HepatoMegab (cm)
*lst leukemic event in BM. +Patients died of sepsis (infection and complications). tPatient was diagnosed as T-lymphoblastic lymphoma but after 6 months developed aggressive T-cell leukemia. -, No data. RM, remission; R, relapse; ABMT, after BM transplantation; D, dead; A, alive; M, male; F, female.
Patient No.
0
3
4
-
0
8
1
SplenoMedy (cm)
15
30
15
26
60
51
24
6.5
28
8.5
21
1st leukemic event* (months)
45
18
1st RM BM (day)
Table 2. Initial characteristics and clinical outcome of the seven relapsed childhood ALL patients with ~53 mutations
40
125
66
31
10.5
9
36
Survival (months)
Last
(2ndARM) (‘QMn D
(3rf: R)
(2ni R) D
(3ri R) D
St&US
Exon5 mutations in the~53 gene
patients, 25 B-lineage and 12 T-lineage, for alterations in the ~53 gene during the diseasecourse, using LOH, SSCPand sequencinganalyzes. We identified seven mutations, three (12%) in the Blineage ALL and four (33%) in the T-lineage ALL patients; overall incidence was 19%. Mutations in the ~53 geneappearto be a very rare event in primary newly diagnosed B and T-lineage ALL patients, but occur more often in the relapse of both lineages with overall frequency of ca 25% in progressedT-ALL. Our results are in concordancewith the relatively high incidence of the ~53 mutations in relapse, but although our seven mutations were identified during the recurrence of the disease, they cannot all be considered as a late event since three of the sevenpatients (Nos 4,5 and 7) had no available BM samplesat diagnosis. PatientsNos 3 and 6 also lacked BM samples at diagnosis but since the mutation in patient No. 3 was acquired only in the secondrelapse it can be consideredas a late event while patient No, 6 inherited a germ line mutation. Only in the T-lineage patient No. 2 the mutation was presentalready at diagnosis (out of 12 patients). This is quite remarkable, since only one mutation was identified out of 116 newly diagnosed T-lineage ALL casesrecently described [ll, 13, 21, 26, 27,281. The cancer history of patient No. 2 implies a possible germ line mutation since another young relative, a teenagefemale maternal first cousin, died of ovarian germ-cell tumor, a possible component tumor of LFS [30]. Only one germ line mutation was identified in a study of 25 childhood ALL patients [20]. Furthermore, no germ line mutation was found in ten familial leukemia pedigrees [38]. Our data indicate an inherited germ line mutation in patient No. 6 and a possible germ line mutation in patient No. 2. No ~53 mutations were identified in any of the other 20 BM samplesobtained at diagnosis. Since we screenedonly exons 5-8 of the ~53 gene, and mutations do not necessarily occur within the conservedregions, a higher incidence of ~53 mutations cannot be excluded. Only three patients (12%) (Nos 1, 7 and 8) underwent LOH in the progressive stage of the disease.Two of them (Nos 1 and 7) harbored also ap53 mutation. These data confirm the low frequency of ~53 LOH in human leukemia comparedto solid tumors. It is of note that in our study four of the seven mutations (57%) (two in B-lineage and two in T-lineage) were identified in exon 5. At least four other reports [24,25, 28,391 have recently shown a frequent incidence of ~53 mutations in exon 5 of ALL patients (17/29, 58%). Moreover, a high frequency of ~53 mutations in exon 5 have also been observed in various lymphoid malignancies, including CLL [ 11,401, ATL [ 181, Burkitt’s lymphoma and its leukemic counterpart B-cell ALL [ll, 401 and non-Hodgkin’s lymphoma [40,41]. Of particular note is our finding that three of the seven
121
mutations (43%), all in exon 5, were found in Arabic patients who comprise only 27% of the studied population. Hamdy et al. [39] studied 47 newly diagnosedALL patients in an Egyptian Arabic population and found three ~53 mutations, all in exon 5 (two of them in codon 175 with the same G to A nucleotide substitution we identified). In an additional Arabic patient who was diagnosedas B-cell ALL and therefore not included in this study group, another ~53 exon 5 mutation was identified in a relapse sample (codon 159). The clinical outcome (indicated by first remission induction, first leukemic event and duration of survival) did not differ significantly between patients with or without ~53 involvement. It should be noted, that the initial clinical data (age, sex, white blood cell count, blasts in blood, FAB (French-American-British), organomegaly and the treatment regimens) were very heterogenous within the patients with ~53 mutations themselves. We assume that there is no correlation between the ~53 status and clinical outcome in this studied group. It also seems unlikely that the ~53 mutations, identified in the relapse samples,are related to a general accumulation of mutations, induced or selected,since each of the six patients was exposedto a different chemo/radiotherapy treatment regimen. The genotypic differentiation stage of the ~53 mutationpositive leukemic blasts was determined by analyzing the immunoglobulin (Ig) and T-cell receptor (TCR) gene configuration (data not shown). The ~53 mutations were identified in every genotypic subset of the B- or Tlineage ALL. In our study, mutations in the ~53 gene were identified in 19% (7/37) of the relapsed ALL children; 12% in B-lineage and 33% in T-lineage. Most of the ~53
~53
alterations, mutations and LOH, were identified in the
relapse phase. In two exceptional cases,one mutation was identified as a germ line mutation and one was already presentat diagnosis.Four of the sevenmutations were located in exon 5. The ~53 mutations were found in all the lymphoid genotypic differentiation stagesin Blineage and T-lineage ALL. We can confirm that in this study, ~53 mutations are seen more frequently in relapse than at diagnosis of childhood ALL. The high incidence, 57%, of the ~53 mutations clustered in exon 5 implicates it as a ‘hot spot’ for relapsed acute lymphoblastic leukemia in children. Acknowledgements-This
work is supportedby the Gilad
Fund and partially supportedby the Israel Cancer Association and Gerson Meerbaum Fund and by the Josefina Maus and Gabriela CesarmanMaus Chair in Pediatric Hematology. This work is in partial fulfillment of the requirementsfor the Ph.D. degree of Orit Blau from the Sackler Faculty of Medicine at Tel Aviv University.
128
0. Blau et al. References
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Exon 5 mutations in the ~53 gene leukemia are assoicated with the relapse phase. Blood, 1994, 83, 2922. 28. Diccianni, M. B., Yu, J., Hsiao, M., Mukhejee, S., Shao, L. -E. and Yu, A. L., Clinical significance of ~53 mutations in relapsed T-cell acute lymphoblastic leukemia. Blood, 1994,84,3105. 29. Cheng, J. and Haas, M., Frequent mutations in the ~53 tumor suppressor gene in human leukemia T-cell lines. Molecular and Cellular Biology, 1990, 10, 5502. 30. Li, F. P., Fraumeni, J. F. Jr, Mulvihill, J. J., Blattner, W. A., Dreyfus, M. G., Tucker, M. A. and Miller, R. W., A cancer family syndrome in twenty-four kindreds. Cancer Research, 1988, 48, 5358. 31. Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning, A Laboratory Manual, 2nd edn, Cold Spring Harbor Laboratory Press, New York, 1989. 32. Hahn, M., Serth, J., Fislage, R., Wolfes, H., Allhoff, E., Jonas, U. and Pingoud, A., Polymerase chain reaction detection of a highly polymorphic VNTR segment in intron 1 of the human ~53 gene (Letter). Clinical Chemistry, 1993, 39, 549. 33. Greenwald, B. D., Harpaz, N., Yin, J., Huang, Y., Tong, Y., Brown, V. L., McDaniel, T., Newkirk, C., Resau, J. H. and Meltzer, S. J., Loss of heterozygosity affecting the ~53, Rb and mcclapc tumor suppressor gene loci in dysplastic and cancerous ulcerative colitis. Cancer Research, 1992, 52, 741. 34. McDaniel, T., Carbone, D., Takahashi, T., Chumakov, P., Chang, E. H., Pirollo, K. F., Yin, J., Huang, Y. and Meltzer, S. J., The MspI polymorphism in intron 6 of ~53 (TP53) detected by digestion of PCR products. Nucleic Acids Research, 1991, 19, 4796. 35. Grita, M., Iwahana, H., Kanazawa, H., Hayashi, K. and
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Sekiya, T., Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proceedings of the National Academy of Science of the U.S.A., 1989, 86, 2766. 36. Barel, D., Avigad, S., Michovitz, S., Cohen, I. J. and Zaizov, R., Infrequent ~53 mutation and mdm-2 gene amplification in childhood brain tumors. The International Journal of Pediatric Hematology/Oncology, 1997, 4, 91100. 37. Serra, A., Gaidano, G. L., Revello, D., Guerrasio, A., Ballerini, P., Dalla Favera, R. and Saglio, G., A new Taql polymorphism in the ~53 gene. Nucleic Acids Research, 1992, 20, 928. 38. Felix, C. A., D’Amico, D., Mitsudomi, T., Nau, M. M., Li, F. P., Fraumeni, J. F. Jr, Cole, D. E., McCalla, J., Reaman, G. H., Whang-Peng, J., Knutsen, T., Minna, J. D. and Poplack, D. G., Absence of hereditary ~53 mutations in 10 familial leukemia pedigrees. The Journal of Clinical Investigations, 1992, 90, 653. 39. Hamdy, N., Bhatia, K., Shaker, H., Kamel, A., El-Mawla, N.-G., Abou-Enein, M., Yassin, D., El-Sharkawy, N. and Magrath, I., Molecular epidemiology of acute lymphoblastic leukemia in Egypt. Leukemia, 1995, 9, 194. 40. Adamson, D. J. A., Thompson, W. D., Dawson, A. A., Bennett, B. and Haites, N. E., ~53 mutation and expression in lymphoma. British Journal of Cancer, 1995, 72, 150. 41. Kocialkowski, S., Pezzella, F., Morrison, H., Jones, M., Laha, S., Harris, A. L., Mason, D. Y. and Gatter, K. C., Mutations in the ~53 gene are not limited to classic “hot spots” and are not predictive of ~53 protein expression in high-grade non-Hodgkin’s lymphoma. British Journal of Haematology, 1995, 89, 55.
Pergamon PII: SO145-2126(97)000386
EXON 5 MUTATIONS IN THE P53 GENE IN RELAPSED CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA Orit Blau, Smadar Avigad, Batia Stark, Yona Kodman, Drorit Luria, Ian J. Cohen and Rina Zaizov Cancer Molecular Genetics, Felsenstein Medical Research Center, Department of Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Beilinson Medical Campus,Petha-Tikva, SackIer Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel (Received 30 December 1996.Accepted 28 February 1997) Abstract-Thirty seven children with relapsed acute lymphoblastic leukemia (ALL), 25 Blineage and 12 T-lineage, were analyzed for p53 alterations at different stages of the disease. Loss of heterozygosity (LOH) was detected in the relapse phase in three patients. ~53 mutations were identified by single strand conformation polymorphism (SSCP) and sequencing analyzes in seven of the 37 ALL patients (19%); three B-lineage (12%) and four T-lineage (33%). Most of the mutations were identified in the relapse phase. In two exceptional cases, one of the mutations was indicated as a germ line and the other was already present at diagnosis. No ~53 mutation was identified in any of the other 20 available bone marrow samples obtained at diagnosis. No correlation between the ~53 status and clinical outcome could be determined. The majority of the mutations (four out of seven, 57%) were clustered at exon 5. Our data implicate that p53 exon 5 is a frequent site of mutations in relapsed childhood ALL. 0 1997 Elsevier Science Ltd Key words: relapse,
childhood,
ALL, ~53, mutations.
Introduction
in MDS, AML and CML patients having 17p monosomy [2,4,6,9, lo]. In lymphoid malignancies, aberrations of the ~53 gene have been reported in B-cell chronic leukemia (B-CLL), especially in the stage of progression known as Richter’s transformation (40%) [ll], in Burl&t’s lymphoma, in its leukemic counterpart B-cell ALL (2040%) [ll-151 and in the transition from lymphoma to acute T-cell leukemia in adults (ATL) (30-50%) [16-181. ~53 mutations in MDS, AML and CLL are known to be poor prognostic factors [19]. In precursor B-cell ALL, mutations of the ~53 gene are relatively rare, ca 3%. Only 11 ~53 mutations were identified among 355 B-lineage ALL patients in five different reports [ 11, 13, 14, 20,211. In the distinct molecular subgroups of B-lineage ALL, different incidences of ~53 mutations have been observed. In Philadelphia chromosome (Ph’)-positive ALL, the ~53 mutations appear more often (17%) in cases accompanied by loss of the distal part or the whole chromosome 17 [22,23]. In ALL patients with translocation t(l:19), the ~53 mutations are infrequent at diagnosis but associated with disease progression and poor clinical outcome [24]. High incidence of ~53 mutation has been reported in cases of B-cell ALL with chromosomal breakpoints at 8q24 (45%) and also with chromosomal breakpoints at 1lq23 (21%) [25]. Only
Acute lymphoblastic leukemia (ALL) is one of the most common childhood neoplasms (30%). The ~53 tumor suppressor gene has been implicated in the pathogenesis of a wide variety (ca 85%) of human cancers, being inactivated by rearrangement, deletion and/or point mutations [ 11. Tbe majority of the p53 mutations cluster in exons 5-8, the four ‘hot spot’ regions of the gene. Alterations of the ~53 gene have been found in nonrandom distribution among the different subtypes of hematological neoplasms. In the myeloid malignancies, a relatively low incidence of structural abnormalities and point mutations has been observed especially, in myelodysplastic syndrome (MDS) (5-10%) [2-4] and in acute myeloid leukemia (AML) (10%) [5-71. A significantly bigher incidence of ~53 mutations was found during the disease course of chronic myeloid leukemia (CML) to blast crisis (20-30%) [8,9, lo] and Abbreviations: ALL, acute lymphoblastic leukemia; LOH, loss of heterozygosity; SSCP, solid phase conformation polymorphism; RFLP, restriction fragment length polymorphism; VNTR, variable number of tandem repeats. Correspondence to: Smadar Avigad, Ph.D., Department Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah-Tikva 49202, Israel (Tel.: 972-39376792;
972-3-9393042). 721
122
0. Blau er al.
Table 1. ~53 Gene involvement in childhood relapsedALL patients Patient No. 1
Lineage
Diseasestage*
T
A.D. Rl R2 R3 RM
LOH
Mutation
Exon
Codon
Nucleotide substitution
Amino acid
132 132 132 -
GGC+GAC GGC+GAC GGC+GAC -
Lys+Arg Lys+Arg Lys+Arg -
275 275
TGT+TAT TGT-tTAT
Cys-rTyr Cys+Tyr
-I-
+
+ -
+ -
5 5 5 -
A.D. Rl
-
+ +
8 8
Rl R2
-
+
-
-
5
175
CGC-XAC
Arg-+His
Rl
-
-
7
248
CGG-+CAG
Arg+Gln
R2
-
+
5
175
CGC-CAC
Arg+His
Rl RM
-
+ +
5 5
154 154
GGC+GAC GGC+GAC
Gly+Asp Gly+Asp
Rl RM
+ -
+ -
7 -
248 -
CGG+CCG -
Arg+Pro -
R2 R4
+
-
-
-
-
-
-
-
+
A.D., at diagnosis; RM, remission; Rl, 1st BM relapse; R2, 2nd BM relapse; R3, 3rd BM relapse; R4, 4th BM relapse; +, harboring a mutation or LOH; -, no mutation nor LOH *: available PBL/BM samplesfor the study.
one ~53 mutation was found in six series of 116 newly diagnosed T-lineage ALL cases [ 11,13, 21, 26-281, although it seems to be a frequent feature in relapsed Tcell ALL (25%) [21,27,28] and in T-cell ALL cell lines (60%) [29]. Since leukemia is considered a component tumor of the Li-Fraumeni syndrome [30], germ line ~53
Patient
mutations might be responsible for the hereditary susceptibility of familial leukemia. To gain insight into the mechanism of leukemogenesis, we studied the involvement of p53 tumor suppressor gene in the initiation and progression of childhood ALL. Thirty-seven relapsed ALL patients
no. 1
Fig. 1. LOH analysis using VNTR in i&on 1 of the ~53 gene. All samples represent different stages of the disease in patient No. 1. LOH was identified in R2 and R3 samples. In R2, residuals of the wild type allele are also seen. A.D., at diagnosis; RM, remission; Rl, 1st BM relapse; R2, 2nd BM relapse; R3, 3rd BM relapse.
123
Exon 5 mutations in the ~53 gene
Fig. 2. SSCP analysis of p53 geneexon 5. Abnormal mobility shifts are detectedin patient No. 1 (in Rl and R3; R2-not shown), in patient No. 3 (only in R2) and in all samples of patients Nos 5 and 6. N, normal control; A.D., at diagnosis; RM, remission; R, single
BM relapse; Rl, 1st BM relapse; R2, 2nd BM relapse; R3, 3rd BM relapse.
were evaluated at different stages of disease by loss of heterozygosity (LOH), single strand conformation polymorphism (SSCP) and sequencing analyzes. Seven missense mutations and three cases of LOH were identified during progression of the disease. Four of the mutations (57%) were detected in exon 5, one of them was indicated as a germ line mutation.
Materials and Methods
to our center during the last 10 years. Thirteen of the Blineage patients and eight of the T-lineage patients were studied at both diagnosis and relapse; the remainder were obtained at subsequent relapses only. In 20 cases (17 B-lineage and 3 T-lineage) bone marrow (BM) cells from the remission period were also available for analysis.
DNA extraction
Patients The study included a group of 37 children with relapsed ALL, 25 B-lineage and 12 T-lineage, referred
High molecular weight genomic DNA was extracted from BM cells or peripheral blood leukocytes (PBL) using standard methods [31].
Patient no. 1 A. D.
23.9.88
RI 1.7.90
RM
R2 15.10.90
19.8.90
Rs 30.7.91
II-I-
\
;: z 2
0
T
T T T T
T T T G
G T A
T A G
p 2 5
1
q Arg A
C Exon 5 - codon
132
Fig. 3. Sequencing analysis of p53 exon 5 in patient No. 1. Mutation in codon 132 was identified in Rl, R2 and R3. A.D., at diagnosis; RM, remission; Rl, 1st BM relapse; R2, 2nd BM relapse; R3, 3rd BM relapse.
724
0. Blau et al. Patient no. 3 RI
R2
18.10.85
20.1.88
-II
G
A
T
C
G
A
T
C
Exon 5-codon
Fig. 4. Sequencing analysis of ~53 exon 5 in patient No. 3. Mutation in codon 175 was identified only in m sample. A.D., at diagnosis; Rh4, remission; Rl, 1st BM relapse; R2, 2nd BM relapse.
LOH analysis
LOH analysis was performed comparing BMIPBL samples of the same patient at least in two different stages of the clinical course of the disease, at diagnosis, remission and subsequent relapses. PCR amplification was carried out using primers for the following three polymorphic loci in the ~1.53gene: variable number of tandem repeats (VNTR)-intron 1 [32]; restriction fragment length polymorphism (RFLP)-exon 4 [33]; and RFLP-intron 6 [34] (custom made by Operon Tech, Inc., Alameda, CA). The digested PCR products of exon 4 and intron 6 were electrophoresed on 3% agarose gel and the VNTR analysis of intron 1 was subjected to 18% polyacrylamide gel. All gels were stained by etbidium bromide. SSCP analysis
SSCP analysis, based on the method of Orita et al. [35], was performed for the detection of ~53 mutations in exons 5-8 of the ~53 gene, using primers as described before [36]. An aliquot of each PCR amplified product was mixed with 0.1% sodium dodecyl sulfate (SDS), 10 mM ethylenediamine-tetra-acetic acid (EDTA) and gel-loading solution (95% formamide, 20 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol), boiled for 2 mm, cooled immediately on ice and loaded on MDE (Mutation Detection Enhancement) gel (FMC BioProduct, Rockland, ME, U.S.A.). The samples were electrophoresed at 3 W for 14-16 h at room temperature and/or 4°C and silver stained. Solid phase sequencing
Samples exhibiting
analysis
SSCP mobility
shifts were sub-
jected to sequencing. The appropriate PCR product, labeled with biotin, was purified with centricon 30, denaturated to single strand PCR using Dynabeads (Dynal) covered with streptavidin and sequenced using the sequenase version 2.0 protocol (United States Biochemical Corp., Cleveland, OH, U.S.A.). Samples that were identified for LOH were also subjected to sequencing analysis. Each sequence was performed by two new independent PCR products and the identified mutations were reconfirmed by sequencing the antistrand. Results LOH analysis
Twenty-four B-lineage patients and 10 T-lineage patients were available for LOH analysis (three patients had only one sample). Three of them (12%) displayed LOH of the ~53 locus during progression of the disease, two B-lineage (Nos 6 and 7) (out of 18 informatives) and one T-lineage (No. 1) (out of eight informatives) (Table 1). Fig. 1 demonstrates LOH analysis using VNTR marker in intron 1 of the ~53 gene. LOH was identified in the second and the third BM relapse samples of the Tlineage patient. The second BM relapse sample contained only 60% leukemic blasts; therefore, residual wild type allele was also present. SSCP analysis
All 37 patients (149 samples) were screened for mutations in exons 5-8 of the ~53 gene using SSCP analysis. Nine electrophoretic mobility shifts were observed in five B-lineage and four T-lineage patients.
125
Exon 5 mutations in the ~53 gene Family
of Patient
no. 6
w ,i.
T ”
C”G
A
T *-I(
C”G
A
T
C’
C
‘A
s 01 I C G G
GUY
Exon 5-codon
Fig. 5. Sequencing analysis of ~53 exon 5 in patient No. 6. Mutation in codon 154 was identified in R, RM and PBL of patient’s father. (sequencing analysis of mouth epithelial cells is not shown). C, normal control; A.D., at diagnosis; RM, remission; R, single BM relapse; F. father.
Four of the shifts were identified in exon 5, two in exon 7, one in exon 8 and two in exon 6. The SSCPmobility shifts in exon 5 are shown in Fig. 2. Sequencing analysis
Sequencinganalysis confirmed sevenmissensemutations (Table l), and two silent mutations. The two silent mutations were detectedin exon 6 codon 213, which has been previously shown to be polymorphic [37]. Four of the seven missensemutations were identified in exon 5. Fig. 3 demonstratesa ~53 mutation in exon 5, codon 132, in a T-lineage patient (No. 1). The mutation was not detected at diagnosis but was identified in all the three relapses. In the sample of the first BM relapse, the presenceof the wild type allele indicates a heterozygous mutation. In the sampleof the secondBM relapse,LOH was identified, but the sample contained only 60% leukemic blasts. Therefore, our findings of mutant and wild type alleles indicate the presenceof a mixture of leukemic cells harboring a hemizygous mutation with normal marrow cells. The hemizygous mutation was clear in the third BM relapsein the leukemic clone from pleural fluid. The same mutation in codon 175 exon 5 was identified in the relapse phase of two different patients, one T-lineage (No. 3) and one B-lineage (No. 5). Fig. 4 demonstrates that the codon 175 mutation developed only in the secondBM relapse of patient No.
3. An additional ~53 mutation in exon 5 was identified in codon 154 in the relapse phase of a B-lineage patient (No. 6) (Fig. 5). This mutation was also present in BM samples from two different remissions which have occurred before the patient had allogeneic BM transplantation. Thus, in order to determine if it is a germ line mutation we analyzed normal tissue (mouth epithelial cells) from the patient and PBL from his father. The mutation was identified in both tissues (Fig. 5). The other three n-&sense mutations were identified two in exon 7, codon 248 (patients Nos 4 and 7) and one in exon 8, codon 275 (patient No. 2). In patient No. 2 the mutation was already identified at diagnosis. Clinical outcome and ~53 involvement
The clinical profile and outcome of all 37 ALL patients were studied in correlation with the ~53 status in the leukemic cells. Table 2 summarizesthe clinical characteristics and the outcome of the three B-lineage and the four T-lineage ALL patients harboring ~53 mutation. No significant difference in outcome was detectedbetween patients with and without ~53 alterations. Discussion In this study we analyzed 37 relapsedchildhood ALL
7+
6
5
4
3”
2+
1
M
54 81
(HLADR+g:9+CD Common
(HLADR+g:9+CD 10”) Pre-B ALL (HLADR+CD19+CDlO+CD20+)
10’)
M
97
M
M
M
194 181
F
M
133
66
Sex
T-ALL (CD7+CD2+CD4+CD3+) Pre T-ALL (CD7’) T-ALL (CD7+CD2+CD8+) T-ALL (CD7+CD3+) Common
Phenotype
Age (months)
Jewish
Moslem
Moslem
Jewish
Moslem
Jewish
Jewish
Ethnic origin
2
4
4.2
50
10
47
14
4
100
4
30
0
90
43
Ll
Ll/L2
Ll
-
-
L2
LlrL2
White blood cell count Blast in (109L-‘) PBL (%) FAB
-
3
3
0
2
4
4
HepatoMegab (cm)
*lst leukemic event in BM. +Patients died of sepsis (infection and complications). tPatient was diagnosed as T-lymphoblastic lymphoma but after 6 months developed aggressive T-cell leukemia. -, No data. RM, remission; R, relapse; ABMT, after BM transplantation; D, dead; A, alive; M, male; F, female.
Patient No.
0
3
4
-
0
8
1
SplenoMedy (cm)
15
30
15
26
60
51
24
6.5
28
8.5
21
1st leukemic event* (months)
45
18
1st RM BM (day)
Table 2. Initial characteristics and clinical outcome of the seven relapsed childhood ALL patients with ~53 mutations
40
125
66
31
10.5
9
36
Survival (months)
Last
(2ndARM) (‘QMn D
(3rf: R)
(2ni R) D
(3ri R) D
St&US
Exon5 mutations in the~53 gene
patients, 25 B-lineage and 12 T-lineage, for alterations in the ~53 gene during the diseasecourse, using LOH, SSCPand sequencinganalyzes. We identified seven mutations, three (12%) in the Blineage ALL and four (33%) in the T-lineage ALL patients; overall incidence was 19%. Mutations in the ~53 geneappearto be a very rare event in primary newly diagnosed B and T-lineage ALL patients, but occur more often in the relapse of both lineages with overall frequency of ca 25% in progressedT-ALL. Our results are in concordancewith the relatively high incidence of the ~53 mutations in relapse, but although our seven mutations were identified during the recurrence of the disease, they cannot all be considered as a late event since three of the sevenpatients (Nos 4,5 and 7) had no available BM samplesat diagnosis. PatientsNos 3 and 6 also lacked BM samples at diagnosis but since the mutation in patient No. 3 was acquired only in the secondrelapse it can be consideredas a late event while patient No, 6 inherited a germ line mutation. Only in the T-lineage patient No. 2 the mutation was presentalready at diagnosis (out of 12 patients). This is quite remarkable, since only one mutation was identified out of 116 newly diagnosed T-lineage ALL casesrecently described [ll, 13, 21, 26, 27,281. The cancer history of patient No. 2 implies a possible germ line mutation since another young relative, a teenagefemale maternal first cousin, died of ovarian germ-cell tumor, a possible component tumor of LFS [30]. Only one germ line mutation was identified in a study of 25 childhood ALL patients [20]. Furthermore, no germ line mutation was found in ten familial leukemia pedigrees [38]. Our data indicate an inherited germ line mutation in patient No. 6 and a possible germ line mutation in patient No. 2. No ~53 mutations were identified in any of the other 20 BM samplesobtained at diagnosis. Since we screenedonly exons 5-8 of the ~53 gene, and mutations do not necessarily occur within the conservedregions, a higher incidence of ~53 mutations cannot be excluded. Only three patients (12%) (Nos 1, 7 and 8) underwent LOH in the progressive stage of the disease.Two of them (Nos 1 and 7) harbored also ap53 mutation. These data confirm the low frequency of ~53 LOH in human leukemia comparedto solid tumors. It is of note that in our study four of the seven mutations (57%) (two in B-lineage and two in T-lineage) were identified in exon 5. At least four other reports [24,25, 28,391 have recently shown a frequent incidence of ~53 mutations in exon 5 of ALL patients (17/29, 58%). Moreover, a high frequency of ~53 mutations in exon 5 have also been observed in various lymphoid malignancies, including CLL [ 11,401, ATL [ 181, Burkitt’s lymphoma and its leukemic counterpart B-cell ALL [ll, 401 and non-Hodgkin’s lymphoma [40,41]. Of particular note is our finding that three of the seven
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mutations (43%), all in exon 5, were found in Arabic patients who comprise only 27% of the studied population. Hamdy et al. [39] studied 47 newly diagnosedALL patients in an Egyptian Arabic population and found three ~53 mutations, all in exon 5 (two of them in codon 175 with the same G to A nucleotide substitution we identified). In an additional Arabic patient who was diagnosedas B-cell ALL and therefore not included in this study group, another ~53 exon 5 mutation was identified in a relapse sample (codon 159). The clinical outcome (indicated by first remission induction, first leukemic event and duration of survival) did not differ significantly between patients with or without ~53 involvement. It should be noted, that the initial clinical data (age, sex, white blood cell count, blasts in blood, FAB (French-American-British), organomegaly and the treatment regimens) were very heterogenous within the patients with ~53 mutations themselves. We assume that there is no correlation between the ~53 status and clinical outcome in this studied group. It also seems unlikely that the ~53 mutations, identified in the relapse samples,are related to a general accumulation of mutations, induced or selected,since each of the six patients was exposedto a different chemo/radiotherapy treatment regimen. The genotypic differentiation stage of the ~53 mutationpositive leukemic blasts was determined by analyzing the immunoglobulin (Ig) and T-cell receptor (TCR) gene configuration (data not shown). The ~53 mutations were identified in every genotypic subset of the B- or Tlineage ALL. In our study, mutations in the ~53 gene were identified in 19% (7/37) of the relapsed ALL children; 12% in B-lineage and 33% in T-lineage. Most of the ~53
~53
alterations, mutations and LOH, were identified in the
relapse phase. In two exceptional cases,one mutation was identified as a germ line mutation and one was already presentat diagnosis.Four of the sevenmutations were located in exon 5. The ~53 mutations were found in all the lymphoid genotypic differentiation stagesin Blineage and T-lineage ALL. We can confirm that in this study, ~53 mutations are seen more frequently in relapse than at diagnosis of childhood ALL. The high incidence, 57%, of the ~53 mutations clustered in exon 5 implicates it as a ‘hot spot’ for relapsed acute lymphoblastic leukemia in children. Acknowledgements-This
work is supportedby the Gilad
Fund and partially supportedby the Israel Cancer Association and Gerson Meerbaum Fund and by the Josefina Maus and Gabriela CesarmanMaus Chair in Pediatric Hematology. This work is in partial fulfillment of the requirementsfor the Ph.D. degree of Orit Blau from the Sackler Faculty of Medicine at Tel Aviv University.
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0. Blau et al. References
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