1. No category

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1995 85: 2546-2552
Mutations of the p53 and ras genes in childhood t(1;19)-acute
lymphoblastic leukemia
M Kawamura, A Kikuchi, S Kobayashi, R Hanada, K Yamamoto, K Horibe, T Shikano, K Ueda, K
Hayashi and T Sekiya
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Mutations of the p53 and ras Genes in Childhood t( l ; 19)-Acute
Lymphoblastic Leukemia
By Machiko Kawamura, Akira Kikuchi, Shigetoshi Kobayashi, Ryoji Hanada, Keiko Yamamoto, Keizou Horibe,
Takaaki Shikano, Kazuhiro Ueda, Kenshi Hayashi, Takao Sekiya, and Yasuhide Hayashi
We have investigated the alterations of p53 and ras genes
including H-, K-, and N-ras genes in 22 acute lymphoblastic
leukemia (ALL) cases and five cell lines carrying t(1;19) by
use of polymerase chain reaction (PCWsingle-strand conformation polymorphism (SSCP) analysis and direct sequencing. The mutations of the p53 gene were found in 2 of 20
t(l;lS)-ALL cases at diagnosis (IO?&),all of4 cases at relapse
1100%), and 4 of the 5 cell lines 180%). Four of the fivepatients who died hadmissense mutations at codons 49, 177.
179, and 248. In cases examined sequentially, one had the
same point mutation at codon 179 at both diagnosis and
relapse, and another had the same p53 gene mutation at
codon 240 both in leukemic cells at relapse and in a cell line
derived at that time. The other case had no mutation at
codon 177 at relapse and
diagnosis but had the mutation at
cell lines derived from blast cells at diagnosis, suggesting
that a small number of leukemic cells with the p53 gene
mutation atdiagnosis might have escaped PCR-SSCP analysis. In cell lines, SCMC-L9 had three point mutations in the
p53 gene at codons 175,248, and 358, whereas SCMC-L10
had frame shift atcodons 209-211. One casehad a rare polymorphism atcodon 11. We found only one mutation of the
N-ras gene that was a 2-bp substitution of GGTiGly) t o
GTC(Val) at codon 13 among 22 t(l;lS)-ALL cases and five
cell lines. This case showed no mutation of the p53 gene
and has had a good course. These results suggest that in
t(1;lS)-ALL, mutations of the p53 and ras genes are infrequent at diagnosis and that p53 gene alterations may be
associated with relapse phase or progression oft(1;lS)-ALL.
0 1995 b y The American Society of Hematology.
C
cancers.” Loss of the normal growth-inhibitoractivity o f
p53protein in most of these tumors occurs as aresult of
point mutationb of the other p53 allele. In myeloid malignancies, p53 gene mutations have been reported in myelodysplastic syndrome,“ blast crisis of chronic myelogenous leukemia,” andacutemyelogenousleukemia
(AML)’‘ at a
lowfrequency. In lymphoidmalignancies,Burkitt’slymphonla,’5,’hR-cell lymphoma,” B-cell ALL.IX multiple myeloma,’” and T-cell-ALL cell line2“ had the p53 mutations
at high frequency whereas early pre-BALL had low frequent
mutations of the p53 gene.” However, there have been few
reports of this gene aberration in t( 1; 19)-ALL with pre-R
phenotype.”
with varying freAltered rus genes havebeendetected
quencies in a number of human malignancies.” N-rus mutations are found in between 25% and 40% cases of AML”.’‘
whereas they were found at lower frequency in ALL.’5
Here we analyze the rearrangement of the E2A gene and
mutations of the p53 and rus genes to investigate the frequency andspectrum
of thesemutations
in aseries
of
t( I ; I9)-ALL. We find that mutations of the p53 and rus
genes were infrequent and that p53 mutations were associated with relapse phase or progression of t( 1 : I WALL.
HROMOSOMAL TRANSLOCATIONS are important
events in the pathogenesis of leukemias and lymphomas. One of the most frequentlyreported
cytogenetic
is the
changes in acutelymphoblasticleukemia(ALL)
t(1; I Y ) ( q 2 3 ; ~ 1 3 ) ’observed
~~
in up to 6% of pediatric ALL
and in approximately 25% of ALL with a pre-B cell phenotype’.’,’ which express cytoplasmic heavy chain Ig (cIg), but
not surface Ig (slg). t(1; 19)-ALL caseshave been reported to
have a poor clinical outcome’.‘ or poor prognostic features.5
The E2A gene, which encodes proteins that bind to enhancer of the kE2, hasbeen isolated,’ mapped to 1 9 ~ 1 3 ,
and shown to be rearranged in t( 1 ; IY)-ALL.XSimilarly, the
breakpoint in chromosome lq23 interrupts a homeobox gene
known as PBXI.”“’Consequently, the production of a chimeric E2A-PBX1 protein may contribute to the development
of t( 1 ; 19)-ALL.”.“’
It has recently been shown that the p53 gene is a tumor
suppressor gene located on chromosome 1 7 ~ 1 3Alterations
.
of the p53geneare
involved in varioustypes of human
From the Department of Pediatrics, Faculty ojMedicine, Universiry of Tokyo, Hongo,Bunkyo-ku,Tokyo; the OncogeneDivision,
National Cancer Center Research Institute; the Depurtment c!f Hematologv/Oncology.SaitamaChildren’sMediculCenter;
the Department of Pediatrics, University of Hokkaido School c$ Medicine;
the Department ($Pediatrics, University of Nagoya School of Medicine; and the Department of Pediutrics. Universiq of Hirmhima
School of Medicine, Hiroshimu, Japan.
Submitted Augusi I , 1994: accepted December 19, 1994.
Supported in parr b y a Grant-in-Aid f?)r Gtncer Research fronl
the Ministty os Health and Wdjure vf Japan, and a Grunt-in-Aid
jiJr Science of Culture (04454568,04454276) P o m the Ministry of
Education of Japun.
Address reprint requests to Yasuhide Hayashi, MD, Departmentof’
Pediatrics, Faculty of Medicine, University of Tokyo, 7-3-I , Hongo.
Bunkyo-ku,Tokyo 113, Japan.
The publication costs of this article were defrayedin part by page
chargepayment. This article must thereforebeherebymarked
“advertisement” in accordance with I8 U.S.C. secrion 1734 solely to
indicate this fact.
6 l995 by The American Society of Hematology.
0006-4Y7//95/8509-0036$3.00/0
2546
MATERIALS ANDMETHODS
Patient .sample,\. From April 1983 t o May 1994. we identified
22 ALL cases with the t ( l ; 19) at diverse institutes in Japan. The
diagnohis of ALL was based on examination of Wright-stained
smears of bone Inarrow (BM) aspirates classified according to the
French-American-British morphologic criteria and negative myel<)peroxidase and a-naphthyl butyrate esterase staining.BM cells from
22 t( 1 ; IY)-ALL cases ~ncluding I8 cases at diagnosis, 2 cases at
relapse, and 2 cases at both diagnosls and relapse were used i n this
study(TableI). In cases 3 and 19, we analyzedperipheral blood
(PB) and BM samples at remission, respectively, and in cabe I O we
analyzed fibroblasts cultured from BM. ‘These included 7 boys and
15 girls whose ages ranged
from 2 to 14 years. These cases were
mainly treated on the TCCSG A1.L protocol.”’
Cell lines. Five cell lines (KMO-90.27 SCMC-L9,’X SCMCLIO.’”.’‘ SCMC-L,I 1.” THP-X”’) with the t( I ; 19) were examined.
KM090 and SCMC-LI I derived from case 21 at diagnosis and from
Blood, Vol 85,No 9 (May l ) , 1995: pp 2546-2552
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2541
p53 GENE MUTATIONS IN t(l;lg)-ALL
Table 1. Clinical Data on 22 ALL Cases With
the t(1;lSI Translocation
Case
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Age
(vrs)
Sex
4
4
11
8
10
4
4
11
3
6
2
4
F
a
F
2
12
14
8
11
4
13
13
8
F
F
M
M
M
F
F
F
F
F
F
M
F
M
F
M
F
F
F
M
WBC
1106/L)
44.9
28.7
6.4
33.5
131.1
8.1
25.8
105.9
1.7
107.1
102.5
42.5
112.6
2.2
10.6
9.7
13.5
23.6
23.9
8.3
20.1
28.3
Survival
E2A Re
+
-
+
+
+
+
+
ND
+
ND
+
+
+
ND
+
+
ND
ND
+
ND
+
+
(mod
C-U
CD10
+
+
+
+
+-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ND
+
ND
ND
+
+
ND
+
ND
+
ND
ND
ND
+
ND
ND
ND
ND
ND
+
ND
75+
73+
62 +
60 +
59 +
55+
54+
48 +
43 +
43
41 +
39 +
36+
36+
28+
28+
IO+
6+
40
18
10
10
+
Abbreviations: WBC, white blood cell count; ND, not done; Re, rearrangement.
case 10 at relapse, respectively. They were cultured in RPM1 1640
medium supplemented with 10% fetal bovine serum (FBS).
Cytogenetic studies. The chromosomes of cell lines and BM or
peripheral samples were analyzed by the regular tripsin-Giemsa- or
Q-banding method as described previo~sly.~’
DNA and Southern blot analysis. High-molecular-weight DNA
of all samples was prepared by proteinase K-phenol-chloroform extraction. DNA was digested with EcoRI, Xba I, BgflI, Hind111 or
BamHI restriction endonuclease, electrophoresed through a 0.8%
agarose gel, and transferred to nylon membrane.”
DNA probes and hybridization. pE47p’ was used inthe Southern
hybridization?’ The insert was isolated from the vector sequence
with Hind111 and EcoRI, and was used as a template to prepare a
labeled probe with Klenow fragment ofDNA polymerase I and
[a-”PIdCTP, after priming with random hexanucleotides. Highly
stringent condition wasused for hybridization and washing. The
filters were exposed to Kodak XAR film (Eastman Kodak, Rochester,
NY) in the presence of intensifying screens. All experiments included control DNA.
Immunophenotyping. Cell surface antigens were detected by a
of
standard indirect immunofluorescence assay3’.’’ withtheuse
monoclonal antibodies to lymphoid-associated antigens, including
CDlO(JS), CD20(B1), and CD19(B4) as well as myeloid antigens
including CD13(MY7) and CD33(MY9). Cells were analyzed for
fluorescence microscopy of flow cytometry (Coulter EPICSC, Hialeah, FL). Results were considered positive if 25% or more of the
cells expressed a particular antigen. Leukemic cells were also tested
for sIg and cIg, which were considered to be positive if greater than
10% of blast cells had fluorescence.
Polymerase chain reaction (PCR) method. Fragments A,B, C,
D, E, F, G , H, and I containing the sequences of the p53 gene
important for the function of the wild-type protein were analyzed
by PCR (Fig 1). Names and sequences of primers for amplification
of the fragments have been described p r e v i o ~ s l y . Mutations
~ ~ , ~ ~ in
codon 12, 13, or 61 of one of the three ras genes, H-, K-, and Nras, convert these genes into active oncogenes. Therefore, amplified
fragments from regions carrying exon 1 or 2 of the K-ras gene (K1
or K2), the Ha-ras 1 gene (H1 or H2) and the N-ras gene (N1 or
N2) were analyzed by PCR. The sequences of primers used for PCR
were the same as reported p r e v i o ~ s l y DNA
. ~ ~ samples (50 ng) in
the mixture ( 5 pL) with appropriate unlabeled primers and [a3’P]
dCTP (20 pCi per tube, 3,000 Cilmmol; Amersham, Buckinghamshire, UK) as one of the nucleotide substrates were subjected to 30
cycles of the reaction.
Single-strand conformation polymorphism (SSCP) analysis.
After addition of 45 pL of formamide denaturing dye mixture (95%
formamide:20 mmoVL EDTA:0.05% xylene cyanol:O.O5% bromophenol blue) PCR mixture was heated at 80°C for 3 minutes, and
then 1 pL ofthe diluted mixture was applied to one lane of 5%
polyacrylamide gel containing 45 mmoVL TRIS-borate (pH 8.3) and
4 mmoVL EDTA.”,35The gel contained 10% glycerol when it was
specified. Electrophoresis was performed at 40 W for 1 to 3 hours
with cooling by fan. The gel was dried on filter paper and exposed
to x-ray film.
Direct sequencing of PCR-amplifiedfragments. Direct sequencing was performed aspreviouslydescribed3‘with some modificat i o n ~A. ~small
~ piece of the gel corresponding to the abnormal band
detected by SSCP analysis was cut out, immersed in 20 pL of water,
heated at 80°C for 15 minutes, and centrifuged. DNA in extract (1
pL) was subjected to 30 cycles of PCR, and the products were
purified with Centricon 30 or Microcon 100 (Amicon, Beverly, MA).
The DNA fragments thus obtained were sequenced by dideoxy chain
termination methodusing5’-’’P-labeled
primers and Taq DNA
polymerase (dsDNA Cycle Sequencing System; GIBCO BRL,
Gaithersburg, MD). Primers for sequencing were the same as those
used for PCR.
Statistical analysis. Cases were classified into two groups on
the basis of the presence or absence of the p53 gene mutations that
their leukemias carried. Significance ofthe different groups with
and without p53 gene mutations was examined by x’ test. The
survival curves of each group of cases were estimated by the KaplanMeier method, and significant differences were determined by the
generalized Wilcoxon test.”
RESULTS
Cytogenetic studies. All cases had t(1;19)(q23;p13) or
der( 19)t(i; 19) with or without additional chromosome abnormalities. An absence or obvious structural abnormalities
of chromosome 17p were not found in any cases.
Exon1
2
Codon
1-25
3
4
33125
5
6
187-P4
7
B I19
262-306
10
332-367
366393
28-32 507-331
225-261
12(1-186
Fig 1. Regions of the p53 gene subjected to PCR-SSCP analysis.
The nine fragments (A through I) carrying the region of the p53 gene
corresponding to exons 2 to 11 are shown. The coding regions
of the
exons era indicated by solidboxes with aminoacidpositions,
whereas noncoding regionsare indicated by open boxes. The numbers in parentheses indicate base pairs.
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2548
KAWAMURA ET AL
Table 2. p53 Gene Mutations in ALL Cases and Cell LinesWith t11;19) Translocation
At the Time of Sampling
Fresh leukemic cells
Case 10
Case 19
Case 20
Exon
Codon
5
4
240
248
179
179
No mutation
177
49
R
D
D
R
Case 21
D
R
R
D*
Case 22
Case 3
Cell lines
SCMC-L9
2
5
7
10
6
7
5
SCMC-L10
SCMC-L11*
KMO-SO§
Nucleotide Substitution
Substitution
Acid
Amino
11
175
248
358
209-211
240
177
AGT to TGT
CGG t o CAG
CAT t o CCT
CAT to CCT
Ser to Cys
Arg to Gln
His to Pro
His to Pro
CCC to CTC
GAT to CAT
GAG to CAG
Pro t o Leu
Asp to His
Glu to Gln
CGC t o TGC
CGG t o CAG
GAG to AAG
Deletionhnsertiont
AGT t o TGT
CCC to CTC
Arg to Cys
Arg to Gln
Glu to Lys
Frame shift
Ser to Cys
Pro to Leu
Abbreviations: D, at diagnosis; R, at relapse.
This mutation was also found inPB of cases in complete remission, suggesting polymorphism.
t AAACAC change t o CCCACACGCA, this line was established from the same patient who had been reported previously.m
Derived from case 10.
§ Derived from case 21 at diagnosis.
*
Renrrrrngement of E2A gene. Eight of 16 cases. I O of
14 cases, and 9 of I2 cases had rearrangements of E2A gene
with BamHI, BgIII, or X h I digestion, respectively. Totally
E2A gene rearrangements were found in all but 1 of 16 cases
tested (Table l ) . E2A gene rearrangements were also found
in all five t( 1 ; 19)-canying cell lines.
Immunophenotying. Leukemia cells from all the cases
expressed CDlO antigen, and eight of them tested had c-p
(Table 1 ). All the five cell lines had CDlO antigen and expression of c-p. None of the 22 fresh leukemias and 5 cell
lines had any myeloid antigens and T-cell antigens.
PCR-SSCP analwis of p53 gene. Fragments A through
I corresponding to the all coding regions of the p53 gene
(Fig I ) were amplified from DNA isolated from tumor samples andanalyzedby
SSCP method.Abnormalmobility
shifts were detected in fragment C from case 22, fragment
D from cases 20 and 2 I , and fragment F from case 19. In
the cell lines, mobility shifts were observed in fragment E
from SCMC-LIO, fragment D from KMO-90, fragments D,
F. and H from SCMC-L9, and fragment F from SCMC-LI 1.
Loss of the other p53 allele wasnot found bythe SSCP
analysis.
Ident$cation of the p53 gene mutations. Nucleotide sequence analysis showed the aberrations of the p53 gene detected by PCR-SSCP analysis in 2 cases at diagnosis. 4 cases
at relapse, and 4 cell lines (Table 2). In freshleukemias.
missense mutations of CGG to CAG at codon 248 in exon
7 and CAT to CCT at codon 179 in exon 5 were identified
in cases 19and 20, respectively. Cases IO. 20.21.and22
atrelapseshowedmissense
mutations of CAT to CCT at
codon 179 in exon 5 (Fig 2), CCC to CTC at codon 177 in
exon 5 (Fig 3 ) , and GAT to CAT at codon 49 in exon 4.
respectively (Table 2).A sample of case 3 at diagnosis
showed a substitution of GAG to CAG at codon I I in exon
2; however, PBof the case in complete remission showed
the same abnormal sequence, suggesting that this basesubstitution is polymorphism. Samples from remissionBM of case
19 and from fibroblasts of case I O showed no aberrations of
the p53 gene.
In the cell lines, SCMC-LIO showed a 6-bp deletion/lO-
0
normal
3'
D R N
179
His
= \
It
T C G A T C G A
3'
pro
f
if
5'
-
20 R
-n
5'
Fig 2. (A) SSCP analysis of the p53 gene. Lanes
D and R show leukemic cells of case 20 at diagnosis
and at relapse, respectively. Lane N shows normal
lymphocyte (negative control).
(B) Direct sequencing
reactions of the p53 gene mutations. 20-R (case 20
at relapse) shows a point mutation at codon179 resulting inchange
a from
CAT(His1 t o CCTfPro).
From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
p53 GENE MUTATIONS IN t(l;lS)-ALL
0
Fig 3. (AI SSCP analysis of
the p53 gene. Lanes D, R, and C
show
21
at diagnosis
leukemic cells
and at
from
relapse,
case
and established cell lines (KMO901, respectively. Lane N shows
normal lymphocytes. (B1 Direct
sequencing reactions of the p53
gene mutations. 21-R (case 21 at
relapse) showed a point mutation at codon 177 resulting in a
change from CCC(Pro1 to
CTC(Leu1.
2549
--
normal
5'
DCRN
Table 3. p53 and ras Gene Atterations and Clinical Outcome
No. of
Cases
Alive without disease
Alive with
1" disease
Dead
Total 5
17
1
4
22
p53 Gene
Alterations
ras Gene
Alterations
0
1
0
4
0
BM relapse after BM transplantation followed by renal tumor.
fi,
5'
T
3'
The E2A gene rearrangements were found in 15 of 16
cases with t( I ; I9)-ALL tested, and in all five cell lines with
Clinical Outcome
~~~
I
DISCUSSION
TCGATCGA
f
i
!
177
177
bp insertion at codon 209-21 I in exon 6; KMO-90 derived
from case 21 at diagnosis showed the same missense mutation of CCC to CTC at codon 177 in exon 5 as leukemic
cells at relapse, and SCMC-L9 showed the multiple missense
mutations of CGC to TGC at codon 175, CGG to CAG at
codon 248, andGAG to AAGat codon 358 (Table 2).
SCMC-L1 I , which derived from case IO at relapse, showed
the same mutation at codon 240 as leukemic cells at relapse.
Correlation between the p53 mutations and clinical outcome. Among 20 cases tested at diagnosis, both of the
cases with p53 mutations have died while only 1 of 18 cases
lacking p53 mutations has died ( P < .02 by x2 test) (Table
3). The Kaplan-Meier analysis of survival times showed a
significant difference between the two groups ( P < .005).
All 4 patients who died had p53 aberrations in the course
of disease. Among 5 patients with p53 aberrations in the
course of disease, 4 patients died and 1 relapsed after BM
transplantation, whereas 17 patients without p53 aberrations
have been alive for 6 to 75 months without any evidence of
recurrence ( P < .W5 by x* test). Two of 20 cases (cases 2
and 17) had central nervous involvement at diagnosis and
had no p53 mutations.
Ras genemutations.
PCR-SSCP analysis of ras gene
showed abnormal mobility shift of fragment NI in case 2.
Nucleotide sequence analysis of this case revealed a 2-bp
substitution of GGT(Gly) to GTC(Va1) at codon 13 ofNras gene. The presence of the wild type of codon 13 suggested that mutations occurred in a smaller portion of leukemic cells.
-
21 R
nn
Leu
3'
t( l ; 19). This frequency is compatible with that of the previous reports8 Case 2 with hyperdiploid chromosome abnormalityhadno rearrangements ofE2A gene andhasbeen
alive for 6 years. Similar cases have been reported thus far."
In lymphoid malignancies frequent mutations of p53 gene
have beenreported in Burkitt's celllinesIsandfresh
tumors," as well as T-cell lines.'" However, there have been
few reports of p53 gene aberrations in non-T,non-B
lymphoid malignancies, especially in t( 1: I9)-ALL with preB phenotype. In this study, alterations of the p53 gene were
found in 2 of 20 t( I ;I9)-ALL cases at diagnosis (10%). 4
of 4 t( 1 ; 19)-ALLcases at relapse ( 100%).and 4 of 5 t( 1;19)ALL cell lines (80%). The frequency of p53 gene mutation
was lower at diagnosis than that at relapse and than that of
cell lines. These findings suggest that t( I ; I9)-ALL cases
with p53 gene aberrations maybe associated withrelapse
phase or progression of disease as observed in B-cell
lymphomas3' and multiple myeloma^,'^ and support the notionthatthe p53 gene ismore frequently mutated in cell
lines than in primary turn or^.^^.^".^'
The occurrence of p53 gene mutations in human cancer
is presumably the consequence of several mutagenic factors
that may act specially in a particular type of tumor. A notable
finding of p53 mutations in human cancer is the fact that
transitions at CpG dinucleotides occur in about one third of
tumors (mainly colon carcinoma); in lung and liver carcinomas, G to T transversions are predominantly found." CpG
dinucleotides represent a target of spontaneously arising mutations in mammalian cells because of S-methylcytosine residues. Itis important to note that 3 of 8 mutations in this
study occurred at CpG dinucleotides in the form of C to T
transition (codon 248 in case 19, codons 175 and248 in
SCMC-L9), as was
seen
in B-ALL," Burkitt's lymphoma,"." T-ALL? T-cell lines,'" and early pre-B ALL."
Whether or not this mutation in lymphoid malignancies was
induced throughexposure to exogenous carcinogens remains
to be investigated in the future.
The mutation at codon 248 (CGG to CAG) seen in case
19 has been reported thus far in a case with pre-B phenotype."3 In respect of codon 248, transversion (CGG to CCG,
arginine to proline) has been reported in an infant with preB ALL at relapse.MCodon 248of p53 gene is a CpG dinucle-
From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
2550
otide site and a hot spot for transitional mutations in other
cancers, both sporadic and hereditary. In Li-Fraumeni syndrome, germ linetransition commonly found at a codon 248
(CGG to TGG, arginine to t r i p t ~ p h a n ) is~ ~different
.~
from
that seen in ALL. Therefore, the mutations at codon 248 in
ALL may be associated with the leukemogenesis of ALL.
Polymorphism at codon 11 found in case 3 has been reported p r e v i o ~ s l y . ~ ~ GtoA CAT
T
transversion at codon 49
(case22)has
beenreported in a case with CML in the
accelerated phase.48However, the same basesubstitution has
been reported in DNA from noncancerous tissue of patients
with sarcoma4' and a patientwith hepatocellular carcinoma.33
It was proposed that this polymorphism is a germinal mutation causing proneness to cancer because the amino acid of
codon49 is relatively well conservedamong species;an
allelewitha histidine at codon 49 is very rareinnormal
individuals, and the patient with this mutation had a family
history of ovarian and breast cancers. The segregations of
this allele in this family remain
to be analyzed. Germ line
configulation of p53 gene was found in normal cells from 3
of S cases with p53 mutations. Moreover, all the S cases did
not have any family history ofcancer. Thesefindings suggest
that p53 gene mutations found in t( 1; I9)-ALL cases are not
associated with germ linemutations suchasLi-Fraumeni
syndrome.
Leukemiccellsfromcase21
atrelapse and celllines
derived from the same case at diagnosis showed the same
aberrationsof p53 gene, whereas the leukemic cells from
the same case at diagnosis had no mutations. This suggested
that a smallnumber of leukemic cellswith p53 genemutation
at diagnosis might have escaped from PCR-SSCP analysis.
Indeed, both the SSCP and direct sequencing techniquefail
to detect mutations that are present in less than 10% of the
cell pop~lation.''~ These results of case 21 are compatible
with a report thata clonal expansionassociated with mutated
p53 was observed in clinical sequential studies of brain tumors in which a low percentage of the cells with p53 gene
mutation were observed at diagn~sis.~"
Alternatively, mutations of the p53 gene in t(1; I9)-ALL would have possibly
occurredin the later stage ofthetumorigenesis
stepand
might havebeeninvolvedinthetumor
progression. The
other possible explanation is that the cells with the mutated
p53 gene may have dominated over the other cells lacking
mutated p53 gene in the process of cell culture.
Cases 19 and 20 with p53 gene mutation at diagnosis did
not obtain complete remission with intensive chemotherapy.
These results may show that leukemic cells with p53 gene
mutations even in the early stage are
very resistant to chemotherapy, as are leukemic cells
withthesemutations
at relapse.*' At the time of diagnosis both of the two patients
with p53 aberrations died whereas 1 of the 18 without the
aberrations died;this isstatisticallysignificant
(P < .02).
Among S patients with p53 gene mutations in the course of
disease,4died
and 1 relapsed; 17 survivors lacking p53
aberrations were freefromdisease (P < ,005)(Table 3).
These results may suggest that the presence of the p53 gene
mutationsisassociated
with a poorclinicaloutcomein
t(1;19)-ALL.In this regard, p53-dependentapoptotic response has recently been shown to modulate the cytotoxity
KAWAMURA ETAL
of anticancer agents.51Mutations of p53 gene couldpossibly
induce drug resistance by interfering with normal apoptotic
pathways in leukemic cells.52
As rus gene mutations,the N-rus gene wasmutated in
only 1 of 22 t(1; I9)-ALL cases and in 0 of 5 cell lines in
this study, suggesting that rus gene mutation is infrequent
in lymphoidmalignancy,
as
indicated
by previous reports."'s7
Case 2 with N-ras mutation, who has been alive
for 73 months since diagnosis, neither
had p53 genemutation
nor E2A gene rearrangement, and had hyperdiploid chromosome abnormality. Therefore, the N-rusmutation in addition
to hyperdiploidy may be involved in the leukemogenesis of
this case. Presence of an N-rus mutationin children with
ALL has been reported to be associated with leukemic prog r e ~ s i o n ~and
~ . ~ 'possibly be anindependentpredictor
of
worse clinical outcome.'“^" Our result is incompatible with
thesereports.Interestingly,
rusgene mutationswereless
frequent than p53 gene mutations in relapsed cases and in
cell lines in this study, suggesting that
rus gene mutations
may not be involved in the development and progression of
t( I ;19)-ALL. The 2-bpsubstitution of GGT to GTC at codon
13 identified in case 3 has not been reported so far in ALL.
We conclude that in t( 1 ; 19)-ALL mutations of the p53
and rus genes are infrequent at diagnosis and that p53 mutations may be associated with relapse phase or progression
of t( 1; 19)-ALL.
ACKNOWLEDGMENT
We thank Drs Masahiro Sako, Osaka Children Health Center;
Satoshi Tashiro and Kazuko Hamamoto, Hiroshima University
School of Medicine; Manabu Sotomatsu, Gunma University School
of Medicine; and Kouichi Ishimoto, Juntendo University School of
Medicine, for providing the samples and clinical data. We also thank
Drs Sinpei Nakazawa, Yamanashi Medical Colledge, and Junichiro
Fujimoto, National Children's Research Center, for phenotyping of
some samples; andDr David Baltimore, Whitehead Institute for
Biomedical Research, Cambridge, MA, for his generous gift of the
pE47p probe.
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