Vol. 7, 303-308, April 1998 Cancer Epidemiology, Biomarkers & Prevention 303
3 The abbreviations used are: SSCA. single-strand conformation analysis: FFPE,
formalin-fixed, paraffin-embedded.
p53 Mutations in Malignant Gliomas’
Yu Li, Robert C. Millikan, Susan Carozza,Beth Newman, Edison Liu, Richard Davis, Rei Miike, andMargaret Wrensch2
Department of Epidemiology, School of Public Health, University of North
Carolina, Chapel Hill, North Carolina 27599-7400 [Y. L., R. C. M., B. NI;
Cancer Registry Division, Texas Department of Health, Austin, Texas 78756
[S. Cl; Division of Clinical Sciences, National Cancer Institute, Bethesda,Maryland 20892-2440 [E. LI; Departments of Neuropathology ER. D.l and
Epidemiology and Biostatistics [R. M., M. W.], School of Medicine,
University of California, San Francisco, Califomia 94143
Abstract
A population-based series of incident cases of malignantglioma were analyzed for mutations in the tumorsuppressor gene p53. Exons 4-8 were screened usingPCR-single-strand conformation analysis and confirmedthrough direct sequencing. Of 62 tumors analyzed, 12(19%) contained mutations in pS3: one 18-bp duplicationin exon 5, five point mutations in exon 4, three pointmutations in exon 7, two point mutations in exon 8, and asplice-site mutation at the exon 6/intron 7 boundary. Incontrast to previous studies of malignant glioma, theprevalence of transversion mutations (56%) was higherthan transition mutations (33%). A large proportion oftransversion mutations occurred in exon 4, a region thatis not routinely screened in gliomas. We present here animproved method for screening exon 4 (and other GC-rich regions) ofp53 using PCR-single-strandconformation analysis. The high frequency oftransversion mutations suggests a role for exogenouscarcinogens in the etiology of malignant glioma.
Introduction
Approximately 34,000 benign and malignant brain tumors arediagnosed every year in the United States (1). The most fre-quent primary malignant brain tumor is cerebral glioma (2, 3).
Gliomas are classified morphologically as astrocytomas, oligo-dendromas, ependymomas, and mixed tumors. Astrocytomas,
the most common category, include a spectrum of tumorsranging from slow-growing juvenile pilocytic astrocytomas to
highly malignant glioblastoma multiforme. The incidence ofmalignant glioma has increased over the past 20 years (4), andthe etiology remains largely unknown (5-7). Even with themost aggressive available treatment, overall survival averages1-3 years (8).
Received 8/7/97; revised 12/1 2/97; accepted 1/8/98.
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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
� Supported by American Cancer Society Grant IRG-15-33 and USPHS Grant
CA 52689.
2 To whom requests for reprints should be addressed, at Department of Epide-
miology and Biostatistics, School of Medicine, University of California, San
Francisco, CA 94143-0560.
Mutations in the tumor suppressor gene p53 are found in-25% ofhuman gliomas (9, 10). Mutations occur in both high-
and low-grade astrocytomas (1 1-14) but are less common innon-astrocytic brain tumors ( 15). The majority of p53 muta-(ions in gliomas are reported to be transitions, including a high
proportion of G:C-�A:T transitions at CpG sites ( 10, 1 1, 16,17). The observed high frequency of transition mutations and
low frequency of transversions have lead some reviewers tosuggest that the etiology of gliomas is most compatible withendogenous mutagenic mechanisms, rather than the influenceof exogenous environmental carcinogens ( 13, 16, 17).
Most previous studies of gliomas have screened only ex-ons 5-8 (14, 18-19) or exons 5-9 (20) of the p53 gene. Only
one study screened introns within the exon 5-8 region (1 1); thesame study analyzed exon 4, but only a portion of this exon was
screened ( 1 1). Exon 4 is not routinely screened using SSCA3because its high GC content and complicated secondary struc-
ture interfere with conformation analysis. However, exon 4contains part of the highly conserved domain 2 of p53, as well
as a portion of the core DNA-binding domain critical for tumorsuppressor activity (21). Mutations in exon 4 ofp53 have been
reported in a wide variety of human tumors using direct se-quencing and other screening techniques (22, 23).
In this study, we screened 62 gliomas for mutations in p53using PCR-SSCA, followed by direct sequencing (24). We
developed methods for screening exon 4 of p53, includingrestriction enzymatic digestion of PCR products before SSCA
and variation in gel conditions.
Subjects and Methods
Human Tumor Samples. As part of the San Francisco BayArea Adult Glioma Study, a population-based, case-controlstudy of brain cancer (6), all histologically confirmed incidentcases of malignant glioma (ICD-0-2 morphology codes 9380-
9481) in adults aged 20 years and older were identified in sixSan Francisco Bay Area counties (Alameda, Contra Costa,Mann, San Mateo, San Francisco, and Santa Clara) for theperiod August 1 , 199 1 to March 3 1 , 1994. FFPE tumor blockswere obtained from a consecutive series of 62 incident cases to
develop techniques for p53 mutation analysis.
Centralized Histological Review. Pathology slides were re-viewed by a single neuropathologist (R. D.), who indicated the
most informative tumor block for molecular studies. Theseblocks were then sectioned and areas of tumor were marked on
an H&E-stained slide, which identified areas of interest forDNA extraction from 10-sm unstained sections.
Interviews. Structured in-person interviews were conducted toobtain personal and family history of cancer, as well as otherinformation. Details for obtaining and validating personal and
family medical histories, as well as the algorithm used for
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304 p53 Mutations in Gliomas
Table I p53 mutational ana lysis: Sequence of oligonucleotide primers for PCR-SSC A and direct DNA-sequencing
Exon” Size (bp) 5-primer” 3-primer
4.1 188 GACCTGGTCCTCTGACTGCT
( IN) CCTCTGACTGCTCTTTTCAC
CGGTGTAGGAGCTGCTGGTG
( IN) CGGTGTAGGAGCTGCTGGTG
4.2 249 TCCAGATGAAGCTCCCAGAA
(IN) AAGCTCCCAGAATGCCAGAG
ACGGCCAGGCATTGAAGTCT
(IN) TCTCATGGAAGCCAGCCCCT
5 294 GCTGCCGTGTTCCAGTTGCT
( IN) CCAGTTTCTTTATCTGTTCA
CCAGCCCTGTCGTCTCTCCA
( IN) TGTCGTCTCTCCAGCCCCAG
6 199 GGCCTCTGATTCCTCACTGA
(IN) CCTCTGATTCCTCACTGATT
GCCACTGACAACCACCCTTA
(IN) ACCACCCTTAACCCCTCCTC
7 196 TGCCACAGGTCTCCCCAAGG
(IN) GCGCACTGGCCTCATCTTGG
AGTGTGCAGGGTGGCAAGTG
(IN) TGTGCAGGGTGGCAAGTGGC
S 225 CCTTACTGCCTCTTGCTTCT ATAACTGCACCCTTGGTCTC
( IN) TCTCCTCCACCGCTTCTTGT( IN) CCTCTTGCTTCTCTTTTCCT
“ Exon 4 is divided into two fragments. For Exon 4-1, the 3-inner and outer primers share the same sequence.
I, (IN). internal primers for direct DNA sequencing.
classifying family history of cancer, have been presented else-where (6).
Extraction of Tumor DNA. For each tumor sample, areas of
tumor tissue were individually microdissected and scraped intoa I .5-ml microcentnfuge tube. Approximately three to fourunstained sections were used from each tumor. Tissues were
deparaffinized by adding 1 ml of xylenes, vortexing, and al-lowing the samples to sit at room temperature for 20 mm. Afterdecanting off the xylenes, samples were precipitated by adding1 ml of 95% ethanol. Samples were then dried by vacuum for2-3 h. After drying, 150 pA of lysis buffer containing 1% TritonX-l00, 1 x PCR reaction buffer (Perkin-Elmer Cetus, Norwalk,
CT), and 3 pA of 10 mg/ml proteinase K were added. Themixture was incubated at 58#{176}Cfor 3 h, followed by incubation
at 95#{176}Cfor 10 mm to inactivate the proteinase. The finalsolution was centrifuged at 12,000 rpm/mm for 10 mm and
stored at 20#{176}C.For analysis of p53 mutations, each tissueextract was divided into two aliquots. The first aliquot was usedto screen for mutations using SSCA and direct sequencing. Thesecond aliquot was used to confirm the presence of mutation in
a separate set of amplification and sequencing PCR reactions(described below).
PCR. The initial PCR amplification was conducted using a100-pA reaction mixture containing 10 mrvi Tris-HC1 (pH 8.3),50 mM KC1, 1 .5 mM MgCl,, 200 �tM deoxynucleotide triphos-phates, 2.5 units of Taq DNA polymerase (Perkin-Elmer Ce-
tus), 0.6 .LM 3’ and 5’ PCR-primers (exons 4-8 of the p53gene), and 1 pA lysis DNA solution. Primer sequences arepresented in Table 1.
Because exon 4 is a larger exon, we designed two over-lapping primer pairs to amplify each half of exon 4 separately
(exons 4- 1 and 4;2, see Table 1 ). The reaction mixture wasoverlaid with 75 pA of mineral oil and subjected to 35 cycles ofPCR amplification using a DNA thermocycler (Perkin-Elmer
Cetus). The first cycle consisted of 5 mm at 95#{176}C,1 mm at55#{176}C,and I mm at 72#{176}C,followed by 33 cycles of I mm at
94#{176}C,1 mm at 55#{176}C,and 1 mm at 72#{176}C.The final cycle was 1mm at 94#{176}Cand 10 mm at 60#{176}C.
SSCA. For SSCA, PCR products were subjected to a secondround of PCR amplification using radioactive labeling. The20-pA reaction mixture contained 10 msi Tris-HC1 (pH 8.3), 50
mM KC1-,, 1 .5 mM MgC1.,, and 120 mrvi of each deoxynucleotidetriphosphate, mixed with 1 pA of PCR product from the pre-
liminary PCR reaction, 0.2 p1 of [a-32P]dCTP (3000 Ci/ml),
0.5 unit of Taq polymerase, and 0.6 �.LM 3’ and 5’ internalprimers for each exon (Table 1 ). The reaction mixture was
subjected to the same cycle sequence as described for the initial
PCR reaction. Two pA of the amplified product were withdrawn
and mixed with 100 pA of 0. 1% SDS and 10 mrsi EDTA. Three
pA of this solution were mixed with 3 pA of 95% formamide, 20mM EDTA, and 0.05% xylene cyanol, heated to 95#{176}Cfor 6 mm,
and then placed on ice. To overcome problems generated by the
GC-rich secondary structure of exon 4-2, 5 pA of the second-round SSCA-PCR product for exon 4-2 were digested by A!uI
(United States Biochemical Corp., Cleveland, OH) in a totalvolume of 25 pA at 37#{176}Covernight.
Electrophoresis for SSCA was performed under two con-ditions for each sample: (a) 6 pA of the denatured PCR products
were electrophoresed for 4 h at 30 W in a 6% nondenaturing
polyacrylamide gel containing 10% glycerol at room tempera-
ture (cooled by a fan); and (b) a second 6-pA aliquot of eachsample was electrophoresed for 2 h at 30 W in a nondenaturing
polyacrylamide gel without glycerol at 4#{176}C(in a refrigerator).
Autoradiographs were exposed at -70#{176}Cfor 1-3 days. DNAfrom FFPE normal spleen was used as a negative control.
Specimens showing electrophoretic mobility shifts at eithertemperature compared with the negative control DNA were
deemed SSCA positive and were submitted to direct DNAsequencing.
Fig. 1 presents an example of SSCA analysis for exon 4-2.
SSCA patterns before and after restriction enzyme digestion areshown, with the electrophoretic mobility shift designated with
an arrow (sample 80).
DNA Sequencing. Direct sequencing of PCR products was
used to confirm positive SSCA results. First-round PCR prod-ucts were purified using Centricon 30 spin columns (Amicon,
Beverly, MA). For sequencing, a second round of amplification
was performed using the primers used for SSCA analysis (in-
ternal primers, Table 1). Primers were used in a ratio of 1 :50 togenerate single-strand amplification of the antisense DNAstrand and in a 50: 1 ratio for single-strand amplification of the
sense strand. Asymmetric PCR cycling conditions were iden-tical to those described for previous PCR reactions. Second-round PCR products were purified using Centricon 30 spin
columns. Sequencing of the purified asymmetric PCR productwas performed using the standard dideoxy-chain termination
approach recommended by the manufacturer (United StatesBiochemical). Samples were electrophoresed on an 8% Se-
quencing gel at 55 W for 2 h. The gel was dried and exposed
to Hyper film (Amersham, Arlington Heights, IL) overnight atroom temperature. Ten % of all SSCA negatives were submit-
ted to direct DNA sequencing as a standard procedure to
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Cancer Epidemiology, Biomarkers & Prevention 305
SamplelD: � � � �
(a)
. --� .1
SamplelD: � � E �
. � � � =�4e a a
I
(b)
Fig. 1. SSCA ofp53. exon 4 (2). a. SSCA-PCR gel electrophoresis. b, SSCA-
PCR gel electrophoresis after AluI restriction enzyme digestion of PCR products.
determine the false-negative rate of SSCA screening. In ourcumulative experience with over 300 samples analyzed for p53
mutations using SSCA, we have not uncovered any false neg-atives.
A sample was scored as a preliminary positive for p53mutation only if the same mutation was observed on both the
sense and antisense DNA strand of PCR products from the firstaliquot of tissue lysate. Preliminary positives were submitted toa second round of PCR amplification and sequencing using the
second stored aliquot of the original tissue lysate. Samples werescored as a final positive only if the same mutation appeared on
both sense and antisense strands of both aliquots of the tissuelysate.
Statistical Analysis. Fisher’s exact test was used to evaluateassociations between the presence of p53 mutation and family
history of any cancer, family history of brain tumors, andpersonal history of other cancers (25). Two-sided Ps werecalculated using SAS (26).
Results
Histological Classification. Tumors were classified into the
following histological categories: glioblastoma multiforme(WHO grade IV; n 39); highly anaplastic astrocytomas(WHO grade III; n = 11); mixed gliomas, including oligoas-
trocytomas and other mixed types (n = 6); and other tumors,
consisting of juvenile pilocytic astrocytomas (n = 2), oligo-dendroglioma (n 1), ependymoma (n = 1), low-grade astro-cytoma (n = 1), and an anaplastic glioma too small to be
classified.
p53 Mutation. Of 62 tumors analyzed, 12 (19%) showed mu-
tations in p53. Histological characteristics of gliomas with andwithout p53 mutations are presented in Table 2. The proportion
of tumors bearing p53 mutations within each histological cat-egory were: 8 of 39 (21%) glioblastomas; 1 of 1 1 (8%) highly
anaplastic astrocytomas; and 2 of 6 (33%) mixed gliomas. Inthe “other” category, the only tumor showing p53 mutation was
an anaplastic glioma too small to be classified.
Patient Characteristics. Characteristics of patients with p53mutation-positive and p53 mutation-negative gliomas are pre-sented in Table 3. Patients were categorized as White or non-
Table 2 San Francisco Bay Area Adult Glioma Study ) 1991-1995):
Histological characteristics of gliomas according to p53 mutation status
J)53 mutationMorphology --------� �-- �- �- -
Negative (%) Positive (‘Ic)
Glioblastoma 31 (62) S (67)
Highly anaplastic astrocytoma 0 (20) I (8)
Mixed glioma 4 (8) 2 ) I7
Other 5” (10) I” (8)
Total 50 (100) 12 (1(8))
“ Includes juvenile pilocytic astrocytoma (n = 2). oligodendroglionia (ii
ependymoma (n = 1 ). and low-grade astrocytoma (ii = I).
I, Anaplastic glioma (too small to be classified).
Table 3 San Francisco Bay Area Adult Glioma Study ( 199 1-1995):
Characteristics of glioma patients according to pSi mutation status
= I
GroupJS53 mutation
Negative (‘/) Positive (‘k)
�1 50 12
Age
Mean (yr) ± SD 54.7 � 16.1 55.5 ± 20.2
20-29 3 (6.0) I (8.3)
30-39 8l6.0) 2(lfl.7
40-49 714.0) 3)25.0)
50-59 13 (26.0) I (8.3)
60-69 6(12.0) I(8.3
70 or over 13 (26.0) 4 (33.3)
Race
White 45 (90.0) 1 1 (91.7)
Non-White” 5 ( 10.0) I (8.3)
Sex
Male 28 (56.0) 7 (58.3)
Female 22(44.0) 5(41.7)
Family history of any cancer
Positive 21 (42.0) 7 (58.3)
Negative 22 (44.0) 4 (25.0)
Missing 7 ( 14.0) 1 ) 16.7)
Family history of brain tumors
Positive I (2.0) 0
Negative 45 (90.0) 1 1 (92.0)
Missing 4 (8.0) I (8.0)
Personal history of other cancers
Positive 4(8.0) 2)16.7)
Negative 46 (92.0) 10 (83.3)
Includes one African-American, three Asian-Americans. and two Hispanics.
White (including African-American, Asian-American, or His-
panic). Differences between those with and without p53mutations were minimal for age, race, and gender. The patientgroups also did not differ significantly according to family
history of other cancers (P = 0.30), family history of braintumors (P = 0.81), or personal history of other cancers (P =
0.33).
Spectrum and Location ofp53 Mutations. The location andspectrum of observed p53 mutations are presented in Table 4.All mutations were single-base pair point mutations except fora duplication of 18 bases (TGAGCGCTGCCCCCACCA) inexon 5. The majority of mutations were transversions (7 of 12,
58%), whereas only 33% (4 of 12) were transitions. Transitionmutations occurred at CpG sites (27) in two patients (cases 3
and 1 83). In three patients, the DNA alteration did not result ina change in amino acid (cases 3, 80, and 142). Excludingpatients with noncoding mutations, the proportion of transver-
sions was 56% (5 of 9), and 33% (3 of 9) contained transitions.
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306 p53 Mutations in Gliomas
Table 4 San Francisco Bay Area Adult Glioma Study (1991-1995): Location and spectrum of p53 mutations
Block number Tumor typeAge at . . . .. . Location Codon Nucleotide change Amino acid Type
diagnosis
Family history
of cancer
3 GBM 77 Exon 4 36 CCG -� CCA Pro -“ Pro G -“ A Transition Yes
65 GBM 49 Exon 4 69 GCT -s GAT Ala -� Asp G - A Transversion Adopted
258 AG 77 Exon 4 53 TGG -“ TGC Trp -‘ Cys G -� C Transversion Yes
80 GBM 85 Exon 4 1 12 GGC -“ GGA Gly -“ GIy C -“ A Transversion Yes
142 GBM 76 Exon 4 1 12 GOC -“ GGA Gly -‘ Gly C -“ A Transversion No
45 GBM 58 Exon 5 TGAGCGCTGCCCCCACCA (18 bps) Duplication No
92 GBM 63 Exon 7 234 TAC -a CAC Tyr -“ His T -a C Transition Yes
238 GBM 47 Exon 7 251 ATC - AGC lIe -� 5cr T -“ G Transversion Yes
183 GBM 39 Exon 8 273 CGT -a CAT Arg -“ His G � A Transition No
159 HAA 40 Exon 6/ Splice gt -“ gg T -“ G Transversion
Intron 7 Site
Yes
84 Mixed 30 Exon 7 234 TAC -* TGC Tyr -� Cys A -“ G Transition Yes
I I I Mixed 25 Exon 8 270 1TI’ -“ TGT Phe -“ Cys T -“ G Transversion No
“ GBM, glioblastom a multiforme; A G, anaplastic glioma (too small to be classified); HAA, highly anaplastic astrocytoma; Mixed, mixed gliomas.
Discussion
We analyzed p53 mutations from 62 incident cases of malig-
nant glioma from a population-based, case-control study. Mu-
tations were found in 12 tumors ( 19%). Previous studies of
glioma report an average mutation prevalence of 25%, with arange of 18-37% (reviewed in Refs. 10, 12, 13, 17, and 28).Most previous studies screened only exons 5-8 of p53. Ouroverall prevalence of p53 mutation is low relative to otherstudies, despite more extensive screening of exon 4 as well as
intron/exon boundaries. This lower prevalence may reflect the
fact that our study is population based. However, the distribu-(ion of gliomas by histological type is similar to previous
hospital-based studies (Table 2). The lower prevalence of mu-
tation could also have resulted from the fact that we repeatedamplification and sequencing on all positive samples, avoidingpotential PCR artifacts.
We observed p53 mutations only in high-grade and mixed
tumors (Table 2). However, the number of low-grade tumorsanalyzed was small. Some previous studies report that theprevalence of p53 mutations is similar in low- and high-grade
gliomas (1 1, 15). However, Fulls et a!. (29) reported a highprevalence ofp53 mutation in high-grade gliomas and absenceof mutation in low-grade gliomas, and Rasheed et a!. (20)reported a higher prevalence of p.53 mutation in patients with
anaplastic astrocytomas. Sidransky et a!. (30) hypothesized thatp53 mutation may be involved in progression of gliomas fromlow to high grade. Van Meir el a!. (31) reported that many
glioblastomas contain only wild-type p53, suggesting that in-activation of p53 is not an obligatory step in formation of
high-grade brain tumors. Additional models for development ofmalignant glioma that combine histology and molecular alter-ations at a variety of loci have been presented (17, 28).
Previous studies suggest that patients with p53 mutation-
positive gliomas are younger than patients with mutation-neg-ative tumors (reviewed in Ref. 12). We compared these twopatient groups in our dataset and did not observe appreciabledifferences based on age, race, or gender (Table 3). Patientswith p53 mutations were slightly more likely to have a historyof cancer in first-degree relatives (58% versus 42%) as well as
a personal history of other cancers (17% versus 8%; Table 3),but these differences were not statistically significant.
The prevalence of germ-line p53 mutation among uns-
elected glioma patients is reported to be quite low (32, 33). Liet a!. (34) detected germ-line p53 mutations in only 1 of 80unselected glioma patients. Prevalence of inherited p53 muta-
tion is reported to be higher among patients with a family
history of cancer, a personal history of other primary malig-
nancies, or both (8, 34). We felt it was important to address thepossibility of germ-line mutation, because some patients in ourstudy reported a positive family history and/or a personal his-
tory of other malignancies (Table 3). However, we were unableto perform p53 mutation screening using germ-line DNA sam-ples because the informed consent for our case-control study
did not grant permission for genetic testing.The CGT-*CAT mutation at codon 273 (case 183) has
been reported previously as an inherited mutation in Li-Frau-
meni syndrome (32, 33). However, codon 273 also represents a
hotspot for somatic alteration in gliomas ( 10, 1 1, 13, 16, 18),
and the CGT-+CAT mutation has been reported as a somaticalteration in sporadic brain tumor patients (9, 14, 27). The
patient with the codon 273 mutation (case 183) did not reporta positive family history. In addition to codon 273, a high
frequency of somatic mutation has been reported in codons 175and 248 ofp53 in gliomas (1 1, 18; reviewed in Refs. 10, 13, and
17). We did not observe mutations at codons 175 or 248 in ourdataset. The CCG-*CCA alteration at codon 36 has beenreported as a germ-line polymorphism (35) and as a somatic
genetic alteration in lung cancer (23, 27). The remaining mu-
tations have been reported previously in tumors from a varietyof sites ( 10, 22-23, 27, 36).
In three patients, the mutations in p53 were not predictedto lead to a change in amino acid sequence (cases 3, 80, and
142). Noncoding mutations in p53 mutations have been re-ported in gliomas (14, 18, 27) and other tumors (27, 36). Some
noncoding mutations in p53 (37) and other genes (38, 39, 40)have the potential to create cryptic splice sites. However, the
functional significance of the noncoding mutations observed incases 3, 80, and 142 is unknown. In the database of Hollstein
et a!. (27), 3% ofp53 mutations in all tumors as well as gliomasare silent. According to Strauss (41), the high frequency ofsilent mutations in p53 suggests that a hypermutability processmay operate on this gene during tumorigenesis. However, it is
possible that silent mutations bear no relationship to tumor
etiology (10, 41). Thus, in the discussion of potential etiologyof glioma (below), we exclude noncoding mutations.
Our observed proportion of transversion mutations (56%
of coding mutations) is higher than previous reports of gliomas
[19% in the p53 databases of both Greenblatt et a!. (10) andHollstein et a!. (27)]. Two of the coding transversion mutations
in our dataset were observed in exon 4. Although exon 4 is one
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Cancer Epidemiology, Biomarkers & Prevention 307
of the larger exons of p53 and contains a portion of the coreDNA-binding domain (21), only 20% of studies that screen for
p53 mutations analyze exons other than 5-8 (41). Studies that
sequence additional exons of p53 often find a different patternof mutation (42, 43). Interestingly, in the database of Hollstein
et a!. (27), of five mutations reported in exon 4 in gliomas, fourwere transversions and one was an insertion mutation.
One reason for failure to screen exon 4 of p53 may betechnical difficulties using the most common screening method,
SSCA. To address the relatively large size of exon 4, as well assecondary structures created by regions of high GC content, we
modified our SSCA protocol to screen for mutations in this
region: (a) to decrease the size of the PCR amplicon, we
designed two overlapping primer pairs within exon 4, ampli-
fying fragments of 1 88 bp (primer set 4- 1) and 249 bp (primer
set 4-2). Sensitivity of SSCA has been shown to decrease withfragment sizes larger than 300 bp (24). We followed protocolsaimed at achieving 90% or greater sensitivity for SSCA screen-ing using FFPE tissue extracts (24); (b) we performed restric-tion enzyme digestion of the second exon 4 fragment (exon 4-2)with A!uI to relieve intrastrand binding and reduce secondarystructures (e.g., hairpin loops) created by regions of high GCcontent; and (c) we modified the gel running conditions, in-
cluding variations in temperature and glycerol content. Thesemodifications increased the number of SSCA bands and facil-itated detection of mobility shifts required for identification of
sequence alterations (Fig. 1).Our finding of a high prevalence of transversion mutations
in p53 suggests that exposure to exogenous environmental
factors should be considered in the etiology of malignant gli-
oma. A high prevalence of transversion mutations has beeninterpreted as suggestive of the action of exogenous carcino-
gens (e.g. , chemical mutagens), whereas a high prevalence oftransitions, particularly at CpG sites, suggests endogenous mu-
tagenic processes (10, 16, 44). Previous studies of gliomasreport a higher prevalence of transition than transversion mu-
tations in p53 (10, 1 1, 18, 27). Our observed prevalence oftransversion mutations (56%) is similar to that of lung tumors
(57%; Ref. 10). Our results must be regarded as preliminary,because our series of glioma patients is small (n = 62). We
suggest that future studies of glioma (and perhaps other malig-nancies) routinely screen for mutations in exon 4 of the p53
gene. The laboratory techniques presented in this report mayprove useful in this regard. In epidemiological studies aimed at
identifying risk factors for glioma, history of exposure to en-vironmental factors (including solvents and other occupationalexposures) should be considered.
Acknowledgments
We thank two anonymous reviewers for helpful comments on the manuscript.
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