Nov 22, 1993 - year.1 It is the second most frequent cancer in de- veloping countries ..... nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide. J. Natl Cancer ...
American Journal ofPathology, Vol. 144, No. 3, March 1994 Copyright © American Society for Investigative Patbology
p53 Alteration in Gastric Precancerous Lesions
Yih-Horng Shiao,* Massimo Rugge,t Pelayo Correa,* H. Peter Lehmann,* and W. Douglas Scheer* From the Department ofPathology,* Louisiana State University Medical Center, New Orleans, Louisiana; and Cattedra di Istochimica ed Immunoistochimica,t Universita degli Studi di Padova-Servizio di Anatomia Patologica,
Cittadella, Padua, Italy
It has been postulated that chronic atrophic gastritis, intestinal metaplasia, and dysplasia are precancerous stages of stomach tumorigenesis. We investigated the timing of p53 alterations in these events ofgastric tumorigenesis. Each of 12 cases ofarchived tissue containing precancerous and cancerous lesions were selected for the detection of p53 alterations. Accumulation of p53 protein was detected by immunohistochemistry. Exons 5 to 8 ofp53 gene were examinedfor mutations by polymerase chain reaction-single strand conformation polymorphism and DNA sequencing. p53 immunoreactivity was detected in 60% ofthe dysplasia cases and in 60% ofthe cases with carcinomas. p53 gene alterations werefound in 37.5% of the metaplasia cases, 58.3% ofthe dysplasia cases, and 66.7% of the cases with carcinomas. In 71 % of the cases, mutations were shown as G:C -- A:T transitiont We conclude that mutation ofthe p53 gene is an early event in stomach tumorigenesis. (AmjPathol 1994, 144:511-
diagnosed at an advanced stage.5 Therefore, an understanding of phenotypic and genotypic events in stomach tumorigenesis is important in terms of early detection, selecting appropriate treatment, and hence preventing the progression of this disease. The most common gastric cancer in populations at high risk is the so called intestinal type, in which the malignant tissue resembles glands of the gastrointestinal tract.6 This type is preceded by a chain of morphological events, namely, chronic gastritis, atrophy, metaplasia of the small intestinal and colonic types, dysplasia, intramucosal (early) carcinoma and invasion.7 p53 gene mutations have been reported in the dysplasial and carcinoma9'10 stages. The accumulation of p53 protein has also been detected in dysplasia1l and carcinoma.12'13 Patients with p53 protein accumulation in gastric carcinoma showed a worse survival than those without p53 accumula-
tion.12.13 To investigate the timing of p53 alterations in the process of gastric tumorigenesis, areas representing
morphologically normal cells (mucosa, lymphocytes, or muscle), intestinal metaplasia, dysplasia, and carcinoma from each gastrectomy specimen were mi-
crodissected from formalin-fixed, paraffin-embedded sections and submitted for detection of p53 gene mutations by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), and DNA sequencing. p53 protein accumulation was detected by immunohistochemistry (IHC).
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Materials and Methods In 1980, stomach cancer was a leading cancer throughout the world, with 682,400 new cases per year.1 It is the second most frequent cancer in developing countries and the fourth most frequent cancer in developed countries.2 In the United States, cancer of the stomach is the eighth most common cause of cancer deaths. Although the mortality rate has decreased from 22.8 per 100,000 in 1950 to 9 per 100,000 in the 1980s in white males and from 12.3 to 4.3 per 100,000 in white women in the United States,3 the overall five-year survival rate is only 5 to 15%.4 The reason for the low survival rate of stomach cancer is related to the fact that the disease is typically
Study Subjects Twelve gastrectomy cases were selected from a group of Italian patients enrolled in a prospective study on gastric epithelial dysplasia.14 Morphologically normal cells (mucosa, lymphocytes, or muscle), gastric epithelial dysplasia, and gastric Supported by NIH grant PO1-CA28842. Accepted for publication November 22, 1993. Address reprint requests to Dr. Pelayo Correa, Department of Pathology, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70112.
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carcinoma were obtained from all 12 patients. Intestinal metaplasia was identified only in eight cases. After performing a 5-p section for hematoxylin and eosin staining, five serial 10-p sections were cut for DNA extraction. An additional 5-p section was prepared for p53 immunohistochemical determination.
IHC Sections were dewaxed in xylene (twice for 5 minutes each) and rehydrated through serial ethanol (100%, 95%; two changes of 3 minutes each) to distilled water (twice for 5 minutes each). A polyclonal antibody against both wild-type and mutant p53 protein (CM-1; Signet Laboratories, Inc., Dedham, MA)15 was used at a working concentration of 1:25 (v/v) dilution at room temperature for 20 minutes. Hydrogen peroxide, normal goat blocking serum, biotinylated immunoglobulins, avidin-biotin complex, and 3-amino-9-ethylcarbazole substrate solution were used according to the instructions from the detection kit (Signet ELITE avidin-biotin detection system; Signet Laboratories, Inc.). Sections were lightly counterstained with Mayer's hematoxylin, mounted with aqueous mounting medium (Crystal/Mount; Biomeda, Foster City, CA), and postmounted with Postmounting Crystal/Mount in Permount (Biomeda). Positive control sections from formalin-fixed, paraffin-embedded colonic cancers were cut and prepared in the same manner as the specimens. In serial sections from each case tissue, primary antibody was omitted as the negative control. In each lesion the nuclear p53 immunoreactivity, identified as a red stain, was determined by an experienced pathologist (MR). Immunoreactivity in any nuclei was considered as positive for p53 protein accumulation.
DNA Extraction Five 10-p sections were dewaxed using a routine histological procedure. Unstained section was overlaid on a hematoxylin-and-eosin-stained section, in which the precancerous and cancerous areas had been circled with marking pen. Each lesion was microdissected from the unstained sections following the mark and were combined in a 1.5-ml microcentrifuge tube for proteinase K digestion as described by Wright and Manos16 with some modifications; use of 1% (w/v) sodium dodecyl sulfate instead of 0.5% (v/v) tween 20 solution and incubation with enzyme for 2 days instead of overnight. After heat in-
activation of the proteinase K, DNA was extracted by adding 160 pl saturated NaCI solution to the proteinase K digests, mixing thoroughly, and centrifuging for 15 minutes at 12,000 rpm in a bench-top Eppendorf Centrifuge at room temperature. The upper aqueous layer was transferred to a fresh 1.5-ml eppendorf tube using a wide-bore pipette to avoid the shearing of DNA. The DNA was then precipitated with 3 volumes of cold absolute ethanol, placed on ice for 30 minutes and centrifuged (12,000 rpm) for 15 minutes at room temperature to pellet the DNA. After careful removal of the supernatant, 500 pl of 70% ethanol was added to wash the DNA. The tubes were centrifuged at 12,000 rpm for 15 minutes and the supernatant was discarded. After drying in an RC 10.10 Concentrator (Jouan, Winchester, VA), DNA was dissolved in 100 pl of double distilled water at 4 C overnight. Finally, the amount of dissolved DNA was quantified on the TKO-100 Minifluorometer according to the manufacturer's procedure (Hoefer Scientific Instruments, San Francisco, CA) and transferred to a 0.5-ml microcentrifuge tube for long-term storage at -20 C. A negative control containing only reagents and no tissue was run in parallel for each DNA extraction.
PCR-SSCP The primers for amplification of p53 exon 5 to exon 8 (Figure 1) from genomic DNA were selected using the "OLIGO" software program.17 The PCR mixture, modified from Prior,18 contains 67 mmol/L Trisbase (pH 8.8), 16.6 mmol/L ammonium sulfate [(NH4)2SO4], 6.7 pmol/L disodium ethylenediaminetetraacetic acid (EDTA), 0.5 pg/pl nuclease-free bovine serum albumin, 1.5 mmol/L MgCI2, 200 pmol/L deoxyribonucleoside triphosphates, 0.5 pmol/L primers, 1.25 units of Taq polymerase (Cetus-Perkin Elmer, Norwalk, CT), and 20 ng of DNA template in a total 50 pl reaction mixture. After adding 50 to 100 pl of mineral oil to cover the top of each reaction mixture, the reactions were carried out in Ericomp Twinblock thermal cycler (Ericomp, San Diego, CA), which had been prewarmed to 90 C. The temperature profile, as described by Wright and Manos,16 denatures the DNA at 94 C for 5 minutes in the first cycle followed by 30 seconds annealing at 55 C, 2 minutes extension at 72 C, and 30 seconds at 94 C for a total of 40 reaction cycles. The extension time for the last cycle was increased to 5 minutes to ensure complete extension. A negative control containing no DNA template was run in parallel for each amplification reaction.
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1
3
3
3
EA E5A
+2-T
5
5B6A 4 E6B+B 6 ESB
E5A(152 bp)
9
7
1
E7 E7
T 8
E8 E8
10 5
1: 5'-TTCCTCTTCCTGCAGTACTC-3'
2: 5'-TCCGTCATGTGCTGTGACTG-3'
E5B6A(167 bp)
3: 5'-GCCATCTACAAGCAGTCACA-3' 4: 5'-GCCAGACCTAAGAGCAATCA-3'
E6B(132 bp)
5: 5'-TTAGGTCTGGCCCCTCCTCA-3' 6: 5'-AGTTGCAAACCAGACCTCAG-3'
E7(136 bp)
7: 5'-TTGTCTCCTAGGTTGGCTCT-3'
8: 5'-CAAGTGGCTCCTGACCTGGA-3'
E8(149 bp)
9: 5'-TGGTAATCTACTGGGACGGA-3'
10: 5'-CTGCTTGCTTACCTCGCTTA-3'
Before carrying out SSCP, 10 pl of PCR product was electrophoresed in 2% (w/v) agarose and visualized with ethidium bromide stain (0.5 pg/ml) to confirm the absence of contamination and to ensure that the PCR product was a single band of the appropriate size. The SSCP method was modified from Ainsworth et al.19 One microliter of each PCR product was mixed with 5 pl of a denaturing solution of 95% (v/v) formamide, 20 mmol/L disodium EDTA, 0.05% (w/v) xylene cyanole, and 0.05% (w/v) bromophenol blue. Immediately before electrophoresis, the samples were heated to 95 C for 5 minutes in a water bath. After heat denaturation, the tubes were immediately placed on ice to prevent renaturation. A 2-pI aliquot of each denatured sample were loaded onto 0.75-mm-thick, 12% (w/v) polyacrylamide gel (29:1 ratio of acrylamide to bisacrylamide) gel with 22.5 mmol/L Tris-borate (pH 8.4) and 2 mmol/L EDTA in Miniprotein 11 Slab cell (BioRad, Richmond, CA). Electrophoresis of gel was carried out at room temperature at 5 mA constant current for 4 hours. Single stands of DNA were visualized with silver stain (Bio-Rad) according to the instructions provided by the manufacturer. The band pattern of each precancerous or cancerous lesion was compared to that of morphologically normal cells from the same patients. Any extra band(s) present in the sample was considered as positive for a mutation and DNA sequencing was performed on those
samples.
Figure 1. Locations and sequences of five primer sets (E5A, E5B6A, E6B, E7, and E8) for the amplification of p53 exons 5 to 8. Arrow primer. Number in parenthesis: base pairs of amplified product.
DNA Sequencing Twenty microliters of PCR product was electrophoresed in 1% (w/v) agarose gel. The target product was excised from the gel and eluted in Tris-EDTA buffer at 55 C for at least 10 hours. After elution, DNA was precipitated with absolute ethanol, washed with 70% (v/v) ethanol, and dried in the RC 10.10 Concentrator (Jouan). DNA was redissolved into 20 pl double-distilled water and 5 pl of this solution was used for DNA sequencing. A dideoxy DNA sequencing method was performed using CircumVent Thermal Cycle Sequencing Kit as described by the manufacturer's instructions (New England BioLabs, Beverly, MA). The detection signal was revealed by incorporation of [a-35S]dATP into the DNA sequence. The sequence ladder was resolved in 7.7 mol/L urea and 6% (w/v) polyacrylamide gel using a 35 x 40 cm electrophoresis chamber (GIBCO, Bethesda Research Laboratories, Gaithersburg, MD). After electrophoresis, the gel was dried in a gel dryer (Bio-Rad) and exposed to x-ray film for 3 to 5 days at room temperature.
Results The results of p53 alterations in precancerous and cancerous lesions detected by IHC, PCR-SSCP, and DNA sequencing are shown in Table 1.
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Table 1. p53 Alterations Detected by IHC, SSCP, and DNA Sequencing
Case 1 2 3 4 5 6 7 8
9 10 11 12
Normal cells IHC SSCP -
-
-
_ _ *E6
_ -
-
-
-
-
-
-
I.T. -
-
-
-
-
*E6B
-
IM IHC SSCP
*E7
IT. -
NP. NP. -
*E6B -
E5B6A *E5B6A NP. -
NP. *E7
IHC
+ +
lT.
GED SSCP *E7 *E7 *E6B E7
IHC
+
-
+
IT.
-
*E5B6A
+ +
*E5B6A
+ +
-
IT.
IT. + +
*E5B6A *E6B *E7
+ +
GC SSCP *E7 *E7 *E6B *E7 *E5A *E5B6A *E5B6A
-
*E5B6A *E6B *E7
DNA sequencing
Codon
*CCATCCTCA deletion *CGG-CAG (missense) Not tested *CGA-*CGG (polymorphism) *TGC-TCC (missense) *CCC-TCC (missense) *T-*C (Intron5) *CGCOCAC (missense) Not tested Not tested *CGCOCAC (missense) *CGA.--CGG (polymorphism) *AAC-*AAT (same A.A.)
250-253 248 213 242 151 175 175 213 247
IM: intestinal metaplasia; GED: gastric epithelial dysplasia; GC: gastric cancer; A: adenosine; C: cytidine; G: guanosine; T: thymidine; A.A.: amino acid; N.P.: lesion not present; IT.: insufficient tissue. * Mutation confirmed by DNA sequencing.
Immunohistochemical results were not obtained in two cases (6 and 10) because of insufficient tissue. A positive p53 immunoreactivity was observed in the stage of dysplasia and carcinoma (Figure 2). p53 immunoreactivity was detected in 60% (6 of 10) of the dysplasia cases, and 60% (6 of 10) of the carcinoma cases. A consistent stain, either positive
Figure 2. p53 immunohistochemical stain with CM-1 antibody. N. normal mucosa; D: dysplasia; arrow: positive immunoreactivity in nuclei (250X).
or negative, in both precancerous and cancerous stages was observed in 60% of cases. However, there were two cases (3 and 8) having a positive p53 stain in dysplasia but not in carcinoma. p53 gene alterations detected by PCR-SSCP only were found in 25% (3 of 12) of the normal, 50% (4 of 8) of the metaplasia, 66.7% (8 of 12) of the dysplasia, and 75% (9 of 12) of the carcinoma cases. When the degree of dysplasia was considered, there were p53 gene alterations detected by PCRSSCP in two cases of mild dysplasia (cases 5 and 7), one case of moderate dysplasia (case 6), and five cases of severe dysplasia (cases 1, 2, 8, 11, and 12). All the alterations were located in the exons 5 to 7 region. No mutations were detected in exon 8. Once a mutation of a particular exon presented in a precancerous stage, the same alteration was also detected in all subsequent stages, including carcinoma (Figure 3), except in case 6. In this case, different mutations were present in metaplasia, dysplasia, and carcinoma. The gene alterations detected by PCR-SSCP were confirmed by DNA sequencing (Figure 4). Samples with the same electrophoretic pattern in SSCP showed the same mutation by DNA sequencing. Case 1 had a 9-base deletion between codons 250 to 253. Five cases (2, 5, 6, 8, and 11) had missense mutations. Case 7 presented a mutation at intron 5. Three cases (4, 11, and 12) had base changes without substitution of amino acids in all stages of tumorigenesis. Base change at codon 213 in cases 4 and 1 1 was a natural polymorphism. G:C -> A:T mutation was detected in 71% (5 of 7) of point mutations. The majority of cases with negative IHC and positive SSCP were found in cases with gene deletion
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Case 1 N
D
C
N
Case 2 D C
N
Case 12 D M
C
Figure 3. PCR-SSCP analysis of exon 7 in cases 1, 2, and 12. N: normal cells; M: metaplasia; D: dysplasia; C: carcinoma; arrow: extra band representing a mutation.
Case 12
(case 1), intron mutation (case 7), or base changes without substitution of amino acids (cases 4, 11, and 12).
31
__C -> T
Discussion Mutations of the p53 gene have been widely studied in primary gastric carcinoma by immunohistochemical and molecular genetic techniques 12,13,20,21 The presence of p53 alterations in precancerous stages has also been reported8 in isolated lesions from different patients. In this study, detection of p53 alterations in samples containing precancerous and cancerous lesions from the same patient provided an opportunity to study the spectrum of p53 alterations in gastric tumorigenesis. The high prevalence of immunoreactivity, by IHC, in dysplasia and carcinoma suggests that p53 protein accumulation is associated with gastric tumorigenesis. Observation of a positive p53 stain starting from dysplasia demonstrates that p53 protein accumulation occurs at a late precancerous stage. Examination of the p53 gene by PCR-SSCP and DNA sequencing revealed mutations that were not detected by immunohistochemistry (cases 1 and 7). This indicates that alterations of the p53 gene may not always result in protein accumulation. Detection of the same type of mutation in precancerous stages and in carcinoma provide evidence for the monoclonal expansion of mutated cells (cases 1, 2, 7, 8, 11, and 12). In case 6, different mutations were detected by SSCP in metaplasia, dysplasia, and carcinoma. In addition, no base change was identified by DNA sequencing in metaplasia and dysplasia. The repeated experiment also showed the same result. This suggests that the instability of the p53 gene during tumor progression22 could initiate gene alterations in a small population of cells, which may not be detected by DNA sequencing. A possible false positive base misincorporation during PCR by Taq polymerase is low because the fragment of PCR product was short.23 The chance for error of amplification in three different lesions from
-A
51
Case 11 A
C
G
T
3' -C -G -> A 5'
Figure 4. Dideoxy DNA sequencing on cases 12 and 11. Case 12: A mutation in codon C -* T mutation in codon 247; case 11: G 175.
the same patient is unlikely. Nucleotide A-,G transition at codon 213 in cases 4 and 11 has been known as a rare polymorphism.24 Base change without amino acid substitution at codon 247 in case 12 may be a background mutation during evolution. Seven out of the nine cases on which DNA sequencing was carried out, gene alterations were localized in exon 5 and exon 7. In addition, mutations
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in codons 175 and 248, which have been frequently detected in gastric cancer,9,25 were observed in three cases (2, 8, and 11). This suggests that the same carcinogen may have been involved, or a particular conformation of sequence-dependent DNA in this region is susceptible to the binding of a specific carcinogen. Seventy-one percent (5 of 7) of the detected mutations were G:C -- A:T transition, which has been reported as a major type of mutation in gastric cancer.2627 This type of mutation has been linked to exposure to nitric oxide.28 29 Nitroso compounds have been experimentally used to induce gastric cancer.3031 Inflammatory cells, in the gastric mucosa, associated with Helicobacter pylori infection have been proposed as source of nitric oxide-related mutagens in gastric carcinogenesis.6 Applications of PCR-SSCP to cases (3 and 9) that showed positive IHC revealed that the detectable accumulation of p53 protein may not result from mutations of p53 gene or that mutations were located outside the exons 5 to 8. It has been shown that agents that damage DNA32 and viral proteins33 can induce the accumulation of wild-type p53 protein. In two cases (3 and 8), positive p53 immunoreactivity detected in dysplasia was not detected in carcinoma. This may represent a false negative result, which has been reported in formalin-fixed, paraffinembedded tissue.34 Another possibility is loss of p53 expression in the progression to carcinoma. A negative p53 immunohistochemical stain was observed in cases with gene deletion, intron mutation, base change without substitution of amino acid, and no detected mutation. This may be caused by either loss of p53 expression or the short half-life of wild-type p53 protein. A positive immunoreactivity detected in dysplasia or carcinoma for cases 7 and 12, which have intron mutation and base change without amino acid substitution, respectively, could be a result of mutations outside exons 5 to 8. All samples with missense mutations showed a positive p53 stain, suggesting that missense mutations result in a stable p53 protein. Associations of p53 protein accumulation with poor survival12'13 have been shown, but a contrary result has also been reported.35 In addition, as we have pointed out that alterations of the p53 gene may not result in the accumulation of p53 protein, and vice versa. To clarify the relationship between p53 alterations and survival requires a study considering both p53 protein accumulation and gene mutations in a larger population. We conclude that the mutation of p53 gene is an early event in stomach tumorigenesis, although its
detection by immunohistochemical stain is first seen at the stage of dysplasia. This also indicates that PCR-SSCP is a sensitive technique for detecting genetic alterations. Early detection of p53 alterations in precancerous gastric lesions will be useful information in terms of prevention and may be relevant in understanding its role in the natural history of the disease.
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