A Comprehensive System to Explore p53 Mutations

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1 A Comprehensive System to Explore p53 Mutations Chizumi Furuwatari,1 Asako Yagi,1 Osamu Yamagami,1 Masayo Ishikawa,1 Eiko Hidaka, Ichiro Verio, PhD,1 Kenichi Furihata, MD, PhD,2 Yoshifumi Ogiso, MD, PhD,1 and Tsutomu Katsuyama, MD, PhD2 Key Words: p53 mutations; Polymerase chain reaction-single-strand conformation polymorphism; PCR-SSCP; Fluorescence in situ hybridization; FISH; Immunohistochemistry To establish an effective and reliable system for the detection of p53 mutations, we evaluated the detection efficiencies of nonisotopic polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), fluorescence in situ hybridization (FISH), and immunohistochemistry. Ten cell lines (AsPcl, BxPc3, Miapaca.2, Panel, Colo32-11, Lovo, MCF, LNCaP, HL-6, and Daudi), a peripheral blood sample from a patient with a p53 germline mutation (p53gml), and a normal peripheral blood sample were used for examination. Direct nucleotide sequencing identified p53 mutations in of 12 samples (AsPcl, BxPc3, Miapaca.2, Panel, Colo3211, HL-6, andp53gml). The nonisotopic PCR-SSCP detected anomalies of the PCR fragments in 5 cell lines. In the FISH analysis, 2 cell lines exhibited loss of heterozygosity of the p53 locus. Immunohistochemistry detected an accumulation of the abnormal p53 in 4 cell lines. The combination of these 3 methods produced no false-negative or false-positive results. This combination may be an excellent and beneficial system for the clinical diagnosis of the various human cancers. 368 Am J Clin Pathol 1998; 11: p53, a tumor suppressor gene product, is an essential molecule for the control of cellular proliferation and apoptosis. The alteration of p53 in somatic cells is closely related to the malignancy of neoplasms. For example, p53 mutations are detectable in more than 5% of colorectal cancers, 3% of breast cancers, and 4% of small cell lung cancers. Moreover, loss of heterozygosity (LOH) in the p53 locus (1pl3) occurred in most malignant tumors with mutated p53.1 For the determination of the malignancy of a tumor, the efficacy of the molecular diagnosis of the p53 gene is widely accepted. Even in histopathologically benign cells, the presence of a p53 mutation heralds the development of a malignant tumor, as in the case of Li-Fraumeni syndrome, which is known to be associated with a p53 mutation in the germline.2 Since the determination of the whole nucleotide sequence of the p53 gene requires much time and cost, several brief methods are used to detect mutations in the p53 gene. They are the polymerase chain reaction (PCR)-mediated singlestrand conformation polymorphism (SSCP), fluorescence in situ hybridization (FISH), and immunohistochemistry ().3"6 Although SSCP is a simple and sensitive technique, its use of a radioisotope limits its application to conventional diagnosis. In some cases, a missense mutation of p53 results in the stabilization of its gene product, so that it can be distinguished from normal protein by. FISH analysis reveals not only the aneuploidy, but also the presence of LOH in the p53 locus, which is one of the characteristics of a malignant tumor. Because of the diversity of p53 mutations, the application of a single molecular procedure to the detection of a p53 mutation is insufficient. The development of a brief and effective system is therefore a key for the clinical application of p53 analysis. In the present study, we examined the detection efficiencies of p53 mutation by SSCP, FISH, and and Downloaded from by guest on 3 October 218 Abstract

2 demonstrated that the combination of the 3 methods is an excellent system for the detection of p53 mutations. Materials and Methods Source of Genomic DN A PCR Genomic DNA was amplified by PCR using 4 sets of primers designed by Mashiyama et al.3 The fragment of p53 gene, containing the portion between exon 5 and exon 9, was divided into 4 segments and amplified independently. The segments were as follows: exon 5 and a part of exon 6 (nucleotides 56 to 64), the rest of exon 6 (nucleotides 56 to 62), exon, and exon 8 and 9. The PCR mixture contained 2-u.mol/L concentrations of each deoxynucleoside triphosphate,.2-u.mol/l concentrations of each primer,.5 U/uL of Ampli Taq DNA polymerase (Perkin-Elmer, Foster, Calif), 5 mmol/l tromethamine hydrochloride (ph 8.3), a 2-mmol/L concentration of magnesium chloride, a.25% concentration of bovine serum albumin, and 5 ng/ul of template DNA. The PCR amplification was performed with denaturing at 96 C for 1 minute, followed by 3 cycles at 96 C for 3 seconds, 6 C for 1 minute, and 2 C for 3 seconds with a final extension at 2 C for 1 minute. Nonradioisotopic SSCP The SSCP assay was performed using the Phast System (Pharmacia Biotech, Uppsala, Sweden).8 An equal volume of PCR product was mixed with loading buffer (.5% bromophenol blue and.5% xylene cyanol in formamide) and denatured at 8 C for 5 minutes. After incubation on ice for 5 minutes, samples were analyzed at 1 C (for the exon 5 and part of exon 6, the rest of exon 6, and the exon PCR products) or at 28 C (the exon 8 and 9 PCR products). After a prerun at 4 V/1 ma/2.5 W/1 avh (ampere x volt per hour), samples were applied at 1 V/2 ma/2.5 W/2 avh and analyzed at 4 V/1 ma/2.5 W/1 avh. DNA fragments were analyzed by PhastGel homogeneous 12.5% (for exon 5 and part of exon 6, exon 8 and exon 9) or PhastGel homogeneous 2% (the rest of exon 6 and exon ). The gels were stained with the PhastGel silver stain kit. Cells were smeared and fixed as described. For the inactivation of internal peroxidase activity, slides were soaked in 3% hydrogen peroxide/methanol for 3 minutes at room temperature. The slides were then stained with 3times diluted mouse monoclonal antihuman p53 oncoprotein antibody (DAKO, Glostrup, Denmark) for 2 hours at room temperature. After the primary immunoreaction, the slides were washed with phosphate-buffered saline 2 times at room temperature. The slides were then stained with A B ^^... A* Downloaded from by guest on 3 October 218 Ten cell lines (AsPcl, BxPc3, Miapaca2, Panel, Colo32-11, Lovo, MCF, LNCaP, HL-6, and Daudi), a peripheral blood sample from a patient with a p53 germline mutation (p53gml), and a normal peripheral blood sample (NL) were used for the examinations. Genomic DNAs were prepared from 15 cultured cells and 5 ul of whole blood with a DNA extractor (WB kit, Wako Pure Chemical Industries, Osaka, Japan). LOH Detection by FISH Analysis Cells were smeared on glass slides coated with 3-aminopropyltriethoxysilane using an autosmear (Sakura, Koshoku, Japan). Air-dried specimens were fixed in 1% ethanol for 24 hours at room temperature. After incubation at 3 C for 3 minutes in.1% Nonidet P-4 and 2x standard saline citrate (SSC), samples were denatured at 5 C for 3 minutes in % formamide/2x SSC. Ten microliters of hybridization solution was applied to each slide, followed by incubation at 3 C for 16 hours. Hybridization solution contains 1 ul of LSI p53 (1pl3.1) SpectramOrange (Vysis, Downers Grove, 111), 1 ul of CEP 1 SpectramGreen (Vysis), ul of LSI Hybridization Buffer (Vysis), and 1 ul of sterile water. After hybridization, slides were washed 3 times with 5% formamide/2x SSC at 45 C for 1 minutes, once with 2x SSC at 45 C for 1 minutes, and once with.1% NP-4/2x SSC at 45 C for 5 minutes. After being washed briefly with 2x SSC, slides were counterstained with DAPI (Vysis). The number of 1p 13.1 (p53 locus) and CEP1 (chromosome 1 centromere) hybridized signals were counted in 1 cells. The LOH was determined when the total number of p53 signals divided by the chromosome 1 centromere signals was a value lower than.6. Figure I I Detection of p53 mutation by polymerase chain reaction (PCR)-single-strand conformation polymorphism with silver staining. A, Exon 5-6. Lane 1, p53 germline mutation (p53gml); lane 2, ASPcl; lane 3, normal peripheral blood sample (NL). B, Exon. Lane 1, Colo32-11; lane 2, NL, 3. Miapaca2. In p53gml, AsPcl, Colo32-11, and Miapaca2, the mobility shifts of PCR-amplified products were distinguishable (arrowheads). Am J Clin Pathol 1998; 11:

3 Furuwatari et al / DETECTION OF P53 MUTATIONS # * * $ %"«% llmage I I Detection of p53 mutations by fluorescence in situ hybridization (FISH) and immunohistochemistry (). In the FISH analysis (A-D), the chromosome 1 centromere signals are green, and the p53 locus signals are red. E and F, immunohistochemistry. A, p53germline mutation; B, HL-6; C, LNCaP; D, Panel; E, Miapaca2; F, AsPcl. 1-times diluted antimouse immunoglobulin (DAKO) of horseradish peroxidase conjugate. After being washed with phosphate-buffered saline 2 times at room temperature, reacted antibody was detected by diaminobenzidine. The slides were then counterstained with hematoxylin. More than 1 cells were examined. The labeling index (LI) was calculated as the proportion of the cells with a positive signal in the nucleus. Direct Nucleotide Sequence of PCR Products The nucleotide sequences of the PCR products were determined by an Applied Biosystem 33A DNA sequencer (Perkin-Elmer) using dye-labeled dideoxynucleotide. Results PCR-SSCP When the PCR-amplified products of exon 5 and part of exon 6 (nucleotides 56 to 64) were analyzed by PCRSSCP, the mobility shifts of the PCR-amplified products 3 Am J Clin Pathol 1998;11: were distinguishable in the AsPcl and p53gml cells Figure 1, Al. In the p53gml cells, we detected 3 bands. The mobility of the upper 2 bands was identical to that of the NL cells, but the other band was not found in the NL cells. In the AsPcl cells, we detected 2 bands. The mobility of both bands was different from that of the NL cells. In the Miapaca2 and Colo32-11 cells, the mobilities of PCRamplified exon were distinguishable from the mobility of the NL cells. In the Miapaca2 and Colo32-U, the upper band had disappeared IFigure 1, Bl. Owing to the deletion in the p53 locus (between exon and exon 9), no detectable band was obtained in the case of HL-6 cells. No significant changes in the mobility shifts of PCR-amplified products were observed in the other samples. FISH In the AsPcl, BxPc3, Colo32-11, p53gml llmage 1, Al, Lovo, Daudi, and NL, 2 chromosome 1 centromere signals (green) and 2 p53 locus signals (red) were detected, and the average numbers of chromosome 1 centromere signals were 2.3, 2.12, 2.3, 2.3, 1.99, 2. and 1.9, respectively ITable II. The average number of chromosome Downloaded from by guest on 3 October 218 I 1

4 Table II Detection of p53 Gene Mutation by PCR-SSCP, FISH, and Direct Sequence Predicted Product Altered Exons 5 6 8,8,9 5 4 T deletion 659 A to G 44 C to T 819 G to A 44 C to T Deletion 51 CG insertion Truncated Tyr 22 to Cys Arg 248 to Trp Arg 23 to His Arg 248b to Trp 5-6 Truncated,8, / FISH* Labeling Index (%).99 (2.2/2.3) 1.(2.13/2.12).99(3.1/3.4).64(1.9/2.96) 1.5(2.14/2.3).51 (1.5/2.3).98(1.98/2.3).98(1.95/1.99) 1.1 (3.6/3.2) 1.(3.91/3.92).99(1.98/2.).99(1.96/1.9) 2/ / PCR-SSCP = polymerase chain reaction-single-strand conformation polymorphism; FISH = fluorescence in situ hybridization; = immunohistochemistry; p53gml = p53 germline mutation; NL = normal peripheral blood sample. *Data are given as the number of p53 signals divided by the number of chromosome 1 centromere signals (mean of p53 signals in 1-cell counts/mean of chromosome 1 centromere signals in 1-cell counts). 1 centromere signals in the HL-6 sample was 2.3; however, the average number of p53 signals was 1.5 (Table 1) Image 1, Bl. In the Miapaca2, MCF, and LNCaP samples Image 1, CI, more than 3 chromosome 1 centromere signals were detected, and the average numbers of chromosome 1 centromere signals were 3.4, 3.2, and 3.92, respectively (Table 1). In the Panel samples I Image 1, Dl, there also were 3 chromosome 1 centromere signals, but there were 2 p53 signals. The total number of p53 signals divided by chromosome 1 centromere signals was.51 in the HL-6 sample and.64 in the Panel sample (Table 1). to exon 9 accompanying LOH was found in HL-6 genomic DNA (Table 1). Detection Efficiency The detection efficiencies obtained by single methods were 5 of alterations by SSCP, 2 of in LOH analysis by FISH, and 4 of by. The combinations of 2 methods showed better results (PCR-SSCP and FISH, 6 of ; FISH and, 5 of ). When PCR-SSCP and or all 3 methods were combined, all of the alterations determined by direct nucleotide sequencing were detected liable 21. In the BxPc3, Miapaca2 Ilma«e 1, E l, Panel, and Colo32-11 samples, positive signals were detected in the nuclei, and their LI values were higher than, whereas no significant signal was detected in the AsPcl I Image 1, Fl or other samples (Table 1). Direct Nucleotide Sequence The somatic cell mutations in all of the cell lines were determined by direct nucleotide sequencing of the PCR fragments. An alteration in the nucleotide sequence was found in of 12 samples (Table 1): the deletion of nucleotide 4 T in the AsPcl, the substitution of nucleotide 659 A to G in BxPc3, the substitution of nucleotide 44 C to T in the Miapaca2, the substitution of nucleotide 819 G to A in Panel, the substitution of nucleotide 44 C to T in the Colo32-11, and the insertion of CG at nucleotide 51 in the p53gml. A LOH also was found in the AsPcl, BxPc3, Miapaca2, Panel, and Colo A deletion from exon Discussion The human p53 gene is a representative tumor suppressor gene that is located on chromosome 1pl3. This gene is composed of 11 exons and produces a 2.2- to 2.5-kilobase (kb) messenger RNA. Many studies have demonstrated that alterations of the p53 gene are involved in the progression of various tumors. The mutation cluster region is between exon 4 (codon 33) and exon 1 (codon 366). Of p53 mutations, 8% cause an amino acid substitution. Hot spots, which account for 4% of the missense mutations, have been shown at codons 15 (exon 5), 248 (exon ), 249 (exon ), 23 (exon 8), and 281 (exon 8). The other cases produce nonsense mutations, large deletions, and frameshift due to small deletions or insertions.1 Redston et al9 reported that the proportion of nonsense mutations or small deletions varies according to tumor type. The rates of nonsense mutations are 4% in sarcoma and 1% in esophagus carcinoma. Those of Am J Clin Pathol 1998;11: Downloaded from by guest on 3 October 218 Nucleotides Altered Exon Samples AsPd BxPc3 Miapaca2 Panel Colo32-11 HL6 p53gml Lovo MCF LNCaP Daudi NL Detection PCR-SSCP

5 Furuwatari et al / DETECTION OF P53 MUTATIONS Table 21 Detection Efficiency of Combinations of Double and Triple Methods Method PCR-SSCP FISH PCR-SSCP and FISH FISH and PCR-SSCP and PCR-SSCP, FISH, and Detection Efficiency 5/ 2/ 4/ 6/ 5/ / / small deletions (1-2 base pairs) are.8% in colon cancers to 1% in pancreatic cancers. Moreover, the alleles of p53 mutations reduce to heterozygosity. In other words, most of the somatic cells with p53 mutations carry LOH. Nucleotide sequencing of the whole gene is the most reliable method to confirm the genetic alterations. We used nucleotide sequencing in the present study and identified the p53 mutations in of 12 samples (Table 1). Whole gene sequencing, however, is not practical for clinical laboratories because it requires much time and cost. In the present study, therefore, we tested the conventional procedures, ie, PCR-SSCP performed on the Phast System, FISH, and. The Phast System may be one of the most suitable PCR-SSCP systems for laboratory use because it is nonradioisotopic and fast, and results are highly reproducible. With the PCR-SSCP analysis, we detected anomalies of the PCR fragments in 5 of samples with mutated p53. One of the anomalies was an absence of the PCR product due to a large deletion, and the others were changes of mobility on the nondegenerative polyacrylamide gel electrophoresis (Phast System). The PCR-SSCP analysis failed to detect 2 missense mutations, in the BxPc3 and Panel cells. Since the maximum mobility of the DNA fragment (gel size) was only 3.5 cm, it occasionally was difficult to obtain sufficient resolution. The mobility also may be affected by the location and change of the nucleotide in the fragment to be analyzed. Even in the case of an undetectable mutation, it is possible to expect that the mobilities of the PCR-amplified fragment using another primer will be distinguishable. Although a PCR-SSCP analysis cannot detect all of the p53 mutations at this time, the detection efficiency of PCR-SSCP may be improved in the future. In the present FISH analysis, the chromosome 1 number showed an abnormality in 4 of 1 cell lines, and 2 of them were found to have LOH (Image 1). Although a FISH analysis is a potent tool for investigating large genetic anomalies like LOH, the probe size must be larger than 2 kb to obtain sufficient sensitivity and reproducibility. FISH analyses may therefore miss deletions smaller than 2 kb. 32 Am J Clin Pathol 1998; 11: In the present analysis, 4 of mutations showed markedly high LI values. These cells possess missense mutations in the coding region of the p53 gene. Finlay et al reported that the metabolism of mutant p53 proteins is generally slower than that of normal p53 protein. They also demonstrated that these mutant p53 proteins were detectable by anti-normal p53 protein antibody. In contrast, the normal p53 is undetectable owing to its prompt metabolism. However, it is frequently impossible to detect the truncated p53 proteins that are encoded by the nonsense, frameshift, or missense mutations, which cause various conformational changes. Clinical samples generally contain a number of normal cells. It is, therefore, necessary to know how those normal cells affect the results. In our hands, PCR-SSCP detected the mutated p53 alleles in cell pools with a minor population (>1%) of p53 mutation-carrying tumor cells (data not shown). FISH and seem to have fewer problems with the cell population than PCR-SSCP, because these methods are feasible for morphologic distinction of the tumor cells. Especially, FISH analysis is applicable for the Papanicolaoustained cells. Although a small part of samples might not be suitable for the mutation analysis, most clinical specimens would be sufficient to search for p53 mutations. Each of the conventional molecular diagnostic systems has the potential to detect false-negative cases. In the present study, we demonstrated that the combination of PCR-SSCP, FISH, and could detect a variety of genetic alterations, eg, point mutations, small deletions or insertions (1 to several bases), large deletions (>1 kilobase pairs), and LOH, Downloaded from by guest on 3 October 218 PCR-SSCP = polymerase chain reaction-single-strand conformation polymorphism; FISH = fluorescence in situ hybridization; = immunohistochemistry. Indeed, the present FISH analysis detected a p53 signal in the HL-6 cells, in which no DNA fragment was amplified between exon and exon 9 by PCR owing to deletion. Even in a case with a deletion of more than 2 kb, the successful detection of LOH depends on the position of the probe in FISH analysis. In contrast to other investigators, we detected the chromosome 1 centromere simultaneously with the p53 locus by a 2-color fluorescent system. Our system detected LOH in the Panel cells, which have 2 mutant alleles but have lost the normal p53 gene. The 2-color fluorescent system detected the presence of 3 chromosome 1s despite the presence of 2 p53 loci. As Gomyo et al6 pointed out in their analysis of LOH in gastric cancer, an internal control not only avoids misjudgment due to technical errors, but also often provides valuable information for chromosome abnormalities in FISH analyses. FISH also is effective for the detection of aneuploidy of the p53 locus. The relationship between p53 gene dosage and the malignancy of the tumor is still under investigation. However, numerous studies have elucidated the change of the gene dosage, including oncogene and suppressor gene effect on the malignancy of tumor. A change in the dosage of weakly dominantnegative p53 probably affects the malignancy of the tumors.

6 From the 'Central Clinical Laboratories, Shinshu University Hospital, Matsumoto, Japan; and the 2Department of LaboratoryMedicine, Shinshu University School of Medicine, Matsumoto, Japan. Address reprint requests to Dr Ogiso: Central Clinical Laboratories of Shinshu University Hospital, Asahi 3-1-1, Matsumoto, 39, Japan. Acknowledgment: Cell lines were provided by Shigeyuki Kawa, MD, and Shigetaka Shimodaira, MD, at the Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan. References 1. Mendelsohn J, Howley PM, Israel MA, et al. The Molecular Basis of Cancer. Philadelphia, Pa: Saunders; 1995: Malkin D, Li FP, Strong LC, et al. Germline mutation in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science. 199;25: Mashiyama S, Murakami Y, Yoshimoto T, et al. Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism of polymerase chain reaction products. Oncogene. 1995;6: Dohner H, Fisher K, Beutz M, et al. p53 gene deletion predicts for poor survival and no response to therapy with purine analog in chronic B-cell leukemias. Blood. 1995;85: Baas IO, Mulder JR, Offerhaus GJA, et al. An evaluation of six antibodies for immunohistochemistry of mutant p53 gene product in archival colorectal neoplasms. ] Pathol 1994; 12: Gomyo Y, Osaki M, Kaibara N, et al. Numerical aberration and point mutation of f>53 gene in human gastric intestinal metaplasia and well-differentiated adenocarcinoma: analysis by fluorescence in situ hybridization (FISH) and PCR-SSCP. lnt) Cancer. 1996;66: Finlay CA, Hinds PW, Tan TH, et al. Activating mutations for transformation by p53 produce a gene product that forms an hsc-p53 complex with an altered half-life. Mol Cell Biol. 1988;8: Mohabeer AJ, Hiti AL, Martin WJ. Non-radioactive single strand conformation polymorphism (SSCP) using the Pharmacia 'PhastSystem'. Nucleic Acids Res. 1991;19: Redston MS, Caldas C, Seyymour AB, et al. P53 mutations in pancreatic carcinoma and evidence of common involvement of homocopolymer tracts in DNA microdeletions. Cancer Res. 1994;54: Nguyen DM, Spitz FR, Yen N, et al. Gene therapy for lung cancer: enhancement of tumor suppression by a combination of sequential systematic cisplatin and adenovirus-mediated p53 gene transfer. ] Thorac Cardiovasc Surg. 1996;112: Am J Clin Pathol 1998;11: Downloaded from by guest on 3 October 218 and there were no false-negative cases. However, we did not obtain any positive data in the cells with no mutated gene, indicating that there were no false-positive cases. The combination of PCR-SSCP, FISH, and could maximize the detection efficiency for p53 mutations. In addition to an excellent accuracy level, this combination of methods saves substantial time and cost in comparison with the direct nucleotide sequencing of the whole gene. Since the p53 mutation is associated with biologic phenotypes of tumors, including drug susceptibility and tumor progression, this combination would provide essential information on the nature of neoplasms.