Detection of the P53 Gene Deletion by Dual Color Fluorescence in Situ Hybridization in Squamous Cell Carcinoma of the Skin

Acta Histochem. Cytochem. Vol.31 No.3 197-202 (1998)

Detection of the p53 Gene Deletion by Dual Color Fluorescence In situ Hybridization in Squamous Cell Carcinoma of the Skin

Morimasa Matsuta1, Mayumi Matsuta2, Hidehiko Suzuki3, Toshihide Akasaka2 and Teruo Kagabu1 Departmentsof1 Obstetrics& Gynecology,2Dermatology, 3PlasticSurgery, Iwate Medical University, 19-1 Uchimaru, Morioka020-8505

Receivedfor publicationJanuary 5, 1998and in revisedform May11, 1998

Dual color fluorescence in situ hybridization 22% to 59% (33.9•}10.9%). The ratio be- (FISH) analysis was applied to 22 squamous tween the copy number of chromosome 17 cell carcinomas (SCC) of the skin in order to centromeres and p53 in the predominant

detect deletion of the p53 tumor suppressor population of the tumor nuclei with p53 gene gene in interphase nuclei using a cosmid deletion was either 2/1 or 1/1. Moreover, probe for p53 and a centromeric probe for the major subfraction was composed of chromosome 17. Fewer p53 signals than chromosome 17 disomic cells in all cases. 17 centromeric signals and zero or one cos- This suggests that either p53 gene deletion of mid signals were observed in 5.6•}4.9% one allele in disomic cells or the loss of an (mean•}SD) of controls. Based on this, 20% entire chromosome 17 in aneusomic cells is deletion was established as the cutoff line to the main mechanism of p53 gene deletion in define deletion of the p53 gene. Twenty of SCC of the skin. Immunohistochemical p53 the 22 SCC cases demonstrated p53 gene expression was frequently associated with deletion according to this criterion. The the p53 gene deletion detected by FISH. percentage of cells with deletion ranged from

Key words: Dual color fluorescence in situ hybridization, p53 deletion, Squamous cell carcinoma, Skin

I. Introduction as a surrogate of p53 mutation. Furthermore over-expression of the p53 protein has been clinicopathologically Tumor suppressor gene p53 is located on chromo- correlated to various tumors [1, 7, 16]. Mutation and some 17p13.1. This gene is involved in transcriptional deletion of the p53 alleles are observed in various solid regulation and codes various cyclin dependent kinase inhi- tumors, including colon cancer, lung cancer, ovarian can- bitors [2, 4]. Many studies have implied that the p53 pro- cer, esophageal cancer, and squamous cell carcinoma of tein plays an important role in arresting cell growth in the the skin [6, 15, 17, 20, 23]. The incidence of these p53 1 phase of the cell cycle in response to DNA damage G [4, mutations and deletions is greater than 50% for each of 23], and that the biological function of p53 assures the these tumors, suggesting that alterations of the p53 gene appropriate internal environment for cell replication and play an important role in the development of tumors. p53 division [11]. The tumor suppressor genes are generally mutations can be detected by polymerase chain reaction believed to be inactivated by mutation of one allele and the single strand conformation polymorphism (SSCP-PCR) . deletion of the second. This theory, known as the •g2 hits The loss of heterozygosity (LOH) at 17p has been detected theory•h, was first proposed by Knudson [9]. Mutation by restriction fragment length polymorphism (RFLP) . and deletion of the p53 gene leads to amino acid changes, A non-isotopic in situ hybridization method has re- and results in the over-expression of mutant-type p53 pro- cently been developed called fluorescence in situ hybridi- tein due to its prolonged half-life. It is widely accepted zation (FISH) technique [3]. This technique enables that immunohistochemical p53 protein expression is used several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of Correspondence to: Dr. Morimasa Matsuta, Department of Ob- specific genetic sequences in single metaphase or inter- stetrics & Gynecology, Iwate Medical University , 19-1 Uchimaru, phase cells [8, 17, 19, 20, 23]. Amplified and deleted loci Morioka 020-8505, Japan. on the target chromosomes can be visualized microscopi-

197 198 Matsuta et al.

cally in single cells by FISH using nucleic acid probes pH 7.0), 2ƒÊl of salmon testis DNA (Sigma) and 1ƒÊl of specific to these loci. Since deletion of the p53 gene is biotinized probe specific for a centromeric repetitive DNA theoretically detectable by FISH in interphase nuclei, sequence on chromosome 17 (D17Z1, Oncor). This mix- allelic loss of the p53 gene is in principle detectable by FISH ture was denatured in a water bath at 73•Ž for 5min and if the loss occurs by deletion [17]. A few studies have immediately placed on ice. examined interphase cytogenetics by dual color hybridi- After mixing the p53 and centromeric probes, 10ƒÊl of zation using chromosome 17 pericentromeric- and p53 the hybridization mixture were added for each square region-specific DNA probes. Those results have suggest- centimeter of the preparation slides. The slides were then ed that the dominant mechanism involved in p53 allelic covered by a coverslip and sealed with rubber cement. loss in breast and bladder cancer is a physical deletion at Hybridization was performed at 37•Ž overnight. After 17p [17, 23]. incubation, the slides were washed in 50% forma- The present study investigates the interphase-cyto- mide/2•~SSC at 45•Ž three times each for 5 min, and then genetic application of dual color FISH to evaluate deletion in 2•~SSC at room temperature (RT) twice each for of the p53 gene in squamous cell carcinoma(SCC) of the 10min. Finally, the slides were washed in PN buffer skin in order to better understand the role of p53 deletion (0.1M NaH2PO4, pH 8, and 0.1% Nonidet P-40) at RT in this tumor. for 2min.

Detection of hybridization signals II Materials and Methods The hybridized probes were detected immuno- Slides preparation histochemically. Each slide was treated for 10min with Tissue samples were obtained at surgery or by biopsy PNM (5% CarnationTM dry milk, 0.02% Na-azide in PN from twenty-two cases of SCC of the skin. Staging and buffer) and then for 30min with 1ƒÊl of Texas Red avidin grading of the tumors were performed using criteria (Vector Laboratories, Burlingame, CA) in 1ml of PBS in published by Lever et al. [13]. Touch preparations were order to detect centromeric signals. The slide was then prepared by touching the freshly cut surfaces of tumors to washed three times in PBS at RT for 3min and once in glass slides. The slides were fixed in 1% paraformalde- PNM for 10min. p53 signals were detected simultane- hyde/phosphate buffered saline, pH 7.2 (PBS) for 30 min. ously. The slide was incubated for 40min in 10ƒÊl/ml After washing in PBS, the slides were treated with pro- anti-digoxigenin-fluorescein isothiocyanate (FITC) (Boeh- teinease K (0.6ƒÊg/ml) in Tris EDTA. Following the ringer Mannheim Corporation, Indianapolis, IN) diluted wash in PBS, the slides were denatured in 70% at 5% in PNM, followed by three PN washes (3min). formamide/2•~SSC (1•~SSC is 0.15M NaCl/0.015M The counterstain was treated with 4•f-6-diamino-2-phenyl- sodium citrate, pH 7.0) for 3 min at 73•Ž. Next, the slides indole (DAPI) in antifade (100mg p-phenylene diamine were dehydrated through an ethanol series (70%, 90%, dihydrochloride/PBS, pH 8.0). 100%) and then air dried. All chemicals were purchased from Sigma Chemicals (St. Lois, MO) unless otherwise Scoring of FISH signals specified. Large cells (nuclei) and those forming groups or Hybridization efficiency was measured by the same sheets were selected for analysis, because the cells were methods in 10 lymph nodes and 5 normal skin tissues that considered to be epithelial in origin. Dissociated small were not exposed to sun of 5 volunteers to provide con- cells (nuclei) and lymphocyte-like cells were disregarded. trols. Lymphocytes on the tumor sample slides served as Copy numbers of p53 and the centromere signals of 100 internal controls. nuclei per case were counted using a Nikon fluorescent Due to instability in the cosmid signal intensity, microscope. The ratio of chromosome 17 centromeric presumably due to variability in the hybridization efficien- copy numbers to those of p53 (chromosome 17/p53) in cy, a higher concentration of proteinase K treatment (up to each nucleus hybridized with a set of p53 and chromosome 4ƒÊg/ml) was used in some cases in order to increase the 17 centromere probes was calculated. The centromeric intensity of the p53 cosmid probe signal. signals were counted using criteria previously published by Hopman et al. [5]: i.e., 1) nuclei should not be overlapped Probe preparation and hybridization 2) signal intensity should be more or less of the same Hybridizations were performed as described by homogeneous staining intensity 3) minor hybridization Kallioniemi et al. [8, 17] with slight modifications. spots, which can be recognized by their smaller size and p53 cosmid probe: Ten ƒÊl of digoxigenin-labeled p53 lower intensity, should be excluded 4) signal may only be cosmid probe (Oncor, Gaithersburg, MD: supplied in for- counted when completely separated from each other and mamide solution) were preincubated in a water bath at 5) paired (split phenomenon in G2 phase) or closely op-

37•Ž for 5min. posed spots count as one signal. Following the criteria Centromeric probe of chromosome 17: The hybridi- proposed by Sauter et al. [23], the percentage of cells with zation mixture consisted of 7ƒÊl of master mixture (5.5 ml deletions detected by FISH was defined as the fraction of formamide, 1g dextran sulfate, and 0.5ml 20•~SSC, all investigated nuclei that had fewer p53 signals than 17 Detection of p53 Deletion by FISH in SCC 199

centromeric signals and that had at least one centromeric goat serum/PBS for 20min at RT to block nonspecific signal and one cosmid signal. If the average centromere binding of antibodies. The primary monoclonal antibody 17 copy number per cell was•†2.3, these cases were de- against p53 (DO7: Novocastra, Claremount Place, UK) fined as polysomy cases according to the criteria of Sauter was employed at a dilution of 1: 50 in PBS. The diluted et al. [23]. If the subfraction of cells with one chromo- antibody was incubated for 90min at RT, and then the some 17 signal was at least 10% of the total number of slides were incubated with biotylated anti-mouse IgG cells, these cases were considered to contain a chromosome antibody (Nichirei, Tokyo, Japan) and streptoavidin-

17 monosomy subfraction, based on the control data. peroxidase (Nichirei). Immunostaining was developed The cases were considered to contain polysomy subfrac- using 3,3•f-diaminobenzidine tetrahydrochloride (Dojin tions if the subfractions of cells with•†3 chromosome 17 Kagaku, Tokyo), and sections were counterstained with constituted at least 10% of the total number of cells. hematoxylin, dehydrated and mounted.

Statistical analysis III. Results Associations among chromosome 17 copy number,

p53 gene copy number and other parameters were evalu- The number of red chromosome 17 centromeric probe ated by the chi-square test and Mann-Whitney U test. signals and green p53 signals observed in the nuclei varied Significance was established at the P<0.05 level. among the nuclei (Fig.1). Centromere signals were bright and clear in all interphase nuclei, but the p53 signals Immunohistochemistry for p53 protein were weaker and smaller than the centromere signals. Tissues were fixed in 20% buffered formalin solution Increased proteinase K treatment tended to improve the and embedded in paraffin in the conventional way. The cosmid signal intensity, although it also tended to induce

sections were cut at 3-4ƒÊm in thickness and mounted on greater morphological cell damage. poly-L-lysine-coated glass slides. Deparaffinized sections were washed in PBS and transferred to a microwave FISH on controls resistant container containing 0.01 M sodium citrate In controls, 93.0•}3.1% (mean•}standard deviation) buffer (pH 6.0). The slides were exposed to microwave of the cells showed two hybridization signals of each

heating 3 times at intervals of 5 min, using a 700-W probe. Only 5.6•}4.9% of the nuclei had fewer p53 sig- Microwave oven (Hitachi Electronics Co., Japan), as nals than chromosome 17 centromeric signals or had zero described previously [18]. A labeled-streptoavidin-biotin- or one cosmid signals (Table 1). Thus, in the present peroxidase method was employed for immunohistochemi- study 20% deletion was established as the cutoff value to cal staining of p53 protein. The slides were immersed in define deletion of the p53 gene. 0.3% H2O2/methanol for 10min to inactivate an endo-

genous peroxidase activity. After washing in PBS for FISH on SCC 5 min twice, the sections were covered with 10% normal The overall percentage of cells with p53 deletion is

A B

Fig.1. Dual color fluorescence in situ hybridization for p53 and chromosome 17 centromere . p53 gene signals appeared in green with fluorescein isothiocyanate (FITC), and chromosome 17 centromere appeared in red with Texas Red . The counterstain was treated with 4•f- 6-diamino-2-phenyl-indole (DAPI). Photographed using dual-bandpass filter . •~1,000 Bar=10ƒÊm. A: Two signals of each probe in a nucleus. B: Two signals of chromosome 17 centromere and one signal of p53 in a nucleus , showing a deletion of p53 gene. 200 Matsuta et al.

Table1. Copy number of p53 gene and chromosome 17 centromere Table 2. Patterns of chromosome 17 and p53 copy number in in controls squamous cell carcinoma of the skin

mere copy number and the percentage of p53 deleted cells is shown in Figure 3. The average centromere 17 copy number ranged from 1.8 to 2.3 (2.0•}0.1) among the cases. There was no significant correlation between the average centromere 17 copy number and the percentage of cells with p53 deletion (p=0.86).

Association between p53 deletion and tumor grade and stage There was no significant correlation between p53 deletion and tumor stage (p=0.45) or grade (p=0.88). Similarly, the average centromere 17 copy number showed no correlation with tumor grad (p=0.88) or stage (p= 0.72). The relationship between FISH and immunohistoche-

Fig.2. Fraction of tumor cells with p53 deletion by fluorescence in mistry: p53 protein was positive in 16 of the cases (73%) situ hybridization. Each bar represents one case. (Fig.4). A deletion of the p53 gene was detected in all p53 protein-positive cases. Six cases were p53 protein- negative,and they included two cases without p53 dele- shown in Figure 2. According to the 20% cutoff value, 20 tion. Positive p53 immunostaining was associated with of the 22 SCC cases (90%) showed a deletion of the p53 the presence of p53 deletions (P<0.05). Expression of gene. The percentage of cells with p53 deletion ranged. p53 was not associated with polysomy 17 (P=0.89). p53 from 22 to 59% (33.9•}10.9%) in these cases. The chro- protein-positive cells were not detected in the normal mosome 17/p53 signal distribution for the largest sub- epidermal cells. population of cells with deletion for each case is shown in Table2. Note that 17 of the 20 cases (85%) with deletion had a ratio of 2/1 (chromosome 17/p53 signals). The percentage of the cells with deletion ranged from 14 to 32% (21.5•}5.8%) in the 2/1 deletion type cases. In the 1/1 deletion type cases, this ranged from 10 to 13%

(11.3•}1.5%). A small fraction of cells with fewer p53 signals than centromere 17 signals was always present, presumably due to incomplete hybridization of the cosmid probe. The major subfraction was composed of chromosome 17 disomic cells in all cases, and 47-92% (77.4•}12.8%) of the cells were chromosome 17 disomic. Chromosome 17 monosomy was detected in thirteen cases, and 10-24%

(16.0•}4.3%) cells showed one signal in these cases. A subfraction of chromosome 17 polysomy was detected in ten cases, in which 10-37% (17.1•}10.3%) cells showed 3 chromosome 17. Six cases had both monosomy •† and polysomy subfractions. Fig.3. Relationship between fractions with p53 deletion and The overall relationship between the average centro- average of 17 centromere. Each square represents one case . 201 Detection of p53 Deletion by FISH in SCC

the predominant population of the tumor nuclei with p53 gene deletion was either 2/1 or 1/1 in the present study, although the ratio was either 4/3 or 4/2 in bladder cancer [25] and in pleomorphic adenocarcinoma (the estimated ratio) [14]. Moreover, the major subfraction was composed of chromosome 17 disomic cells in all cases. This suggested that either p53 gene deletion of one allele in disomic cells or the loss of an entire chromosome 17 in aneusomic cells is the main mechanism of p53 gene deletion in SCC of the skin. Nuclear p53 staining was frequently overexpressed in the present cases. Since the incidence of p53 positive cases has been reported to be nearly 50% of the cases of cutaneous SCCs [7-9, 12, 21, 22], this high level p53 positivity in our study may reflect the p53 antigen retrieval process [18]. In the present study, almost all of the cases with p53 over-expression had deletion of the p53 gene, and there was a good correlation between p53 over-expression Fig.4. Immunohistochemical detection of p53. Positivity is ap- and p53 gene deletion detected by FISH. However, the proximately 40%. The average of chromosome 17 copy number is 2.0. The percentage of cells with p53 deletion is 42% in this case. present results revealed no significant correlations between The chromosome 17/p53 signal distribution for the largest sub- the incidence of p53 deletion and tumor grade, stage, and

population of cells with deletion is a ratio of 2/1. •~250, 17 polysomy. Furthermore, no correlation was detected Bar=50ƒÊm. Counterstained with hematoxylin. between 17 polysomy and stage, and grade. As previous reports have suggested that stage and grade are associated with polysomy 17 detected by FISH [14, 23], this may be unique to SCC of the skin. IV. Discussion FISH analysis of deletions appears to provide a rapid, Dual color FISH has been used to detect numerical simple and sensitive approach to study chromosomal abnormalities of chromosomes and their loci [8, 17, 23, 24] aberrations. However, further examination is necessary (Fig.5). The major advantage of FISH as compared to confirm its efficiency in comparison to LOH by RFLP with other methods is the ability to visualize quantitative analysis. A higher incidence of deletion of the p53 gene genomic alternations on a cell-by-cell basis [8, 25], which detected in gastric and bladder cancers by FISH compared enables us to measure the average level of deletion in a with that by LOH was reported [10, 23]. Although LOH tumor as well as the actual number and distribution of p53 data were not available in the present cases, the difference gene in individual, morphologically defined cells. Analy- in the results obtained by these two methods may be ex- sis of numerical aberrations of chromosome 17 and p53 plained by the large number of non-neoplastic cells which gene deletion in the same nuclei of SCC has not yet been may mask the true allelic loss when total DNA is examined reported. The present study examined the deletion of by RFLP analysis [23]. p53 gene in 22 SCC cases of the skin by dual color FISH analysis using a cosmid probe for p53 and a centromeric V. Acknowledgment probe for chromosome 17. The ratio between the copy number of chromosome 17 centromeres and p53 in Part of this study was presented at the 28th Annual

Dual color FISH using a combination of centromeric and locus-specific probes

Fig.5. Dual color FISH using a combination of centromeric and locus specific probes in disomic cells (Modified from Pinkel et al: Reference 23) 202 Matsuta et al.

Meeting of the Clinical Electron Microscopy Society of 7th ed., Lippincott, Philadelphia, 1990, pp. 552-560.

Japan, Osaka, October 17-19, 1996. 14. Li, X., Tsuji, T., Wen, S., Mimura, Y., Sasaki, K. and Shinozaki, F.: Detection of numeric abnoramalities of chro- Supported in part by a Grant-in-Aid for Scientific mosome 17 and p53 deletions by fluorescence in situ hybridiza- Research (C) from the Ministry of Education, Science, tion in pleomorphic adenomas and carcinomas in pleomorphic Sports and Culture (No.40157326). adenoma. Correlation with p53 expression. Cancer 79; 2314- 2319, 1997.

VI. References 15. Malkin, D., Li, F.P., Strong, L.C., Fraumeni, J.F., Nelson, C.E., Kim, D.H., Kassel, J., Gryca, M.A., Bischoff, F.Z., 1. Bartek, J., Bartkova, J., Vojtesek, B., Staskova, Z., Lukas, J., Tainsky, M.A. and Strong, L.C.: Germ line mutations of the Rejthar, A., Kovarik, J., Midgley, C.A., Gannon, J.V. and p53 mutations in a familial syndrome of breast cancer, sarcomas Lane, D.P.: Aberrant expression of the p53 oncoprotein is a and other neoplasm. Science 250; 1233-1238, 1990. common feature of a wide spectrum of human malignancies. 16. Marks, J., Davidoff, A., Kerns, B., Hummphrey, P., Pence, J., Oncogene 6; 1699-1703, 1991. Dodge, R., Clarke-Pearson, D., JD, I., Bast, R.J. and Ber- 2. Fields, S. and Jang, S.: Presence of a potent transcription ac- chuck, A.: Overexpression and mutation of p53 in epithelial tivating sequence in the p53 protein. Science 249; 1046-1049, ovarian cancer. Cancer Res. 51; 2979-2984, 1991. 1990. 17. Matsumura, K., Kallioniemi, A., Kallioniemi, O., Chen, L., 3. Gray, J.W., Kuo, W.L., Lucas, J., Pinkel, D., Weier, H. Ul. Smith, H.S., Pinkel, D., Gray, J.W. and Waldman, F.M.: and Yu, L.C.: Molecular cytogenetics using fluorescence in Deletion of chromosome 17p loci in breast cancer cells detected situ hybridization. In •gFlow Cytometry and Image Analysis for by fluorescence in situ hybridization. Cancer Res. 52; 3474- Clinical Applications•h, ed. by I. Nishiya, L.S. Cram and J.W. 3477, 1992. Gray, Excerpta Medica, Amsterdam, New York, Oxford, Else- 18. Matsuta, M., Kon, S., Sasaki, K. and Matsuta, M.: Immuno- vier Science Publishers B.V., Amsterdam, 1991, pp. 73-82. histochemical detection of p21WAF1/CIP1and p53 proteins in for- 4. Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K. and malin-fixed paraffin-embedded tissue sections of squamous Elledge, S.J.: The p21 cdk-interacting protein cip 1 is a potent cell carcinoma of the skin. J. Dermatol. Sci. 14; 233-239, 1997. inhibitor of G1 cyclin-dependent kinases. Cell 75; 805-816, 19. Matsuta, M., Imammura, Y., Matsuta, M., Kon, S. and Sasaki, 1993. K.: Applications of DNA flow cytometry and fluorescence in 5. Hopman, A.H., Poddighe, P.J., Smeets, A.W., Moesker, situ hybridization using a chromosome-specific DNA probe on Om., Beck, J.L., Vooijs, G.P. and Ramaekers, F.C.: Detec- paraffin-embedded tissue sections of primary malignant mela- tion of numerical chromosome aberrations in bladder cancer by nomas. J. Dermatol. 21; 14-19, 1994. in situ hybridization. Am. J. Pathol. 135; 1105-1117, 1989. 20. Matsuta, M., Matsuta, M., Kon, S., Thompson, C., LeBoit, 6. Imazeki, F., Omata, M., Nose, H., Ohto, M. and Isono, K.: P.E., Weier, H. -U. and Gray, J.W.: Interphase cytogenetics

p53 gene mutations in gastric and esophageal cancers. Gas- of melanocytic neoplasm: numerical aberrations of chromo- troenterol. 103; 892-896, 1992. somes can be detected in interphase nuclei using centromeric 7. Iwaya, K., Tsuda, H., Hiraide, H., Tamaki, K., Tamakuma, S., DNA probes. J. Cut. Pathol. 21; 1-6, 1994. Fukutomi, T., Mukai, K. and Hirohashi, S.: Nuclear p53 im- 21. Nagano, T., Ueda, M., Ichihashi, M.: Expression of p53 munoreaction associated with poor prognosis of breast cancer. protein is an early event in ultraviolet light-induced cutaneous Jpn. J. Cancer Res. 82; 835-840, 1991. squamous cell carcinogenesis. Arch. Dermatol. 129; 1157- 8. Kallioniemi, O.P., Kallioniemi, A., Kurisu, W., Thor, A., Yu, 1161, 1993. L.C., Smith, H.S., Waldman, F.M., Pinkel, D. and Gray, 22. Nelson, M.A., Einspahr, J.G., Alberts, D.S., Balfour, C.A., J.W.: ERBB2 amplification in breast cancer analyzed by Wymer, J.A., Welch, K.L., Salasche, S.J., Bangert, J.L., fluorescence in situ hybridization. Proc. Natl. Acad. Sci. USA Grogan, T.M. and Bozzo, P.O.: Analysis of the p53 gene in 89; 5321-5325, 1992. human precancerous actinic keratosis lesions and squamous cell 9. Knudson, A.G.: Mutation and cancer, Statistical study of cancers. Cancer Lett. 85; 23-29, 1994. retinoblastoma. Proc. Natl. Acad. Sci. USA 68; 820-823, 23. Pines, J.: p21 inhibits cyclin shock. Science 269; 520-521, 1971. 1994. 10. Kobayashi, M., Kawashima, A., Mai, M. and Ooi, A.: Analy- 24. Pinkel, D., Sakamoto, M., Matsumura, K., Kallioniemi, A., sis of chromosome 17p13 (p53 locus) alterations in gastric carci- Kallioniemi, O., Waldman, F. and Gray, J.: Application of noma cells by dual-color fluorescence in situ hybridization. fluorescence in situ hybridization to detect of chromosomal Am.J.Pathol. 149; 1575-1584, 1996. aberrations. Cytometry Res. 2; 1-11, 1992. 11. Lane, D.: p53, guardian of the genome. Nature 358; 15-16, 25. Sauter, G., Deng, G., Moch, H., Kerschmann, R., Matsumura, 1992. K., De Vries, S., George, T., Fuentes, J., Carroll, P., Mihatsch, 12. Lee, Y.S. and Teh, M.: p53 expression in pseudoepithelioma- M.J. and Waldman, F.M.: Physical deletion of the p53 gene tous hyperplasia, keratoacanthoma, and squamous cell carci- in bladder cancer. Detection by fluorescence in situ hybridiza- noma of skin. Cancer 73; 2317-2323, 1994. tion. Am. J. Pathol. 144; 756-66, 1994. 13. Lever, W.F. and Lever, G.S.: Histopathology of the Skin,