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Clonal Nature of Seborrheic Keratosis Demonstrated by Using the Polymorphism of the Human Androgen Receptor Locus as a Marker

2001; Elsevier BV; Volume: 116; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1747.2001.01289.x

1523-1747

Harumi Nakamura, Seiichi Hirota, Shiro Adachi, Keiichiro Ozaki, Hideo Asada, Yukihiko Kitamura,

Cutaneous Melanoma Detection and Management

We evaluated the clonality of seborrheic keratoses using a polymorphism due to the random inactivation of one of two X chromosomes in females. Thirty-eight seborrheic keratoses obtained from the skin of females with polymorphism of the human androgen receptor (HUMARA) locus were examined by a fluorescent polymerase chain reaction procedure, which allowed accurate measurement of the peak intensities of each HUMARA allele. The epithelial portion of seborrheic keratosis and normal control epidermis adjacent to the seborrheic keratosis were removed by laser capture microdissection. As biopsied specimens of seborrheic keratoses contained small amounts of normal epidermis, the effect of digestion by a restriction enzyme (HhaI) recognizing the nonmethylated active sites was compared between seborrheic keratoses and normal control epidermis in only five seborrheic keratosis cases. Disappearance or significant reduction in intensity of one of two HUMARA alleles was observed after HhaI digestion in seborrheic keratoses, but not in the normal control epidermis. Although the skewing of the polymorphism was not corrected by the normal control epidermis in the remaining 33 seborrheic keratosis cases, one of two HUMARA peaks practically disappeared after HhaI digestion in 20 of 33 seborrheic keratosis cases. In total, 25 of 38 seborrheic keratoses were considered to be monoclonal. The histologic type of seborrheic keratoses did not affect clonality. We evaluated the clonality of seborrheic keratoses using a polymorphism due to the random inactivation of one of two X chromosomes in females. Thirty-eight seborrheic keratoses obtained from the skin of females with polymorphism of the human androgen receptor (HUMARA) locus were examined by a fluorescent polymerase chain reaction procedure, which allowed accurate measurement of the peak intensities of each HUMARA allele. The epithelial portion of seborrheic keratosis and normal control epidermis adjacent to the seborrheic keratosis were removed by laser capture microdissection. As biopsied specimens of seborrheic keratoses contained small amounts of normal epidermis, the effect of digestion by a restriction enzyme (HhaI) recognizing the nonmethylated active sites was compared between seborrheic keratoses and normal control epidermis in only five seborrheic keratosis cases. Disappearance or significant reduction in intensity of one of two HUMARA alleles was observed after HhaI digestion in seborrheic keratoses, but not in the normal control epidermis. Although the skewing of the polymorphism was not corrected by the normal control epidermis in the remaining 33 seborrheic keratosis cases, one of two HUMARA peaks practically disappeared after HhaI digestion in 20 of 33 seborrheic keratosis cases. In total, 25 of 38 seborrheic keratoses were considered to be monoclonal. The histologic type of seborrheic keratoses did not affect clonality. corrected ratio laser capture miction seborrheic keratosis Seborrheic keratosis (SK) is one of the most common benign lesions of the human epidermis (Stewart et al., 1978Stewart W.D. Danto J.L. Maddin S. Seborrheic keratosis.in: Dermatology-Diagnosis and Treatment of Cutaneous Disorders. 4th edn. CV Mosby, St Louis, MO1978: 508Google Scholar;Mackie, 1992Mackie R.M. Seborrheic keratosis.in: Champion R.H. Burton J.L. Ebling F.J.G. Textbook of Dermatology. 5th edn. Blackwell, Scientific Publications, Oxford1992: 1465-1467Google Scholar;Ho and McLean, 1993Ho V.C.Y. McLean D.I. Seborrheic keratosis.in: Fitzpatrick T.B. Eisen A.Z. Wolff K. Freedberg I.M. Austen K.F. Dermatology in General Medicine. 4th edn. McGraw-Hill, New York1993: 855-858Google Scholar;Kirkham, 1997Kirkham N. Seborrheic keratosis.in: Elder D. Elenitsas R. Jaworsky C. Johnson B. Lever's Histopathology of the Skin. 8th edn. Lippincott-Raven Publishers, Philadelphia1997: 689-693Google Scholar). SK do not usually appear before middle age, and occur mainly on the trunk and face, but also on the extremities, with the exception of the palms and soles (Kirkham, 1997Kirkham N. Seborrheic keratosis.in: Elder D. Elenitsas R. Jaworsky C. Johnson B. Lever's Histopathology of the Skin. 8th edn. Lippincott-Raven Publishers, Philadelphia1997: 689-693Google Scholar). They show a considerable variety of histologic appearances, and six types are recognized: acanthotic, hyperkeratotic, reticulated, clonal, irritated, and melanoacanthoma. Often, more than one type is found in the same lesion, and all types show hyperkeratosis, acanthosis, and papillomatosis (Kirkham, 1997Kirkham N. Seborrheic keratosis.in: Elder D. Elenitsas R. Jaworsky C. Johnson B. Lever's Histopathology of the Skin. 8th edn. Lippincott-Raven Publishers, Philadelphia1997: 689-693Google Scholar). The etiology of SK is unknown. Whether SK are hyperplastic or neoplastic has not yet been determined. If SK is a hyperplasia of the epidermis, it does not originate from a single epithelial cell and therefore is polyclonal; however, if it is a neoplasia, SK is monoclonal. As an examination of clonality appeared to promote understanding of the etiology of SK, we investigated this point. As epithelial tumors of the skin, verrucae vulgaris were first demonstrated to be monoclonal (Murray et al., 1971Murray R.F. Hobbs J. Payne B. Possible clonal origin of common warts (Verruca vulgaris).Nature. 1971; 232: 51-52Crossref PubMed Scopus (38) Google Scholar). Basal cell carcinomas are also shown to be monoclonal (Walsh et al., 1996Walsh D.S. Tsou H.C. Harrington A. James W.D. Peacocke M. Clonality of basal cell carcinoma—molecular analysis of an interesting case.J Invest Dermatol. 1996; 106: 579-582Crossref PubMed Scopus (10) Google Scholar,Walsh et al., 1998Walsh D.S. Peacocke M. Harrington A. James W.D. Tsou H.C. Patterns of X chromosome inactivation in sporadic basal cell carcinomas: Evidence for clonality.J Am Acad Dermatol. 1998; 38: 49-55Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Although the former is benign and the latter malignant and, although the former is caused by a human papillomavirus, both of these epithelial lesions are now considered to be neoplasia. To examine the clonality of human samples, random inactivation of one of two female X chromosomes is most commonly used (Vogelstein et al., 1985Vogelstein B. Fearon E.R. Hamilton S.R. Feinberg A.P. Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors.Science. 1985; 227: 642-645Crossref PubMed Scopus (321) Google Scholar,Vogelstein et al., 1987Vogelstein B. Fearon E.R. Hamilton S.R. et al.Clonal analysis using recombinant DNA probes from the X-chromosome.Cancer Res. 1987; 47: 4806-4813PubMed Google Scholar). Currently, the polymorphism of the human androgen receptor (HUMARA) locus, which is located on the X chromosome, is most frequently used as it has a highly polymorphic tandem repeat (Allen et al., 1992Allen R.C. Zoghbi H.Y. Moseley A.B. Rosenblatt H.M. Belmont J.W. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar). Approximately 90% of females are heterozygous for the number of CAG trinucleotide repeats (Allen et al., 1992Allen R.C. Zoghbi H.Y. Moseley A.B. Rosenblatt H.M. Belmont J.W. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar). Moreover, methylation-sensitive restriction sites of HpaII and HhaI are within the HUMARA locus (Allen et al., 1992Allen R.C. Zoghbi H.Y. Moseley A.B. Rosenblatt H.M. Belmont J.W. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar), and the base pairs amplified by polymerase chain reaction (PCR) are relatively small (Mashal et al., 1993Mashal R.D. Lester S.C. Sklar J. Clonal analysis by study of X chromosome inactivation in formalin-fixed paraffin-embedded tissue.Cancer Res. 1993; 53: 4676-4679PubMed Google Scholar). We obtained pure SK lesions using a laser capture microdissection (LCM) system (Emmert-Buck et al., 1996Emmert-Buck M.R. Bonner R.F. Smith P.D. et al.Laser capture microdissection.Science. 1996; 274: 998-1001https://doi.org/10.1126/science.274.5289.998Crossref PubMed Scopus (2027) Google Scholar;Simone et al., 1998Simone N.L. Bonner R.F. Gillespie J.W. Emmert-Buck M.R. Liotta L.A. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Review.Trends Genet. 1998; 14: 272-276https://doi.org/10.1016/s0168-9525(98)01489-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). A clonality assay using the polymorphism of the HUMARA locus indicated that more than half the SK were monoclonal in nature. SK lesions were biopsied from nongenital regions of 40 female patients, ranging in age from 29 to 90 y. Diagnosis was made under a microscope by examining paraffin sections stained with hematoxylin and eosin. Of the 32 specimens in which the location was specified, 12 were from the trunk, seven from the face, six from the neck, five from the upper limbs, and two from the lower limbs. Forty-four SK obtained from 40 patients were histologically classified into the following four types: acanthotic (27), hyperkeratotic (14), reticulated (two), and irritated (one). DNA samples were extracted from the formalin-fixed, paraffin-embedded tissues according to a previous method (Mashal et al., 1993Mashal R.D. Lester S.C. Sklar J. Clonal analysis by study of X chromosome inactivation in formalin-fixed paraffin-embedded tissue.Cancer Res. 1993; 53: 4676-4679PubMed Google Scholar). The SK lesion and normal control epidermis adjacent to the SK lesion were microdissected from 6 μm sections stained with hematoxylin and eosin using an LCM system (LM200, Arcturus Engineering, Mountain View, CA). A representative SK sample is shown in Figure 1. A total volume of 20 μl of PK buffer containing 1.0% proteinase K, 10 mM Tris–HCl (pH 8.0), 1 mM ethylenediamine tetraacetic acid, and 1% Tween 20 was mounted on a microdissected specimen attached to a piece of LCM Transfer Film (Arcturus Engineering). Then, a 0.5 ml micro test-tube (Eppendorf-Nethtler-Hinz-GmbH, Hamburg, Germany) was placed over it. After 16 h incubation at 37°C, proteinase K was inactivated by heating at 95°C for 10 min. The lysis mixture was centrifuged for 5 min to remove undigested tissue fragments. We used a quantitative fluorescent PCR procedure that enables accurate measurement of the peak intensities of each allele according to a previous report (Wu et al., 1999Wu C.D. Wickert R.S. Williamson J.E. Sun N.C.J. Brynes R.K. Chan W.C. Using fluorescence-based human androgen receptor gene assay to analyze the clonality of microdissected dendritic cell tumors.Am J Clin Pathol. 1999; 111: 105-110PubMed Google Scholar). The HUMARA gene includes a polymorphic [(CAG)n] repeat located at 3′ of the methylation-sensitive HhaI restriction-enzyme sites (Allen et al., 1992Allen R.C. Zoghbi H.Y. Moseley A.B. Rosenblatt H.M. Belmont J.W. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar). The PCR assay used primers, the product of which spanned both the HhaI sites and the [(CAG)n] polymorphism. Variations in length of the [(CAG)n] repeats on the paternal and maternal X chromosomes yield HUMARA alleles of different lengths. Methylation of the HhaI sites distinguishes the active (nonmethylated) from the inactive (methylated) X chromosome. It is only the undigested inactive methylated allele that is subsequently amplified by PCR (Allen et al., 1992Allen R.C. Zoghbi H.Y. Moseley A.B. Rosenblatt H.M. Belmont J.W. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am J Hum Genet. 1992; 51: 1229-1239PubMed Google Scholar). Each SK DNA sample or nonlesional epidermis (7.5 μl) was digested overnight at 37°C in a 2.5 μl reaction mixture containing 0.5 μl of 16 units of HhaI (TOYOBO, Osaka, Japan), 1 μl of concentrated (× 10) TA buffer (330 mM Tris-acetate, 660 mM KOAc, 100 mM MgOAc2, 5 mM dithiothreitol), and 1 μl of concentrated (× 10) bovine serum albumin. For each case, a control sample containing only a restriction-enzyme buffer was run simultaneously. The restriction enzyme was then inactivated by heating at 95°C for 10 min. For the PCR, 1 μl of each digested DNA sample was added to 24 μl of a PCR reaction mixture containing 2.5 μl of concentrated (× 10) PCR buffer, 1.5 μl of deoxyribonucleoside triphosphate (200 μM), 0.2 μl of primers 1A and 1B (10 pM each), 0.1 μl of Taq polymerase (Boehringer Mannheim GmbH, Mannheim, Germany) and 19.7 μl of deionized H2O. The DNA samples were amplified using a sandwiched primer approach (Fujita et al., 1996Fujita M. Enomoto T. Wada H. Inoue M. Okudaira Y. Shroyer K.R. Application of clonal analysis: differential diagnosis for synchronous primary ovarian and endometrial cancers and metastatic cancer.Am J Clin Pathol. 1996; 105: 350-359Crossref PubMed Scopus (79) Google Scholar). The first step was performed using outer primers 1A (5′-GCT GTG AAG GTT GCT GTT CCT CAT-3′) and 1B (5′-CGT CCA AGA CCT ACC GAG GAG CTT-3′). The second step was performed with inner primers 2A (5′-TCC AGA ATC TGT TCC AGA GCG TGC-3′) and 2B (5′-ATG GGC TTG GGG AGA ACC ATC CTC-3′). Primer 2A was labeled at the 5′ end with 6-carboxyfluorescein. Initial denaturation was performed for 10 min at 95°C, followed by 30 cycles of 1 min at 95°C, 1 min at 60°C, and 1 min at 72°C. In the final cycle, extension at 72°C was prolonged for 10 min. The second-step PCR profile was the same as the first-step PCR. Following the second-step amplification, 5 μl of the PCR product was assessed using 2.0% agarose gel electrophoresis to confirm the amplification of the HUMARA target. After amplification, 1 μl of the PCR products was mixed with 12 μl of a Template Suppression Reagent (Applied Biosystems, Foster City, CA) and 0.5 μl of internal size standards [GENESCAN-500 (TAMRA), Applied Biosystems). The mixture was denatured at 95°C for 2 min, and analyzed through a DNA Sequencing Polymer (Applied Biosystems) with an ABI PRISM Genetic Analyzer (Applied Biosystems), according to a previous method (George et al., 1997George K.S. Zhao X. Gallahan D. Shirkey A. Zareh A. Esmaeli-Azad B. Capillary electrophoresis methodology for identification of cancer related gene expression patterns of fluorescent differential display polymerase chain reaction.J Chromatogr B. 1997; 695: 93-102Crossref PubMed Scopus (16) Google Scholar). Data were analyzed using Genescan 310 Software (Applied Biosystems). Amplification of each HUMARA allele usually generated a set of multiple peaks, including one major peak and a few associated peaks of lesser intensity as described previously (Paradis et al., 1997Paradis V. Laurent A. Flejou J.-F. Vidaud M. Bedossa P. Evidence for the polyclonal nature of focal nodular hyperplasia of the liver by the study of X-chromosome inactivation.Hepatology. 1997; 26: 891-895Crossref PubMed Scopus (142) Google Scholar). Clonality assessment was based on the major peak generated from each allele. Patients were considered heterozygous when PCR amplification of undigested DNA showed two major peaks of almost equal intensity. This suggested that maternal and paternal X chromosomes have HUMARA alleles of different molecular weights. PCR products showing a single major peak suggested that maternal and paternal X chromosomes have HUMARA alleles of the same molecular weight. Such patients were considered to be homozygous for the HUMARA gene, and thus uninformative for the analysis. For each sample, the peak intensities of two alleles (the allele with the lower molecular weight and the allele with the higher molecular weight) were measured. A corrected ratio (CR) was assessed by dividing the ratio of the HhaI-digested sample (the allele with the lower molecular weight/the allele with the higher molecular weight) by the ratio of the undigested sample. The use of CR corrects for the preferential amplification of one allele that can occur if the alleles differ markedly in molecular weight (Paradis et al., 1997Paradis V. Laurent A. Flejou J.-F. Vidaud M. Bedossa P. Evidence for the polyclonal nature of focal nodular hyperplasia of the liver by the study of X-chromosome inactivation.Hepatology. 1997; 26: 891-895Crossref PubMed Scopus (142) Google Scholar). The ratio was inverted, if necessary, to obtain a value of over 1.0. When enough DNA was obtained from the normal epidermis adjacent to the SK lesion, the final clonality ratio for each SK was determined by dividing the CR of the lesional DNA by the CR of the nonlesional DNA. This final clonality ratio corrects for any potential skewed lyonization. We assumed that SK with a final clonality ratio of ≥ 1.5 were monoclonal, according to a previous report (Paradis et al., 1997Paradis V. Laurent A. Flejou J.-F. Vidaud M. Bedossa P. Evidence for the polyclonal nature of focal nodular hyperplasia of the liver by the study of X-chromosome inactivation.Hepatology. 1997; 26: 891-895Crossref PubMed Scopus (142) Google Scholar). Statistical analysis was performed using Microsoft Excel (Microsoft, WA). The data are presented as mean and SEM. Differences between groups were evaluated by Student's t test. SK specimens were obtained from 40 women. Sections from the SK lesions were stained with hematoxylin and eosin, and histologic types were determined. Then, the epithelial portions of the individual SK lesions were carefully removed by LCM (Figure 1). DNA was extracted from each sample, and the portion of the HUMARA locus containing the trinucleotide repeats was amplified by PCR. Samples derived from six of 40 patients could not be used because the polymorphism of the HUMARA locus was not detectable. The remaining 34 patients were considered to be informative. As three of 34 informative patients (12, 17, and 27) had two or three SK, 38 SK lesions were analyzed in total (Table I).Table IHistologic types, size of alleles, corrected ratios and final ratios of SK casesPatient no.Histologic typeaAC, acanthotic; HY, hyperkeratotic; RE, reticulated; IR, irritated.Size of alleles (difference in bases)CRFinal ratio (SK/normal epidermis)SKNormal epidermis1AC222/225 (3)1.52AC219/222 (3)1.63AC231/234 (3)1.74AC225/228 (3)2.55AC228/231 (3)2.96AC216/219 (3)3.17AC237/240 (3)3.88AC228/231 (3)4.39AC225/228 (3)8.110ACbConsidered monoclonal because the final ratio is ≥ 1.5.216/222 (6)2.71.12.511AC225/231 (6)3.812AC225/231 (6)8.7ACcConsidered monoclonal because CR is ≥ 10.0.225/231 (6)> 1013ACcConsidered monoclonal because CR is ≥ 10.0.228/234 (6)> 1014ACcConsidered monoclonal because CR is ≥ 10.0.222/228 (6)> 1015ACcConsidered monoclonal because CR is ≥ 10.0.222/231 (9)10.016ACbConsidered monoclonal because the final ratio is ≥ 1.5.231/243 (12)2.51.02.517ACcConsidered monoclonal because CR is ≥ 10.0.234/246 (12)> 10ACcConsidered monoclonal because CR is ≥ 10.0.234/246 (12)> 1018ACbConsidered monoclonal because the final ratio is ≥ 1.5.222/237 (15)6.01.15.519ACcConsidered monoclonal because CR is ≥ 10.0.207/222 (15)> 1020ACcConsidered monoclonal because CR is ≥ 10.0.222/237 (15)> 1021ACcConsidered monoclonal because CR is ≥ 10.0.228/246 (18)> 1022ACcConsidered monoclonal because CR is ≥ 10.0.204/222 (18)> 1023HY231/237 (6)2.024HYbConsidered monoclonal because the final ratio is ≥ 1.5.225/231 (6)4.21.13.825HYcConsidered monoclonal because CR is ≥ 10.0.234/240 (6)> 1026HYb,c228/237 (9)> 101.0> 1027HYcConsidered monoclonal because CR is ≥ 10.0.228/237 (9)> 10HYcConsidered monoclonal because CR is ≥ 10.0.228/237 (9)> 10REcConsidered monoclonal because CR is ≥ 10.0.228/237 (9)> 1028HYcConsidered monoclonal because CR is ≥ 10.0.234/243 (9)> 1029HYcConsidered monoclonal because CR is ≥ 10.0.237/246 (9)> 1030HYcConsidered monoclonal because CR is ≥ 10.0.216/228 (12)> 1031HYcConsidered monoclonal because CR is ≥ 10.0.225/237 (12)> 1032HYcConsidered monoclonal because CR is ≥ 10.0.222/237 (15)> 1033RE228/231 (3)1.634IRcConsidered monoclonal because CR is ≥ 10.0.228/234 (6)> 10a AC, acanthotic; HY, hyperkeratotic; RE, reticulated; IR, irritated.b Considered monoclonal because the final ratio is ≥ 1.5.c Considered monoclonal because CR is ≥ 10.0. Open table in a new tab Histologic types, allelic sizes, and CR values of informative SK cases are shown in Table I. As biopsied SK specimens contained relatively small amounts of normal epidermis adjacent to the SK, CR values of the normal control epidermis were obtained in only five SK cases, and the final clonality ratio was calculated by dividing the CR of each SK by the CR of the normal control epidermis (Table I). The final clonality ratio of these five SK was > 1.5. CR values were plotted against numbers of bases that were different between two HUMARA alleles of individual SK (Figure 2). The CR values appeared to be dependent on the number of bases, being different between alleles of lower molecular weight and those of higher molecular weight. CR values were ≥ 10 in none of the 10 SK in which 3 bp were different between the two alleles, in five of 10 SK in which 6 bp were different between the two alleles, and in 16 of 18 SK in which ≥ 9 bp were different between the two alleles (Figure 2). In total, CR values were ≥ 10 of 21 of the 38 SK examined. When the number of bases that differed between the two alleles was 3 or 6, the variation in CR values was significant among individual SK (Figure 2). The following tendency was observed. CR values were higher when the HUMARA alleles with higher molecular weights were activated and digested than when the allele with a lower molecular weight were activated and digested (Figure 3). For example, compare Figure 4(4a1) (CR = 8.1) and Figure 4(4a2) (CR = 1.7), and Figure 4(4b1) (CR > 10) and Figure 4(4b2) (CR = 3.8). The number of bases that differed between the two HUMARA alleles was three in the cases shown in Figure 4(4a1, 4a2). Also, the number of bases that differed between the two alleles was six in the cases shown in Figure 4(4b1, 4b2).Figure 4Comparison of clonal analysis patterns between the SK in which the HUMARA allele with higher molecular weight was digested and those in which the allele with lower molecular weight was digested. The peaks to be digested are painted out. Clonal analysis pattern of a normal control epidermis is also shown.View Large Image Figure ViewerDownload (PPT) In this study, we examined the clonality of SK. Biopsy specimens of SK contained only small amounts of normal epidermis. Therefore, we obtained enough normal control epidermis in only five SK cases; however, in these five SK cases, the CR values of the normal epidermis were ≤ 1.1. In other words, no significantly skewed lyonization of the normal epidermis was detected in these five cases. The final clonality ratio was calculated by dividing the CR value of each SK by that of the control normal epidermis. As a final ratio of ≥ 1.5 has been used as a criterion for monoclonality (Paradis et al., 1997Paradis V. Laurent A. Flejou J.-F. Vidaud M. Bedossa P. Evidence for the polyclonal nature of focal nodular hyperplasia of the liver by the study of X-chromosome inactivation.Hepatology. 1997; 26: 891-895Crossref PubMed Scopus (142) Google Scholar), we considered that at least these five SK were monoclonal. The skewing was different among various tissues from each patient. We dissected the normal epidermis using an LCM system; such skewing of the normal human epidermis has not been reported to our knowledge. To establish the normal range of CR values for the normal human epidermis, the examination of more skin samples using the present method appears to be necessary. The slippage phenomenon observed in Taq amplification of trinucleotide repeats may result in minor peaks trailing the major peak at 3 bp intervals (Mutter and Boynton, 1995Mutter G.L. Boynton K.A. PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies.Nucleic Acids Res. 1995; 23: 1411-1418Crossref PubMed Scopus (173) Google Scholar). This interference is insignificant when two alleles differ by 9 bp or more in size. The two alleles differed by 9 bp or more in the 18 SK, which were examined in this study. In 16 of these 18 SK, one of two HUMARA peaks practically disappeared after digestion by HhaI (i.e., CR values ≥ 10.0). Therefore, at least these 16 SK were considered to be clonal. When the two alleles differed by 6 bp, one of two HUMARA peaks also disappeared after HhaI digestion (CR > 10.0) in five of 10 SK cases (Figure 2). We concluded that these five cases were also monoclonal. Overall, five SK were considered to be monoclonal because the final ratios were > 1.5, and 21 SK were judged to be monoclonal because their CR values were ≥ 10.0. As both criteria were applicable to only one SK (patient 26), 25 of 38 SK examined were judged to be monoclonal (Table I). This does not imply that the remaining 13 SK were polyclonal. Although they were not determined to be monoclonal using the present method, some of them may have been monoclonal if genes on the X chromosome other than the HUMARA gene had been used as a marker of clonality. The clonality of various skin tumors has been reported. As for epidermal tumors, verrucae vulgaris (Murray et al., 1971Murray R.F. Hobbs J. Payne B. Possible clonal origin of common warts (Verruca vulgaris).Nature. 1971; 232: 51-52Crossref PubMed Scopus (38) Google Scholar) and basal cell carcinomas (Walsh et al., 1996Walsh D.S. Tsou H.C. Harrington A. James W.D. Peacocke M. Clonality of basal cell carcinoma—molecular analysis of an interesting case.J Invest Dermatol. 1996; 106: 579-582Crossref PubMed Scopus (10) Google Scholar,Walsh et al., 1998Walsh D.S. Peacocke M. Harrington A. James W.D. Tsou H.C. Patterns of X chromosome inactivation in sporadic basal cell carcinomas: Evidence for clonality.J Am Acad Dermatol. 1998; 38: 49-55Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) have been shown to be monoclonal. In this study, we demonstrated that more than half of the SK were also monoclonal. Regarding mesenchymal tumors, dermatofibromas were reported to be monoclonal (Chen et al., 2000Chen T.C. Kuo T. Chan H.L. Dermatofibroma is a clonal proliferative disease.J Clin Pathol. 2000; 27: 36-39Google Scholar). Although the monoclonality of benign neurofibromas is controversial (Rey et al., 1987Rey J.A. Bello M.J. de Campos J.M. Benitez J. Sarasa J.L. Boixados J.R. Sanchez Cascos A. Cytogenetic clones in a recurrent neurofibroma.Cancer Genet Cytogenet. 1987; 26: 157-163Abstract Full Text PDF PubMed Scopus (26) Google Scholar;Skuse et al., 1991Skuse G.R. Kosciolek B.A. Rowley P.T. The neurofibroma in von Recklinghausen neurofibromatosis has a unicellular origin.Am J Hum Genet. 1991; 49: 600-607PubMed Google Scholar;Daschner et al., 1997Daschner K. Assume G. Eisenbarth I. et al.Clonal origin of tumor cells in a plexiform neurofibroma with LOH in NF1 intron 38 and in dermal neurofibromas without LOH of the NF1 gene.Biochem Biophys Res Commun. 1997; 234: 346-350https://doi.org/10.1006/bbrc.1997.6645Crossref PubMed Scopus (53) Google Scholar), neurofibrosarcomas that developed from neurofibromas were monoclonal (Friedman et al., 1982Friedman J.M. Fialkow P.J. Greene C.L. Weinberg M.N. Probable clonal origin of neurofibrosarcoma in a patient with hereditary neurofibromatosis.J Natl Cancer Inst. 1982; 69: 1289-1291PubMed Google Scholar). With Kaposi's sarcoma, controversial results have been reported (Rabkin et al., 1995Rabkin C.S. Bedi G. Musaba E. Sunkutu R. Mwansa N. Sidransky D. Biggar R.J. AIDS-related Kaposi's sarcoma is a clonal neoplasm.Clin Cancer Res. 1995; 1: 257-260PubMed Google Scholar,Rabkin et al., 1997Rabkin C.S. Janz S. Lash A. et al.Monoclonal origin of multicentric Kaposi's sarcoma lesions.N Engl J Med. 1997; 36: 988-993Crossref Scopus (196) Google Scholar;Delabesse et al., 1997Delabesse E. Oksenhendler E. Lebbe C. Verola O. Varet B. Turhan A.G. Molecular analysis of clonality in Kaposi's sarcoma.J Clin Pathol. 1997; 50: 664-668Crossref PubMed Scopus (71) Google Scholar;Gill et al., 1998Gill P.S. Tsai Y.C. Rao A.P. et al.Evidence for multiclonality in multicentric Kaposi's sarcoma.Proc Natl Acad Sci USA. 1998; 95: 8257-8261https://doi.org/10.1073/pnas.95.14.8257Crossref PubMed Scopus (110) Google Scholar). Although melanomas have been shown to be monoclonal, results regarding nevocellular neavus are controversial (Harada et al., 1997Harada M. Suzuki M. Ikeda T. Kaneko T. Hharada S. Fukayama M. Clonality in nevocellular nevus and melanoma: an expression-based clonality analysis at the X-linked genes by polymerase chain reaction.J Invest Dermatol. 1997; 109: 656-660Crossref PubMed Scopus (30) Google Scholar;Robinson et al., 1998Robinson W.A. Lemon M. Elefanty A. Harrison-Smith M. Markham N. Norris D. Human acquired naevi are clonal.Melanoma Res. 1998; 8: 499-503Crossref PubMed Scopus (48) Google Scholar). Many nontumor cells may be present within tumors. Microdissection of tissue fragments that contain tumor cells alone, and examination of clonality by a sensitive and quantitative method may be necessary to reach definite conclusions. As mentioned previously, one artifact that may prevent accurate quantitative analysis using the present method is the slippage phenomenon observed in Taq amplification of trinucleotide repeats. When alleles differ by 3 or 6 bp in size, the presence of a secondary or tertiary peak may preclude the accurate analysis (Wu et al., 1999Wu C.D. Wickert R.S. Williamson J.E. Sun N.C.J. Brynes R.K. Chan W.C. Using fluorescence-based human androgen receptor gene assay to analyze the clonality of microdissected dendritic cell tumors.Am J Clin Pathol. 1999; 111: 105-110PubMed Google Scholar). We pooled the CR values of SK in which the two alleles differed by 3 and 6 bp in size. The CR values were significantly higher when alleles with higher molecular weights were digested than when alleles with lower molecular weights were digested (Figure 3). If alleles with higher molecular weights were digested, the intensity of the undigested alleles with lower molecular weights was unaffected (Figure 4 4a1, 4b1). In contrast, even if an allele with a lower molecular weight was digested, a secondary or tertiary peak of an undigested allele with higher molecular weight remained at the position where the digested peak had been situated. Thirty-eight informative SK were histologically classified into acanthotic, hyperkeratotic, reticulated, or irritated types. SK that were demonstrated to be monoclonal were found in all histologic types. There was no correlation between histologic type and clonality (Table I). In this study, we showed the monoclonal origin of more than half the SK in the skin of human females, suggesting that SK are a benign neoplasia. Although the biology of SK has not been studied in depth, it is possible that further investigation may be useful in understanding the development and progression of benign tumors in human skin.