erbB-2 Overexpression But No Activation of β-Catenin Gene in Extramammary Paget's Disease
1999; Elsevier BV; Volume: 113; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.1999.00634.x
ISSN1523-1747
AutoresMinoru Takata, Akihide Fujimoto, Hiromasa Aoki, Naohito Hatta, Kazuhiko Takehara, Akishi Ooi,
Tópico(s)Cancer-related Molecular Pathways
ResumoOur previous study in extramammary Paget's disease showed neither p53 mutations nor allelic loss at selected loci implicated in other cancers, suggesting a pathogenesis of this skin cancer different from other common epithelial malignancies. To examine further the genetic defects in extramammary Paget's disease, we carried out molecular genetic analyses in 31 tumor samples obtained from 27 cases of extramammary Paget's disease without underlying malignancies. Immunohistochemistry using CB-11 monoclonal antibody revealed either membrane or cytoplasmic erbB-2 oncoprotein overexpression in none of the 13 primary in situ tumors, but in one recurrent in situ tumor, 10 of 13 invasive primary tumors and two of four lymph node metastases. Sensitive dual color fluorescence in situ hybridization analysis using probes for erbB-2 gene locus and chromosome 17 pericentromere, however, revealed different erbB-2 gene status in the erbB-2 overexpressing tumors. One recurrent in situ tumor and one lymph node metastasis showed definite gene amplification characterized by multiple scattered signals or a few large clustered erbB-2 signals, whereas four tumors with predominantly cytoplasmic erbB-2 overexpression were thought to have low-grade gene amplification. The remaining six tumors overexpressing erbB-2 showed no increase of erbB-2 copy numbers. No evidence of abnormal activation of the β-catenin gene, a critical mediator of Wnt signaling pathway, in any tumor by immunohistochemical staining and by direct sequencing and reverse transcription–polymerase chain reaction analysis was found. Frequent overexpression of erbB-2 by either gene amplification or possible transcriptional activation in invasive primary tumors and metastases suggests an important part for this oncogene in the progression of extramammary Paget's disease. Our previous study in extramammary Paget's disease showed neither p53 mutations nor allelic loss at selected loci implicated in other cancers, suggesting a pathogenesis of this skin cancer different from other common epithelial malignancies. To examine further the genetic defects in extramammary Paget's disease, we carried out molecular genetic analyses in 31 tumor samples obtained from 27 cases of extramammary Paget's disease without underlying malignancies. Immunohistochemistry using CB-11 monoclonal antibody revealed either membrane or cytoplasmic erbB-2 oncoprotein overexpression in none of the 13 primary in situ tumors, but in one recurrent in situ tumor, 10 of 13 invasive primary tumors and two of four lymph node metastases. Sensitive dual color fluorescence in situ hybridization analysis using probes for erbB-2 gene locus and chromosome 17 pericentromere, however, revealed different erbB-2 gene status in the erbB-2 overexpressing tumors. One recurrent in situ tumor and one lymph node metastasis showed definite gene amplification characterized by multiple scattered signals or a few large clustered erbB-2 signals, whereas four tumors with predominantly cytoplasmic erbB-2 overexpression were thought to have low-grade gene amplification. The remaining six tumors overexpressing erbB-2 showed no increase of erbB-2 copy numbers. No evidence of abnormal activation of the β-catenin gene, a critical mediator of Wnt signaling pathway, in any tumor by immunohistochemical staining and by direct sequencing and reverse transcription–polymerase chain reaction analysis was found. Frequent overexpression of erbB-2 by either gene amplification or possible transcriptional activation in invasive primary tumors and metastases suggests an important part for this oncogene in the progression of extramammary Paget's disease. adenomatous polyposis coli extramammary Paget's disease fluorescence in situ hybridization Extramammary Paget's disease (EMPD) is a distinct form of skin cancer of unknown histogenesis. Although in rare instances EMPD is a secondary event caused by extension of an adenocarcinoma either of the rectum to the perianal region, of the cervix to the vulvar region, or of the urinary bladder to the urethra and glans penis, the cells of origin in EMPD without underlying genitourinary or gastrointestinal carcinomas are unknown (Kirkham, 1997Kirkham N. Tumors and cysts of the epidermis.in: Elder D. Elenitsas R. Jaworsky C. Johnson JB. Lever's Histopathology of the Skin. 8th edn. Philadelphia, Lippincott-Raven1997: 685-746Google Scholar). Furthermore, genetic abnormalities underlying the pathogenesis of this skin tumor are poorly understood. In our previous molecular genetic study of 14 cases of EMPD, neither loss of heterozygosity of several selected chromosome arms (i.e., 3p, 9p, 9q, 13q, 16q, 17p, and 17q) nor p53 gene mutation were detected, although a subset of tumors expressed erbB-2 oncoprotein (Takata et al., 1997Takata M. Hatta N. Takehara K. Tumour cells of extramammary Paget's disease do not show either p53 mutation or allelic loss at several selected loci implicated in other cancers.Br J Cancer. 1997; 76: 904-908Crossref PubMed Scopus (24) Google Scholar). These results suggested that underlying genetic defects in EMPD are different from those in other common epithelial malignancies, and that certain oncogenes or tumor suppressor genes not examined in that study may be important. To explore further the genetic changes in this particular form of skin cancer, we collected primary and metastatic tumor samples from a total of 27 EMPD patients. Using these samples, we examined: (i) the frequency of erbB-2 protein overexpression in different stages of progression in EMPD; (ii) whether the overexpression is associated with the erbB-2 gene amplification; and (iii) whether the abnormal activation of Wnt signaling pathway, which has recently been show to play an important part in the pathogenesis of other human cancers (Pennisi, 1998Pennisi E. How a growth control path takes wrong turn to cancer.Science. 1998; 281: 1438-1441Crossref PubMed Scopus (83) Google Scholar), is involved in the pathogenesis of EMPD. Because the abnormal activation of Wnt signaling pathway was mainly reported in adenocarcinomas including colon cancers (Korinek et al., 1997Korinek V. Barker N. Morin P.J. et al.Constitutive transcriptional activation by a β-catenin-Tcf complex in APC-/- colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2843) Google Scholar;Morin et al., 1997Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or.APC. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3375) Google Scholar), and because EMPD is an adenocarcinoma (Roth et al., 1977Roth L.M. Lee S.C. Ehrlich C.E. Paget's disease of the vulva. A histogenetic study of five cases including ultrastructural observations and review of the literature.Am J Surg Pathol. 1977; 1: 193-206Crossref PubMed Scopus (54) Google Scholar), we were interested in examining this pathway in EMPD. The subjects included 27 patients with EMPD (21 men and six women) who were admitted to the Department of Dermatology, Kanazawa University Hospital from 1987 to 1998 (Table I). The median age at diagnosis was 72 y old (range 48–85 y). The disease was entirely intraepidermal (in situ carcinoma) in 14 patients, and was accompanied by synchronous dermal invasion (invasive carcinoma) in 13 patients. Eight of the 13 invasive carcinomas were associated with distant and/or lymph node metastases. None of the cases were associated with underlying genitourinary or gastrointestinal malignancies. Paraffin-embedded tissue blocks of all 27 cutaneous lesions and four lymph node metastases were available. Fresh frozen tumor and peripheral blood samples were also obtained from 12 patients, and these samples were employed for analysis of β-catenin gene status. DNA was isolated from tumor and blood samples according to standard methods by proteinase K digestion and phenol–chloroform extraction. Total RNA was also isolated from each frozen tumor sample with Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's protocol, and were reverse transcribed to generate single-stranded cDNA using an RNA PCR Kit (Takara, Tokyo, Japan).Table IExtramammary Paget's disease show erbB-2 overexpression associated with or without gene amplification, but show no mutational activation of the β-catenin geneerbB-2β-cateninGeneCase no.Age/sexTumorbIn situ, in situ primary tumor; In situ, R, recurrent in situ tumor; Invasive, invasive primary tumor; LN meta, lymph node metastasis.Protein expressionc-, no staining or weak staining in 80% of cells; M, membrane staining; C, cytoplasmic staining.Gene amplificationdMSS, multiple scattered signals; NA, no amplification; LGA, low-grade amplification; CR, cluster regions.Protein expressionc-, no staining or weak staining in 80% of cells; M, membrane staining; C, cytoplasmic staining.MutationDeletion1aCases partly examined in the previous paper (Takata et al. 1997).86/FIn situ--280/MIn situ-3aCases partly examined in the previous paper (Takata et al. 1997).72/MIn situ-++/M--4aCases partly examined in the previous paper (Takata et al. 1997).70/FIn situ-++/M--5aCases partly examined in the previous paper (Takata et al. 1997).72/FIn situ-+/M--6aCases partly examined in the previous paper (Takata et al. 1997).71/MIn situ-++/M-772/MIn situ-++/M885/FIn situ-++/M975/MIn situ---1071/MIn situ-++/M--1152/FIn situ-+/M1268/MIn situ-++/M1370/MIn situ-+/M1477/FIn situ, R++/MMSS-15aCases partly examined in the previous paper (Takata et al. 1997).63/MInvasive-++/MLN meta-+/M16aCases partly examined in the previous paper (Takata et al. 1997).48/MInvasive++/MNA++/M17aCases partly examined in the previous paper (Takata et al. 1997).78/MInvasive++/MNA+/M18aCases partly examined in the previous paper (Takata et al. 1997).69/MInvasive++/CNA1974/MInvasive+/CLGA2084/MInvasive++/CLGA-21aCases partly examined in the previous paper (Takata et al. 1997).70/MInvasive++/MNA+/M22aCases partly examined in the previous paper (Takata et al. 1997).76/MInvasive-+/M-23aCases partly examined in the previous paper (Takata et al. 1997).78/MInvasive---24aCases partly examined in the previous paper (Takata et al. 1997).58/MInvasive-+/M--25aCases partly examined in the previous paper (Takata et al. 1997).76/MInvasive++/CLGA+/M--LN meta+/C+/M--2664/MInvasive+/MNA++/M-LN meta++/MCR+/M-2772/MInvasive+/CLGA+/M--LN meta----a Cases partly examined in the previous paper (Takata et al., 1997Takata M. Hatta N. Takehara K. Tumour cells of extramammary Paget's disease do not show either p53 mutation or allelic loss at several selected loci implicated in other cancers.Br J Cancer. 1997; 76: 904-908Crossref PubMed Scopus (24) Google Scholar).b In situ, in situ primary tumor; In situ, R, recurrent in situ tumor; Invasive, invasive primary tumor; LN meta, lymph node metastasis.c -, no staining or weak staining in 80% of cells; M, membrane staining; C, cytoplasmic staining.d MSS, multiple scattered signals; NA, no amplification; LGA, low-grade amplification; CR, cluster regions. Open table in a new tab Paraffin sections 4 μm thick were cut from each block and stained by the biotin–streptavidin–peroxidase method as described previously (Takata et al., 1997Takata M. Hatta N. Takehara K. Tumour cells of extramammary Paget's disease do not show either p53 mutation or allelic loss at several selected loci implicated in other cancers.Br J Cancer. 1997; 76: 904-908Crossref PubMed Scopus (24) Google Scholar). The primary antibodies used were anti-erbB-2 (CB-11; Novocastra, Newcastle, U.K.; working dilution, 1:40) and anti-β-catenin (Transduction Laboratories, Lexington, KY; working dilution 1:100) mouse monoclonal antibodies. Antigen retrieval was performed prior to staining for β-catenin by immersing slides in sodium citrate buffer (pH 6.0) and heating for 10 min in a conventional microwave oven. Stained sections were scored for the proportion of positively stained tumor cells and for the intensity of staining, and were graded as follows: –, no staining or weak staining in less than 10% of cells; +, moderate to strong staining in 10%–80% of cells; + +, moderate to strong staining over 80% of cells. The pattern of staining (i.e., membrane, cytoplasmic, or nuclear) was also recorded. Scoring was carried out independently by two observers and a consensus reached when results were discrepant. Amplification of the erbB-2 gene was detected in paraffin-embedded tissue sections by a sensitive FISH technique recently developed by one of the authors (Ooi et al., 1998Ooi A. Kobayashi M. Mai M. Nakanishi I. Amplification of c-erbB-2 in gastric cancer; detection in formalin- fixed, paraffin-embedded tissue by fluorescence in situ hybridization.Lab Invest. 1998; 78: 345-351PubMed Google Scholar). The sections adjacent to those used for immunostaining were mounted on glass slides and the representative areas containing erbB-2 overexpressing cells were selected for FISH analysis. Cells of human gastric adenocarcinoma line MKN-7, which had amplified erbB-2 DNA (30–50-fold of normal) and showed clusters of erbB-2 signals on FISH (Ishikawa et al., 1997Ishikawa T. Kobayashi M. Mai M. Suzuki T. Ooi A. Amplification of the erbB-2 (HER-2/neu) gene in gastric cancer cells. Detection by fluorescence in situ hybridization.Am J Pathol. 1997; 151: 761-768PubMed Google Scholar), was also stained for comparison. A dual color probe mixture consisting of Spectrum Orange LSI HER-2/neu probe (band region 17q11.2–12) and Spectrum Green CEP 17 (band region 17p11.1–q11.1, locus D17Z1) was purchased from Vysis (Downers Grove, IL). Deparaffinized sections were incubated in 20% sodium bisulfite/2 × sodium citrate/chloride buffer at 43°C for 20 min. After washing in 2 × sodium citrate/chloride buffer, the tissues were digested with 25 ng per ml of proteinase K (Boehringer Mannheim, Mannheim, Germany) at 37°C for 30 min. After washing in 2 × sodium citrate/chloride buffer and dehydration by passage through a graded ethanol series, the slides were dried and denatured with the probes on a hot plate at 69°C for 5 min, and then hybridized overnight at 37°C. After washing in 0.4% sodium citrate/chloride buffer/0.3% NP-40 solution at 75°C for 2 min and followed by rinsing with 2 × sodium citrate/chloride buffer/0.1% NP-40 solution at room temperature, the slides were then counterstained with 4′6-diamidine-2′-phenylindol dihydrochloride in anti-fade solution (Oncor, Gaithersburg, ML) and examined with an epifluorescence microscope (Olympus, Tokyo, Japan) equipped with a triple band pass filter. Based on our previous results in gastric cancer (Ishikawa et al., 1997Ishikawa T. Kobayashi M. Mai M. Suzuki T. Ooi A. Amplification of the erbB-2 (HER-2/neu) gene in gastric cancer cells. Detection by fluorescence in situ hybridization.Am J Pathol. 1997; 151: 761-768PubMed Google Scholar;Ooi et al., 1998Ooi A. Kobayashi M. Mai M. Nakanishi I. Amplification of c-erbB-2 in gastric cancer; detection in formalin- fixed, paraffin-embedded tissue by fluorescence in situ hybridization.Lab Invest. 1998; 78: 345-351PubMed Google Scholar), a tumor mainly consisted of cells with clustered erbB-2 signals or multiple scattered signal were considered to have erbB-2 gene amplification. In equivocal cases, copy numbers of centromere 17 (green) and erbB-2 signals (orange) were, respectively, counted for more than 50 nonoverlapping tumor cells. The nuclei from normal epidermis in each specimen were used as controls of hybridization efficiency and specificity. Although the expected number of centromere 17 (and also erbB-2) signals in a normal keratinocyte varies from 2 to 4 depending on the phase of the cell cycle, the actual mean number of signals in normal epidermis in individual cases ranged from 1.3 to 1.5 due to nuclear truncation by sectioning. The mean copy numbers of centromere 17 and erbB-2 signals in tumor cells were statistically compared by Student's t test in each tumor. The mean erbB-2 copy numbers was also compared by Student's t test between tumor cells and normal keratinocytes in each specimen. p < 0.05 was regarded as significant. When the mean number of erbB-2 signals was significantly greater than that of centromere 17 signals in tumor cells and the mean number of erbB-2 signals was significantly greater in tumor cells than in normal keratinocytes, the tumor was considered as having low-grade erbB-2 gene amplification. Because the previous studies in other cancer systems showed that all the activating β-catenin mis-sense mutations were found in serine or threonine residues in exon 3 which encodes the NH2-terminal regulatory domain (Sparks et al., 1998Sparks A.B. Morin P.J. Vogelstein B. Kinzler K.W. Mutational analysis of the APC/β-catenin/Tcf pathway in colorectal cancer.Cancer Res. 1998; 58: 1130-1134PubMed Google Scholar), exon 3 was amplified from genomic DNA using primers 5′-ATTTGATGGAGTTGGACATGGC-3′ (forward) and 5′-CCAGCTACTTGTTCTTGAGTGAAGG-3′ (reverse) (Kitaeva et al., 1997Kitaeva M.N. Grogan L. Williams J.P. et al.Mutations in β-catenin are uncommon in colorectal cancer occurring in occasional replication error-positive tumors.Cancer Res. 1997; 57: 4478-4481PubMed Google Scholar). The polymerase chain reaction (PCR) products were purified using a DNA affinity spin columns (Wizard PCR Preps, Promega, Madison, WI) and directly sequenced by automated sequencing with fluorescently labeled dideoxy chain-terminating nucleotides and Taq DNA polymerase using a Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Foster City, CA). The PCR primers were also used for sequencing. To exclude the possibility of the β-catenin gene activation by large interstitial deletions involving entire exon 3 such as those reported in colon cancers and hepatocellular carcinomas (Iwao et al., 1998Iwao K. Nakamori S. Kameyama M. et al.Activation of the β-catenin gene by interstitial deletions involving exon 3 in primary colorectal carcinomas without adenomatous polyposis coli mutations.Cancer Res. 1998; 58: 1021-1026PubMed Google Scholar;Miyoshi et al., 1998Miyoshi Y. Iwao K. Nagasawa Y. et al.Activation of the β-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3.Cancer Res. 1998; 58: 2524-2527PubMed Google Scholar), we also performed reverse transcription–PCR using single-stranded cDNA as a template. Primers used were C-F (5′-CCAGCGTGGACAATGGCTAC-3′) and C-R (5′-TGAGCTCGAGTCATTGCATAC-3′), which corresponded to DNA sequences of exons 2 and 4, respectively, and we are expected to yield a 287 bp product (Iwao et al., 1998Iwao K. Nakamori S. Kameyama M. et al.Activation of the β-catenin gene by interstitial deletions involving exon 3 in primary colorectal carcinomas without adenomatous polyposis coli mutations.Cancer Res. 1998; 58: 1021-1026PubMed Google Scholar). The PCR products were resolved in 1.5% agarose gels and visualized with ethidium bromide. No erbB-2 overexpression was detected in any of the primary in situ carcinomas, except in one recurrent in situ carcinoma. In contrast, 10 of 13 (77%) invasive primary tumors, and two of four (50%) lymph node metastases overexpressed erbB-2. The pattern of erbB-2 immunostaining and the erbB-2 gene status, however, revealed by FISH analysis were variable (Table I). A recurrent in situ carcinoma (case 14) showed strong membrane staining for erbB-2 in intraepidermal tumor cells most of which had more than five multiple scattered erbB-2 signals (Figure 1a, b). Three invasive primary carcinomas (cases 16, 17, and 21) also showed distinct membrane staining for erbB-2 protein in nearly 100% of tumor cells (Figure 1c). FISH analysis, however, showed no increase of erbB-2 copy number in these tumors (Figure 1d). In case 26, only tumor cells in the peripheral region of the primary tumor showed membrane overexpression of erbB-2, which were not associated with the erbB-2 gene amplification. Most of the metastatic tumor cells in the regional lymph node in this case, however, overexpressed erbB-2 (Figure 1e) and were found to have one to several large clusters of amplified erbB-2 signals that are identical to those observed in interface MKN-7 cells (Figure 1f). The remaining five primary invasive tumors (cases 18, 19, 20, 25, and 27) showed cytoplasmic rather than membrane staining for erbB-2 protein (Figure 1g), although the corresponding lymph node metastases in two of these cases (25 and 27) contained less or no erbB-2 expressing cells. Interestingly, four of these five tumors showed low-grade erbB-2 gene amplification (Figure 1h). In normal epithelial cells, β-catenin is localized at the cell membrane. Many adenomatous polyposis coli (APC) gene mutations resulting in truncated proteins with loss of β-catenin regulatory activity, however, cause cytoplasmic and nuclear accumulation of β-catenin protein (Korinek et al., 1997Korinek V. Barker N. Morin P.J. et al.Constitutive transcriptional activation by a β-catenin-Tcf complex in APC-/- colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2843) Google Scholar). Mutations of the serine/threonine residues on exon 3 of the β-catenin gene also result in stabilization and accumulation of this protein (Morin et al., 1997Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or.APC. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3375) Google Scholar). This abnormal accumulation of β-catenin protein can be detected by immunohistochemistry, which may serve as a useful method of screening for the disruption and abnormal activation of Wnt signaling pathway (Fukuchi et al., 1998Fukuchi T. Sakamoto M. Tsuda H. Maruyama K. Nozawa S. Hirohashi S. β-catenin mutation in carcinoma of the uterine endometrium.Cancer Res. 1998; 58: 3526-3528PubMed Google Scholar;Palacios and Gamallo, 1998Palacios J. Gamallo C. Mutations in the catenin gene (CTNNB1) in endometrioid ovarian carcinomas.Cancer Res. 1998; 58: 1344-1347PubMed Google Scholar). Thus, we first examined the expression and subcellular localization of β-catenin protein in 24 primary or recurrent skin tumors and four lymph node metastases. Normal keratinocytes showed distinct membrane staining for β-catenin protein, which served as an internal positive control. Although the staining was mostly negative in four skin tumors and one lymph node metastasis, all the other 22 tumors showed β-catenin expression localized at the cellular membrane of Paget's cells (Table I and Figure 2). No cytoplasmic and nuclear accumulation of β-catenin protein that suggests disruption of the Wnt signaling was observed in any of the skin tumors and metastases examined. To confirm further the absence of β-catenin mutations, we sequenced exon 3 of the β-catenin gene in 12 primary tumors (six in situ carcinomas and six invasive carcinomas) and three lymph node metastases. All the 15 samples showed wild-type sequence, and neither missense mutations nor small deletions affecting codons 29–45 in exon 3 were found. In reverse transcription–PCR analysis using a primer pair spanning exon 2 through exon 4, all of the 10 tested cDNA samples showed normal-sized transcripts of 287 bp, and no smaller aberrant bands suggesting large interstitial deletions were detected. These results confirmed absence of the activating β-catenin gene mutations in EMPD. In many human cancers including breast, ovarian, and gastric adenocarcinomas, erbB-2 gene amplification and/or erbB-2 protein overexpression are common and are thought to play an important part in tumorigenesis (Hynes and Stern, 1994Hynes N.E. Stern D.F. The biology of erbB-2/neu/HER-2 and its role in cancer.Biochim Biophys Acta. 1994; 1198: 165-184Crossref PubMed Scopus (977) Google Scholar). In mammary Paget's disease which is phenotypically similar to EMPD, nearly all cases examined by immunohistochemistry showed distinct membrane staining of erbB-2 antigen in both intraepidermal Paget's cells and underlying ductal carcinoma cells, indicating erbB-2 overexpression (Keatings et al., 1990Keatings L. Sinclair J. Wright C. et al.erbB-2 oncoprotein expression in mammary and extramammary Paget's disease: an immunohistochemical study.Histopathology. 1990; 17: 243-247Crossref PubMed Scopus (71) Google Scholar;Meissner et al., 1990Meissner K. Riviere A. Haupt G. Loning T. Study of neu-protein expression in mammary Paget's disease with and without underlying breast carcinoma and in extramammary Paget's disease.Am J Pathol. 1990; 137: 1305-1309PubMed Google Scholar;Wolber et al., 1991Wolber R.A. Dupuis B.A. Wick M.R. Expression of erbB-2 oncoprotein in mammary and extramammary Paget's disease.Am J Clin Pathol. 1991; 96: 243-247PubMed Google Scholar). Early small-scale studies, however, that examined overexpression of erbB-2 protein in EMPD reported variable frequencies of positivity ranging from 0% to 43% (Keatings et al., 1990Keatings L. Sinclair J. Wright C. et al.erbB-2 oncoprotein expression in mammary and extramammary Paget's disease: an immunohistochemical study.Histopathology. 1990; 17: 243-247Crossref PubMed Scopus (71) Google Scholar;Meissner et al., 1990Meissner K. Riviere A. Haupt G. Loning T. Study of neu-protein expression in mammary Paget's disease with and without underlying breast carcinoma and in extramammary Paget's disease.Am J Pathol. 1990; 137: 1305-1309PubMed Google Scholar;Wolber et al., 1991Wolber R.A. Dupuis B.A. Wick M.R. Expression of erbB-2 oncoprotein in mammary and extramammary Paget's disease.Am J Clin Pathol. 1991; 96: 243-247PubMed Google Scholar;Nishi et al., 1994Nishi M. Yoshida H. Setoyama M. Tashiro M. Immunohistochemical study of erbB-2 oncoprotein expression in extramammary Paget's disease.Dermatology. 1994; 188: 100-102Crossref PubMed Scopus (16) Google Scholar;Nakamura et al., 1995Nakamura G. Shikata N. Shoji T. Hatano T. Hioki K. Tsubura A. Immunohistochemical study of mammary and extramammary Paget's disease.Anticancer Res. 1995; 15: 467-470PubMed Google Scholar;Takata et al., 1997Takata M. Hatta N. Takehara K. Tumour cells of extramammary Paget's disease do not show either p53 mutation or allelic loss at several selected loci implicated in other cancers.Br J Cancer. 1997; 76: 904-908Crossref PubMed Scopus (24) Google Scholar), and the role of this oncoprotein in the pathogenesis of EMPD was uncertain. This study clearly showed that erbB-2 overexpression was absent in in situ carcinomas but was common in invasive primary tumors, and suggests selection for erbB-2 overexpression at the invasive stage. This is in contrast to the results in breast cancer in which erbB-2 alterations are homogeneously detected in different stages of tumor progression from in situ carcinomas to metastases and are thought to be important for initiation rather than progression (Iglehart et al., 1990Iglehart J.D. Kraus M.H. Langton B.C. Huper G. Kerns B.J. Marks J.R. Increased erbB-2 gene copies and expression in multiple stages of breast cancer.Cancer Res. 1990; 50: 6701-6707PubMed Google Scholar). The variability of reported positivity rates of erbB-2 expression in EMPD is most likely explained by the difference of study population, as none of the previous studies recorded the type of EMPD tumors examined (i.e., in situ or invasive) (Keatings et al., 1990Keatings L. Sinclair J. Wright C. et al.erbB-2 oncoprotein expression in mammary and extramammary Paget's disease: an immunohistochemical study.Histopathology. 1990; 17: 243-247Crossref PubMed Scopus (71) Google Scholar;Meissner et al., 1990Meissner K. Riviere A. Haupt G. Loning T. Study of neu-protein expression in mammary Paget's disease with and without underlying breast carcinoma and in extramammary Paget's disease.Am J Pathol. 1990; 137: 1305-1309PubMed Google Scholar;Wolber et al., 1991Wolber R.A. Dupuis B.A. Wick M.R. Expression of erbB-2 oncoprotein in mammary and extramammary Paget's disease.Am J Clin Pathol. 1991; 96: 243-247PubMed Google Scholar;Nishi et al., 1994Nishi M. Yoshida H. Setoyama M. Tashiro M. Immunohistochemical study of erbB-2 oncoprotein expression in extramammary Paget's disease.Dermatology. 1994; 188: 100-102Crossref PubMed Scopus (16) Google Scholar;Nakamura et al., 1995Nakamura G. Shikata N. Shoji T. Hatano T. Hioki K. Tsubura A. Immunohistochemical study of mammary and extramammary Paget's disease.Anticancer Res. 1995; 15: 467-470PubMed Google Scholar). In other cancer systems such as breast, ovarian, and gastric cancers, erbB-2 gene amplification is the most common mechanism leading to high erbB-2 protein expression (Hynes and Stern, 1994Hynes N.E. Stern D.F. The biology of erbB-2/neu/HER-2 and its role in cancer.Biochim Biophys Acta. 1994; 1198: 165-184Crossref PubMed Scopus (977) Google Scholar). In gastric cancers, an absolute correlation between overexpression and the gene amplification was found, suggesting that the erbB-2 protein overexpression is tightly and principally controlled by gene amplification (Ishikawa et al., 1997Ishikawa T. Kobayashi M. Mai M. Suzuki T. Ooi A. Amplification of the erbB-2 (HER-2/neu) gene in gastric cancer cells. Detection by fluorescence in situ hybridization.Am J Pathol. 1997; 151: 761-768PubMed Google Scholar;Ooi et al., 1998Ooi A. Kobayashi M. Mai M. Nakanishi I. Amplification of c-erbB-2 in gastric cancer; detection in formalin- fixed, paraffin-embedded tissue by fluorescence in situ hybridization.Lab Invest. 1998; 78: 345-351PubMed Google Scholar). In this series of EMPD, however, only two of six tumors that showed strong membrane overexpression of erbB-2 protein had definite gene amplification. It was reported that some tumors that have elevated levels of erbB-2 RNA and protein have no gene amplification such as in breast cancer, suggesting that there are alternative transcriptional and post-transcriptional mechanisms controlling erbB-2 expression (Iglehart et al., 1990Iglehart J.D. Kraus M.H. Langton B.C. Huper G. Kerns B.J. Marks J.R. Increased erbB-2 gene copies and expression in multiple stages of breast cancer.Cancer Res. 1990; 50: 6701-6707PubMed Google Scholar). Furthermore, recent studies in breast cancer cell lines showed that erbB-2 expression was regulated at the transcriptional level rather than the translational level (Pasleau et al., 1993Pasleau F. Grooteclaes M. Gol-Winkler R. Expression of the c-erbB2 gene in the BT474 human mammary tumor cell line: measurement of c-erbB2 mRNA half-life.Oncogene. 1993; 8: 849-854PubMed Google Scholar), and that increased levels of the transcription factor AP-2 are responsible for erbB-2 overexpression without gene amplification (Bosher et al., 1995Bosher J.M. Williams T. Hurst H.C. The developmentally regulated transcription factor AP-2 is involved in c-erbB-2 overexpression in human mammary carcinoma.Proc Natl Acad Sci USA. 1995; 92: 744-749Crossref PubMed Scopus (207) Google Scholar). Thus, it seems reasonable to speculate that similar transcriptional activation of erbB-2 leading to the protein overexpression may also be involved in the pathogenesis of EMPD. It is generally agreed that amplification of proto-oncogenes including myc and erbB-2 occurs late in the progression of many human tumors. It is uncertain, however, whether or not the tumor cell clones with gene amplification are specifically selected for progression (Schwab and Amler, 1990Schwab M. Amler L.C. Amplification of cellular oncogenes: a predictor of clinical outcome in human cancer.Genes Chromosomes Cancer. 1990; 1: 181-193Crossref PubMed Scopus (176) Google Scholar;Brison, 1993Brison O. Gene amplification and tumor progression.Biochim Biophys Acta. 1993; 1155: 25-41PubMed Google Scholar). In this context, the observation in case 26 was interesting where the successive progression stages were clearly demonstrated from tumor cells with no erbB-2 expression to those overexpressing erbB-2 without gene amplification in the primary lesion, and finally to metastatic cells with definite erbB-2 gene amplification in the regional lymph node. This appears to indicate that erbB-2 overexpression and amplification, respectively, played a crucial part in each progression step. This observation is also keeping with several recent studies in breast cancer showing correlation between erbB-2 overexpression and random cell migration (Verbeek et al., 1998Verbeek B.S. Adriaansen-Slot S.S. Vroom T.M. Beckers T. Rijksen G. Overexpression of EGFR and c-erbB2 causes enhanced cell migration in human breast cancer cells and NIH3T3 fibroblasts.FEBS Lett. 1998; 425: 145-150Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), enhancement of metastatic potential by erbB-2 overexpression (Tan et al., 1997Tan M. Yao J. Yu D. Overexpression of the c-erbB-2 gene enhanced intrinsic metastasis potential in human breast cancer cells without increasing their transformation abilities.Cancer Res. 1997; 57: 1199-1205PubMed Google Scholar), and the demonstration of potentially metastatic cell subpopulations expressing erbB-2 within the individual cancer tissue (Roetger et al., 1998Roetger A. Merschjann A. Dittmar T. Jackisch C. Barnekow A. Brandt B. Selection of potentially metastatic subpopulations expressing c-erbB-2 from breast cancer tissue by use of an extravasation model.Am J Pathol. 1998; 153: 1797-1806Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Although the possibility remains that overexpression and/or amplification of erbB-2 occurred secondarily to other genetic changes such as loss of wild-type p53 that leads to genetic instability (Brison, 1993Brison O. Gene amplification and tumor progression.Biochim Biophys Acta. 1993; 1155: 25-41PubMed Google Scholar), direct sequencing of exons 5–8 of the p53 gene in both primary tumor and metastasis in this case did not reveal any mutation (data not shown). In contrast to the distinct membrane expression of erbB-2 in five cases, the remaining five invasive primary tumors predominantly showed cytoplasmic erbB-2 expression. Cytoplasmic staining with the CB11 monoclonal antibody used in this study was reported to reflect 150 kDa and 130 kDa proteins which could represent precursor forms of the erbB-2 oncoprotein (Corbett et al., 1990Corbett I.P. Henry J.A. Angus B. et al.NCL-CB11, a new monoclonal antibody recognizing the internal domain of the c-erbB-2 oncogene protein effective for use on formalin-fixed, paraffin-embedded tissue.J Pathol. 1990; 16: 15-25Crossref Scopus (115) Google Scholar). Interestingly, this study showed that the four of five tumors with cytoplasmic erbB-2 expression were associated with low-grade amplification of the erbB-2 gene, suggesting the possible link between the presence of additional erbB-2 and cytoplasmic accumulation of aberrant erbB-2 oncoprotein. Although the exact chromosomal localization of additional erbB-2 is unknown,Hara et al., 1998Hara T. Ooi A. Kobayashi M. Mai M. Yanagihara K. Nakanishi I. Amplification of c-myc, k-sam, and c-met in gastric cancers: Detection by fluorescence in situ hybridization.Lab Invest. 1998; 78: 1143-1153PubMed Google Scholar, who observed similar low-grade amplification of c-myc and c-met oncogenes in gastric cancer, speculated that the additional copies might represent translocated genes. Whether the observed low-grade erbB-2 amplification in EMPD also involves translocation and its relationship to the cytoplasmic erbB-2 expression are currently unknown. The biologic significance of cytoplasmic erbB-2 expression is also uncertain. In breast cancer, moderate to strong erbB-2 oncoprotein cytoplasmic positivity measured with CB-11 antibody was found to be associated with poor prognosis (Keshgegian and Cnaan, 1997Keshgegian A.A. Cnaan A. erbB-2 oncoprotein expression in breast carcinoma. Poor prognosis associated with high degree of cytoplasmic positivity using CB-11 antibody.Am J Clin Pathol. 1997; 108: 456-463PubMed Google Scholar). Tumor cells with this phenotype, however, may not be selected for metastatic progression in EMPD, because the corresponding lymph node metastases in cases 25 and 27 contained less or no tumor cells with cytoplasmic erbB-2 expression. Recent investigations have revealed an important role of the Wnt signaling pathway, one of the cell's key developmental and growth regulatory pathways, in cancer development (Pennisi, 1998Pennisi E. How a growth control path takes wrong turn to cancer.Science. 1998; 281: 1438-1441Crossref PubMed Scopus (83) Google Scholar). The protein β-catenin is a key downstream transcriptional activator of the Wnt signaling pathway, forming complexes with the DNA-binding protein Tcf and lef-1 which finally activate c-myc oncogene and induce cell proliferation (He et al., 1998He T.-C. Sparks A.B. Rago C. et al.Identification of c-MYC as a target of the APC pathway.Science. 1998; 281: 1509-1512Crossref PubMed Scopus (3913) Google Scholar). The APC tumor suppressor gene product regulates β-catenin levels by co-operating with GSK-3β via multiple serine/threonine residues encoded on exon 3 of the β-catenin gene. In colon cancers, disruption of the Wnt signaling pathway by mutation of either the APC or the β-catenin gene plays a crucial part in the early stage of tumorigenesis (Morin et al., 1997Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or.APC. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3375) Google Scholar). Mutations in the β-catenin gene have also been found in liver, prostate, and endometrial cancer as well as in melanomas and meduloblastomas (Pennisi, 1998Pennisi E. How a growth control path takes wrong turn to cancer.Science. 1998; 281: 1438-1441Crossref PubMed Scopus (83) Google Scholar). Our results, however, show that abnormal activation of β-catenin in Wnt signaling pathway is unlikely to be involved in EMPD. The authors are grateful to Yuko Yamada for excellent technical assistance in immunohistochemistry. This work was partly supported by the Hokkoku Cancer Research Fund.
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