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Loss of cytokeratin 14 expression is related to human papillomavirus type and lesion grade in squamous intraepithelial lesions of the cervix
Loss of Cytokeratin 14 Expression Is Related to Human Papillomavirus Type and Lesion Grade in Squamous Intraepithelial Lesions of the Cervix SHIRLEY A. SOUTHERN, PHD, IAN W. MCDICKEN, MD, MRCPATH, AND C. SIMON HERRINGTON, DPHIL, MRCP, MRCPATH In a recent study of low-grade cervical squamous intraepithelial lesions (SILs), we reported that infection with both low- and high-risk human papillomaviruses (HPVs) upregulated cyclin A, B, E, and Ki67 expression in basal and suprabasal cells. In view of the intricate link between cell cycle exit, proliferation, and differentiation, we examined the morphologic distribution of cytokeratins 13 and 14 and involucrin expression in 49 low-grade SILs infected with HPV types 6, 11, 16, 18, 31, 33, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66; 2 lesions contained both low- and high-risk HPVs. The findings were compared with 30 high-grade SILs infected with HPV types 16, 31, 33, 51, 58, 66, and 67; 3 of these were infected with 2 different HPVs. In low-grade lesions, the differentiation markers were expressed normally, showing that differentiation proceeds despite upregulation of cell cycle–associated proteins. Loss of involucrin (3 of 33) and cytokeratin 13 (8 of 33) expression occurred only in the high-grade lesions and was therefore related to lesion grade. Loss of cytokeratin
14 expression was also significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P < .01). In addition, cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low-grade SILs infected with highrisk HPVs than in those infected with low-risk HPVs (3 of 27 v 14 of 24; P < .001). These findings are consistent with in vitro data and suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation. HUM PATHOL 32:1351-1355. Copyright © 2001 by W.B. Saunders Company Key words: papillomavirus, cytokeratin, cervix, squamous intraepithelial lesion. Abbreviations: HPV, human papillomavirus; SIL, squamous intraepithelial lesion; AP1, activator protein 1; PCR, polymerase chain reaction; ISH, in situ hybridization; SSC, saline sodium citrate.
Human papillomaviruses (HPVs) are small epitheliotropic DNA viruses that are regarded as low- or highrisk types depending on their association with invasive carcinoma. Because of the small size of their genome (7.9kb), HPVs cannot encode essential proteins required for viral replication. Thus they have developed ways to hijack key host regulatory factors that control cellular replication. In a previous study of low-grade cervical squamous intraepithelial lesions (SILs),1 we reported that both low- and high-risk HPVs upregulate cyclin A, B, and E and Ki-67 expression in low-grade SILs. Cyclin D1 was overexpressed in almost all lesions infected with low-risk HPVs (92%) but absent in most lesions infected with high- risk HPVs (87%). The morphologic pattern of cyclin expression is consistent with the suggestion that upregulation/stabilization of cellular replication machinery has occurred to facilitate viral DNA replication.2 However, the HPV life cycle is also dependent on host cellular differentiation. There is in
vitro evidence that HPV E5 and E7 proteins indirectly alter the regulation of the activator protein 1 (AP1) transcription factor,3-5 which is required for transactivation of the keratin 14 promoter,6 and that inhibitors of AP1 activation block neoplastic transformation.7 The E7 oncoprotein has also been shown to uncouple cellular differentiation and proliferation in normal foreskin keratinocytes by abrogating p21cip1-mediated inhibition of cdk2, which interacts with cyclins A and E.8 Moreover, raft cultures transfected with HPV 16 show altered differentiation and acquisition of chromosome abnormalities on immortalization.9 However, organotypic culture studies are limited to a small number of HPV types, and the cultures undergo transformation at a higher rate than naturally occurring lesions. Therefore, these models may not accurately reflect events during in vivo infection. In this study, we examined the potential role of abnormalities of squamous differentiation in the development of intraepithelial lesions by comparing the morphologic distribution of cytokeratins 13 and 14 and involucrin in 49 naturally occurring low-grade SILs and 30 high-grade SILs infected with a variety of HPV types. The absence of cytokeratin 14 expression was associated with high-risk HPV infection and lesion grade, and dedifferentiation, identified by loss of cytokeratin 13 or involucrin expression, was confined to high-grade SILs. These findings are consistent with in vitro data and taken together with our previous data suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation.
From the Department of Pathology, University of Liverpool, Royal Liverpool University Hospital, Liverpool, England. Accepted for publication July 19, 2001. Supported by grants from Wellbeing and the Royal College of Obstetricians and Gynaecologists (H1/96) and the University of Liverpool. Address correspondence and reprint requests to C. Simon Herrington, Department of Pathology, University of Liverpool, Royal Liverpool University Hospital, Daulby St, Liverpool L69 3GA, England. Copyright © 2001 by W.B. Saunders Company 0046-8177/01/3212-0010$35.00/0 doi:10.1053/hupa.2001.29656
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MATERIALS AND METHODS Choice of Specimens
Statistical Analysis
Ten control normal cervical tissues from patients with no history of SIL and 49 low-grade SIL specimens were from a previously reported series that examined cyclin expression.1 Ten additional normal cervical biopsy specimens and 30 highgrade SILs were identified from the routine diagnostic files of the Department of Pathology of Royal Liverpool University Hospital, and diagnoses were confirmed by 2 gynecologic pathologists (C.S.H. and I.W.M.). Six-micrometer parallel formalin-fixed paraffin sections were cut for immunohistochemistry and HPV in situ hybridization, and 3 other sections were cut for HPV polymerase chain reaction (PCR) typing.
HPV Typing The GP5⫹/6⫹ HPV PCR system was used to amplify a 140 – to 150 – base pair (bp) fragment (depending on HPV type) in the L1 gene, as previously described.10 This was followed by hybridization with HPV type-specific 5⬘ digoxigenin–labeled oligonucleotide probes to HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66. Type specificity of these probes was verified using plasmid clones and sequenced PCR products. DNA quality was assessed using PCO3 and PCO5 primers, which amplify a 209-bp -globin fragment.
Distributions were compared using the 2 test with a significance level of P ⬍ .05
RESULTS HPV Typing The 209-bp -globin fragment was amplified, showing adequate DNA quality from all tissues. All normal cervical tissue controls were negative for HPV. Twentyseven low-grade SILs1 were infected with high-risk HPV types (HPV 16, n ⫽ 5; 18, n ⫽ 4; 31, n ⫽ 1; 33, n ⫽ 1; 39, n ⫽ 2; 51, n ⫽ 2; 52, n ⫽ 3; 56, n ⫽ 1; 58, n ⫽ 5; 66, n ⫽ 3), and 24 were infected with low-risk HPV types (HPV 6, n ⫽ 12; 11, n ⫽ 6; 42, n ⫽ 3; 43, n ⫽ 2; 44, n ⫽ 1). Double infections (included in the above figures) were identified in 2 specimens: 1 contained HPV 6 and 18, and the other HPV 18 and 31. All high-grade lesions contained high-risk HPV types (HPV 16, n ⫽ 21; 31, n ⫽ 6; 33, n ⫽ 2; 51, n ⫽ 1; 58, n ⫽ 1; 66, n ⫽ 1). Dual infections (included in the above figures) were identified in 3 specimens: 1 contained HPV 16 and 66, 1 contained HPV 16 and 31, and 1 contained HPV 31 and 51. HPV 67 was identified in 1 lesion by direct sequencing of the PCR product that did not hybridize with any of the probes.
HPV Localization
HPV Localization
In situ hybridization (ISH) was performed as previously described10 using a cocktail probe mixture containing digoxigenin-labeled genomic probes for HPV 6, 11, 16, 18, 31, and 33. These probes and hybridization conditions allow crosshybridization with many HPV types,11 thereby establishing viral localization. By altering the posthybridization wash to 50% formamide in 0.1⫻ saline sodium citrate (SSC) at 35°C, the stringency of ISH was increased to achieve specificity in the specimens containing dual infections as detected by PCR. Hela cells and tissues containing known HPV types were used as positive controls.
The use of a cocktail probe established morphologic localization of HPV infection. Further high-stringency in situ hybridization with individual probes was used to discriminate between the different HPV-infected areas in the dual infections. In 2 of the highgrade lesions containing dual infection, an HPV probe type 16 was used to discriminate between areas infected with HPV 16 and 66 and an HPV 31 probe was used for HPV 31 and 51 areas. In all other dual infections, specific probe types were available. All control tissues were negative for HPV. Therefore, morphologic localization of HPV was established in all cases, allowing accurate assessment of cytokeratin expression.
Immunohistochemistry Parallel 6-m formalin-fixed paraffin sections were dewaxed in xylene and rehydrated through graded alcohols. Microwave heat treatment in 0.01 mol/L citric acid buffer (pH 6.0) for 15 minutes on a high setting (800 W) was required for antigen retrieval of cytokeratin 14. No pretreatment was required for cytokeratin 13 and involucrin. Endogenous peroxidase was blocked using 1.5% (vol/vol) hydrogen peroxide in methanol for 10 minutes, followed by 5% normal goat serum for 20 minutes. Monoclonal antibodies (all from Novocastra, Newcastle, England) were then applied for 1 hour at room temperature: anti– cytokeratin 13 (clone DE-K13), 1:75; anti– cytokeratin 14 (clone LL002), 1:20; anti-involucrin (clone SY5), 1:100. The sections were then incubated in biotinylated sheep anti-mouse, followed by horseradish peroxidase– conjugated avidin– biotin complex (ABC-HRP; Dako, Ely, England). Signal was developed with diaminobenzidine and hydrogen peroxide (DAB/H2O2). Immunohistochemistry was assessed by evaluation of the morphologic distribution of positive cells within the basal/parabasal, intermediate, and superficial layers of the epithelium for each lesion.
Cytokeratin Expression In all 51 HPV-infected areas in the low-grade SILs, cytokeratin 13 and involucrin expression (Fig 1A, B) extended throughout the suprabasal layers which was similar to that present in the normal controls. Cytokeratin 14 expression varied, with lesions falling into 3 categories: type 1, cytokeratin expression in the basal, intermediate, and superficial layers of the epithelium; type 2, cytokeratin distribution as in type 1, but with focal areas where expression was restricted to basal and parabasal cells; and type 3, cytokeratin distribution as in types 1 or 2, but with areas of complete cytokeratin loss. In most low-grade SILs infected with low-risk HPVs, cytokeratin 14 expression was present in all epithelial layers (58% [14 of 24] type 1; Fig 1C), with fewer lesions showing expression confined to the basal/parabasal layers (29% [7 of 24] type 2) and focal loss of
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FIGURE 1. A low-grade SIL infected with HPV 6 shows retention of expression of (A) cytokeratin 13, (B) involucrin, and (C) cytokeratin 14. (D) A type 2 cytokeratin 14 expression pattern is present in a low-grade SIL infected with both HPV 6 (arrowhead) and HPV 18 (arrow). (E) Cytokeratin 13 expression is restricted to superficial cells in a high-grade SIL infected with HPV 16 and is associated with (F) loss of involucrin expression but (G) retention of cytokeratin 14. (H) Cytokeratin 14 expression is focally absent in a high-grade lesion containing HPV 16.
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FIGURE 2. Histogram showing the distribution of different cytokeratin 14 expression patterns in low-grade lesions infected with low-risk (LG-LR) and high-risk (LG-HR) HPV and high-grade lesions infected with high-risk (HG-HR) HPV. The cytokeratin 14 patterns are defined as follows: type 1, expression in the basal, intermediate, and superficial epithelial layers; type 2, as in type 1, but with focal areas where expression is restricted to basal and parabasal cells; type 3, as in type 1 or 2, but with areas of complete cytokeratin loss.
expression (13% [3 of 24] type 3). By contrast, cytokeratin 14 expression was present in all epithelial layers in only 11% (3 of 27 type 1) of low-grade SILs infected with high-risk HPVs. Most of these lesions showed cytokeratin 14 expression confined to the basal/parabasal layers (56% [15 of 27] type 2), with a number of lesions showing focal loss of expression (33% [9 of 27] type 3). Cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low grade SILs infected with high-risk HPVs than in those infected with low-risk HPVs (2 ⫽ 12.75; df ⫽ 1; P ⬍ .001; Figs 1D and 2). None of the normal controls showed cytokeratin 14 loss in either metaplastic or ectocervical squamous epithelium. In the high-grade SILs (Fig 1E through G), all of which were infected with high-risk HPVs, cytokeratin 13 and involucrin expression was present in the suprabasal epithelium in the majority of lesions. In 8 lesions (24%), cytokeratin 13 expression was lost from suprabasal keratinocytes; in 3 lesions (9%), involucrin expression was absent. Loss of cytokeratin 14 expression was significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P ⬍ .01). Compared with the low-grade SILs, fewer high-grade lesions showed cytokeratin 14 expression throughout the epithelium (18% [6 of 33] type 1) and confined to the basal/parabasal layers (24% [8 of 33] type 2). Moreover, loss of cytokeratin 14 expression (type 3) was observed in 19 (58%) of 33 lesions (Figs 1H and 2). Signal intensity was similar for all proteins. DISCUSSION There were 2 main findings in this study: (1) absence of cytokeratin 14 expression is associated with high-risk HPV infection and occurs more frequently in high-grade SILs; and (2) dedifferentiation, with loss of
cytokeratin 13 or involucrin expression, occurs only in high-grade lesions. Overall, disruption of squamous differentiation occurred more often in high-grade than in low-grade lesions and was associated with high-risk HPV infection. In most of the low-grade SILs, cytokeratin 14 expression was present throughout the full epithelial thickness, although expression was focally restricted to basal and parabasal cells in some. In the high-grade SILs, absence of cytokeratin 14 expression was significantly more frequent than in low-grade SILs and was associated with high-risk HPV infection. This is consistent with an in vitro study showing that down-regulation of cytokeratin 14 expression (messenger RNA [mRNA] and protein) occurred in cell lines naturally transformed by HPV 16, ie, Caski and SiHa cells and other cell lines derived from cervical squamous carcinomas. However, in HPV 16 –immortalized keratinocytes, downregulation of cytokeratin 14 expression occurred at a transcriptional level but protein synthesis remained normal. Transformation of these cells by cotransfection of HPV 16 and v-Ha-ras resulted in low levels or absence of both mRNA and protein.12 This suggests that loss/ reduction of cytokeratin 14 protein expression is associated with transformation but not immortalization. Similar findings have been reported using differential display methodology to analyze transformed and nontransformed HPV 16 keratinocyte cell lines.13 Cytokeratin 14 gene expression was downregulated in the transformed cell line only. Moreover, cells infected with SV40, also a member of the papovaviridae family, showed no significant changes in cytokeratin 14 expression in early passage but lacked expression at later passage.14 Downregulation is thought to occur through direct competition for AP2 binding sites by SV40; AP2 is involved in keratinocyte-specific expression of the cytokeratin 14 gene.15 These findings are consistent with
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our observation and with other in vivo studies; variable but low levels of cytokeratin 14 expression have been observed in squamous cell carcinomas of the skin. Moreover, the lowest levels of expression occurred in poorly differentiated tumors.16 The exact mechanism by which cytokeratin 14 expression is disrupted is unknown but may depend on the interplay between upregulation and downregulation of AP1 by HPV E5 and E7. AP1 transcription factor regulates cellular growth and differentiation. Overexpression of c-fos and c-jun, components of AP1, individually induces the cytokeratin 14 promoter 4-fold while synergistically a 9-fold upregulation occurs.6 HPV E5 mediates upregulation of AP1, and E7 mediates inhibition of c-jun. Conversely, AP1 has been described as the predominant determinant of HPV 31 enhancer activity, which directs E6/E7 expression.4 Therefore, a complex regulatory loop appears to exist between HPV and AP1. In a recent study of SILs, HPV E6/E7 mRNA and AP1 expression increased with lesion grade and colocalized throughout the epithelium in high-grade lesions.4 Downregulation of cytokeratin 14 was observed in the high-grade lesions in our study, suggesting that this effect is either (1) independent of AP1, possibly related to secondary genetic alteration, a hypothesis consistent with the observed heterogeneity of cytokeratin 14 expression; or (2) due to direct competition for AP1 binding sites by HPV proteins, similar to suggested mechanisms relating to SV40. In all of the low-grade-SILs and normal controls, uniform cytokeratin 13 and involucrin expression extended from parabasal to superficial layers, but in the high-grade SILs focal loss of cytokeratin 13 and involucrin was identified. Dedifferentiation was therefore confined to high-grade lesions but did not correspond to abnormalities of cytokeratin 14 expression, which appeared to occur independently. No relationship was observed between cytokeratin/involucrin expression patterns and previously reported cyclin expression.1 Overall, the changes identified in this study are in concordance with results of in vitro studies and provide further in vivo evidence that the ability of high-risk HPVs to induce abnormalities of squamous differentiation and cell cycle control, as previously demonstrated, are central to the biologic differences between low- and high-risk HPVs. Acknowledgment. The authors thank Dr E.-M. de Villiers (Deutches Krebsforschungszentrum, Heidelberg, Germany),
Dr G. Orth (Institut Pasteur, Paris, France), Dr Y. Matsukura (National Institute of Health, Tokyo, Japan), and A. T. Lorincz (DiGene Diagnostics, Gaithersburg, MD) for providing HPV plasmid clones.
REFERENCES 1. Southern AS, Herrington CS: Differential cell cycle regulation by low- and high-risk human papillomaviruses in low-grade squamous intraepithelial lesions of the cervix. Cancer Res 58:2941-2945, 1998 2. Chow LT, Broker TR: Papillomavirus DNA replication. Intervirology 37:150-158, 1994 3. Li JJ, Rhim JS, Schlegel R, et al: Expression of dominant negative Jun inhibits elevated AP-1 and NF-kappa B transactivation and suppresses anchorage independent growth of HPV immortalized human keratinocytes. Oncogene 16:2711-2721, 1998 4. Kyo S, Klumpp DJ, Inoue M, et al: Expression of AP1 during cellular differentiation determines human papillomavirus E6/E7 expression in stratified epithelial cells. J Gen Virol 78:401-411, 1997 5. Nead MA, Baglia LA, Ludlow MJ, et al: Rb binds c-Jun and activates transcription. EMBO J 17:2342-2352, 1998 6. Ma S, Rao L, Freedberg IM, et al: Transcriptional control of K5, K6, K14, and K17 keratin genes by AP-1 and NF-kB family members. Gene Exp 6:361-370, 1997 7. Li JJ, Westergaard C, Ghosh P, et al: Inhibitors of both nuclear factor-kappaB and activator protein-1 activation block the neoplastic transformation response. Cancer Res 57:3569-3576, 1997 8. Jones DL, Alani RD, Mu¨nger K: The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2. Genes Dev 11:2101-2111, 1997 9. McCance DJ, Kopan R, Fuchs E, et al: Human papillomavirus type 16 alters human epithelial cell differentiation in vitro. Proc Natl Acad Sci U S A 85:7169-7173, 1988 10. Southern SA, Herrington CS: Basal cell tetrasomy occurs in low-grade cervical squamous intraepithelial lesions infected with high-risk human papillomaviruses. Cancer Res 57:4210-4213, 1997 11. Herrington CS, Anderson SM, Graham AK, et al: The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction. Histochem J 25:191-198, 1993 12. Bowden PE, Woodworth CD, Doniger J, et al: Down-regulation of keratin 14 gene expression after v-Ha-ras transfection of human papillomavirus-immortalised human cervical epithelial cells. Cancer Res 52:5865-5871, 1992 13. Nees M, van Wijngaarden E, Bakos E, et al: Identification of novel molecular markers which correlate with HPV-induced tumor progression. Oncogene 16:2447-2458, 1998 14. Morris A, Steinberg ML, Defendi V: Keratin gene expression in simian virus 40-transformed human keratinocytes. Proc Natl Acad Sci U S A 82:8498-8502, 1985 15. Leask A, Byrne C, Fuchs E: Transcriptional factor AP2 and its role in epidermal-specific gene expression. Proc Natl Acad Sci U S A 88:7948-7952, 1991 16. Stoler A, Kopan R, Duvic M, et al: Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation. J Cell Biol 107:427-446, 1988
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14 expression was also significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P < .01). In addition, cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low-grade SILs infected with highrisk HPVs than in those infected with low-risk HPVs (3 of 27 v 14 of 24; P < .001). These findings are consistent with in vitro data and suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation. HUM PATHOL 32:1351-1355. Copyright © 2001 by W.B. Saunders Company Key words: papillomavirus, cytokeratin, cervix, squamous intraepithelial lesion. Abbreviations: HPV, human papillomavirus; SIL, squamous intraepithelial lesion; AP1, activator protein 1; PCR, polymerase chain reaction; ISH, in situ hybridization; SSC, saline sodium citrate.
Human papillomaviruses (HPVs) are small epitheliotropic DNA viruses that are regarded as low- or highrisk types depending on their association with invasive carcinoma. Because of the small size of their genome (7.9kb), HPVs cannot encode essential proteins required for viral replication. Thus they have developed ways to hijack key host regulatory factors that control cellular replication. In a previous study of low-grade cervical squamous intraepithelial lesions (SILs),1 we reported that both low- and high-risk HPVs upregulate cyclin A, B, and E and Ki-67 expression in low-grade SILs. Cyclin D1 was overexpressed in almost all lesions infected with low-risk HPVs (92%) but absent in most lesions infected with high- risk HPVs (87%). The morphologic pattern of cyclin expression is consistent with the suggestion that upregulation/stabilization of cellular replication machinery has occurred to facilitate viral DNA replication.2 However, the HPV life cycle is also dependent on host cellular differentiation. There is in
vitro evidence that HPV E5 and E7 proteins indirectly alter the regulation of the activator protein 1 (AP1) transcription factor,3-5 which is required for transactivation of the keratin 14 promoter,6 and that inhibitors of AP1 activation block neoplastic transformation.7 The E7 oncoprotein has also been shown to uncouple cellular differentiation and proliferation in normal foreskin keratinocytes by abrogating p21cip1-mediated inhibition of cdk2, which interacts with cyclins A and E.8 Moreover, raft cultures transfected with HPV 16 show altered differentiation and acquisition of chromosome abnormalities on immortalization.9 However, organotypic culture studies are limited to a small number of HPV types, and the cultures undergo transformation at a higher rate than naturally occurring lesions. Therefore, these models may not accurately reflect events during in vivo infection. In this study, we examined the potential role of abnormalities of squamous differentiation in the development of intraepithelial lesions by comparing the morphologic distribution of cytokeratins 13 and 14 and involucrin in 49 naturally occurring low-grade SILs and 30 high-grade SILs infected with a variety of HPV types. The absence of cytokeratin 14 expression was associated with high-risk HPV infection and lesion grade, and dedifferentiation, identified by loss of cytokeratin 13 or involucrin expression, was confined to high-grade SILs. These findings are consistent with in vitro data and taken together with our previous data suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation.
From the Department of Pathology, University of Liverpool, Royal Liverpool University Hospital, Liverpool, England. Accepted for publication July 19, 2001. Supported by grants from Wellbeing and the Royal College of Obstetricians and Gynaecologists (H1/96) and the University of Liverpool. Address correspondence and reprint requests to C. Simon Herrington, Department of Pathology, University of Liverpool, Royal Liverpool University Hospital, Daulby St, Liverpool L69 3GA, England. Copyright © 2001 by W.B. Saunders Company 0046-8177/01/3212-0010$35.00/0 doi:10.1053/hupa.2001.29656
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MATERIALS AND METHODS Choice of Specimens
Statistical Analysis
Ten control normal cervical tissues from patients with no history of SIL and 49 low-grade SIL specimens were from a previously reported series that examined cyclin expression.1 Ten additional normal cervical biopsy specimens and 30 highgrade SILs were identified from the routine diagnostic files of the Department of Pathology of Royal Liverpool University Hospital, and diagnoses were confirmed by 2 gynecologic pathologists (C.S.H. and I.W.M.). Six-micrometer parallel formalin-fixed paraffin sections were cut for immunohistochemistry and HPV in situ hybridization, and 3 other sections were cut for HPV polymerase chain reaction (PCR) typing.
HPV Typing The GP5⫹/6⫹ HPV PCR system was used to amplify a 140 – to 150 – base pair (bp) fragment (depending on HPV type) in the L1 gene, as previously described.10 This was followed by hybridization with HPV type-specific 5⬘ digoxigenin–labeled oligonucleotide probes to HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66. Type specificity of these probes was verified using plasmid clones and sequenced PCR products. DNA quality was assessed using PCO3 and PCO5 primers, which amplify a 209-bp -globin fragment.
Distributions were compared using the 2 test with a significance level of P ⬍ .05
RESULTS HPV Typing The 209-bp -globin fragment was amplified, showing adequate DNA quality from all tissues. All normal cervical tissue controls were negative for HPV. Twentyseven low-grade SILs1 were infected with high-risk HPV types (HPV 16, n ⫽ 5; 18, n ⫽ 4; 31, n ⫽ 1; 33, n ⫽ 1; 39, n ⫽ 2; 51, n ⫽ 2; 52, n ⫽ 3; 56, n ⫽ 1; 58, n ⫽ 5; 66, n ⫽ 3), and 24 were infected with low-risk HPV types (HPV 6, n ⫽ 12; 11, n ⫽ 6; 42, n ⫽ 3; 43, n ⫽ 2; 44, n ⫽ 1). Double infections (included in the above figures) were identified in 2 specimens: 1 contained HPV 6 and 18, and the other HPV 18 and 31. All high-grade lesions contained high-risk HPV types (HPV 16, n ⫽ 21; 31, n ⫽ 6; 33, n ⫽ 2; 51, n ⫽ 1; 58, n ⫽ 1; 66, n ⫽ 1). Dual infections (included in the above figures) were identified in 3 specimens: 1 contained HPV 16 and 66, 1 contained HPV 16 and 31, and 1 contained HPV 31 and 51. HPV 67 was identified in 1 lesion by direct sequencing of the PCR product that did not hybridize with any of the probes.
HPV Localization
HPV Localization
In situ hybridization (ISH) was performed as previously described10 using a cocktail probe mixture containing digoxigenin-labeled genomic probes for HPV 6, 11, 16, 18, 31, and 33. These probes and hybridization conditions allow crosshybridization with many HPV types,11 thereby establishing viral localization. By altering the posthybridization wash to 50% formamide in 0.1⫻ saline sodium citrate (SSC) at 35°C, the stringency of ISH was increased to achieve specificity in the specimens containing dual infections as detected by PCR. Hela cells and tissues containing known HPV types were used as positive controls.
The use of a cocktail probe established morphologic localization of HPV infection. Further high-stringency in situ hybridization with individual probes was used to discriminate between the different HPV-infected areas in the dual infections. In 2 of the highgrade lesions containing dual infection, an HPV probe type 16 was used to discriminate between areas infected with HPV 16 and 66 and an HPV 31 probe was used for HPV 31 and 51 areas. In all other dual infections, specific probe types were available. All control tissues were negative for HPV. Therefore, morphologic localization of HPV was established in all cases, allowing accurate assessment of cytokeratin expression.
Immunohistochemistry Parallel 6-m formalin-fixed paraffin sections were dewaxed in xylene and rehydrated through graded alcohols. Microwave heat treatment in 0.01 mol/L citric acid buffer (pH 6.0) for 15 minutes on a high setting (800 W) was required for antigen retrieval of cytokeratin 14. No pretreatment was required for cytokeratin 13 and involucrin. Endogenous peroxidase was blocked using 1.5% (vol/vol) hydrogen peroxide in methanol for 10 minutes, followed by 5% normal goat serum for 20 minutes. Monoclonal antibodies (all from Novocastra, Newcastle, England) were then applied for 1 hour at room temperature: anti– cytokeratin 13 (clone DE-K13), 1:75; anti– cytokeratin 14 (clone LL002), 1:20; anti-involucrin (clone SY5), 1:100. The sections were then incubated in biotinylated sheep anti-mouse, followed by horseradish peroxidase– conjugated avidin– biotin complex (ABC-HRP; Dako, Ely, England). Signal was developed with diaminobenzidine and hydrogen peroxide (DAB/H2O2). Immunohistochemistry was assessed by evaluation of the morphologic distribution of positive cells within the basal/parabasal, intermediate, and superficial layers of the epithelium for each lesion.
Cytokeratin Expression In all 51 HPV-infected areas in the low-grade SILs, cytokeratin 13 and involucrin expression (Fig 1A, B) extended throughout the suprabasal layers which was similar to that present in the normal controls. Cytokeratin 14 expression varied, with lesions falling into 3 categories: type 1, cytokeratin expression in the basal, intermediate, and superficial layers of the epithelium; type 2, cytokeratin distribution as in type 1, but with focal areas where expression was restricted to basal and parabasal cells; and type 3, cytokeratin distribution as in types 1 or 2, but with areas of complete cytokeratin loss. In most low-grade SILs infected with low-risk HPVs, cytokeratin 14 expression was present in all epithelial layers (58% [14 of 24] type 1; Fig 1C), with fewer lesions showing expression confined to the basal/parabasal layers (29% [7 of 24] type 2) and focal loss of
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FIGURE 1. A low-grade SIL infected with HPV 6 shows retention of expression of (A) cytokeratin 13, (B) involucrin, and (C) cytokeratin 14. (D) A type 2 cytokeratin 14 expression pattern is present in a low-grade SIL infected with both HPV 6 (arrowhead) and HPV 18 (arrow). (E) Cytokeratin 13 expression is restricted to superficial cells in a high-grade SIL infected with HPV 16 and is associated with (F) loss of involucrin expression but (G) retention of cytokeratin 14. (H) Cytokeratin 14 expression is focally absent in a high-grade lesion containing HPV 16.
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Volume 32, No. 12 (December 2001)
FIGURE 2. Histogram showing the distribution of different cytokeratin 14 expression patterns in low-grade lesions infected with low-risk (LG-LR) and high-risk (LG-HR) HPV and high-grade lesions infected with high-risk (HG-HR) HPV. The cytokeratin 14 patterns are defined as follows: type 1, expression in the basal, intermediate, and superficial epithelial layers; type 2, as in type 1, but with focal areas where expression is restricted to basal and parabasal cells; type 3, as in type 1 or 2, but with areas of complete cytokeratin loss.
expression (13% [3 of 24] type 3). By contrast, cytokeratin 14 expression was present in all epithelial layers in only 11% (3 of 27 type 1) of low-grade SILs infected with high-risk HPVs. Most of these lesions showed cytokeratin 14 expression confined to the basal/parabasal layers (56% [15 of 27] type 2), with a number of lesions showing focal loss of expression (33% [9 of 27] type 3). Cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low grade SILs infected with high-risk HPVs than in those infected with low-risk HPVs (2 ⫽ 12.75; df ⫽ 1; P ⬍ .001; Figs 1D and 2). None of the normal controls showed cytokeratin 14 loss in either metaplastic or ectocervical squamous epithelium. In the high-grade SILs (Fig 1E through G), all of which were infected with high-risk HPVs, cytokeratin 13 and involucrin expression was present in the suprabasal epithelium in the majority of lesions. In 8 lesions (24%), cytokeratin 13 expression was lost from suprabasal keratinocytes; in 3 lesions (9%), involucrin expression was absent. Loss of cytokeratin 14 expression was significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P ⬍ .01). Compared with the low-grade SILs, fewer high-grade lesions showed cytokeratin 14 expression throughout the epithelium (18% [6 of 33] type 1) and confined to the basal/parabasal layers (24% [8 of 33] type 2). Moreover, loss of cytokeratin 14 expression (type 3) was observed in 19 (58%) of 33 lesions (Figs 1H and 2). Signal intensity was similar for all proteins. DISCUSSION There were 2 main findings in this study: (1) absence of cytokeratin 14 expression is associated with high-risk HPV infection and occurs more frequently in high-grade SILs; and (2) dedifferentiation, with loss of
cytokeratin 13 or involucrin expression, occurs only in high-grade lesions. Overall, disruption of squamous differentiation occurred more often in high-grade than in low-grade lesions and was associated with high-risk HPV infection. In most of the low-grade SILs, cytokeratin 14 expression was present throughout the full epithelial thickness, although expression was focally restricted to basal and parabasal cells in some. In the high-grade SILs, absence of cytokeratin 14 expression was significantly more frequent than in low-grade SILs and was associated with high-risk HPV infection. This is consistent with an in vitro study showing that down-regulation of cytokeratin 14 expression (messenger RNA [mRNA] and protein) occurred in cell lines naturally transformed by HPV 16, ie, Caski and SiHa cells and other cell lines derived from cervical squamous carcinomas. However, in HPV 16 –immortalized keratinocytes, downregulation of cytokeratin 14 expression occurred at a transcriptional level but protein synthesis remained normal. Transformation of these cells by cotransfection of HPV 16 and v-Ha-ras resulted in low levels or absence of both mRNA and protein.12 This suggests that loss/ reduction of cytokeratin 14 protein expression is associated with transformation but not immortalization. Similar findings have been reported using differential display methodology to analyze transformed and nontransformed HPV 16 keratinocyte cell lines.13 Cytokeratin 14 gene expression was downregulated in the transformed cell line only. Moreover, cells infected with SV40, also a member of the papovaviridae family, showed no significant changes in cytokeratin 14 expression in early passage but lacked expression at later passage.14 Downregulation is thought to occur through direct competition for AP2 binding sites by SV40; AP2 is involved in keratinocyte-specific expression of the cytokeratin 14 gene.15 These findings are consistent with
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CYTOKERATIN 14 IN SILs (Southern et al)
our observation and with other in vivo studies; variable but low levels of cytokeratin 14 expression have been observed in squamous cell carcinomas of the skin. Moreover, the lowest levels of expression occurred in poorly differentiated tumors.16 The exact mechanism by which cytokeratin 14 expression is disrupted is unknown but may depend on the interplay between upregulation and downregulation of AP1 by HPV E5 and E7. AP1 transcription factor regulates cellular growth and differentiation. Overexpression of c-fos and c-jun, components of AP1, individually induces the cytokeratin 14 promoter 4-fold while synergistically a 9-fold upregulation occurs.6 HPV E5 mediates upregulation of AP1, and E7 mediates inhibition of c-jun. Conversely, AP1 has been described as the predominant determinant of HPV 31 enhancer activity, which directs E6/E7 expression.4 Therefore, a complex regulatory loop appears to exist between HPV and AP1. In a recent study of SILs, HPV E6/E7 mRNA and AP1 expression increased with lesion grade and colocalized throughout the epithelium in high-grade lesions.4 Downregulation of cytokeratin 14 was observed in the high-grade lesions in our study, suggesting that this effect is either (1) independent of AP1, possibly related to secondary genetic alteration, a hypothesis consistent with the observed heterogeneity of cytokeratin 14 expression; or (2) due to direct competition for AP1 binding sites by HPV proteins, similar to suggested mechanisms relating to SV40. In all of the low-grade-SILs and normal controls, uniform cytokeratin 13 and involucrin expression extended from parabasal to superficial layers, but in the high-grade SILs focal loss of cytokeratin 13 and involucrin was identified. Dedifferentiation was therefore confined to high-grade lesions but did not correspond to abnormalities of cytokeratin 14 expression, which appeared to occur independently. No relationship was observed between cytokeratin/involucrin expression patterns and previously reported cyclin expression.1 Overall, the changes identified in this study are in concordance with results of in vitro studies and provide further in vivo evidence that the ability of high-risk HPVs to induce abnormalities of squamous differentiation and cell cycle control, as previously demonstrated, are central to the biologic differences between low- and high-risk HPVs. Acknowledgment. The authors thank Dr E.-M. de Villiers (Deutches Krebsforschungszentrum, Heidelberg, Germany),
Dr G. Orth (Institut Pasteur, Paris, France), Dr Y. Matsukura (National Institute of Health, Tokyo, Japan), and A. T. Lorincz (DiGene Diagnostics, Gaithersburg, MD) for providing HPV plasmid clones.
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