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p16INK4A expression in cervical premalignant and malignant lesions
Experimental and Molecular Pathology 80 (2006) 192 – 196 www.elsevier.com/locate/yexmp
p16INK4A expression in cervical premalignant and malignant lesions Ana Paula Franco Lambert a,*, Fernando Anschau b, Virgı´nia Minghelli Schmitt a,c a
Laborato´rio de Biologia Molecular do Instituto de Pesquisas Biome´dicas, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, Rua Prof. Cristiano Fischer 1118/201, Jardim do Salso, Porto Alegre/RS, CEP 91410-000, Brazil b Servic¸o de Ginecologia e Obstetrı´cia do HSL, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, Porto Alegre, Brazil c Faculdade de Farma´cia, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, Porto Alegre, Brazil Received 1 August 2005 Available online 28 October 2005
Abstract p16INK4a is a cyclin-dependent kinase (CDK) inhibitor which decelerates cell cycle by inactivating CDKs that phosphorylate pRb. Human Papillomavirus persistent infection plays an important role on cervical carcinogenesis, mainly by the action of two viral oncoproteins, E6 and E7, which interact with p53 and pRb, respectively. Increasing expression of E6 and E7 in dysplastic cervical cells might thus be reflected by increased expression of p16INK4a. Recent studies revealed that p16INK4a expression could be a marker for dysplastic and neoplastic cervical cells. The aim of this study was to analyze p16INK4a expression in cervical preneoplastic and neoplastic lesions and correlate with lesion grade. Expression of p16INK4a was analyzed by immunohistochemistry. A total of 6 low-grade squamous intraepithelial lesion (LSIL), 21 high-grade squamous intraepithelial lesions (HSIL) and 27 cancer samples were studied. In HPV-positive cervical samples (n = 48), p16INK4a expression was observed in 1 of 3 LSIL, in 18 of 19 HSIL and in all 26 cancer cases. These results are in accordance with the hypothesis that functional inactivation of pRb by HPV-E7 protein induces p16INK4a expression in cervical lesions. In our study, a statistically significant association was observed between cervical lesion grade and p16INK4a expression (P < 0.001). D 2005 Elsevier Inc. All rights reserved. Keywords: HPV; p16INK4a; Cervical cancer; LSIL; HSIL; Immunohistochemistry
Introduction Cervical cancer is one of the most common cancers in women worldwide, with about 510,000 new cases and nearly 288,000 deaths each year. Many studies have shown that human Papillomavirus (HPV) persistent infection plays an important role in cervical carcinogenesis (Berek and Novak, 1998; DiSaia and Creasman, 1997; Jay and Moscicki, 2000; Sano et al., 1998). In fact, HPV infection has been detected in almost all preneoplastic (high-grade squamous intraepithelial lesions – HSIL – and low-grade squamous intraepithelial lesions – LSIL) and neoplastic lesions of the cervix. Recent extensive studies have revealed the existence of more than 80 types of HPV, classified into two categories, high and low oncogenic risk, according to the frequency they appear associated with cervical cancer (Berek and Novak, 1998; Sano * Corresponding author. Fax: +55 51 33341634. E-mail addresses: [email protected], [email protected], [email protected] (A.P.F. Lambert). 0014-4800/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2005.08.005
et al., 1998; Murakami et al., 1999). High-risk HPV types (HRHPV), particularly HPV type 16 and 18, have been identified in the majority of cervical cancer (Walboomers et al., 1999). HPV contributes to neoplastic progression through the action of predominantly two viral oncoproteins, namely, E6 and E7, which interact with various cell cycle regulatory proteins (Doeberitz, 2002; Waggoner, 2003). The E6 oncoprotein induces premature degradation of the p53 tumor suppressor protein, via the ubiquitin – proteasome pathway. The E7 oncoprotein binds to the tumor suppressor protein pRb, which normally inhibits progression of cell cycle into the S phase by complexing the E2F-like transcription factors. Progressive and prolonged phosphorylation of the Rb protein, performed by the cyclin/cyclin-dependent kinase (CDK) complexes, leads to its inactivation with consequently reduction of its growth suppressive activity. HPV E7 binding to pRb favors the release of E2F-like transcription factors, activating the transcription of Rb-regulated genes, which consequently promotes G1/S phase progression of the cell cycle (Doeberitz, 2002; Klaes et al., 2001; Murphy et al., 2003).
A.P.F. Lambert et al. / Experimental and Molecular Pathology 80 (2006) 192 – 196
In normal cells, the activity of CDKs is tightly regulated by several CDK inhibitors, including p16INK4a. This CDK inhibitor is a tumor suppressor protein that inhibits CDK4 and CDK6, which phosphorylate the Rb protein. A reciprocal relation between p16INK4a and pRb expression has been observed, suggesting the presence of a negative feedback loop allowing pRb to limit the concentration of p16INK4a. p16INK4a is inactivated in many cancers by mutation, deletion of the gene or hypermethylation of its promoter, resulting in reduced or absent expression of the p16INK4a gene product (Klaes et al., 2001; Murphy et al., 2003; Piccinin et al., 1997; Zhao et al., 2003; Zhou et al., 2002; Langosch et al., 2001). In the case of HPV-positive cervical cancer, it has been demonstrated an overexpression of p16INK4a as a result of functional inactivation of pRb by the HPV E7 protein. Doeberitz proposed that the loss of pRb function should result in the release of the p16INK4a gene from negative transcriptional feedback control. As a consequence of the downstream block of pRb by HPV E7, the functional effect of p16INK4a overexpression is not observed (Doeberitz, 2002). Due to the overexpression of p16INK4a in HPV-positive cervical cancers, a possible potential of p16INK4a as a marker for cervical intraepithelial lesions and cervical cancer was suggested (Sano et al., 1998; Murphy et al., 2003; Langosch et al., 2001). In the present work, we report the analysis of p16INK4a expression in cervical preneoplastic and neoplastic lesions and compare p16 INK4a expression levels with lesion grade.
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peroxidase conjugate (Novostain Universal Detection Kit) for 10 min. Slides were developed with diaminobenzidine tetrahydrochloride (DAB) for 10 min and counterstained lightly with hematoxylin.
Interpretation of p16
INK4A
staining
Immunostaining of the formalin-fixed, paraffin-embedded sections was reviewed tree times to confirm the results, and strong nuclear as well as cytoplasmic staining was considered a positive reaction. The distribution of p16INK4a positive was scored on a semiquantitative scale as follows: ( ) none, (+) less than 5% of the cells, (++) 5 – 50% of the cells and (+++) more than 50% of the cells stained for p16INK4a (Skomedal et al., 1999).
DNA extraction and HPV detection DNA from cervical samples was extracted according to proteinase K standard protocol (Sambrook et al., 1999). For HPV detection, a polymerase chain reaction using consensus degenerate primers My09 and My11 was performed as described elsewhere (Bauer and Manos, 1993). DNA viability of the negative samples was checked by a PCR with primers complementary to the human betaglobin gene (Bauer and Manos, 1993). PCR products were electrophoresed on a 2% agarose gel containing 0.5 Ag/ml ethidium bromide in TAE buffer (1) and analyzed under UV light. All HPV-positive samples were tested by a typespecific PCR for HPV-16, -18, -31 and -33, the most frequent HR-HPVs. PCR conditions and primers used are described in Bauer and Manos (1993). Samples positive for HPV but negative for the tested HPV types were referred as ‘‘not identified HPV type’’ (NI).
Statistical analysis Statistical analysis was performed using the Chi-square test (Microsoft Office Excel, version 97). A P value of 0.05 or less was considered statistically significant (95% confidence).
Material and methods Paraffin-embedded cervical samples from 54 women with histological results for LSIL, HSIL or cancer were analyzed for p16INK4a expression. Cervical samples were obtained from subjects with colposcopic referral to biopsy of the uterine cervix attending the Gynecology Service of Sa˜o Lucas Hospital – PUCRS, from December 1999 to December 2001, with a complete medical history report. This study was approved by the local ethical committee (Protocol no. 288/03-CEP-PUCRS).
Immunohistochemistry Novostain Universal Detection Kit reagents (Novocastra Laboratories Ltd., Benton Lane, Newcastle upon Tyne, UK) were used for immunohistochemistry. Cervical biopsy samples were selected from pathology files of Sa˜o Lucas Hospital – PUCRS. All samples were fixed in formalin and embedded in paraffin by conventional techniques. Hematoxylin and eosin stained slides of all samples were classified by a certified pathologist. Serial sections (5 Am thick) of formalin-fixed and paraffin-embedded biopsy samples were cut and mounted on Novobond (Novocastra Laboratories Ltd.) slide tissue adhesive-coated glass slides. Sections were dewaxed by passage through xylene and then rehydrated in graded alcohol (100%, 90%, 80% and 70%). Endogenous peroxidase activity was blocked by incubating the section in 3% H2O2 for 10 min. Slides were then rehydrated with 0.01 M citrate buffer (pH 6) and microwaved 3 times, without boiling, for antigen retrieval. After rinsing with 0.01 mol/L phosphate-buffered saline (PBS), pH 7.4, nonspecific antibody binding was reduced by incubating the sections with prediluted blocking serum (normal horse serum) for 10 min. After decanting excess serum, sections were incubated overnight at 4-C with a mouse antihuman p16INK4A monoclonal antibody (Novocastra Laboratories Ltd.) at a 1:150 dilution in PBS. After washing thoroughly with PBS, sections were incubated with biotinylated universal secondary antibody (Novostain Universal Detection Kit) for 10 min, followed by incubation with Ready-to-use streptavidin –
Results A total of 54 samples from patients with cervical lesions were analyzed for p16 expression consisting of 6 cases of LSIL (11.1%), 21 of HSIL (38.9%) and 27 (50%) of cancer (23 cervical squamous cell carcinoma and 4 adenocarcinoma). Patients’ age ranged from 17 to 59 (40.9 T 10.86) years. Mean age of patients with LSIL was 30.3 (T12.48), HSIL was 38.2 (T10.41), and cancer was 45.4 (T8.66). In all p16-positive samples, both nuclear and cytoplasmic staining were observed. Relation of patient’s HPV status with lesion grade is presented in Fig. 1. HPV types detected in cases of multiple infection (n = 15) were 7 cases of coinfection of HPV-16 and -31 (1 LSIL, 3 HSIL and 3 cancer); 4 of HPV-16, -18 and -31 (1 HSIL and 3 cancer); 2 of HPV-16 and -18 (1 HSIL and 1 cancer); 1 of HPV-18 and -31 (cancer); 1 of HPV-16, -18 and -33 (cancer). Results of p16 expression and HPV status according to lesion grade are summarized in Table 1. Among HPV-16-positive patients, 1 was negative, and 7 were positive (1++ and 6+++) for p16 expression. Among HPV-18-positive patients, 4 were positive (+++) for p16. Five HPV-31-positive patients were positive (2+ and 3+++) for p16. In cases with multiple HPV types (n = 15), p16 expression was detected in 14 patients (1+, 4++, 9+++). In the group of patients with non-identified HPV type (n = 16), 15 were positive (2+, 2++, 11+++) for p16 expression.
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Fig. 2. Distribution of different lesion grades according to p16 INK4a expression level. LSIL—low-grade squamous intraepithelial lesions, HSIL—high-grade squamous intraepithelial lesions. v 2 = 39.74; P = 0.00000051; CI 95%.
Fig. 1. Frequency of HPV types in different lesion grades. NI*—not identified HPV type; **Infection with multiple HPV types; LSIL—low-grade squamous intraepithelial lesions; HSIL—high-grade squamous intraepithelial lesions.
Analyzing levels of p16 expression in different lesion grades, it was observed that 96.3% of cancer cases presented p16 +++ expression. On the other hand, the majority of p16 +++ expression (74.3%) corresponded to cancer cases. A statistically significant association was observed between lesion grade and p16 expression (v 2 = 39.74, P < 0.001, CI 95%) (Fig. 2). Discussion According to the Brazilian National Institute of Cancer (Instituto Nacional do Caˆncer—INCA), women most frequently develop cervical cancer at the age of 45 to 49 years (INCA, 2005). In North America, the mean age at cervical cancer diagnosis is 47 years, and nearly half of the cases are diagnosed before the age of 35 (Waggoner, 2003). The mean age of women with SIL is 15.6 years younger than the one of women with invasive cancer, suggesting a slow progression of SIL to invasive cancer (DeVita et al., 2001). Our study showed a mean age of 30.3 years for patients with LSIL, 38.2 years for HSIL and 45.4 years for cancer. The difference between mean age of cancer and LSIL was 15.1 years, similar to North American data, suggesting the same slow progression to cancer. Table 1 Distribution of p16 expression level according to lesion grade and HPV status p16 expression Negative
+
++
+++
HPV
LSIL (n = 6) HSIL (n = 21) Cancer (n = 27)
Neg
Pos
Neg
Pos
Neg
Pos
Neg
Pos
1 0 0
2 1 0
0 0 0
0 5 0
1 2 0
1 5 1
1 0 1
0 8 25
LSIL—low-grade squamous intraepithelial lesions; HSIL—high-grade squamous intraepithelial lesions.
HPV-16 is the most prevalent type worldwide. It is frequently detected in SIL and cancer cases. In our study, HPV-16 was the most frequent HPV type, representing 45.8% of all HPV infections, including single or multiple type infections. Among HPV-16-positive samples, 59.1% corresponded to cancer cases. HPV-18 is considered the most aggressive HPV type and may be a negative prognostic factor (DiSaia and Creasman, 1997). HPV-18 has been associated with poorly differentiated carcinomas and with a high rate of disease recurrence (DeVita et al., 2001). In our study, all 4 HPV-18 single infection cases were cancer, but when single and multiple type infections including HPV-18 (n = 12) were considered, 83.3% corresponded to cancer cases. According to Schellekens et al., in cases of infections with multiple HPV types, it is difficult to determine which HPV type(s) is responsible for the development of cancer, but it also cannot be excluded that only one type was the responsible, while the other(s) merely represent HPV infection with no involvement in cancer development (Schellekens et al., 2004). In our study, the majority of multiple HPV type infections were cancer cases (9/15), followed by HSIL (5/15) and LSIL (1/15). Among HPV-positive cases with non-identified HPV types (n = 16), most of them were HSIL (n = 8) and cancer (n = 7), which could suggest high-risk HPV types, different from the tested ones (16, 18, 31, 33), were involved. The pRb and p53 pathways of the cell cycle regulatory cascade are central to the regulation of the G1 to S phase transition and to the understanding of human cancer (Alberts et al., 1999). HPV infection is critically involved in cervical carcinogenesis and plays an important role in this regulatory cascade by the binding of its oncoproteins E6 and E7 to the tumor suppressor genes p53 and pRb, respectively (Schellekens et al., 2004). According to Walboomers et al., 99.7% of all cervical cancer are HPV-positive suggesting that HPV is a necessary but not sufficient cause of cervical cancer (Walboomers et al., 1999). In our study, only one cancer case, out of a total of 27, was negative for HPV. p16INK4a plays an important role in the regulation of cell cycle. CDK4/6 form complexes with cyclin D, which regulates the progression of the cell cycle. pRb binds to E2F transcription factors and suppresses their role in transcription during the G1 phase. pRb is inactivated and dissociated from
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the pRb-E2F complex after phosphorylation by the cyclin DCDK4/6 complex during G1 phase. The activity of cyclin DCDK4/6 is negatively regulated by inhibitors of the cyclin/ CDK complex, such as p16INK4a, which bind directly to CDK4/ 6 or the cyclin D-CDK4/6 complexes. Therefore, lack of functional p16INK4a results in abnormal cell cycle progression, which is associated with carcinogenesis (Bringold and Serrano, 2000; Masumoto et al., 2003; Serrano, 1997). On the other hand, overexpression of p16INK4a can result from inactivation of pRb, and transcriptional activity of p16INK4a seems to be repressed by pRb. The overexpression of p16INK4a in cervical neoplasia has been reported to be associated with HPV, and an inverse correlation has been found between pRb function and overexpression of p16INK4a. In fact, HPV E7 protein is responsible for an increase in p16INK4a level (Doeberitz, 2002; Masumoto et al., 2003). It was reported that p16INK4a mRNA was strongly induced by E2F, and accumulation of E2F might induce the overexpression of p16INK4a (Masumoto et al., 2003). According to these hypothesis, dysfunction of pRb through HPV E7 protein association should increase free E2F, resulting in both p16INK4a overexpression and abnormal cell cycle progression (Cho et al., 2002). Our results are in accordance with this hypothesis showing expression of p16INK4a in 93.75% of HPV-positive samples (45/48). Although 5 cervical lesions negative for HPV were positive for p16INK4a expression (2 LSIL, 2 HSIL and 1 cancer). As mentioned earlier in this paper, overexpression of p16INK4a could result from pRb inactivation. So, we could conjecture that in these HPV-negative cervical lesions, p16INK4a expression was a result of pRb inactivation by means of mechanisms independent from HPV infection. In a study conducted by Tsuda et al., mutation and deletion of p16 in cervical neoplasm were rare events, and methylation of the p16 was thought to be one of the important mechanisms of p16 loss. They also suggest that there may exist an alternative translational mechanism, or posttranslational degradative mechanism, for the loss of p16 protein expression (Tsuda et al., 2003). Our work showed negative p16 expression in 2 LSIL and 1 HSIL HPV-positive cases. One of the mechanisms suggested by Tsuda et al. could be the responsible for these results. Klaes et al. demonstrated that p16-specific immunohistochemical staining allows the sensitive and specific identification of dysplastic cervical cells in tissue sections or cervical smear (Klaes et al., 2001). Murphy et al. suggest that p16INK4a marks dysplastic squamous and glandular cells of the cervix with a sensitivity of 99.9% and specificity of 100% (Murphy et al., 2003). In our study, p16INK4a expression was observed in precursor lesions, with the higher expression in cervical cancer samples. A statistically significant association was observed between lesion grade and p16 expression (v 2 = 39.74, P < 0.001, CI 95%). A study conducted by Sano et al. concluded that p16INK4a is overexpressed in most cervical lesions, and the status of its immunoreactivity allows differentiation between infection with low-risk HPV and high-risk HPV (Sano et al., 2002). In our study, among HPV-positive samples, p16INK4a expression was
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observed in 1 LSIL (n = 3), 18 HSIL (n = 19) and in all cancer cases (n = 26) (Table 1). These results are in accordance with the hypothesis that functional inactivation of pRb by HPV-E7 protein induces p16INK4a expression in cervical lesions (Langosch et al., 2001). In conclusion, a good correlation was observed between cervical lesion grade and p16INK4a expression. Despite the reduced number of sample analyzed, our results indicate that p16INK4a expression would be a good marker for cervical dysplastic lesions and cancer, but further studies are needed to reinforce this statement. Acknowledgments We thank Dr. Emilio Antoˆnio Jeckel Neto, Dr. Denise Cantarelli Machado and Dr. Vinicius Duval da Silva for scientific support; Vinı´cius S. Michaelsen and Raquel Mattos de Oliveira for technical assistance. Ana Paula Franco Lambert was a fellow of CAPES. References Alberts, B., Bray, D., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., 1999. Fundamentos de Biologia Celular. Uma Introduc¸a˜o a Biologia Molecular da Ce´lula. Editora Artes Me´dicas. Porto Alegre. Bauer, H.M., Manos, M.M., 1993. PCR detection of genital human Papillomavirus. In: Persing, D. (Ed.), Diagnostic Molecular Microbiology. American Society for Microbiology, pp. 407 – 413. Berek, J.S., 1998. Tratado de Ginecologia. 12a edic¸a˜o.: Guanabara Koogam, Rio de Janeiro. Bringold, F., Serrano, M., 2000. Tumor suppressors and oncogenes in cellular senescence. Exp. Gerontol. 35, 317 – 329. Cho, N.H., Kim, Y.T., Kim, J.W., 2002. Alteration of cell cycle in cervical tumor associated with human Papillomavirus-cyclin-dependent kinase inhibitors. Yonsei Med. J. 43 (6), 722 – 728. DeVita, V.T., Hellman, S., Rosenberg, S.A., 2001. Cancer: Principles and Practice of Oncology. Lippincott Williams and Wilkins, Philadelphia. DiSaia, P.J., Creasman, W.T., 1997. Clinical Ginecologic Oncology, 5th edR Library of Congress. Doeberitz, M.V.K., 2002. Searching for new biomarkers for cervical cancer: molecular accidents and the interplay of Papillomavirus oncogenes and epithelial differentiation: Part I and II. Papillomavirus Rep. 13 (2), 35 – 74. INCA (Instituto Nacional do Caˆncer)-Ministe´rio da Sau´de (captured in 2005). Available at: http://www.inca.gov/. accessed January 21, 2005. Jay, N., Moscicki, A.B., 2000. Human Papillomavirus infections in women with HIV disease: prevalence, risk, and management. AIDS Read. 10 (11), 659 – 668. Klaes, R., Friedrich, T., Spitkovsky, D., Ridder, R., Rudy, W., Petry, U., 2001. Overexpression of p16INK4a as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri. Int. J. Cancer 92, 276 – 284. Langosch, K.M., Riethdorf, S., Po¨ppighaus, A.K., Riethdorf, L., Lo¨ning, T., 2001. Expression of cyclin-dependent kinase inhibitors p16MTS1, p21WAF1 and p27KIP1 in HPV-positive and HPV-negative cervical adenocarcinomas. Virchows Arch. 439, 55 – 61. Masumoto, N., Fujii, T., Ishikawa, M., Saito, M., Nozawa, S., 2003. p16INK4a overexpression and human Papillomavirus infection in small cell carcinoma of the uterine cervix. Hum. Pathol. 34 (8), 778 – 783. Murakami, M., Gurski, K.J., Steller, M.A., 1999. Human Papillomavirus vaccines for cervical cancer. J. Immunother. 22 (3), 212 – 218. Murphy, N., Ring, M., Killalea, A.G., Uhlmann, V., O’Donovan, M., Mulcahy, F., 2003. p16INK4a as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrepTM smear. J. Clin. Pathol. 56, 56 – 63.
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Piccinin, S., Doglioni, C., Maestro, R., Vukosavljevic, T., Gasparotto, D., D’Orazi, C., Boiocchi, M., 1997. p16/CDKN2 and CDK4 gene mutations in sporadic melanoma development and progression. Int. J. Cancer 74, 26 – 30. Sambrook, J., Fritsch, E.F., Maniatis, T., 1999. Appendix E: commonly used techniques in molecular cloning. Purification of nucleic acids. Molecular Cloning: A Laboratory Manual, 3rd edR Cold Spring Harbor, New York, pp. E.3 – E.4 (3v). Sano, T., Oyama, T., Kashiwabara, K., Fukuda, T., Nakajima, T., 1998. Expression status of p16 protein is associated with human Papillomavirus oncogenic potential in cervical and genital lesions. Am. J. Pathol. 153 (6), 1741 – 1748. Sano, T., Masuda, N., Oyama, T., Nakajima, T., 2002. Overexpression of p16 and p14ARF is associated with human Papillomavirus infection in cervical squamous cell carcinoma and dysplasia. Pathol. Int. 52, 375 – 383. Schellekens, A.C., Dijkman, A., Aziz, M.F., Siregar, B., Cornain, S., Fleuren, G.J., 2004. Prevalence of single and multiple HPV types in cervical carcinomas in Jakarta, Indonesia. Gynecol. Oncol. 93, 49 – 53. Serrano, M., 1997. The tumor suppressor protein p16INK4a. Exp. Cell Res. 237, 7 – 13.
Skomedal, H., Kristensen, G.B., Lie, A.K., Holm, R., 1999. Aberrant expression of the cell cycle associated proteins TP53, MDM2, p21, p27, cdk4, cyclin D1, RB, and EGFR in cervical carcinomas. Gynecol. Oncol. 73 (2), 223 – 228. Tsuda, H., Hashiguchi, Y., Nishimura, S., Kawamura, N., Inoue, T., Yamamoto, K., 2003. Relationship between HPV typing and abnormality of G1 cell cycle regulators in cervical neoplasm. Gynecol. Oncol. 91, 476 – 485. Waggoner, S.E., 2003. Cervical Cancer. The Lancet 361, 2217 – 2225. Walboomers, J.M., Jacobs, M.V., Manos, M.M., Bosch, F.X., Kummer, J.A., Sh, K.V., 1999. Human Papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189 (1), 12 – 19. Zhao, P., Hu, Y.C., Talbot, I., 2003. Expressing patterns of p16 and CDK4 correlated to prognosis in colorectal carcinoma. World J. Gastroenterol. 9 (10), 2202 – 2206. Zhou, Y., Gao, S.S., Li, Y.X., Fan, Z.M., Zhao, X., 2002. Tumor suppressor gene p16 and Rb expression in gastric cardia precancerous lesions from subjects at a high incidence area in northern China. World J. Gastroenterol. 8 (3), 423 – 425.
p16INK4A expression in cervical premalignant and malignant lesions Ana Paula Franco Lambert a,*, Fernando Anschau b, Virgı´nia Minghelli Schmitt a,c a
Laborato´rio de Biologia Molecular do Instituto de Pesquisas Biome´dicas, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, Rua Prof. Cristiano Fischer 1118/201, Jardim do Salso, Porto Alegre/RS, CEP 91410-000, Brazil b Servic¸o de Ginecologia e Obstetrı´cia do HSL, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, Porto Alegre, Brazil c Faculdade de Farma´cia, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, Porto Alegre, Brazil Received 1 August 2005 Available online 28 October 2005
Abstract p16INK4a is a cyclin-dependent kinase (CDK) inhibitor which decelerates cell cycle by inactivating CDKs that phosphorylate pRb. Human Papillomavirus persistent infection plays an important role on cervical carcinogenesis, mainly by the action of two viral oncoproteins, E6 and E7, which interact with p53 and pRb, respectively. Increasing expression of E6 and E7 in dysplastic cervical cells might thus be reflected by increased expression of p16INK4a. Recent studies revealed that p16INK4a expression could be a marker for dysplastic and neoplastic cervical cells. The aim of this study was to analyze p16INK4a expression in cervical preneoplastic and neoplastic lesions and correlate with lesion grade. Expression of p16INK4a was analyzed by immunohistochemistry. A total of 6 low-grade squamous intraepithelial lesion (LSIL), 21 high-grade squamous intraepithelial lesions (HSIL) and 27 cancer samples were studied. In HPV-positive cervical samples (n = 48), p16INK4a expression was observed in 1 of 3 LSIL, in 18 of 19 HSIL and in all 26 cancer cases. These results are in accordance with the hypothesis that functional inactivation of pRb by HPV-E7 protein induces p16INK4a expression in cervical lesions. In our study, a statistically significant association was observed between cervical lesion grade and p16INK4a expression (P < 0.001). D 2005 Elsevier Inc. All rights reserved. Keywords: HPV; p16INK4a; Cervical cancer; LSIL; HSIL; Immunohistochemistry
Introduction Cervical cancer is one of the most common cancers in women worldwide, with about 510,000 new cases and nearly 288,000 deaths each year. Many studies have shown that human Papillomavirus (HPV) persistent infection plays an important role in cervical carcinogenesis (Berek and Novak, 1998; DiSaia and Creasman, 1997; Jay and Moscicki, 2000; Sano et al., 1998). In fact, HPV infection has been detected in almost all preneoplastic (high-grade squamous intraepithelial lesions – HSIL – and low-grade squamous intraepithelial lesions – LSIL) and neoplastic lesions of the cervix. Recent extensive studies have revealed the existence of more than 80 types of HPV, classified into two categories, high and low oncogenic risk, according to the frequency they appear associated with cervical cancer (Berek and Novak, 1998; Sano * Corresponding author. Fax: +55 51 33341634. E-mail addresses: [email protected], [email protected], [email protected] (A.P.F. Lambert). 0014-4800/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2005.08.005
et al., 1998; Murakami et al., 1999). High-risk HPV types (HRHPV), particularly HPV type 16 and 18, have been identified in the majority of cervical cancer (Walboomers et al., 1999). HPV contributes to neoplastic progression through the action of predominantly two viral oncoproteins, namely, E6 and E7, which interact with various cell cycle regulatory proteins (Doeberitz, 2002; Waggoner, 2003). The E6 oncoprotein induces premature degradation of the p53 tumor suppressor protein, via the ubiquitin – proteasome pathway. The E7 oncoprotein binds to the tumor suppressor protein pRb, which normally inhibits progression of cell cycle into the S phase by complexing the E2F-like transcription factors. Progressive and prolonged phosphorylation of the Rb protein, performed by the cyclin/cyclin-dependent kinase (CDK) complexes, leads to its inactivation with consequently reduction of its growth suppressive activity. HPV E7 binding to pRb favors the release of E2F-like transcription factors, activating the transcription of Rb-regulated genes, which consequently promotes G1/S phase progression of the cell cycle (Doeberitz, 2002; Klaes et al., 2001; Murphy et al., 2003).
A.P.F. Lambert et al. / Experimental and Molecular Pathology 80 (2006) 192 – 196
In normal cells, the activity of CDKs is tightly regulated by several CDK inhibitors, including p16INK4a. This CDK inhibitor is a tumor suppressor protein that inhibits CDK4 and CDK6, which phosphorylate the Rb protein. A reciprocal relation between p16INK4a and pRb expression has been observed, suggesting the presence of a negative feedback loop allowing pRb to limit the concentration of p16INK4a. p16INK4a is inactivated in many cancers by mutation, deletion of the gene or hypermethylation of its promoter, resulting in reduced or absent expression of the p16INK4a gene product (Klaes et al., 2001; Murphy et al., 2003; Piccinin et al., 1997; Zhao et al., 2003; Zhou et al., 2002; Langosch et al., 2001). In the case of HPV-positive cervical cancer, it has been demonstrated an overexpression of p16INK4a as a result of functional inactivation of pRb by the HPV E7 protein. Doeberitz proposed that the loss of pRb function should result in the release of the p16INK4a gene from negative transcriptional feedback control. As a consequence of the downstream block of pRb by HPV E7, the functional effect of p16INK4a overexpression is not observed (Doeberitz, 2002). Due to the overexpression of p16INK4a in HPV-positive cervical cancers, a possible potential of p16INK4a as a marker for cervical intraepithelial lesions and cervical cancer was suggested (Sano et al., 1998; Murphy et al., 2003; Langosch et al., 2001). In the present work, we report the analysis of p16INK4a expression in cervical preneoplastic and neoplastic lesions and compare p16 INK4a expression levels with lesion grade.
193
peroxidase conjugate (Novostain Universal Detection Kit) for 10 min. Slides were developed with diaminobenzidine tetrahydrochloride (DAB) for 10 min and counterstained lightly with hematoxylin.
Interpretation of p16
INK4A
staining
Immunostaining of the formalin-fixed, paraffin-embedded sections was reviewed tree times to confirm the results, and strong nuclear as well as cytoplasmic staining was considered a positive reaction. The distribution of p16INK4a positive was scored on a semiquantitative scale as follows: ( ) none, (+) less than 5% of the cells, (++) 5 – 50% of the cells and (+++) more than 50% of the cells stained for p16INK4a (Skomedal et al., 1999).
DNA extraction and HPV detection DNA from cervical samples was extracted according to proteinase K standard protocol (Sambrook et al., 1999). For HPV detection, a polymerase chain reaction using consensus degenerate primers My09 and My11 was performed as described elsewhere (Bauer and Manos, 1993). DNA viability of the negative samples was checked by a PCR with primers complementary to the human betaglobin gene (Bauer and Manos, 1993). PCR products were electrophoresed on a 2% agarose gel containing 0.5 Ag/ml ethidium bromide in TAE buffer (1) and analyzed under UV light. All HPV-positive samples were tested by a typespecific PCR for HPV-16, -18, -31 and -33, the most frequent HR-HPVs. PCR conditions and primers used are described in Bauer and Manos (1993). Samples positive for HPV but negative for the tested HPV types were referred as ‘‘not identified HPV type’’ (NI).
Statistical analysis Statistical analysis was performed using the Chi-square test (Microsoft Office Excel, version 97). A P value of 0.05 or less was considered statistically significant (95% confidence).
Material and methods Paraffin-embedded cervical samples from 54 women with histological results for LSIL, HSIL or cancer were analyzed for p16INK4a expression. Cervical samples were obtained from subjects with colposcopic referral to biopsy of the uterine cervix attending the Gynecology Service of Sa˜o Lucas Hospital – PUCRS, from December 1999 to December 2001, with a complete medical history report. This study was approved by the local ethical committee (Protocol no. 288/03-CEP-PUCRS).
Immunohistochemistry Novostain Universal Detection Kit reagents (Novocastra Laboratories Ltd., Benton Lane, Newcastle upon Tyne, UK) were used for immunohistochemistry. Cervical biopsy samples were selected from pathology files of Sa˜o Lucas Hospital – PUCRS. All samples were fixed in formalin and embedded in paraffin by conventional techniques. Hematoxylin and eosin stained slides of all samples were classified by a certified pathologist. Serial sections (5 Am thick) of formalin-fixed and paraffin-embedded biopsy samples were cut and mounted on Novobond (Novocastra Laboratories Ltd.) slide tissue adhesive-coated glass slides. Sections were dewaxed by passage through xylene and then rehydrated in graded alcohol (100%, 90%, 80% and 70%). Endogenous peroxidase activity was blocked by incubating the section in 3% H2O2 for 10 min. Slides were then rehydrated with 0.01 M citrate buffer (pH 6) and microwaved 3 times, without boiling, for antigen retrieval. After rinsing with 0.01 mol/L phosphate-buffered saline (PBS), pH 7.4, nonspecific antibody binding was reduced by incubating the sections with prediluted blocking serum (normal horse serum) for 10 min. After decanting excess serum, sections were incubated overnight at 4-C with a mouse antihuman p16INK4A monoclonal antibody (Novocastra Laboratories Ltd.) at a 1:150 dilution in PBS. After washing thoroughly with PBS, sections were incubated with biotinylated universal secondary antibody (Novostain Universal Detection Kit) for 10 min, followed by incubation with Ready-to-use streptavidin –
Results A total of 54 samples from patients with cervical lesions were analyzed for p16 expression consisting of 6 cases of LSIL (11.1%), 21 of HSIL (38.9%) and 27 (50%) of cancer (23 cervical squamous cell carcinoma and 4 adenocarcinoma). Patients’ age ranged from 17 to 59 (40.9 T 10.86) years. Mean age of patients with LSIL was 30.3 (T12.48), HSIL was 38.2 (T10.41), and cancer was 45.4 (T8.66). In all p16-positive samples, both nuclear and cytoplasmic staining were observed. Relation of patient’s HPV status with lesion grade is presented in Fig. 1. HPV types detected in cases of multiple infection (n = 15) were 7 cases of coinfection of HPV-16 and -31 (1 LSIL, 3 HSIL and 3 cancer); 4 of HPV-16, -18 and -31 (1 HSIL and 3 cancer); 2 of HPV-16 and -18 (1 HSIL and 1 cancer); 1 of HPV-18 and -31 (cancer); 1 of HPV-16, -18 and -33 (cancer). Results of p16 expression and HPV status according to lesion grade are summarized in Table 1. Among HPV-16-positive patients, 1 was negative, and 7 were positive (1++ and 6+++) for p16 expression. Among HPV-18-positive patients, 4 were positive (+++) for p16. Five HPV-31-positive patients were positive (2+ and 3+++) for p16. In cases with multiple HPV types (n = 15), p16 expression was detected in 14 patients (1+, 4++, 9+++). In the group of patients with non-identified HPV type (n = 16), 15 were positive (2+, 2++, 11+++) for p16 expression.
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Fig. 2. Distribution of different lesion grades according to p16 INK4a expression level. LSIL—low-grade squamous intraepithelial lesions, HSIL—high-grade squamous intraepithelial lesions. v 2 = 39.74; P = 0.00000051; CI 95%.
Fig. 1. Frequency of HPV types in different lesion grades. NI*—not identified HPV type; **Infection with multiple HPV types; LSIL—low-grade squamous intraepithelial lesions; HSIL—high-grade squamous intraepithelial lesions.
Analyzing levels of p16 expression in different lesion grades, it was observed that 96.3% of cancer cases presented p16 +++ expression. On the other hand, the majority of p16 +++ expression (74.3%) corresponded to cancer cases. A statistically significant association was observed between lesion grade and p16 expression (v 2 = 39.74, P < 0.001, CI 95%) (Fig. 2). Discussion According to the Brazilian National Institute of Cancer (Instituto Nacional do Caˆncer—INCA), women most frequently develop cervical cancer at the age of 45 to 49 years (INCA, 2005). In North America, the mean age at cervical cancer diagnosis is 47 years, and nearly half of the cases are diagnosed before the age of 35 (Waggoner, 2003). The mean age of women with SIL is 15.6 years younger than the one of women with invasive cancer, suggesting a slow progression of SIL to invasive cancer (DeVita et al., 2001). Our study showed a mean age of 30.3 years for patients with LSIL, 38.2 years for HSIL and 45.4 years for cancer. The difference between mean age of cancer and LSIL was 15.1 years, similar to North American data, suggesting the same slow progression to cancer. Table 1 Distribution of p16 expression level according to lesion grade and HPV status p16 expression Negative
+
++
+++
HPV
LSIL (n = 6) HSIL (n = 21) Cancer (n = 27)
Neg
Pos
Neg
Pos
Neg
Pos
Neg
Pos
1 0 0
2 1 0
0 0 0
0 5 0
1 2 0
1 5 1
1 0 1
0 8 25
LSIL—low-grade squamous intraepithelial lesions; HSIL—high-grade squamous intraepithelial lesions.
HPV-16 is the most prevalent type worldwide. It is frequently detected in SIL and cancer cases. In our study, HPV-16 was the most frequent HPV type, representing 45.8% of all HPV infections, including single or multiple type infections. Among HPV-16-positive samples, 59.1% corresponded to cancer cases. HPV-18 is considered the most aggressive HPV type and may be a negative prognostic factor (DiSaia and Creasman, 1997). HPV-18 has been associated with poorly differentiated carcinomas and with a high rate of disease recurrence (DeVita et al., 2001). In our study, all 4 HPV-18 single infection cases were cancer, but when single and multiple type infections including HPV-18 (n = 12) were considered, 83.3% corresponded to cancer cases. According to Schellekens et al., in cases of infections with multiple HPV types, it is difficult to determine which HPV type(s) is responsible for the development of cancer, but it also cannot be excluded that only one type was the responsible, while the other(s) merely represent HPV infection with no involvement in cancer development (Schellekens et al., 2004). In our study, the majority of multiple HPV type infections were cancer cases (9/15), followed by HSIL (5/15) and LSIL (1/15). Among HPV-positive cases with non-identified HPV types (n = 16), most of them were HSIL (n = 8) and cancer (n = 7), which could suggest high-risk HPV types, different from the tested ones (16, 18, 31, 33), were involved. The pRb and p53 pathways of the cell cycle regulatory cascade are central to the regulation of the G1 to S phase transition and to the understanding of human cancer (Alberts et al., 1999). HPV infection is critically involved in cervical carcinogenesis and plays an important role in this regulatory cascade by the binding of its oncoproteins E6 and E7 to the tumor suppressor genes p53 and pRb, respectively (Schellekens et al., 2004). According to Walboomers et al., 99.7% of all cervical cancer are HPV-positive suggesting that HPV is a necessary but not sufficient cause of cervical cancer (Walboomers et al., 1999). In our study, only one cancer case, out of a total of 27, was negative for HPV. p16INK4a plays an important role in the regulation of cell cycle. CDK4/6 form complexes with cyclin D, which regulates the progression of the cell cycle. pRb binds to E2F transcription factors and suppresses their role in transcription during the G1 phase. pRb is inactivated and dissociated from
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the pRb-E2F complex after phosphorylation by the cyclin DCDK4/6 complex during G1 phase. The activity of cyclin DCDK4/6 is negatively regulated by inhibitors of the cyclin/ CDK complex, such as p16INK4a, which bind directly to CDK4/ 6 or the cyclin D-CDK4/6 complexes. Therefore, lack of functional p16INK4a results in abnormal cell cycle progression, which is associated with carcinogenesis (Bringold and Serrano, 2000; Masumoto et al., 2003; Serrano, 1997). On the other hand, overexpression of p16INK4a can result from inactivation of pRb, and transcriptional activity of p16INK4a seems to be repressed by pRb. The overexpression of p16INK4a in cervical neoplasia has been reported to be associated with HPV, and an inverse correlation has been found between pRb function and overexpression of p16INK4a. In fact, HPV E7 protein is responsible for an increase in p16INK4a level (Doeberitz, 2002; Masumoto et al., 2003). It was reported that p16INK4a mRNA was strongly induced by E2F, and accumulation of E2F might induce the overexpression of p16INK4a (Masumoto et al., 2003). According to these hypothesis, dysfunction of pRb through HPV E7 protein association should increase free E2F, resulting in both p16INK4a overexpression and abnormal cell cycle progression (Cho et al., 2002). Our results are in accordance with this hypothesis showing expression of p16INK4a in 93.75% of HPV-positive samples (45/48). Although 5 cervical lesions negative for HPV were positive for p16INK4a expression (2 LSIL, 2 HSIL and 1 cancer). As mentioned earlier in this paper, overexpression of p16INK4a could result from pRb inactivation. So, we could conjecture that in these HPV-negative cervical lesions, p16INK4a expression was a result of pRb inactivation by means of mechanisms independent from HPV infection. In a study conducted by Tsuda et al., mutation and deletion of p16 in cervical neoplasm were rare events, and methylation of the p16 was thought to be one of the important mechanisms of p16 loss. They also suggest that there may exist an alternative translational mechanism, or posttranslational degradative mechanism, for the loss of p16 protein expression (Tsuda et al., 2003). Our work showed negative p16 expression in 2 LSIL and 1 HSIL HPV-positive cases. One of the mechanisms suggested by Tsuda et al. could be the responsible for these results. Klaes et al. demonstrated that p16-specific immunohistochemical staining allows the sensitive and specific identification of dysplastic cervical cells in tissue sections or cervical smear (Klaes et al., 2001). Murphy et al. suggest that p16INK4a marks dysplastic squamous and glandular cells of the cervix with a sensitivity of 99.9% and specificity of 100% (Murphy et al., 2003). In our study, p16INK4a expression was observed in precursor lesions, with the higher expression in cervical cancer samples. A statistically significant association was observed between lesion grade and p16 expression (v 2 = 39.74, P < 0.001, CI 95%). A study conducted by Sano et al. concluded that p16INK4a is overexpressed in most cervical lesions, and the status of its immunoreactivity allows differentiation between infection with low-risk HPV and high-risk HPV (Sano et al., 2002). In our study, among HPV-positive samples, p16INK4a expression was
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observed in 1 LSIL (n = 3), 18 HSIL (n = 19) and in all cancer cases (n = 26) (Table 1). These results are in accordance with the hypothesis that functional inactivation of pRb by HPV-E7 protein induces p16INK4a expression in cervical lesions (Langosch et al., 2001). In conclusion, a good correlation was observed between cervical lesion grade and p16INK4a expression. Despite the reduced number of sample analyzed, our results indicate that p16INK4a expression would be a good marker for cervical dysplastic lesions and cancer, but further studies are needed to reinforce this statement. Acknowledgments We thank Dr. Emilio Antoˆnio Jeckel Neto, Dr. Denise Cantarelli Machado and Dr. Vinicius Duval da Silva for scientific support; Vinı´cius S. Michaelsen and Raquel Mattos de Oliveira for technical assistance. Ana Paula Franco Lambert was a fellow of CAPES. References Alberts, B., Bray, D., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., 1999. Fundamentos de Biologia Celular. Uma Introduc¸a˜o a Biologia Molecular da Ce´lula. Editora Artes Me´dicas. Porto Alegre. Bauer, H.M., Manos, M.M., 1993. PCR detection of genital human Papillomavirus. In: Persing, D. (Ed.), Diagnostic Molecular Microbiology. American Society for Microbiology, pp. 407 – 413. Berek, J.S., 1998. Tratado de Ginecologia. 12a edic¸a˜o.: Guanabara Koogam, Rio de Janeiro. Bringold, F., Serrano, M., 2000. Tumor suppressors and oncogenes in cellular senescence. Exp. Gerontol. 35, 317 – 329. Cho, N.H., Kim, Y.T., Kim, J.W., 2002. Alteration of cell cycle in cervical tumor associated with human Papillomavirus-cyclin-dependent kinase inhibitors. Yonsei Med. J. 43 (6), 722 – 728. DeVita, V.T., Hellman, S., Rosenberg, S.A., 2001. Cancer: Principles and Practice of Oncology. Lippincott Williams and Wilkins, Philadelphia. DiSaia, P.J., Creasman, W.T., 1997. Clinical Ginecologic Oncology, 5th edR Library of Congress. Doeberitz, M.V.K., 2002. Searching for new biomarkers for cervical cancer: molecular accidents and the interplay of Papillomavirus oncogenes and epithelial differentiation: Part I and II. Papillomavirus Rep. 13 (2), 35 – 74. INCA (Instituto Nacional do Caˆncer)-Ministe´rio da Sau´de (captured in 2005). Available at: http://www.inca.gov/. accessed January 21, 2005. Jay, N., Moscicki, A.B., 2000. Human Papillomavirus infections in women with HIV disease: prevalence, risk, and management. AIDS Read. 10 (11), 659 – 668. Klaes, R., Friedrich, T., Spitkovsky, D., Ridder, R., Rudy, W., Petry, U., 2001. Overexpression of p16INK4a as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri. Int. J. Cancer 92, 276 – 284. Langosch, K.M., Riethdorf, S., Po¨ppighaus, A.K., Riethdorf, L., Lo¨ning, T., 2001. Expression of cyclin-dependent kinase inhibitors p16MTS1, p21WAF1 and p27KIP1 in HPV-positive and HPV-negative cervical adenocarcinomas. Virchows Arch. 439, 55 – 61. Masumoto, N., Fujii, T., Ishikawa, M., Saito, M., Nozawa, S., 2003. p16INK4a overexpression and human Papillomavirus infection in small cell carcinoma of the uterine cervix. Hum. Pathol. 34 (8), 778 – 783. Murakami, M., Gurski, K.J., Steller, M.A., 1999. Human Papillomavirus vaccines for cervical cancer. J. Immunother. 22 (3), 212 – 218. Murphy, N., Ring, M., Killalea, A.G., Uhlmann, V., O’Donovan, M., Mulcahy, F., 2003. p16INK4a as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrepTM smear. J. Clin. Pathol. 56, 56 – 63.
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