ARTICOLE ORIGINALE
IMMUNOCHEMICAL ASSESSMENT OF P16 AND HPV L1 CAPSID PROTEIN IN CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS
Raluca Balan1, Simona Giuºcã1, Irina Draga Cãruntu1, V. Gheorghiþã2, D. Neacºu3, Cornelia Amãlinei1
Gr.T. Popa University of Medicine and Pharmacy Iaºi School of Medicine
1. Department of Morphology
Elena Doamnã Clinical Hospital of Obstetrics and Gynecology Iaºi 2. Department of Obstetrics and Gynecology
County Emergency Hospital Buzãu 3. Department of Obstetrics and Gynecology
IMMUNOCHEMICAL ASSESSMENT OF p16 AND HPV L1 CAPSID PROTEIN IN CERVICAL INTRAEPITHELIAL LESIONS (Abstract): The behavior of the cervical squa- mous intraepithelial lesions cannot be predicted, many of them, particularly of the low grade type, may disappear without treatment. Invasive cervical carcinoma occurs in approximately 10% of the intraepithelial precursor lesions, being strongly associated with HPV infection. The aim of this study was to make a comparative assessment between immunohistochemical and immunocytochemical expression of p16 and L1 HPV capsid protein respectively, in low grade and high grade cervical squamous intraepithelial lesions. Material and method: The study involved 20 patients with cytological diagnosis of LSIL/CIN1 (low grade squamous intra- epithelial lesion/cervical intraepithelial neoplasia) and HSIL (CIN2 and CIN3) (high grade squamous intraepithelial lesion), which underwent a subsequent cervical biopsy. The conven- tional smears were evaluated for the immunoexpression of L1HPV protein and the correspond- ing biopsies for the immunoexpression of p16. Results: The HPV L1 capsid protein was expressed in 46% of LSIL and 24% of HSIL. P16 was positive in 68% of LSIL, 84% of CIN2 and 100% of CIN3. The correlative analysis of p16 status and protein L1HPV expres- sion can be very useful in the assessment of progression risk of cervical squamous intraepithelial lesions. Key words: HPV, L1 CAPSID PROTEIN, p16, LSIL, HSIL
It is now accepted that invasive cervical squamous carcinomas and the correspon- dent precursor lesions are determined by specific types of Human papillomavirus (HPV), especially high oncogenic risk types, which infect the anogenital tract. At pre- sent, the proportion of cervical carcinoma attributed to HPV infection is estimated to 99%. Many observations have indicated the importance of immune response in HPV infection. Antibodies against capsid pro- teins of different types of HPV have been previously studied, using fusion bacterial proteins or chemically synthesized peptides
(1,2). As an essential condition of these studies, HPV infecting type has to be de- tected. Until recently, the detection of HPV type in a certain tissue was performed by hybridization arrays with nucleic acids. Poly- merase chain reaction (PCR) was developed as a more specific and sensitive method of HPV DNA amplification, used either for general detection or for the HPV typing, particularly in genital infections. The issue of quantification is the main limit of the procedure, the distinction between latent subclinical infection and evident clinical lesions being difficult. As an alternative to
hybridization and PCR, the immunologic detection of viral capsid antigens can be used for the diagnostic of productive HPV infections.
The tumor suppressor protein p16 is a cyclin-dependent kinase inhibitor that regu- lates transition from the G1 to the S phase of the cell cycle (3,4).
The high immunoexpression of the p16 was previously reported to be characteristic for dysplastic and neoplastic epithelium of the cervix (5,6,7). P16 overexpression slows down the cell cycle by inactivating the cyclin- -dependent kinases that phosphorylate retino- blastoma protein (pRb) (8,9). The viral onco- genes E6 and E7 of HPV, whose expression is associated with malignant transformation of the cervical epithelial cells (10,11), have the ability to bind and inactivate pRb which, in turn, influence the p16 expression in cer- vical intraepithelial squamous lesions (SIL) (12). Recent studies have proposed that p16 is a useful marker for HR (high risk)-HPV type related cervical neoplasia (9,13) and for predicting SIL progression (14,15).
The behavior of the cervical squamous intraepithelial lesions cannot be predicted, many of them, particularly of the low grade type, may disappear without treatment. Inva- sive cervical carcinoma occurs in approxi- mately 10% of the intraepithelial precursor lesions, being strongly associated with HPV infection (16,17,18). The aim of this study was to make a comparative assessment between immunohistochemical and immunocytoche- mical expression of p16 and L1 HPV capsid protein respectively, in low grade and high grade cervical squamous intraepithelial lesions in order to determine, by clinico-morpholo- gical correlations, their practical applicabi- lity in patients diagnosis and follow-up.
MATERIALS AND METHODS Twenty patients were included in our study, 10 cases with cytological diagnosis of LSIL (low grade squamous intraepithelial lesion) and 10 cases with HSIL (high grade
squamous intraepithelial lesion), which under- went a subsequent cervical biopsy. The cer- vico-vaginal smears were fixed and stained using Papanicolaou method. The conven- tional smears were evaluated for the immuno- expression of L1HPV capsid protein and the corresponding biopsies for the immuno- expression of p16.
After Pap cytodiagnosis, the cervico-va- ginal smears were used to detect HPV L1 capsid protein by immunocytochemistry, using the monoclonal antibodies (Cytoactiv HPV L1 High Risk Set REF SCA0850, Cyto- immun Diagnostics GmbH) in a standar- dized protocol. Epithelial cells with positi- ve nuclear staining were scored as positive, considering one stained nucleus enough for scoring.
The cervical biopsies were investigated by routine histopathological exam and by immunohistochemistry, using p16-D25 anti- bodies. Collected tissues were fixed for 24 hours in buffered formalin and processed for paraffin embedding. Serial sections of 4-5 mm were dewaxed and stained with Hema- toxylin-Eosin. After performing the stan- dard histopathologic exam, supplementary sections were used for immunohistochemical exam. Heat-induced epitope retrieval tech- nique was performed using Target Retrieval Solution pH 6 (code S1700, DAKO, Den- mark). After blocking the endogenous per- oxidase and non-specific binding, the sections were incubated with one of the primary antibodies, anti-p16 mouse monoclonal anti- body (clone D25, code sc-81613, Santa Cruz, USA), dilution range 1:100. The immune reaction was amplified using the appropriate secondary antibody and the Streptavidin
Biotin Peroxidase HRP complex (code K5001, DAKO, Denmark). Sections were then deve- loped using 3,3-diaminobenzidine tetrahy- drocloride chromogen (DAB) (code K5001, DAKO, Denmark), under microscope con- trol. The sections were finally counterstained with Mayers Hematoxylin. Negative con- trol was also performed.
The quality control represented by exter- nal and internal negative and positive con- trols was necessary to monitor the accuracy of tissue processing, staining procedures and reagents effectiveness. The primary anti- body specificity sought to be assesed by their negative controls.
Protein p16 was scored based upon the estimated proportion of immunopositive cells (table I).
RESULTS
In our study, the histopathological diag- noses were consistent with the cytodiag- noses, as follows: 10 cases with LSIL (CIN1) and 10 cases with HSIL (4 cases CIN2 and 6 cases CIN3).
HPV infection was morphologically con- firmed by the presence of cytopatic HPV effect (koilocytes) in the smears and biopsies.
The p16 staining pattern was predomi- nantly nuclear with occasional cytoplasmic positivity. Most cases presented heterogeneity of staining with positive cells admixed with negative cells.
From all cervical biopsies, p16 was po- sitive in 68% of LSIL, 84% of CIN2 and 100% of CIN3. The proportion of biopsies with intense immunoexpression of p16 in- creased with the severity of cytological ab- normality. In HSIL cases, the staining dis- tribution was as follows: 65% full thickness (figure 3), 35% basal and intermediate. The staining intensity for HSIL cases was strong in 80% (figures 1, 3), moderate in 16%, and weak in 4% accordingly. Regarding LSIL category, the staining distribution was basal in 75% of cases and occasional in the
remaining 25%. There was no LSIL case with full thickness of p16 positivity. The staining intensity of LSIL cases was strong in 20% (figures 2, 4, 5), moderate in 13%, and weak in 67%.
From all cervical smears, the HPV L1 capsid protein was expressed in 46% of LSIL and 24% of HSIL. Expression of L1-capsid proteins was significantly reduced for HPV positive HSIL. In HPV positive LSIL, no significant reduction of L1 capsid protein expression could be demonstrated.
Positive reaction was characterized by the strong staining of the whole nucleus, surrounded by a cytoplasm with no back- ground. In most cases, positive reaction for HR-HPV L1 was positive in typical koilo- cytes or in dyskeratocytes, presenting nuclear characteristics for HSIL (CIN 2 or CIN 3).
In LSIL cases, the positivity of the nuclei was presented only in typical koilocytes (fi- gure 6).
All HPV-positive cases (with morpho- logic evidence of HPV infection) were also p16-positive, but no significant relationship between HPV infection immunopositivity (active infection) and the intensity and dis- tribution of p16 was found.
DISCUSSIONS
L1 capsid protein is expressed in the active phase of HPV infection and is ne- cessary in viral cellular cycle completion.
Consequently, viral protein detection, by immunohistochemical reaction is an evi- dence of active HPV infection in examined tissue (19). L1 viral capsid protein is consi- dered a major target of the cellular immune TABLE I
p16 Immunohistochemical score
Score p16
0 none
1 Weak (<25% immunopositivity) 2 Moderate (25-75% immunopositivity) 3 Intense (75-100% immunopositivity)
response (20). LSIL and moderate SIL without immunohistochemically detected L1 are corre- lated, in more than 80% of cases, with dys- plasia progression. Griesser et al. certify these aspects, highlighting that minor and moderate lesions without L1 capsid protein expression are significantly more exposed to a progression in comparison to L1 posi- tive cases (21). Most probably, the lack of HPV antigen is determined by a weak pro- tein synthesis, under immunohistochemical test minimum level. As L1 represents the major target of the immune cellular response (22), its deficient translation may result in an inefficient depuration of the infected cells, promoting viral DNA integration in host cellular genome and the transformation of immature epithelial cells. The observa-
tion that the decrease of the HPV16 capsid positivity in cervical cancer patients serum is an indicator of a poor prognosis sustains the importance of a specific humoral res- ponse. Immunohistochemical detection of L1 capsid, on conventional Papanicolau smears, may consequently indicate the locally in- duced defence status on HPV infection and may offer prognosis information, mainly in LSIL lesions.
From all cervical smears, our data re- vealed that the HPV L1 capsid protein was expressed in 46% of LSIL and 24% of HSIL.
Expression of L1-capsid proteins was signi- ficantly reduced for HPV positive HSIL. In HPV positive LSIL, no significant reduc- tion of L1 capsid protein expression could be demonstrated. Because of the low rate of Fig. 1. HSIL (CIN2), strong p16
immunostaining (x10) Fig. 2. LSIL, strong p16
immunostaining (x10)
Fig. 3. HSIL (CIN3), strong full thickness p16
immunostaining (x10) Fig. 4. LSIL, strong p16
immunostaining (x10)
HR-HPV L1 positivity found in LSIL cases in our study, we can admit that HPV is not helpful in grading cervical SIL, which is in accordance with the literature data (12).
In our study, patients mean age was 34 years. This fact is consistent with the lite- rature data, where it has been demonstrated that the prevalence of HPV infection varies with age and geographical region, reaching highest rates below 35 years of age (23).
The presence of L1 HPV positively stained nuclei of squamous epithelial cells could be correlated with the clinical course. Ex- pression of L1-capsid proteins was signi- ficantly reduced for HPV positive HSIL. In HPV positive LSIL, no significant reduc- tion of L1 capsid protein expression could be demonstrated. Mild and moderate dys- plastic cervical lesions without immuno- histochemical positive reaction of HPV L1 capsid protein are more likely to progress as compared to positive cases (21). Researches consider that lack of detectable HPV anti- gen in the Pap smears is due to low protein synthesis in squamous epithelial cells below the limit of the immunocytochemical test.
The loss of L1 capsid protein immunoex- pression can be the result of the integration of the viral DNA into the human genome.
Although most of cervical carcinomas show integration of viral DNA, it is detectable only in a small proportion of LSIL and
HSIL (24). As was already reported in the literature (20), perhaps there are additional control mechanisms which lead to the L1-ne- gative immunoexpression in the mentioned cases.
The development of viral capsid antigen L1 depends upon transcriptional factors which only can be expressed during maturation process from basal epithelial cell to superfi- cial epithelial cell (19). In HSIL, the natural structure as well as maturation of the epi- thelium are disturbed, thus the dysplastic basal squamous cells represent the predo- minant cell type with reduced L1 capsid protein expression.
P16 is a highly sensitive marker for cer- vical epithelial dysplasia. Strong and full thickness staining of p16 in the cervix epi- thelium is highly supportive of HSIL, while weak and basal/rare staining favors LSIL (4).
In our study, from all cervical biopsies, p16 was positive in 68% of LSIL, 84% of CIN2 and 100% of CIN3. The proportion of biopsies with intense immunoexpression of p16 increased with the severity of cyto- logical abnormality. Our results are consis- tent with the data reported in the literature (25, 26, 27, 28).
As previously reported (14), we also found that all cases morphological HPV positive expressed the p16 protein. No significant relationship between HPV infection immu- Fig. 5. LSIL, strong p16
immunostaining (x20) Fig. 6. LSIL, HPV L1
positive (x100)
nopositivity (active infection) and the in- tensity and distribution of p16 was found.
This is also supported by Lin et al. (29), who admitted that p16 expression in cervical squamous and glandular epithelium is the marker of dysplasia or malignancy irres- pective of the HPV infection status.
Other studies reported the existence of a significant association between cervical squa- mous intraepithelial lesion grade and p16 intensity and distribution of the immuno- staining (30, 31). Regarding the localization of p16 staining, there are authors who re- port predominant cytoplasmic staining (7) as well as predominant nuclear staining (32, 33), or a combination of both patterns (34).
According to our results, we cannot for- mulate any definitive statements regarding the applications of p16 in routine practice,
excepting its recognized value in certifying the diagnosis of doubtful cases.
CONCLUSIONS
The immunoexpression of L1 HPV has clinical application in the progression asses- sment of the cervical precancerous lesions without a correlation to the grade of the cervical SIL. The analysis of p16 status, in parallel with protein expression L1 HPV, can be very useful in the assessment of pro- gression risk of cervical squamous intraepi- thelial lesions.
ACKNOWLEDGEMENTS
The research was funded by the National Council for Scientific Research in Higher Education, grant IDEI 2036/2008.
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