HPV L1 capsid protein is expressed together with the production of infectious viral particles, but its expression and relation to p16 expression, which has been a surrogate marker for human papilloma virus (HPV) infection in cervix, are little studied in cytology samples. The authors aimed to elucidate the relation between L1 capsid protein and p16 protein expressions in liquid-based samples from uterine cervical lesions.
Immunochemical analyses using antibodies against L1 capsid protein and p16 protein were carried out on cytological materials obtained from uterine cervical lesions of low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs), and squamous cell carcinomas (SCCs).
L1 capsid protein was positive in 30% of LSILs and 12% of HSILs, but in 0% of SCCs. In contrast, p16 protein was positive in 55% of LSILs, 100% of HSILs, and 100% of SCCs. L1-positive cells were only observed in the superficial layer, whereas p16-positive cells were seen throughout the full thickness of the epithelium. The relation between L1 capsid protein and p16 protein, p16(−)/L1(+) cases represented 44% of LSILs, but 0% of HSILs, and 0% of SCCs, whereas p16(+)/L1(−) cases represented 82% of LSILs, 88% of HSILs, and 100% of SCCs.
Expression of L1 capsid protein decreased with lesion progression from LSILs to HSILs and SCCs, whereas p16 protein was positive in all HSILs and SCCs. The correlation between L1 and p16 expressions suggests that L1(−)/p16(+) cases have the potential for progression, whereas L1(+)/p16(−) and L1(−)/p16(−) cases may be nonprogressive lesions or potentially in remission. Cancer (Cancer Cytopathol) 2008. ? 2008 American Cancer Society.
Uterine cervical cancer is the second largest cause of cancer deaths in women worldwide.1 Persistent infection with human papilloma virus (HPV) is a necessary, but not a sufficient, cause of progression from low-grade squamous intraepithelial lesions (LSILs) to high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas (SCCs). The life cycle of HPV infection consists of 2 phases. The first phase is a “productive” phase for establishing the infection. In this phase, HPV infects long-living basal cells, in which a copy number of 50–100 genomes per cell is maintained.2 A high proportion of infections associated with LSILs also regress spontaneously.2 After the initial infection of basal keratinocytes, the early HPV genes E1, E2, E5, E6, and E7 are expressed, and the viral DNA replicates. LSILs support productive viral replication. In the upper layers of the epithelium, the viral genome is replicated further. L1 and L2 encapsulate the viral genomes to form progeny virions in the nucleus. These virions shed the virus, which then reinitiates infection.3 Therefore, the productive phase refers to the early stage of HPV infection with L1 capsid protein expression. In contrast, the second phase is the “transformation” phase, in which HPV DNA is integrated into the host DNA. The progression of untreated lesions to microinvasive and overtly invasive cancers is associated with integration of the HPV genome into the host chromosomes.3 Persistent cervical infection and integration with an oncogenic HPVs, such as HPV 16 and HPV 18, are the most important risk factors for progression of LSILs to HSILs or SCCs.4
The number of cases of malignant disease of the cervix is, however, small compared with the number of women infected with HPV.2 Therefore, the main problem is how to identify individuals who are at risk of progressive disease among the large number of individuals infected with HPV. We previously revealed that p16 protein (p16) immunostaining in liquid-based smears, with simultaneously obtained biopsies, was a useful new prognostic biomarker.5 The CDKN2A gene product, p16, has been found to be overexpressed in cervical preneoplastic and neoplastic lesions where high-risk HPV subtypes exist.6 In cervical carcinogenesis, oncogenes E6 and E7 of HPV cause inactivation of the tumor-suppressor gene-protein products p53 and Rb, thereby resulting in disruption of the p53 and Rb pathways at the G1 checkpoint, respectively.7 In cervical lesions, overexpression of p16 is thought to result from increased levels of the transcription factor E2F-1, which is released from Rb protein after binding to the oncogenic HPV E7 protein instead of Rb protein phosphorylated by cyclin-dependent kinases.8, 9 Therefore, p16 is upregulated according to the potential of Rb inactivation by HPV oncogenes.
Immunochemical expression of L1 capsid protein is known to be associated with the productive phase of HPV infection. In the present study, we immunochemically analyzed the association of L1 capsid protein and p16 expressions in liquid-based cytology (LBC) samples from uterine cervical lesions.
MATERIALS AND METHODS
Patient and Study Design
A total of 63 LBC specimens from 20 LSIL cases, 40 HSIL cases, and 3 SCC cases were collected from patients who first visited the Department of Obstetrics and Gynecology, Gunma University Hospital, for a routine gynecological examination between June 2006 and January 2007 and whose cytological diagnosis was confirmed by histological examination. The age of the patients ranged from 24 to 74 years (mean, 50 years) at the time of the cytological examination. Before the beginning of the study, the protocol was approved by the Gunma University Hospital Institutional Review Board. Histological samples from CIN2 and SCC cases, in which HPV type 16 infection was identified by the polymerase chain reaction– restriction fragment-length polymorphism (PCR-RFLP) method,10 were used as positive controls.
LBC samples were obtained from the patients by using a Cervex-Brush (Rovers Medical Devices, Oss, Netherlands) and immediately immersed in fixative solution (SurePath Preservative Fluid; TriPath Imaging, Burlington, NC). The cells were then washed from the Cervex-Brush into the fixative. The LBC specimens were subsequently processed according to the manufacturer's protocol and stained by the Papanicolaou method. The Bethesda System was used for assessment of the thin-layer specimens.
Immunostaining for p16 and L1 Capsid Protein
For immunostaining of p16 and L1 capsid protein, endogenous peroxidase activity was blocked with 3% H2O2 for 30 minutes at room temperature, and antigen retrieval was performed by boiling in 0.01 M citric acid phosphate buffer (pH 8.0) for 20 minutes. Nonspecific staining was eliminated by incubating the tissues with normal bovine serum for 30 minutes at room temperature. The specimens were then reacted with mouse monoclonal antibodies against p16 (JC8; 1:50; Neomarkers, Fremont, Calif) and L1 capsid protein (ready to use; HPV High Risk Pur [VAHP]; Viroactiv & Virofem, Wiesbaden, Germany) for 30 minutes at room temperature in a humidified chamber, washed thoroughly, and incubated with ENVISION reagent (Dako Cytomation, Kyoto, Japan) for 30 minutes at room temperature. The mouse monoclonal antibody recognizes the major L1 capsid proteins of high-risk HPV types 16, 18, 33, 35, 39, 45, 56, and 58. The specimens could then be observed after subjecting them to benzidine reaction and light counterstaining with Mayer's hematoxylin.
Evaluation of p16 and L1 Capsid Protein Immunostaining
Immunostaining results were evaluated as positive (+) when the smear included atypical cells that showed specific immunoreactivity for p16 in both the nucleus and cytoplasm. Nuclear staining was interpreted as positive for L1 capsid protein. The results were analyzed statistically by using the Fisher exact test.
p16 Expression in LBC Samples
Expression of p16 was detected in 11 of 20 (55%) LSILs, 40 of 40 (100%) HSILs, and 3 of 3 (100%) SCCs (Fig. 1Top). The dysplastic epithelia of p16-positive LBC samples and histological controls showed nuclear staining with or without cytoplasmic staining. Cytoplasmic stainings were observed only in a subset of cases with specific nuclear staining. Immunochemically, p16 positivity in dysplastic cells was observed from the basal layer to the superficial epithelial layer (Fig. 2).
L1 Capsid Protein Expression in LBC Samples
Expression of L1 capsid protein was detected in 6 of 20 (30%) LSILs, 5 of 40 (12%) HSILs, and 0 of 3 (0%) SCCs (Fig. 1Bottom). Immunochemically, L1 capsid protein positivity was observed in the cells of the upper epithelial layers but not in basal and parabasal cells (Fig. 2).
Relation Between p16 and L1 Capsid Protein Expressions
When the expression of L1 capsid protein was analyzed in relation to p16 expression status, the staining pattern was divided into the following 4 groups: L1(−)/p16(−); L1(+)/p16(−); L1(+)/p16(+); and L1(−)/p16(+). L1(−)/p16(−) cases represented 5% of all lesions, including 25%, 0%, and 0% of LSILs, HSILs, and SCCs, respectively. L1(+)/p16(−) represented 6.3% of all lesions, including 20%, 0%, and 0% of LSILs, HSILs, and SCCs, respectively. L1(+)/p16(+) represented 11.1% of all lesions, including 10%, 12%, and 0% of LSILs, HSILs, and SCCs, respectively. L1(−)/p16(+) represented 74.6% of all lesions, including 45%, 88%, and 100% of LSILs, HSILs, and SCCs, respectively (Fig. 3).
Previous studies have revealed that interobserver reproducibility for cytological and histological interpretation is lower for low-grade cervical lesions than for high-grade lesions.11, 12 Recently, a study on interobserver reproducibility for histological diagnosis of cervical intraepithelial neoplasia revealed that overexpression of p16, a tumor suppressor protein, was frequently detected in precursors and in carcinoma.13 Therefore, p16 is a new biomarker for dysplastic or carcinoma cells of the cervix and useful for making an exact diagnosis and evaluating the patient prognosis. Sano et al14, 15 reported that the status of p16 immunoreactivity may help to differentiate low-risk HPV infections from high-risk or intermediate-risk HPV infections. We previously reported that immunochemical detection of p16 was more sensitive and specific than clarification of the HPV status in cervical lesions by using a liquid-based method as well as tissue samples,5 thereby suggesting that p16 should represent a good biomarker for primary screening of cervical cytology. Klaes et al13 also reported that p16 expression can be used to identify dysplastic and cancer cells. In the present study, expression of p16 was seen in 55% of LSILs, but in 100% of HSILs and SCCs, thus indicating that p16 expression significantly increased with increasing severity of the cervical lesions.
Recently, there have been some reports of immunostaining of L1 capsid protein as a new powerful and useful marker for revealing the status of productive and/or active HPV infections.16–18 The HPV L1 capsid protein is expressed together with the production of infectious viral particles. Conversely, positive staining for L1 capsid protein was only detected in the productive phase. In contrast, persistent infection without production of HPV viral particles is usually attributed to the viral genomes in the long-living basal epithelial cells, which do not express L1 capsid protein. Loss of L1 capsid protein expression may show the 2 statuses of viral DNA; one is the integration of viral DNA into the host genome and the other is latent infection with low or no synthesis of HPV oncoprotein and no HPV production. Melsheimer et al16 demonstrated that expression of L1 capsid protein is significantly reduced in HPV 16- and in other high-risk HPV-type positive HSILs. These findings are consistent with our present data that show expression of L1 capsid protein in 30% of LSILs, 12% of HSILs, and 0% of SCCs, thus indicating that L1 capsid protein expression tends to decline with increasing severity of the lesions. In the present study, the lack of HPV L1 capsid protein in higher grade lesions was likely to indicate a latent viral infection or integration of HPV DNA.
DNA tests, such as hybrid capture19 or HPV screening kits for the detection of HPV-DNA, are routine tests and useful screening tools, but they cannot differentiate among latent, subclinical, and clinically relevant infections. However, discrimination of the infectious status is important for the diagnosis of precancerous lesions. Therefore, we consider that the combination of p16 and L1 capsid protein immunostaining is a very useful and powerful test as a prognostic marker. Hence, we propose to introduce a combination of L1 capsid protein and p16 immunostaining in LBC for clinical use and divide the results into 4 groups on the assumption that the HPV DNA test is positive. The L1(−)/p16(−) pattern means that viral DNA is present without either viral replication or alteration of the cell cycle, thus indicating that the lesion is in a latent and nondysplastic state. The L1(+)/p16(−) pattern means that viral DNA is present as a productive and/or active form without alteration of the cell cycle, which indicates that the lesion is in a virus-producing but nondysplastic state. The L1(+)/p16(+) pattern means alteration of the cell cycle with a productive and/or active form of HPV, which indicates that the lesion is in an early dysplastic status. The L1(−)/p16(+) pattern means either latent viral infection or integration of HPV DNA into the host genome with alteration of the cell cycle, thus indicating that the lesion is in a late or more advanced dysplastic state. Furthermore, with a positive result for the HPV DNA test, the L1(−)/p16(−) pattern indicates low or no synthesis of HPV oncoprotein, resulting in no alteration of the cell cycle, an early infectious stage, and a possible long duration before progression. Thus, the sequence of the L1/p16 expression status could change in the order of L1(−)/p16(−), L1(+)/p16(−), L1(+)/p16(+), and L1(−)/p16(+) with increasing severity of the cervical lesion (Fig. 4).
Lack of L1 capsid protein may, therefore, reflect abnormalities in transcription-factor pathways that could be responsible for the disturbed basal epithelial cell maturation.17 Moreover, Hasegawa et al20 and McMurray et al2 reported that because L1 capsid protein is also the major target of cellular immune responses, its loss at early stages of the transformation process may lead to ineffective stimulation of immune responses. Therefore, lack of L1 capsid protein may occur to reduce the cellular immune responses, thereby promoting further transformation of immature epithelial cells.
The combination of L1 capsid protein and p16 immunostaining in LBC appears to be useful for an early diagnosis of precancerous lesions, because the L1/p16 expression status may be able to identify individuals at risk of lesion progression and may also be helpful for subsequent follow-up of patients. Clinically, LSIL cases with L1(+)/p16(+) and L1(−)/p16(+) patterns need strict follow-up with colposcopy and biopsy, because p16 positivity is a indicator of dysplastic lesions. LSIL cases with L1(−)/p16(−) pattern can be followed with a longer time interval than L1(+)/p16(−) cases, because the L1(−)/p16(−) pattern means that the lesion is in a latent and nondysplastic state. In contrast, L1(+)/p16(−) cases need follow-up within a short time interval, because this pattern means a productive and/or active form of HPV that may produce a dysplastic lesion in the near future.