Human papillomavirus (HPV) is etiologically involved in most cervical cancers (CCs) and has been identified in approximately 95% of these tumors. ^[1] HPV is also involved in a subset of tumors of the vagina, vulva, penis, anal canal and head and neck. ^[2] More than 200 HPV types have been identified, and 20 HPV types are known to cause cervical cancer. ^[3] Clinically relevant HPV genera such as α-9 and α-7 are the main groups associated not only with invasive CC and its precursor lesion, cervical intraepithelial neoplasia, but also with other cancers associated with this viral infection. HPV 16 and 18 are the most prevalent genotypes of each genus, respectively. ^[4]

Almost all studies, even those using highly sensitive HPV tests, identify a small proportion of CCs negative for HPV. ^[5] Remarkably, HPV infection has shown to be of prognostic significance in carcinomas of the vagina, vulva and head and neck, and in all these anatomical locations, HPV-independent tumors have shown an impaired prognosis compared with HPV-associated carcinomas. This link has also been described in the uterine cervix 12, and the recognition of the biological and clinical relevance of dividing CC into HPV associated and independent was recognized in the last WHO classification. ^[6]

p16 is a cyclin-dependent kinase-4 inhibitor that is expressed in a limited range of normal tissues and tumors. In recent years, immunohistochemistry with p16 antibodies has been used as a diagnostic aid in various scenarios in gynecologic pathology. Diffuse (as opposed to focal) positivity with p16 in the cervix can be regarded as a surrogate marker of the presence of high-risk human papillomavirus (HPV). ^[7]

The immunohistochemical overexpression of p16 has been considered to be a good surrogate for HPV infection. Diffuse and strong p16 staining is consistently present in almost all HPV-associated cancers, whereas HPV-independent tumors are typically p16 negative. Due to this strong association between p16 overexpression and HPV status, in all the above-mentioned anatomical sites, p16-negative tumors behave more aggressively than p16-positive tumors. ^[8]

This is a prospective study consist of 60 patients analyzed had squamous cell carcinoma. Normal and tumour tissue was embedded in paraffin and five-micrometers sections were cut and stained in hematoxylin and eosin. This method made it possible to identify viable, morphologically representative areas of the specimen from which core biopsies were then taken. Three tissue cores with a diameter of 0.2 cm were punched from each specimen and arrayed on a recipient paraffin block. Sections of five micrometers thick were cut from these tissue array blocks and placed on charged poly-lisine-coated slides. These sections were used for immunohistochemistry and hybridization in situ.

HPV RNA-In situ hybridization

In situ hybridization was performed according to the method described by Breitschopf et al. (18) applying probes against HPV6,11 (low risk) and HPV16,18 (high risk). In brief, the tissue sections were deparaffinized, rehydrated in serial dilutions of ethanol, and postfixed in 4% TBS buffered paraformaldehyde. Samples were permeabilized using proteinase K (10 Ìg/ml) at 37ÆC for 30 min. Digestion was stopped by washing the samples in phosphate-buffered saline (pH 7.4). The samples were then dehydrated in serial dilutions of ethanol. Digoxigenine-labeled riboprobes were diluted in hybridization buffer. After sense or antisense probes were applied, the samples were covered with sterile coverslips and placed on a hot plate at 85ÆC for 5 min in order to denaturate the probe. Hybridization was performed overnight at 45ÆC in a sealed humidified chamber containing 50% formamide. Non-specific binding or unbound probes were removed by the following posthybridization washes: 1xSSC/0,1%SDS at room temperature (2x5 min) and 0.2xSSC/0,1%SDS at 60ÆC (2x10 min) followed by RNase digestion at 37ÆC for 30 min. Finally, the sections were washed in TBS containing 0.1% Tween-20.

Hybridization signals were detected using a sheep polyclonal antibody F(ab)2-fragment against Digoxigenine conjugated with horseradish peroxidase (1:300, Boehringer). After washing in Tris-buffered saline Tween-20 (TBST), the slides were incubated in streptavidin-horseradish peroxidase (HRP) complex (diluted 1:100 in trinitrobenzol-TNB) for an additional 30 min. Three washes with TBS were followed by a 15 min application of biotintyramide diluted 1:50 in amplification diluent. Reactive sites were detected with streptavidin-HRP (1:100 in TBS, 30 min, RT). Peroxidase activity was visualized by using diaminobenzidine (DAB) as chromogen. Sense riboprobes served as controls for each tissue section.

p16 Immunohistochemistry.

Immunohistochemistry was performed by using 5 Ìm paraffin sections which were deparaffinized in xylene (three times for five minutes) and rehydrated in decreasing concentrations of ethanol (100%, 96%; two times each for ten minutes) followed by washing in deionised H2O for one minute. To unmask the p16 antigen the slides were covered with 0.01 M sodium citrate buffer (pH 6.0) and placed on a hot plate (95ÆC) for 10 minutes. After cooling down the specimens were rinsed briefly in deionised H2O (three times). The specific primary (mouse monoclonal) antibody was applied in a dilution of 1:50 overnight at 4ÆC.

The reagents I-V used on the second day were supplied in the ImmunoCruz Staining System. Each of these reagents was pre-diluted and ready to use at room temperature. After extensive washing with 0.02M Tris/phosphate buffer (TPBS, pH 7.2) the immunoreactivity was detected with a biotinylated secondary antibody (I) by incubating the specimens for 30 min at room temperature. Slides were rinsed with TPBS for 5 min before a horseradish peroxidase (HRP)streptavidin complex (II) was added for 30 min. Washing in TPBS for 5 min followed. Subsequently colour was developed by using a HRP substrate (mixture of 1.6 ml deionised H2O, 250 Ìl 10x substrate buffer (III), 50 Ìl 50x DAB chromogen (IV) and 50 Ìl 50x peroxidase substrate(V) which was applied on the sections until light brown staining was visible (approx. 10 min). Samples were washed again in deionised H2O, then counterstained with hematoxylin, dehydrated (increasing concentrations of ethanol: 96% and 100%, followed by xylene, each two times for 10 sec) and mounted in DPX. For negative control staining the primary antibodies were omitted. For quantification of the tumour cells with positive staining the CAS200 image analyzer was used and the results were expressed as percentages of positive cells.

Statistics

The data were analyzed using the statistical analysis system (SAS, version 7.5) on an IBM-compatible PC running on Windows. First, they were scanned into a spreadsheet (Microsoft Excel XP); from Excel, they were fed to the statistics program via an ODBC driver (open database connectivity).

The average of the index values was obtained from three single cores. In general, no means or standard deviations were calculated because the parameters investigated did not involve metrical data. Medians, quartiles, minima and maxima were calculated for all parameters. The Mann-Whitney U-test was used for group comparisons.

Table-1: Patient and tumour characteristics

Table-2: Characteristics of tumour

Table-3: p16 expression in relation to HPV status and different HPV types

 In In table 3, HPV types and status in correlation with clinical parameters and expression of p16. Fifty five out of 60 patients with PCV could be evaluated for HPV status. 16 were positive for high-risk HPV and 39 were HPV negative. The majority (11 out of 16, 68.75%) of HPV-positive patients were positive for HPV16. The others were positive for HPV45 (2 patients, 12.5%), HPV18 (2 patient, 12.5%), HPV35 (1 patient), HPV56 (1 patient), and HPV68 (1 patient). Human papillomavirus positivity was significantly correlated with strong p16 expression (p= 0.045). In all, 3 out of the 39 HPV-negative patients were negative for p16 immunostaining, while the remaining 83% showed varying expression: 29 out of 39 (74.4%) showed moderate or strong p16 expression.

This is, to the best of our knowledge, the first research study from our area of the prognostic significance of HPV and p16 in vaginal cancer. Altogether 12 studies were included in the present review. Of seven studies reporting on HPV status as a prognostic factor, the majority found an improved survival for women with HPV- positive tumors. ^[12] For p16 expression status, three out of four studies found an improved survival among women with p16- positive tumors. Most of the studies included small study populations, reflecting the rarity of the disease, and several studies did not adjust for important confounders such as age and tumor stage.

The findings of other review study, with an improved survival for women with HPV- positive vaginal cancer, are in line with results found in similar studies investigating survival after other HPV- related cancers. Within penile, vulvar, anal, and oropharyngeal cancer, HPV is relatively well- established as a prognostic marker with HPV positivity signifying improved prognosis. Our findings are also in agreement with a previous review by Gadducci, they included three studies (of which one was excluded from our review because no formal survival analysis was conducted) and concluded that HPV positivity is associated with improved survival.

Block- type overexpression of the tumor suppressor protein p16 has been established as a prognostic factor in penile, vulvar, anal, and oropharyngeal cancer. ^[13] Our findings are in line with this, indicating that also in vaginal cancer, p16 positivity is a predictor of improved prognosis. Overexpression of p16 is established as a surrogate marker for a transforming HPV infection, which can explain why p16 can be used as a prognostic marker for HPV- related cancers. Studies of HPV- related cancers other than vaginal cancer, including oropharyngeal and anal cancers, have demonstrated that a combination of HPV and p16 testing is a better prognostic marker than using either HPV or p16 separately. Unfortunately, none of the studies included in our systematic review investigated prognosis for these markers combined. ^[14]

Several hypotheses on the reasons for the difference in survival among HPV- related and non- HPV- related cancers have been investigated. In penile cancer, it has been indicated that the viral infection causes an increased immune surveillance, leading to a less aggressive development of the HPV- positive cancers. ^[15] In head- and- neck cancers, it has been shown that HPV positive cancers might possess a lower degree of gross genetic alterations or that the HPV infection in the tumor might influence the molecular profile of the cancer, leading to an increased sensitivity to radiotherapy. ^[16] The same has been proposed for vulvar cancer, but the results are conflicting. With surgical intervention having a limited role in the treatment of vaginal cancer, most patients are treated with external radiotherapy and brachytherapy in combination with concurrent chemotherapy. ^[17] Therefore, a possible higher sensitivity to radiation therapy in HPV- related vaginal cancers could be of great clinical relevance when planning treatment and follow- up strategies as it could potentially be possible to reduce the radiation dose in HPV- positive cancers, thereby minimizing potential side effects.  ^{[18, 19]}

This study suggests that p16 expression is directly proportional to presence of HPV. A high number of SCCVs are related to HPV infection and may be identified by immunohistochemistry for p16. However, because of the limited sample size of existing studies, the current scientific evidence does not support any firm conclusions. Furthermore, studies combining testing for HPV and p16 in vaginal cancer might also prove useful in the prognostication, as has been shown in tonsillar and base of tongue cancer. Such knowledge could potentially contribute to a more personalized and targeted treatment for vaginal cancer, as is being investigated in head and neck cancer, thereby maximizing treatment effectiveness while minimizing side effects and long- term treatment sequelae.

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