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  Table of Contents  
Year : 2015  |  Volume : 52  |  Issue : 3  |  Page : 270-275

Conventional cytology, visual tests and evaluation of P16INK4A as a biomarker in cervical intraepithelial neoplasia

1 Department of Obstetrics and Gynecology, The Oxford Medical College, Hospital and Research Center, Bengaluru, India
2 Department of Obstetrics and Gynecology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India
3 Department of Pathology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India

Date of Web Publication18-Feb-2016

Correspondence Address:
S Rajaram
Department of Obstetrics and Gynecology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0019-509X.176729

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 » Abstract 

Objectives: (1) To detect cervical intraepithelial neoplasia (CIN) using Papanicolaou test (PAP test), visual tests (visual inspection after the application of acetic acid [VIA], visual inspection after the application of Lugol's iodine [VILI]), colposcopy, and biopsy. (2) To study the biomarker p16INK4A expression by immunostaining. Materials And Methods: Experimental study was conducted from November 2009 to April 2011. 1500 women were screened for cancer cervix using conventional PAP test, VIA, and VILI. Sensitivity, specificity, positive, and negative predictive values of these tests were calculated individually, sequentially, and in parallel. Women having positive results underwent colposcopy and biopsy if required. p16INK4Aexpression in biopsy samples was studied using immunohistochemistry. Results: All test positive cases (n = 235) underwent colposcopy. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of PAP with atypical squamous cells of undetermined significance (ASCUS) as cut-off was 40%, 99.25%, 35.25%, and 99.39%; VIA was 60%, 93.06%, 8.03%, and 99.56% and VILI was 80%, 86.06%, 5.4%, and 99.76%, respectively. When PAP, VIA, and VILI were used in parallel sensitivity, specificity, PPV, and NPV improved to 100%, 85.18%, 6.38%, and 100%, respectively. Colposcopic abnormalities were detected in 83 and biopsy proven CIN in 15. p16INK4A expression was seen in eight of 15 CIN cases. Conclusions:(1) PAP test and visual techniques are complementary. (2) p16INK4Aexpression was seen in majority of CIN 2 lesions suggesting a higher grade lesion.

Keywords: Cervical intraepithelial neoplasia, p16INK4A, Papanicolaou smear, visual inspection after the application of acetic acid, visual inspection after the application of Lugol's iodine

How to cite this article:
Kava S, Rajaram S, Arora V K, Goel N, Aggarwal S, Mehta S. Conventional cytology, visual tests and evaluation of P16INK4A as a biomarker in cervical intraepithelial neoplasia. Indian J Cancer 2015;52:270-5

How to cite this URL:
Kava S, Rajaram S, Arora V K, Goel N, Aggarwal S, Mehta S. Conventional cytology, visual tests and evaluation of P16INK4A as a biomarker in cervical intraepithelial neoplasia. Indian J Cancer [serial online] 2015 [cited 2022 May 21];52:270-5. Available from:

 » Introduction Top

Cervical carcinoma is one of the most common cancers among women worldwide and second only to breast cancer in incidence and mortality.[1] Current cervical cancer screening tests include Papanicolaou (PAP) test; visual inspection after the application of acetic acid (VIA); visual inspection after the application of Lugol's iodine (VILI); and Human Papillomavirus (HPV)-DNA testing.

The PAP test has decreased cervical cancer incidence substantially in the countries with regular screening programs.[2] However, the suboptimal reproducibility of the PAP test has to be compensated by frequent retesting.

In developing countries, simple, inexpensive visual-based screening programs can be applied to a large population.[3],[4] VIA is a valuable screening tool in low-resource settings; however, it has low sensitivity and specificity.[5]

Randomized controlled trials published recently have demonstrated that HPV testing can be efficiently integrated into primary screening, either as an adjunct to cytology or as a sole primary test.[6],[7] A single HPV DNA test although confirms infection by the virus, it does not discriminate between transient and persistent infection.[8] The discrimination between the two types of infection is crucial as persistent infections can progress to cervical neoplasia.[9]

Thus, cervical screening would benefit from a test based on a disease-specific biomarker that identifies high-grade lesions. Such a marker would be useful if it could also indicate the presence of early pre-cancerous lesions that have a high risk of progression to cancer. One such potential biomarker is p16INK4A. Some preliminary studies suggest that p16INK4A positive low-grade lesions showed a greater frequency of progression than p16INK4A negative lesions.[10]

p16INK4A is a gene that is expressed by host cells in response to infection, and is not normally expressed in non-transformed cells.[11] In cervical carcinomas, the viral DNA of hrHPV (i.e., HPV 16 and 18) is integrated into the host genome at the E2 region, resulting in overexpression of the oncoproteins E6 and E7. The E6 binds with the host p53 tumor suppressor gene protein product and degrades it, thereby disrupting its cell regulatory role.[10] The E7 binds to and inactivates the tumor suppressor retinoblastoma protein (pRB) that inhibits the progression of cells into the S-phase. Consequently, loss of pRB function should result in the release of the p16INK4A gene from negative transcriptional feedback control in the respective cells and in marked overexpression of p16INK4A gene product.

Several properties of p16INK4A make this protein a promising biomarker for HPV-related cancers; expression is directly linked to the HPV oncogene action, since continuous expression of E7 is necessary to maintain a malignant phenotype in HPV-associated cancer.[12] The expression of p16INK4A seems to be independent of the HPV type causing the oncogenic infection, obviating the need to detect different HPV types in DNA and RNA assays. Thus, the objectives of this study were to evaluate the time tested PAP test with cost-effective visual-based tests in the diagnosis of cervical intraepithelial neoplasia (CIN) and to study the biomarker p16INK4A in biopsy-positive cases.

 » Materials and Methods Top

This was an experimental study, conducted at a tertiary care hospital, India, between November 2009 and April 2011. 1500 women were screened for cancer cervix using conventional PAP test, VIA, and VILI. Women who had positive results in any of the above tests underwent colposcopy. Biopsy was taken from the abnormal areas detected at colposcopy. p16INK4A expression in the biopsy samples was studied using immunohistochemistry (IHC). A written informed consent was taken from all patients.

Inclusion criteria: Sexually active women in the age group of 20-50 years.

Exclusion criteria: Women with an obvious cervical growth, acute cervicitis, prior surgery on the cervix, post-menopausal, and pregnant women.

A detailed history including marital, sexual, obstetric, menstrual, and personal history was taken. This was followed by a detailed examination which included general physical and systemic examinations. Prior to per vaginal examination, following screening tests were performed.

Screening tests

Papanicolaou test

Conventional PAP test was performed and results were reported according to the Bethesda 2001 System of reporting cervico-vaginal smears. A PAP test of atypical squamous cells of undetermined significance (ASCUS) and above was taken as positive.


5% acetic acid was applied liberally using a cotton swab soaked in acetic acid over the exposed cervix. The findings were read after 1 min of application. After carefully recording the findings, Lugol'siodine was applied with a cotton swab on the cervix. The cervix was examined for any iodine non-uptake areas. The outcome of VIA and VILI was interpreted as per the international agency for research on cancer guidelines.[13]


All women having positive result through either PAP test, VIA, or VILI underwent colposcopy. In dorsal position, cervix was exposed and using the colposcope, the transformation zone of the cervix was visualized for any abnormal areas. The abnormal areas included acetowhitening, atypical vessels, punctations, mosaic pattern, and iodine negativity. A biopsy was then taken from these abnormal areas.


A colposcopy directed single/multiple punch biopsy of the suspicious areas was taken and transported to the pathology lab in 10% formalin. Results of the biopsy were reported as (a) Chronic cervicitis (b) CIN1

(c) CIN2 (d) CIN3 (e) Microinvasive cancer, (f) Invasive cancer.

Immunostaining for p16INK4A

For IHC, representative sections from paraffin block of cervical biopsy were taken on poly-L-lysinate-coated slides. Antigen was retrieved by microwave heat method using citric acid buffer at pH 6.0. IHC was performed using standard technique by Streptavidin biotin system using DAB as chromogen.

Patterns of p16INK4A were categorized as positive or negative. Positive p16INK4A was defined as presence of nuclear staining or diffuse cytoplasmic staining. Negative p16INK4A was defined as absence of any staining or presence of focal cytoplasmic staining. Specifically, diffuse staining was defined as a continuous staining of cells in the basal and parabasal layers (with or without staining of superficial squamous cell layers). Focal staining was defined as non-continuous staining of isolated cells or small cell clusters, usually not located in the basal and parabasal layers. Degree of nuclear p16INK4A expression in positive cases was expressed as percentage of positive cells. Strength of positivity was compared to a positive control which could be run with each batch of immunostaining. The strength of reaction was graded as 1+, 2+, and 3+.

Positive control: Squamous cell carcinoma of esophagus

Negative control: Obtained by omitting the application of primary antibodies during the immunostaining process and using Tris Buffer Saline instead.

Statistical analysis

Sensitivity, specificity, positive predictive value, and negative predictive value were calculated using 4 × 4 tables. The sensitivity of PAP test was calculated using ASCUS (atypical squamous cells of undetermined significance) as cut-off. For VIA, positive results were those with distinct well-defined dense acetowhite areas on transformation zone, whereas VILI positives were those with dense, thick, bright, mustard yellow iodine non-uptake areas. The gold standard was biopsy positive CIN 1, 2, or 3 lesions, carcinoma in situ, or cervical cancer. Sensitivity, specificity, PPV, and NPV of each of the screening tests were calculated individually, in parallel (result was taken as positive when either test was positive) and sequentially (result was taken as positive when both tests were positive).

 » Results Top

Demographic profile of the study population is given in [Table 1].
Table 1: Demographic profile of study population

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Screening tests

PAP test: Positive result was seen in 17 out of 1500 screened i.e., ASCUS was seen in four cases, low gade squamous intraepithelial lesion (LSIL) in 11 cases, and high grade squamous intraepithelial lesion (HSIL) in two cases. Thus, the percentage of positive PAP smears was 1.13%. 100 women were found to have bacterial vaginosis on PAP test and two were found to have Trichomonas vaginalis. Two were found to have granulomatous cervicitis suggesting tuberculosis. The sensitivity of PAP test with ASCUS as cut-off was 40%, specificity, 99.25%; PPV, 35.29%; and NPV, 99.39%.

VIA and VILI: 112 women out of 1500 i.e., 7.46% were VIA positive. 219 women out of 1500 i.e., 14.6% were VILI positive. Number of women who were either VIA or VILI positive was 226, i.e., 15.06%. Number of women who were positive for both VIA and VILI was 104 i.e., 6.93%.

The sensitivity of VIA was 60%, specificity 93.06%, PPV 8.03%, and NPV 99.56%.

The sensitivity of VILI was 80%, specificity 86.06%, PPV 5.4%, and NPV 99.76%.

When VIA and VILI were used in parallel, the sensitivity was 86.66%, specificity 85.65%, PPV 5.75%, and NPV 99.84%.

PAP, VIA/VILI in parallel: When PAP and VIA/VILI were used in parallel, the sensitivity was 100%, specificity 85.18%, PPV 6.38%, and NPV 100%. Thus, sensitivity increases with slight loss of specificity [Table 2].
Table 2: Accuracy of screening tests individually and in combination for the detection of cervical intraepithelial neoplasia/cancer

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Colposcopy was performed in 235 women who were either VIA, VILI, or PAP positive. Abnormal areas were detected in 83 of these 235 women and were biopsied. Histopathology showed chronic cervicitis (n = 68), CIN-1 (n = 6), CIN-2 (n = 5), CIN-3 (n = 3), and invasive carcinoma (n = 1).

p16INK4A: One out of the six samples of CIN-1 was damaged during the staining process. p16INK4A was negative in four of the five CIN-1 biopsy samples that were stained. One CIN-1 biopsy showed positive p16INK4A; with no cytoplasmic staining, 9% nuclear staining, and strength of positivity 1+ [Figure 1].
Figure 1:(a) H and E, ×200: CIN I, (b) p16INK4A Immunoreactivity seen in lower 1/3 epithelium with some nuclei showing positivity in middle 1/3

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p16INK4A was positive in four of the five CIN-2 biopsy samples that were stained, with diffuse cytoplasmic staining. Nuclear staining in these cases ranged from 16% to 27% and strength of positivity was 2+ in three cases and 1+ to 2+ in one case. One CIN-2 biopsy sample showed negative p16INK4A staining, with only focal cytoplasmic staining and negative nuclear staining. Positivity of p16INK4A in CIN-2 was 80% [Figure 2].
Figure 2:(a) H and E, ×200: CIN II, (b) p16INK4A Immunoreactivity: Nuclear positivity present throughout the epithelium with nuclei showing intense positivity in focal areas

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p16INK4A was positive in two of the three CIN-3 biopsy samples that were stained, with diffuse cytoplasmic staining. Nuclear staining in these two cases was 32% and 64% and strength of positivity was 2+ and 3+, respectively. One CIN-3 biopsy sample showed negative p16INK4A stain [Figure 3].
Figure 3: CIN III: P16INK4A Immunoreactivity seen in nuclei and cytoplasm

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p16INK4A was positive in one case of squamous cell carcinoma that was detected in this study, with diffuse cytoplasmic staining, 90% nuclear positivity, and strength of positivity 3+ [Figure 4].
Figure 4: Squamous cell carcinoma. P16INK4A Immunohistochemistry showing intense nuclear and cytoplasmic positivity

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p16INK4A expression in the various cases of CIN/Squamous cell carcinoma (SCC) has been shown in [Table 3].
Table 3: Cytoplasmic and nuclear p16INK4A expression in cervical intraepithelial neoplasia and squamous cell carcinoma cases

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p16INK4A immunostaining was also performed in 10 histopathologically proven cases of chronic cervicitis. p16INK4A was negative in eight of the 10 cases [Figure 5]. In the two cases of chronic cervicitis in which p16INK4A was positive, there was diffuse cytoplasmic staining and no nuclear staining.
Figure 5: Immunohistochemistry ×20 negative p16INK4A in chronic cervicitis

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 » Discussion Top

In our study, overall positivity of PAP test with ASCUS as cut-off was 1.13% which was lower than that reported in previous studies such as that by Shastri et al.,[14] where the positivity was 2.7% and in the study by Sarian et al.,[15] where the positivity was 2.2%.

In this study, the sensitivity of PAP test was 40%, specificity 99.25%, PPV 35.29%, and NPV 99.39%. A wide range of sensitivities and specificities have been reported for cervical cytology. In the eight cross-sectional studies from developing countries,[4],[16],[17],[18],[19],[20],[21],[22] the reported sensitivity varied from 28.9% to 76.9%, the specificity varied between 90.2% and 98.4%. On the basis of these reviews, the mean sensitivity and specificity for cytology was 59% and 75%, respectively. Thus, a single PAP test is not useful in diagnosis of pre-invasive/invasive cancer but repeated testing as is done in developed countries improves test performance.

In this study, overall positivity of VIA was 7.46% and that of VILI was 14.6%. When VIA and VILI were used together, the positivity was 15.06%. These figures are comparable to previous studies by Sankaranarayanan et al.,[23] where VIA positivity was 8.3% and VILI positivity was 11.4% and in the study by Shastri et al.,[14] VIA and VILI positivity were 12.7% and 17%, respectively.

The sensitivity of VIA in this study was 60%, specificity 93.06%, PPV 8.03%, and NPV 99.56%. The sensitivity of VILI was 80%, specificity 86.06%, PPV 5.4%, and NPV 99.76%. These findings are consistent with the observations by Sankaranarayanan et al.,[23] In their study, sensitivity of VIA was 71.0%, specificity 93.3%, PPV 11.7%, and NPV 99.6%. For VILI, the sensitivity was 75.8%, specificity 90.6%, PPV 9.2%, and NPV 99.7%. Similar results were reported byShastri et al.[14]

As is seen in our study, VILI had higher sensitivity and lower specificity than VIA. Sankaranarayanan et al. also reported similar findings in their study.[23] VILI was a better visual test because the “yellow” color of the abnormal areas was more readily recognized than the acetowhite area by VIA, which takes time to appear.

It is also important to note that in our study, VIA alone failed to identify four cases of CIN which were picked up by VILI and VILI missed one case of CIN which was identified by VIA. Therefore, parallel testing is better than testing in sequence as was reported by Shastri et al.[14]

Also in comparison to PAP test, VIA and VILI individually and when used together had better sensitivity. In this study, PAP test missed nine cases of CIN which were identified by visual techniques, whereas VIA/VILI missed two cases of CIN which were identified by PAP. Thus, PAP and visual techniques are complementary and should be used together (parallel testing) to improve sensitivity and specificity. However, given the resource-poor conditions in developing countries, this suggestion may not be ideal in terms of cost and logistics.

Our results indicate that visual screening tests are promising methods for the early detection of cervical cancer. A logistic advantage of the visual tests is the immediate availability of results, which allows diagnostic investigations and treatment in the same sitting as screening. However, the lower specificity implies that a large number of women will need to be subjected to investigations and/or treatment.

When the screening tests were used in combination (sequential or parallel), the performance in terms of sensitivity improved. VIA and VILI showed better sensitivity when used in parallel as compared to when used individually or in sequence. This, however, reduced the specificity slightly. Likewise, when PAP was used in parallel with either VIA or VILI, the sensitivity improved with marginal decrease in specificity. If one assumes that detecting true-positive cases is a priority for screening programs, our results suggest that parallel testing with a visual-based test and PAP test may be worth considering. It remains to be established whether the additional costs incurred using a second test can be compensated by increasing the interval between screening rounds. These findings are in alignment with the observations of Shastri et al.[14] and Sarian et al.[15]

In this study, it was seen that colposcopy findings were normal in 59.82% of the VIA-positive cases and 63.47% of the VILI-positive cases. More importantly, 40% (n = 6) of the women considered having a normal cervix on VIA actually had a significant abnormality detected at colposcopy. Four out of these six cases were picked up by VILI and the remaining two by PAP test. This finding raises a question against the time honored teaching that one could do away with Lugol's iodine application if acetic acid application shows negative findings. Similar findings were also reported by Sarian et al.[15] Like all studies where less than 100% of the women are examined by the test used as the gold standard, this study suffers from verification bias. Only those women who were referred to colposcopy on the basis of a positive screening test (PAP, VIA, and VILI) had the chance to be examined by the agreed gold standard (i.e., cervical biopsy). Thus, some women with true cervical abnormalities who tested negative with VIA, VILI, and PAP smear might still remain undisclosed, resulting in over-optimistic performance indicators of the screening tests used.

p16INK4A: In this study, it was seen that p16INK4A was positive in 20% (n = 1) of the CIN-1 cases, 80% (n = 4) of CIN-2 cases, and 66.66% (n = 2) of CIN 3 cases. In the single CIN-1-positive case, there was 9% nuclear positivity and strength of positivity was 1+. Whereas, in the CIN-2 cases which were p16INK4A positive, there was diffuse cytoplasmic staining, nuclear positivity varied from 16% to 27% and strength of positivity was 2+ in almost all cases. Among the CIN-3 cases, one was found to be p16INK4A negative. Out of the other two which were p16INK4A positive, both showed diffuse cytoplasmic staining, but they differed in their nuclear staining with one showing 32% and the other 64% nuclear positivity, strength of positivity was 2+ in the first one and 3+ in the second. The single case with squamous cell carcinoma showed diffuse nuclear staining, with 90% nuclear positivity and strength of positivity 3+.

A serious disadvantage of the grading by conventional histopathology is that the three distinct grades used in CIN can easily give a faulty impression of a solidified sculpture, as if CIN were a static event, whereas in reality, a CIN lesion is a dynamic process that can progress and persist but also regress. Compounding the above are the well-known issues of intra- and inter-observer reproducibility, which, for grading of CIN, is far from perfect.[24] It is also difficult to distinguish CIN reliably from non-neoplastic lesions, resulting in either overtreatment or under treatment. These points emphasize the need for adjuvant methods to interpret the actual morphological impression of a CIN lesion in dynamic terms rather than in static morphological grades.

Without doubt, p16INK4A is the most widely available, robust, stable, and strong predictive biomarker currently available for prognosticating CIN lesions. p16INK4A overexpression has been demonstrated in the vast majority of cervical pre-cancers and cancers while in normal tissue, p16INK4A expression is found only rarely.[11] This was also shown in our study where among the 10 morphologically proven cases of chronic cervicitis p16INK4A, only two showed diffuse cytoplasmic staining and no nuclear staining.

Until now, despite several proposed evaluation strategies of p16INK4A in both cytology and histology, there is no general consensus for establishing threshold values above which a sample becomes “p16INK4A positive.”[25] Klaes et al.[11] proposed a system which scored the distribution of p16 positivity on a semi-quantitative scale as follows: Negative (<1% of the cells were positive), sporadic (isolated cells were positive, but <5%), focal (small cell clusters, but <25% of the cells were positive), and diffuse (>25% of cells stained positive).

In this study, patterns of p16INK4A staining defined was a modification of that of Klaes et al.,[11] where we reported cases as negative, focal or diffuse, and also graded nuclear positivity as a percentage. Recently, the American Society of Colposcopists and Cervical Pathology (ASCCP) in its College of American Pathologists (CAP)-ASCCP Lower Anogenital Squamous Terminology (LAST) Standardization project recommended p16 as the only biomarker to be used to distinguish pre-cancer from its mimics. Diffuse, block positive p16 staining supports categorization as a pre-invasive lesion. It is particularly useful in CIN2 lesions to distinguish a low-grade lesion from one that is pre-cancerous and therefore requiring treatment.[26]

 » References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3]

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