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Table of Contents
Year : 2020  |  Volume : 17  |  Issue : 4  |  Page : 240-245

Cytological evaluation of p16Ink4a in precancerous lesions of the cervix: Conventional papanicolaou smears

1 Department of Pathology, Indraprastha Apollo Hospital, New Delhi, India
2 Department of Pathology, University College of Medical Sciences and GTB Hospital, New Delhi, India

Date of Submission23-Mar-2020
Date of Acceptance28-Oct-2020
Date of Web Publication28-Dec-2020

Correspondence Address:
Dr. Seema Singhal
Department of Pathology, Indraprastha Apollo Hospital, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/am.am_17_20

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Background: Cancer of the uterine cervix is the second most common cancer among women worldwide. More than three fourths of these patients are diagnosed at advanced stages, leading to poor prospects of long term survival and cure. Introduction of Papanicolaou (Pap test) cytological screening for cervical precancerous lesions has significantly reduced the incidence and mortality of cervical cancer. Human papillomavirus (HPV) DNA testing has been recently demonstrated to be efficient to be integrated into screening programs, so it can be used to triage women with equivocal cytological abnormalities and also identifies women at risk of residual or recurrent disease after treatment of cervical intraepithelial neoplasia. However, it fails into triage low grade lesions. HPV DNA test confirms infection by the virus, present in 99% of cases. However, it does not discriminate between transient and persistent infection as it is crucial because it is the persistent infection which progresses to neoplasia. Histological assessment of cervical biopsy that is considered as “gold standard” can be hampered by intra and inter observer variability. A more specific triage marker is required to identify women who would need colposcopy. Hence, p16INK4a has emerged as a new diagnostic and prognostic biomarker. Aims and objectives: This study was conducted to evaluate the utility of staining of p16INK4a on conventional Pap smears and its comparison with corresponding biopsies. Material and Methods: 50 cases of conventional Papanicolaou stained cervical smears cases were randomly selected from the archive of cytopathology laboratory. The cervical smears were re-evaluated for adequacy, preservation of cells, cytomorphology and various lesions were categorized according to The Bethesda system 2001 (TBS) classification. Consecutive such cases were selected for which both cytological and histological material were available. Immunostaining of cervical cytological specimens for p16 was performed using monoclonal murine antibody, clone 16P04, JC2. Ready to use antibody was used for immunostaining as per manufacturer's protocol. Results: Out of the 50 smears of preneoplastic and invasive lesions, ASCUS was seen in 5 cases, LSIL in 10 cases, HSIL in 20 cases, SCC in 20 cases and AGUS in 5 cases. Of the 5 cases of ASCUS, histopathology showed 3 chronic cervicitis and 1 each in CIN-1 and CIN-2. Histopathological diagnosis of 10 cases of LSIL showed 6 CIN-1 and 2 each in CIN-2 and chronic cervicitis. Similarly, for 10 cases of HSIL, 3 were CIN-2, 5 CIN-3 and 2 SCC. All 20 cases of carcinoma showed SCC while 5 cases of AGUS showed 4 chronic cervicitis and one adenocarcinoma. Immunohistochemical staining of p16INK4A showed weak positivity in 3 cases of chronic cervicitis and 4 cases of CIN-1. In CIN-2 cases, 66.67% showed strong positivity, CIN-3 showed 80% while both carcinoma and adenocarcinoma showed 100% strong positivity. The sensitivity of immunohistochemical staining of p16INK4A was 77.5%, specificity was 100%, PPV 100% and NPV 52.6%. The relation between histological and cytological immunostaining of p16INK4A. Conclusion: p16INK4A is a reliable marker for dysplastic squamous and glandular cervical cells both in tissue sections and in cervical smears. p16 immunostaining can be easily performed on CPS, and there is high concordance of positivity on smears and tissue sections.

Keywords: Cervical cancer, conventional Papanicolaou, p16INK4a immunocytochemistry

How to cite this article:
Singhal S, Arora VK. Cytological evaluation of p16Ink4a in precancerous lesions of the cervix: Conventional papanicolaou smears. Apollo Med 2020;17:240-5

How to cite this URL:
Singhal S, Arora VK. Cytological evaluation of p16Ink4a in precancerous lesions of the cervix: Conventional papanicolaou smears. Apollo Med [serial online] 2020 [cited 2021 May 14];17:240-5. Available from: https://www.apollomedicine.org/text.asp?2020/17/4/240/303608

  Introduction Top

Nowadays, research is focused on the biomarkers that implicate the persistence of human papillomavirus (HPV) DNA and progression of disease. Expression of these biomarkers is an indirect evidence of HPV DNA integration in the host cell genome. p16INK4a has been identified as a marker for transforming HPV infections.

A reciprocal relationship between p16INK4a and pRb expression has been seen, suggesting the presence of a negative feedback loop allowing pRb to limit the concentration of p16INK4a. Over time, P16 accumulates in the nucleus and cytoplasm of affected cells and can be detected by immunostaining.[1]

Immunoreactivity of p16 has been assessed on cytological specimens[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25],[26] also with histological correlation[2],[3],[4],[11],[15],[17] and only on histological samples.[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36],[37],[38],[39],[40],[41],[42],[43],[44],[45],[46],[47],[48],[49],[50],[51],[52],[53],[54],[55] These studies provide fair evidence that p16Ink4a immunostaining correlates with the severity of cytological/histological abnormalities. Most of the cytological studies have been done on liquid-based specimens. Only a few studies have been done on conventional smears.

Conventional Papanicolaou smears (CPSs) are the most common mode of gynecological screening and diagnostics. Since p16INK4a has emerged as a new promising biomarker for diagnostic and prognostic use in cervical precancerous lesions, a retrospective study was designed with the following.

Aims and objectives using CPS:

  • To study the expression of p16INK4a in various premalignant lesions and cervical carcinomas on the cervical smears
  • To correlate its expression in corresponding biopsies or resection specimens
  • To study the usefulness of its expression in distinguishing low-grade squamous intraepithelial lesion (LSIL) and reactive changes.

  Materials and Methods Top

Conventional Pap-stained cervical smears of 50 cases reported during the last 2 years with abnormal uterine cervix and their corresponding biopsies were retrieved from archival material. All the cases with both cytological and histological material were included in the study.

The cervical smears were re-evaluated for adequacy, preservation of cells, and cytomorphology, and various lesions were categorized according to The Bethesda System 2001 Classification. Abnormal cell clusters/cells in each case were photographed along with documenting both the axis on the microscopic stage before immunostaining was carried out on the same smears. The technique of immunochemistry for p16INK4a was standardized using p16 mouse monoclonal antibody, clone 16P04, JC2 ready-to-use antibody.

Pap cytological smears were left in xylene overnight for removal of the coverslip. The smears were destained briefly in 1% acid alcohol. Antigen retrieval was done in microwave using 0.01 M sodium citrate buffer for 10 min at 800 W. Immunocytochemistry was performed as per the standard protocol.

The intensity of positive staining on the smears was graded as (Klaes).

  • Negative (−): no stain
  • Weakly or sporadically positive (+): less than 50% of abnormal cell clusters or atypical cells showing immunoreactivity [Figure 1]
  • Strongly positive (++): more than 50% of cell clusters or atypical cells showing immunoreactivity [Figure 2] and [Figure 3].
Figure 1: (a) A cluster of metaplastic cells with few cells showing high grade changes (Pap, ×400). (b) p16Ink4a immunocytochemistry showing intense nuclear and cytoplasmic positivity in <50% cells showing high grade changes. The metaplastic cells are negative for the immunostain

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Figure 2: (a) High-grade intraepithelial lesion (Pap, ×400). (b) p16Ink4a immunocytochemistry showing intense nuclear and cytoplasmic positivity in >50% cells showing high grade changes

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Figure 3: (a) Keratinizing squamous cell carcinoma; single malignant dyscohesive cells (PAP, ×400). (b) p16Ink4a Immunocytochemistry showing intense nuclear positivity in >50% cells

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Histopathological specimens were reviewed and classified as negative, cervical intraepithelial neoplasia 1 (CIN1), CIN2, and CIN3 according to the morphological criteria described (Klaes).

Histopathological blocks were retrieved, and fresh multiple sections were taken on lysine-coated slides. Immunohistochemistry was done as per the standard protocol using the same antibody.

Scoring of p16Ink4a immunostaining on biopsies was done quantitatively and qualitatively (Klaes).

Qualitative and Quantitative

  • Negative staining: When there was the presence of focal cytoplasmic or only cytoplasmic staining

There might be the presence of focal glandular cell or few metaplastic cells giving cytopasmic positivity which was considered as negative.

  • Positive staining: Diffuse positivity or presence of both cytoplasmic and nuclear positivity.

Quantitative analysis

  • Negative, <1% of the cells were positive
  • Sporadic, isolated cells were positive but constituted <5% of cells
  • Focal, small clusters of positive cells, constituting <25% of cells
  • Diffuse, >25% of cells were positive.

Strong nuclear with or without cytoplasmic staining was considered as a positive reaction. Percentage of stained neoplastic cells was evaluated. Four microphotographs of the four best large magnification fields (×400) were taken with a light microscope and printed on a paper. Each photograph was divided into quadrants, and a manual cell count was performed to obtain the percentage.

  Results Top

Of the 50 smears containing preneoplastic and invasive lesion, five cases were atypical squamous cells of undetermined significance (ASCUS), 10 LSIL, 10 hIL, 20 squamous cell carcinoma (SCC), and 5 as atypical glandular cells of undetermined significance (AGUS). Follow-up biopsies of these cases revealed a total of 9 cases of chronic cervicitis, 7 cases of CIN1, 6 cases of CIN2, 5 cases of CIN3, 22 cases of SCC, and 1 case of adenocarcinoma [Table 1].
Table 1: Cyto-histological correlation (n = 50)

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The p16 immunocytochemical stain was reactive in 40 (80%) of 50 smears: either weakly/sporadic (9 cases, 18%) or strongly positive (31 cases 62%). Ten smears (20%) were negative for staining. These included 3 cases of ASCUS, 4 cases of LSIL, and 3 cases of AGUS [Table 2]. The ssensitivity of p16INK4a immunostaining on smears was 75.6%, specificity was 100%, negative predictive value (NPV) was 47.4% while positive predictive value (PPV) was 100% (only strong positivity was taken as true positive for statistical analysis).
Table 2: p16INK4a expression in precursor cervical cancer lesions and carcinoma (n = 50)

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Ten cases of negative for intraepithelial malignancy were taken as control. None of the control cases showed positivity.

Immunohistochemically, 3 cases of chronic cervicitis (total 9) and 4 cases of CIN1 (total 7) showed weak positivity, while rest of them were negative. Of the 6 cases of CIN2, 4 showed strong positivity, 1 had weak positivity, while 1 case was negative. Of CIN3 (5 cases), 4 were strongly positive while 1 was weakly positive. All 22 cases of carcinoma and 1 case of adenocarcinoma were strongly positive [Table 3].
Table 3: p16INK4a expression in precursor lesions and carcinoma on biopsy specimens (n = 50)

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Two cases of ASCUS category (total 5) were positive for p16 immunostaining cytologically. Histologically, the 5 ASCUS cases were 3 chronic cervicitis, 1 CIN1, and 2 CIN2. Both cases of CIN2 were showing weak positivity on smears while rest was negative.

Of 10 LSIL cases, 4 cases were negative for staining 4 showed weak immunopositivity while 2 were strongly positive. Histologically, 6 of them were CIN1, 2 were CIN2, and 2 were CIN3. Two cases of CIN1 and 2 cases of CIN2 showed weak positivity, while the 2 cases of CIN3 were strongly positive. Four cases of CIN1 were negative [Table 4]. Sensitivity of p16INK4a immunostaining on sections was 77.5%, specificity was 100%, NPV was 52.6%, while PPV was 100%.
Table 4: p16INK4a expression in precursor lesions and carcinoma on biopsy specimens and conventional papanicolaou smears

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  Discussion Top

Pap staining of the cervical smears is an inexpensive worldwide used technique used for cervical screening. Despite the success of this screening program, the reliability of conventional cervical cytology and histology is debated. It has some limitations. The false-negative rate for cervical premalignant lesions and cervical cancer is between 15 and 50% while 30% are false positives. The failure of the Pap test to eradicate a potentially preventable disease characterizes the limitations of current cervical cancer screening programs and emphasizes the need for more sensitive and specific detection methods. This concern underscores the need for improved screening technologies.

There are several studies performed on cytological specimens[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36],[37],[38] mostly are being performed on liquid-based cytology. Conventional Pap-stained smears though most prevalently used in developing countries for screening have been utilized in only few studies.

Since Pap test is the more commonly used screening test in developing countries in comparison to liquid-based test, this study was conducted to evaluate the applicability and usefulness of the potent biomarker p16INK4a in predicting behavior of preneoplastic cervical lesions using CPS. Corresponding tissue biopsies from the same cases were then taken out and evaluated morphologically and immunocytologically with same antibody.

P16 is a nuclear protein, and hence, immunohistochemistry should show nuclear staining. However, in dysplasias, both nuclear and cytoplasmic staining with p16INK4a is observed possibly because of posttranscriptional modification or overproduction of p16 protein, forcing its transfer into the cytoplasm.

In our study, diffuse staining both nuclear and cytoplasmic involving more than 25% cells was taken as positive for statistical analysis. On smears also the criterion of strong positivity was taken for statistical analysis. Statistically, p16INK4a immunostaining on central venous pressure (CVP) and on tissue sections is comparable with immunohistochemistry having bit higher sensitivity and NPV (Histopathology sensitivity: 77.5%, NPV: 52.6%. cyto sensitivity: 75.6%, NPV: 47.4%). However, specificity and PPV of both came out to be 100% in this study. P value was significant in both cases (<0.001).

On smears, weak positivity was seen in chronic cervicitis, CIN1, and CIN2. As the lesions progressed, so did the intensity of staining.

Cytologically, there was complete concordance between immunostaining in carcinoma cases on smears and tissue sections.

Two cases of HSIL stained weakly on smears while these were CIN2 on cytology. CIN2 lesion showed variable staining.

In the LSIL category, only 2 cases showed strong positivity and both of these were CIN3.

CIN1 and CIN2 lesions indicate the epigenetic phase of HPV. Both of these are reversible and usually do not progress and do not need a long follow-up. However, both of them would be HPV positive and might put a clinician in the dilemma. p16INK4a immunostaining triages these cases at this point because p16INK4a protein expression increases only when HPV is integrated into the host genome, and there is risk of disease progression. p16INK4a protein localization is thought to be nuclear. The presence of p16INK4a in the cytoplasm may result from a type of posttranscriptional modification or, more simply, overproduction of the protein may force its transfer into the cytoplasm. These findings clearly support previous studies confirming the hypothesis that p16INK4a is overexpressed in dysplastic cells of the cervix.

Criterion of choosing strong positivity as true positivity on smears might be significant. All three cases of CIN3 were correctly picked up even when two of them were morphologically LSIL.

This study however comprises a small group of patients, but it focuses on the major aspect of the clinical problem that is to categorize various intraepithelial lesions and carcinoma on CPS with the advent of p16INK4a immunostaining. A larger study comprising more number of ASCUS and LSIL cases might be helpful.

  Conclusion Top

p16INK4A is a reliable marker for dysplastic squamous and glandular cervical cells both in tissue sections and in cervical smears.

p16 immunostaining can be easily performed on CPS, and there is high concordance of positivity on smears and tissue sections. Strong positivity on smears is significant and often indicates CIN3 or further.

p16 immunostaining may be used on CVP for triaging ASCUS, LSIL, and HSIL based on the degree of positivity.

Financial support and sponsorship

Project was funded by CSIR.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4]


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