The authors have declared that no competing interests exist.
P16INK4a and Ki
A retrospective cross-sectional study of 153 cases of cervical biopsies diagnosed as CIN and benign cervical lesions between 2006 and 2013 at the University College Hospital, Ibadan, Nigeria. Slides and tissue blocks of all the selected cases were retrieved and classified using the 2003 WHO classification for intraepithelial and benign cervical lesions and were stained with p16INK4a and Ki-67 immunohistochemical stains following heat-induced antigen retrieval. Results were evaluated and compared with histologic diagnosis.
Cases were classified as chronic cervicitis (12.3%), squamous Metaplasia (0.7%), CIN 1 (47.1%), CIN 2 (36.6%) and CIN 3 (3.3%). Majority of the non-dysplastic cervical lesions (including chronic cervicitis cases) showed low P16INK4a reactivity. Positive P16INK4a reactivity was seen in 80% of CIN 3 cases, 83.9% of CIN 2 cases, and, surprisingly, in 97.2% of CIN 1 cases. Ki-67 positivity was seen in 36.6% of cases (75% CIN 2 and 60% CIN 3). There was a significant correlation between the H&E diagnoses of CIN and P16INK4a/Ki-67 immunoreactivities.
Majority of the CIN 1 cases showing low grade p16INK4a immunereactivity strongly suggesting that cervical squamous intraepithelial neoplasia in this environment is likely associated with high grade HPV subtype infections and may predict possible progression to high grade squamous intraepithelial neoplasia. The use of P16INK4a and Ki-67 in the evaluation of cervical biopsies for benign mimics of high grade intraepithelial lesion will aid proper single Pathologist evaluation and help in patients triaging for follow up.
The incidence of cervical cancer is increasing yearly and it is only second to breast cancer among women.
P16INK4a and Ki-67 have gained increasing popularity as adjuncts in the prognostication of cervical cancer because they may serve as markers indicating the presence of high risk HPV subtypes in this lesion. The E6 and E7 proteins of the high-risk HPV inhibit the p53 and pRb proteins respectively, which are cell cycle regulatory proteins controlling G1-S transition.
Ki-67 is a well-known cell proliferation marker andcan be used in the grading of Cervical intraepithelial lesion (CIN)/SIL,
This study aimed to evaluate p16INK4a and Ki-67 expression in SIL and other benign cervical lesions in order to increase the diagnostic accuracy in equivocal cases.
A retrospective cross-sectional study was done involving the review of all the histologically diagnosed cases of SIL and other benign cervical lesions seen in the Pathology Department of a large tertiary hospital in South Western Nigeria between January 2006 and December 2013. Information was extracted from the departmental records and request forms.
The slides and tissue blocks of all the selected cases were retrieved. The primary antibody for p16INK4a was a monoclonal mouse anti-p16INK4a antibody (1E12E10, San Diego, CA 92124) in 1:200 dilutions while that of Ki-67 was monoclonal rabbit anti Ki-67 antibody (Thermo RM-9106-R7, UK) in 1:100 dilutions. The staining was validated with the respective negative and positive controls.
All the Haematoxylin and Eosin (H&E) slides were reviewed and classified using the 2003 World Health Organization classification for intraepithelial and benign lesions of the cervix.
The p16INK4a immunostaining was nuclear and/or cytoplasmic staining in the epithelium and was graded based on the pattern of distribution and levels of epithelial involvement as negative (0), 1+, 2+, and 3+. Cases with no epithelial staining were grouped as negative for p16INK4a, 1+ was staining of basal/parabasal up to one-third of the epithelium in a focal or diffuse nuclear and/or cytoplasmic stain, 2+ involves up to two-third of the epithelium and 3+ which involves above two-third to full epithelial thickness. The pattern of Ki-67 immunostain was graded as positive and negative based on the pattern of distribution and the level of involvement of the cervical epithelium. Negative Ki-67 was determined by scattered epithelial staining limited to the basal cells or a complete absence of staining while positive Ki-67 immunostain was accepted when the pattern was that of scattered or diffuse epithelial staining above the basal and parabasal cells. The Immunohistochemistry slides were subsequently viewed and analysed. The relationship between the age, H&E diagnoses, P16INK4a and Ki-67 reactivity were determined by correlation and the statistical significance was obtained using the Statistical Package for Social Sciences version 18 (SPSS-18). The chi-square test was used for discrete variables, with the level of significance set at p ≤ 0.05 and kappa was used to test for the strength of the associations. All ethical issues were duly considerfed and ethical approval was obtained from appropriate Local Ethical Committee.
Exclusion criteria:
Cases whose blocks and/or slides could not be retrieved were excluded from this study.
On the basis of their initial diagnoses, the 153 cases of SIL and benign cervical lesions, were classified as CIN 1 (49%), CIN 2 (25.5%) and CIN 3 (3.3%) while the clearly benign cases were basically chronic cervicitis (20.3%) and squamous metaplasia (2.0%).
All the cases were reviewed and re-grouped based on consensus H&E diagnoses as 47.1% CIN 1, 36.6% CIN 2 and 3.3% CIN 3 i.e. some cases of CIN 1 were upgraded to CIN 2 likely because severe chronic inflammation associated with some of the CIN 1 masked the severity of the squamous intraepithelial neoplasia in the initial evaluation. Also, we allotted chronic cervicitis to 12.3% and Squamous metaplasia were 0.7%. Also noted were various degrees of stromal lymphocytic infiltrations. The overall agreement between previous and consensus diagnosis was 60%, and their correlation was statistically significant (p<0.0001).
The cases ranged in age from 23 years to 80 years (mean: 46.3 years). The largest proportion of cases (41.8%) was within the age group 40-49 years while the lowest proportion, (2.6%) were within the 20-29 years age group (
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The p16INK4a distribution pattern was such that 1+ was seen in 92 (60.1%) cases, 2+ seen in 51 (33.3%), 3+ in 8 (5.2%) while only 2 (1.3%) were negative.
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Ki-67 immunoreactivity was seen in fewer cases (36.6%) with the greater proportion seen in CIN 2 (75%) and CIN 3 (60%), while the majority (64.1%) of the cases showed negative Ki-67expression. Ki-67 positivity was also seen in some cases of chronic cervicitis and in the only case of squamous metaplasia.
(p<0.0001) respectively.
Majority of the grade 2+ (76.5%) and grade 3+ (87.5%) p16INK4a positive cases were positive for Ki-67. However, 8.7% of grade 1+ p16INK4a positive cases were also positive for Ki-67. The correlation between the p16INK4a and Ki-67 immunoreactivities seen in this study is significant (p<0.0001). (
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The overall agreement between the p16INK4a and the Ki-67 immunostain reactivity was 68.7% (kappa 52.4%, p<0.0001).
Due to the subjectivity in the CIN evaluation with intra- and inter-observer variations of histomorphological characteristics, the consensus pathologist’s review resulted in variations between the initial H&E assessment and final diagnoses. There was concordance in up to about 60%, slightly less than the 67.5% correlation observed in an Iranian study.
This variation between the initial and final diagnoses seen in the present study may be partly explained by the chronic cervicitis and tissue reaction observed with most dysplastic changes
The majority of cases reviewed in this study were CIN 1 (47.1%) and a significant proportion of chronic cervicitis (12.4%) was also observed to be associated with CIN 1 as compared to the 36.6% of the CIN 2 and the 3.3% of CIN 3. The preponderance of CIN 1 observed in this study (47.1%) is slightly lower than those observed in some previous studies done by Sari et al
The highest proportion of all the reviewed cases in this study was in the age group 40-49 years (mean age: 46.3 years) which is slightly higher than what was obtained in the studies done in Greece by Haidopoulos et al
The patterns of p16INK4a expressivity in this study revealed that majority of the cases (60.1%) had low grade (+1) p16INK4a immunostain reactivity, both in extent and in intensity, 38.5% had high grade (+2 or +3) while less than 5% cases remaining were negative.
There are variations in the pattern of expression of p16INK4a among different grades of cervical intraepithelial neoplasia in several previous studies.
The correlation between the H&E and p16INK4a immunostain showed that high grade dysplastic changes are more associated with high grade p16INK4a immunostain and vice versa. This is also seen in most of the previous studies done
Majority of the CIN 1 cases show low grade p16INK4a immunoreactivity in this study and this is similar to what was observed in previous studies by Klaes et al,
The use of a cocktail of both p16INK4a and Ki-67 antibodies along with the H&E staining has been shown in various studies to aid the proper evaluation of cervical squamous intraepithelial lesions and other benign lesions and for possible prognostication of the progression of a low grade to a high grade squamous intraepithelial neoplasia.
In this study, only a few cases with low p16INK4a positivity (5.2%) showed Ki-67 positivity but there was a significant correlation between p16INK4a and Ki-67 immunostaining with an overall agreement of 68.7%. A high correlation was seen between Ki-67 positivity and high grade p16INK4a immunostaining. This high correlation between p16INK4a and Ki-67 in high grade squamous intraepithelial neoplasia is similar to what was reported in several previous studies,
Thus, the findings in this study further affirm the importance of these stains in the evaluation of the squamous intraepithelial and other benign lesions.
This study revealed the pattern of distribution of the squamous intraepithelial lesions and benign lesions usually encountered in this environment with a higher incidence of CIN 1 cases as against CIN 2 or 3. The majority of these CIN 1 cases have associated cervicitis.
A high proportion of low grade squamous intraepithelial neoplasia (CIN 1) with varying degrees of p16INK4a and Ki-67 expression seen strongly suggests that cervical squamous intraepithelial neoplasia in this environment are likely associated with high grade HPV subtype infections and may predict possible progression to high grade squamous intraepithelial neoplasia.
The use of P16INK4a and Ki-67 in the evaluation of cervical biopsies for benign mimics of high grade intraepithelial lesion will aid proper single Pathologist evaluation and help in patients triaging for follow up.
P16INK4a - P16 Inhibitor of Cyclin Dependent Kinase 4a
Ki
HPV – Human Papillomavirus
E6 and E7 - “Early” genes 6 and 7 oncoprotein (nonstructural)
CIN – Cervical Intraepithelial Neoplasm
H&E – Haematoxylin and Eosin
SIL - Squamous Intraepithelial Lesion
E2F – E2 transcription factor
DAB - Diaminobenzidine
SPSS-