Abstract
Introduction Immunostaining with p16(INK4a) (p16), a tumor-suppressor surrogate protein biomarker for high-risk human papillomavirus (hrHPV) oncogenic activity, may complement standard hematoxylin and eosin (H&E) histology review, and provide more objective criteria to support the cervical intraepithelial neoplasia (CIN) diagnosis. With this study we assessed the impact of p16 immunohistochemistry on CIN grading in an hrHPV-based screening setting. Material and methods In this post-hoc analysis, 326 histology follow-up samples from a group of hrHPV-positive women were stained with p16 immunohistochemistry. All H&E samples were centrally revised. The pathologists reported their level of confidence in classifying the CIN lesion. Results Combining H&E and p16 staining resulted in a change of diagnosis in 27.3% (n = 89) of cases compared with the revised H&E samples, with a decrease of 34.5% (n = 18) in CIN1 and 22.7% (n = 15) in CIN2 classifications, and an increase of 18.3% (n = 19) in no CIN and 20.7% (n = 19) in CIN3 diagnoses. The level of confidence in CIN grading by the pathologist increased with adjunctive use of p16 immunohistochemistry to standard H&E. Conclusions This study shows that adjunctive use of p16 immunohistochemistry to H&E morphology reduces the number of CIN1 and CIN2 classifications with a proportional increase in no CIN and CIN3 diagnoses, compared with standard H&E-based CIN diagnosis alone. The pathologists felt more confident in classifying the material with H&E and p16 immunohistochemistry than by using H&E alone, particularly during assessment of small biopsies. Adjunctive use of p16 immunohistochemistry to standard H&E assessment of CIN would be valuable for the diagnostic accuracy, thereby optimizing CIN management and possibly decreasing overtreatment.
| Original language | English |
|---|---|
| Pages (from-to) | 1328-1336 |
| Number of pages | 9 |
| Journal | Acta Obstetricia et Gynecologica Scandinavica |
| Volume | 101 |
| Issue number | 11 |
| Early online date | 30 Sept 2022 |
| DOIs | |
| Publication status | Published - Nov 2022 |
Bibliographical note
Funding Information:Kits for p16 staining were kindly donated by Roche. Paraffin-embedded tissue blocks were obtained from all histology samples. In the case of multiple blocks, the tissue block containing the CIN lesion that corresponded with the worst H&E assessment was selected. From this tissue block, a 4-μm-thick slice was cut and its slide was stained according to the instructions of the manufacturer with the p16 Histology kit (Roche mtm Laboratories AG). Staining was performed on a Ventana benchmark ultra (Roche mtm Laboratories AG), and each run included one control specimen. All p16 slides were examined by a trained general pathologist. p16 slides were scored as no CIN when either no p16 positivity or focally scattered positive cells, or small positive clusters were seen. Slides were scored as CIN1 when diffuse positivity for p16 was seen in dysplastic cells in the lower third of the cervical epithelium and koilocytotic atypia were present as a sign of hrHPV infection. A CIN2 diagnosis was given when p16 positivity was evenly distributed across the lower two-thirds of the epithelium. Slides were scored as CIN3 when diffuse strong nuclear and cytoplasmic p16 block staining was seen in from two-thirds up to the whole width of the epithelium,5 (Figure 1). The combined H&E and p16 staining score was obtained according to the opinion of the pathologist, taking into account the H&E staining, and p16 staining as described above. Pathologists were asked to score the confidence in their diagnosis differentiating between confident in their diagnosis, rather confident, and unconfident. This study is a post-hoc analysis on histology samples from the PROHTECT-3B trial (Protection by Offering HPV Testing on self-sampled cervicovaginal specimens Trial-3B).12 In the PROHTECT-3B trial, former non-responders to the cervical cancer screening program, aged 30–60 years, were invited to participate in cervical cancer screening, by offering self-sampling for hrHPV DNA testing (GP5+/6+ polymerase chain reaction; EIA HPV GP HR kit; LBP). Three laboratories performed the hrHPV tests. Women who tested hrHPV positive on their self-sample were advised to have a cervical smear taken by a physician for Papanicolaou-cytology triage testing. Women with abnormal cytology results (defined as atypical cells of undetermined significance or worse) were referred for a colposcopy-directed biopsy, whereas women with a normal cytology result (defined by negative for intraepithelial lesion or malignancy cytology result) were re-invited for an exit test with Papanicolaou cytology and hrHPV co-testing 6 months later. Women with a positive exit test, defined as atypical cells of undetermined significance or worse cytology and/or high-risk HPV-positive test results, were in the second instance referred for histological examination by taking a colposcopy-directed biopsy, endocervical curettage, or LLETZ. Colposcopists were aware of the hrHPV-positive status and colposcopy was performed according to the Dutch national guidelines. If no abnormalities were seen at colposcopy, it was advised to take two random biopsies according to the study protocol. The database was closed with a mean follow up of 15 months (range: 6–18 months). Further details of the PROHTECT-3B trial design are reported elsewhere.12 Original H&E diagnoses were retrieved from the Dutch nationwide computerized registry of histopathology and cytopathology (PALGA). The histology outcomes were classified as no CIN, CIN1, CIN2, CIN3, and invasive carcinoma. For revision, all original H&E slides from the worst histology samples collected during colposcopy procedures were obtained and subjected to blinded central review by a general pathologist. If the review diagnosis of the general pathologist was not consistent with the original H&E diagnosis, a second specialized gynecopathologist, with extensive experience in gynecopathology blindly adjudicated the case, resulting in a consensus diagnosis. Revised H&E diagnoses were assessed for all available cervical tissue specimens. Pathologists were blinded to all previous study results. Pathologists were asked to score the confidence in their diagnosis differentiating between confident in their diagnosis, rather confident and unconfident. Paraffin-embedded tissue blocks were obtained from all histology samples. In the case of multiple blocks, the tissue block containing the CIN lesion that corresponded with the worst H&E assessment was selected. From this tissue block, a 4-μm-thick slice was cut and its slide was stained according to the instructions of the manufacturer with the p16 Histology kit (Roche mtm Laboratories AG). Staining was performed on a Ventana benchmark ultra (Roche mtm Laboratories AG), and each run included one control specimen. All p16 slides were examined by a trained general pathologist. p16 slides were scored as no CIN when either no p16 positivity or focally scattered positive cells, or small positive clusters were seen. Slides were scored as CIN1 when diffuse positivity for p16 was seen in dysplastic cells in the lower third of the cervical epithelium and koilocytotic atypia were present as a sign of hrHPV infection. A CIN2 diagnosis was given when p16 positivity was evenly distributed across the lower two-thirds of the epithelium. Slides were scored as CIN3 when diffuse strong nuclear and cytoplasmic p16 block staining was seen in from two-thirds up to the whole width of the epithelium,5 (Figure 1). The combined H&E and p16 staining score was obtained according to the opinion of the pathologist, taking into account the H&E staining, and p16 staining as described above. Pathologists were asked to score the confidence in their diagnosis differentiating between confident in their diagnosis, rather confident, and unconfident. Different scoring strategies including original H&E diagnosis, revised H&E diagnosis, sole p16 diagnosis, and combined revised H&E and p16 diagnosis, were compared. Descriptive statistics were used to calculate numbers and percentages of confidence. Sensitivity and specificity, with 90% confidence intervals (90% CI) were calculated for the original H&E diagnosis, the revised H&E diagnosis, and the stand-alone p16 diagnosis. The combined revised H&E and p16 diagnosis was used as reference standard. McNemar's test was used to calculate p values. Statistical analyses were conducted using SPSS version 20.0.1 for Windows (IBM). All women provided written informed consent. The Ministry of Health gave ethical approval for the PROHTECT3b study on August 31, 2010 (No. 2010/WBO04), and the regional institutional review board approved the protocol for this post-hoc analysis on March 16, 2012.
Publisher Copyright:
© 2022 The Authors. Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).