Use of p53 immunohistochemistry can improve diagnostic agreement for differentiated vulvar intraepithelial neoplasia (dVIN): an international reproducibility study

Abstract

Aims: Differentiated or HPV-independent vulvar intraepithelial neoplasia (dVIN) can progress rapidly to invasive cancer and accurate pathological diagnosis is essential to facilitate appropriate interventions. Histological similarities of dVIN with non-neoplastic lesions, however, often make the diagnosis less reproducible. We investigated among a diverse group of pathologists whether the diagnostic agreement improves with the use of p53 immunohistochemistry (IHC) interpreted using the pattern-based schema.

Methods and results: Fifty haematoxylin-eosin (HE) stained archival slides (30 dVIN and 20 non-dysplastic vulvar lesions) were selected and p53-IHC was performed. Twenty-four board-certified pathologists from eight countries first assessed the HE slides alone, and after a washout period, re-evaluated them alongside the p53-IHC slides. During both rounds, slides were diagnosed as dVIN, favour dVIN, favour no-VIN or no-VIN. p53-IHC was scored as wild-type or mutant (diffuse, basal, cytoplasmic or null). Kappa (κ) statistics and McNemar's test were used for statistical analyses. Overall diagnostic agreement for dVIN saw a significant increase in the Kappa value (κ = 0.6 vs. κ = 0.4, P = 0.002) when HE and p53-IHC slides were assessed together compared with histology assessment alone, although the level of agreement remained moderate. For p53-IHC assessment, overall agreement was substantial (κ = 0.7). Diagnoses changing from no-VIN/favour no-VIN to dVIN correlated significantly with the identification of a p53-mutant pattern (P < 0.001).

Conclusions: Our findings indicate that p53-IHC is a robust ancillary tool that can be reproducibly interpreted by pathologists with varying experience levels and supports the routine use of p53-IHC in cases where dVIN is considered in the differential diagnosis.

Keywords: carcinoma‐in‐situ; histology; immunohistochemistry; lower genital tract; observer variation; tumour suppressor protein p53; vulvar neoplasm.

Figure 1

Inter‐observer agreement for the diagnosis of dVIN for both rounds of assessment. Heatmaps represent the diagnoses rendered for each case (rows) per participant (columns), during the (A) first round (HE‐slide only) and (B) second round (HE and p53‐IHC slides) of assessment. The final column in the heatmap represents p53‐IHC interpretation for each case by the investigating pathologists. P1 through P24 represent the participating pathologists, and IP represents the investigating pathologists. (C) Bar charts depict the percentages of the cases (n = 50) that were judged as dVIN/favor dVIN or no‐VIN/favor no‐VIN by each participating pathologist during each round of assessment, and (D) violin plots represent median, 25th and 75th percentiles of the percentages of agreement for the diagnosis of dVIN/favor dVIN and no‐VIN/favor no‐VIN, during the first (HE) and second (HE + p53‐IHC) rounds of assessment.

Figure 2

(A) Heatmap represents the p53‐patterns for case (rows) judged by each participant columns. The final column in the heatmap represents p53‐IHC interpretation for each case by the investigating pathologists. P1 through P24 represent the participating pathologists and IP represent the investigating pathologists. (B) Violin plots represent the median, 25th and 75th percentiles of the percentages of agreement for interpreting the different patterns of p53‐expression, when computed separately and (C) when binarized into mutant and wild‐type patterns. (D) Sankey plot depicting the changes in majority diagnoses between first (left) and second (right) rounds of assessment.

Figure 3

Example of a case (Slide_25) judged as dVIN with >90% agreement during both rounds of assessment. (A) Histologic features associated with dVIN (thickened epithelium, eosinophilic appearance, hyper and parakeratosis) can be observed under low magnification. Examination under higher magnification (B, C) shows heterogeneity in nuclear size and shape, mitotic figure, individual cell keratinization and cobblestone appearance (D) p53‐IHC was judged as diffuse overexpression with >90% agreement.

Figure 4

Example of a case (Slide_14) judged as dVIN with >90% agreement during both rounds of agreement. (A) Histologic features associated with dVIN (thickened epithelium, eosinophilic appearance, hyper and parakeratosis) can be observed under low magnification. Examination under higher magnification (B, C) shows extensive nuclear atypia, hyperchromatic nuclei and individual cell keratinization (D) p53‐IHC was judged as cytoplasmic expression with >90% agreement.

Figure 5

Case (Slide_2) that did not have a majority diagnosis during the first round of assessment (50% diagnosed as dVIN or favor dVIN and 50% diagnosed as no‐VIN or favor no‐VIN). (A) Histological features of atypia are barely observable under low magnification. (B, C) Higher magnification examination reveals subtle nuclear atypia, that is, nuclear angulation and hyperchromatic nuclei (D) p53‐IHC was judged as null‐pattern with 100% agreement, and the majority diagnosis changed to dVIN/favor dVIN during the second round of assessment; inset shows an area with stromal p53‐expression.

Figure 6

Example of a case (Slide_1) where the majority diagnosis changed from no‐VIN/favor no‐VIN to dVIN/favor dVIN during the second round of assessment (A) Low power examination shows prominent parakeratosis, eosinophilic appearance of the epithelial cells, and occasional hyperchromatic nuclei (B, C) Higher magnification examination reveals features of nuclear atypia and individual cell keratinization (D) p53‐IHC was judged as diffuse overexpression >90% agreement.