Chromogenic in situ hybridization and p16/Ki67 dual staining on formalin-fixed paraffin-embedded cervical specimens: correlation with HPV-DNA test, E6/E7 mRNA test, and potential clinical applications

Abstract

Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16(INK4a)/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16(INK4a)/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16(INK4a)/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

Figure 1

CISH positive signals. Diffuse pattern, where nuclei are completely stained ((a), 20x magnification). Punctated pattern in invasive squamous cervical cancer: distinct dot-like intranuclear signals were noted within cells infiltrating the stroma ((b), 100x magnification). Mixed patterns, where both diffuse and punctated signals are noted ((c), 40x magnification).

Figure 2

Correlation between CISH results and histological diagnosis (P < 0.0001). P: punctate pattern; D/P: diffuse and punctated (mixed) pattern; D: diffuse pattern. CIN2+: Cervical intraepithelial neoplasia grade 2 or greater (including CIN2, CIN3, and invasive squamous cell carcinoma); CIN2−: less than Cervical Intraepithelial Neoplasia grade 2 (including CIN1 and negative for dysplasia.

Figure 3

Correlation between CISH signal and results from molecular tests (k = 0.20, P < 0.05).

Figure 4

Receiving operating characteristic curves (ROC), comparing CISH, HPV-DNA test, and E6/E7 mRNA diagnostic performances. The red line indicates a reference threshold with area under the ROC curve of 0.5.

Figure 5

Correlation between CISH signal and HPV viral load, as detected by HC2 test (P = 0.01). RLU/CO value provided an estimation of the number of HPV-DNA copies of each sample. RLU/CO: ratio between relative light units and control.

Figure 6

p16/Ki67 dual stain (40x magnification). Red square: brown chromogen marked cytoplasmic/nuclear p16 expression. Black arrows: red chromogen marked Ki-67 expression within nuclei. Black square: simultaneous expressions of both markers were revealed within the same cells.