High-Risk HPV CISH Detection in Cervical Biopsies with Weak and/or Focal p16 Immunohistochemical Positivity

Abstract

In cervical biopsies, for diagnosis of Human Papilloma Virus (HPV) related conditions, the immunohistochemical staining for p16 has a diagnostic value only if diffusely and strongly positive, pattern named "block-like". "Weak and/or focal (w/f) p16 expression" is commonly considered nonspecific. In our previous study, we demonstrated the presence of high-risk HPV (hrHPV) DNA by LiPa method in biopsies showing w/f p16 positivity. The aim of the present study was to investigate the presence of hrHPV-DNA by CISH in the areas showing w/f p16 expression. We assessed the presence of hrHPV16, 18, 31, 33, 51 by CISH in a group of 20 cervical biopsies showing w/f p16 expression, some with increased Ki67, and in 10 cases of block-like expression, employed as control. The immunohistochemical p16 expression was also assessed by digital pathology. hrHPV-CISH nuclear positivity was encountered in 12/20 cases of w/f p16 expression (60%). Different patterns of nuclear positivity were identified, classified as punctate, diffuse and mixed, with different epithelial distributions. Our results, albeit in a limited casuistry, show the presence of HPV in an integrated status highlighted by CISH in w/f p16 positive cases. This could suggest the necessity of a careful follow-up of the patients with "weak" and/or "focal" immunohistochemical patterns of p16, mainly in cases of increased Ki67 cell proliferation index, supplemented with molecular biology examinations.

Keywords: CISH; high-risk HPV; immunohistochemistry; in situ hybridization; p16.

Figure 1

Group 1 case: serial sections of w/f p16 positive/HPV51 positive case with H-SIL. (A) Biopsy shows H-SIL (CIN2). (B) Focal expression of p16 in a few basal/parabasal cells. (C) Ki67 immunohistochemical staining highlighting a high proliferation rate in the basal and intermediate squamous layers. (D) Mixed pattern of CISH signals: strong, diffuse pattern in the nuclei of the superficial squamous layer and punctate pattern in the nuclei of the basal layer, suggesting a different state of HPV integration. Original magnification (A–D) 100×, figure inserted in (D): 400×; (A) hematoxylin and eosin staining; (B,C) immunoperoxidase staining with 3,3’-diaminobenzidine (DAB) chromogen; (D) and insert, chromogenic in situ hybridization staining (CISH).

Figure 2

Cervical biopsy with mild epithelial atypia, mixed pattern of P16 expression and Ki67 expression: serial sections. (A) The biopsy shows mild atypia. (B) On the left weak/focal p16 expression; on the right block-like p16 expression positivity, diffuse in all the squamous layers. (C) Ki67 immunohistochemical staining showed a mild increase of proliferating cells in w/f p16 area and a strong increase in block-like p16 positive area. Original magnifications: 100×; (A) hematoxylin and Eosin staining; (B,C) immunoperoxidase staining with 3,3’-diaminobenzidine (DAB) chromogen. Adapted from Cabibi et al., Diagnostics (Basel), 2021 [16].

Figure 3

Group 2 case: serial sections of cervical biopsy with H-SIL, block-like p16 expression, and HPV16. (A) Biopsy shows H-SIL. (B) Block-like p16 positivity, strong and diffuse in all the squamous layers. (C) Ki67 immunohistochemical staining highlighting a high proliferation index with widespread positivity extended to the upper epithelial layers. (D) CISH showed an isolate punctate pattern of positivity in rare nuclei of the lower third of the epithelium, suggesting the integration of HPV. Atypical mitoses are visible (arrows). Original magnifications: (A–C) 200×; (D) 400×; (A) hematoxylin and eosin staining; (B,C) immunoperoxidase staining with 3,3’-diaminobenzidine (DAB) chromogen; (D) chromogenic in situ hybridization staining (CISH).

Figure 4

CISH pattern in group 1.

Figure 5

CISH pattern in group 2.

Figure 6

HPV-DNA CISH patterns and their proposed meaning. hrHPV infection starts in basal cells of epithelium. The first phases of infection should result in an isolated punctate pattern of CISH, limited exclusively to this type of cells (top). The stages of infection could evolve in three different patterns of CISH, the diffuse, the widespread punctate and the mixed. A diffuse pattern indicates the presence of the virus in the squamous cells, and it is a pattern associated to a potential successful intervention of immune system, definitively eliminating HPV (left). Conversely, a widespread punctate pattern in all epithelial layers could be observed if HPV is stably integrated in the almost totality of cells. This is the pattern more likely to evolve toward progression (right). Lastly, the mixed pattern presents intermediate features, with isolated punctate pattern in basal and para-basal cells, becoming diffuse in the superficial layers (bottom). The mixed pattern could evolve toward either diffuse or widespread punctate and retains potential features of clearance or progression.

Figure 7

Representative images of “digital pathology evaluation” performed with QuPath software on p16 digital slides, with original DAB-stained images (left) and the relative digital detection of DAB signal in red (right). (A) Block-like positive case. (B,C) Weak and/or focal positive cases.