In Situ Hybridization Signal Patterns in Recurrent Laryngeal Squamous Papillomas Indicate that HPV Integration Occurs at an Early Stage

Abstract

Laryngeal papillomas are benign tumors that frequently recur and can compromise airways. We investigated HPV genotype, physical status, and protein expression in juveniles versus adults. Thirty-five laryngeal papilloma specimens were obtained from ten juveniles (1-16 years) and eleven adults (24-67 years). In cases of recurrent papillomatosis (7 juveniles, 7 adults), the first and last papillomas were assayed. HPV type was determined by GP5?/6? PCR and dot blot hybridization. In situ hybridization (ISH) was performed on 34 specimens; the data were recorded in terms of diffuse (episomal HPV) and punctate (integrated HPV) signal patterns. Immunohistochemistry for the HPV L1 capsid protein, a marker of HPV productive status, was performed on 32 samples. All samples tested HPV positive: HPV 11 in 2/10 (20.0%) juveniles and 5/11 (45.5%) adults; HPV 6 in 7/10 (70%) juveniles and 5/11 (45.5%) adults; and HPV 6/11 double infection was noted in one juvenile and one adult. ISH signals (punctate ± diffuse) were detected among 7/10 (70.0%) juveniles and 7/11 (63.6%) adults. L1 staining was detected in 1/9 (11.1%) juveniles and 6/10 (60.0%) adults (P = 0.06). These data support the idea that integration of low-risk HPV types into the cell genome is an early and common event in the etiology of juvenile and adult recurrent laryngeal papillomas. Productive HPV infections may be more common in adults; accordingly, constant laryngeal re-infection by HPV shed from a productive lesion may contribute to adult recurrent lesions, whereas the mechanism of papilloma recurrence in juveniles may be more attributable to HPV integration.

Viral load and mRNA expression of HPV type 6 among cases with recurrent respiratory papillomatosis

“…30 However, based on in situ hybridization studies, Brooks et al suggested that HPV integration may occur at an early stage of infection and may be an important factor contributing to the recurrent respiratory papillomatosis phenotype. 31 Interestingly, although HPV6 mRNA was present in all of the 21 HPV6-positive samples analyzed, we did not detect an increased expression of the early E7 mRNA per HPV6 copy in the papilloma, as compared with the paired healthy tissues; therefore, our quantitative analysis of the HPV6 E7 mRNA per viral genome seems to be of limited value in assessing disease activity. Further studies are required to investigate whether there is a difference in splicing patterns of HPV6 mRNAs in laryngeal papilloma and those of healthy mucosa.…”

“…The absence of integration of HPV6 could partly explain the lack of association between low‐risk HPV types (e.g., HPV6/11) and development of laryngeal squamous intraepithelial neoplasia . However, based on in situ hybridization studies, Brooks et al suggested that HPV integration may occur at an early stage of infection and may be an important factor contributing to the recurrent respiratory papillomatosis phenotype …”

HPV RNA in‐situ hybridization as a diagnostic aid in papillary laryngeal lesions

“…But routine high‐risk HPV testing is not recommended in laryngeal lesions due to the low prevalence of HPV and unclear prognostic implications of HPV involvement in non‐oropharyngeal sites . To compound all of these limitations, variable detection rates for both low‐risk and high‐risk HPV have been reported depending on the methodology used, raising concern about the sensitivity of this testing for diagnostic purposes.…”