HPV Strain Predicts Severity of Juvenile-Onset Recurrent Respiratory Papillomatosis with Implications for Disease Screening

Abstract

Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is the most common benign neoplasm of the larynx in children, presenting with significant variation in clinical course and potential for progression to malignancy. Since JoRRP is driven by human papillomavirus (HPV), we evaluated viral factors in a prospective cohort to identify predictive factors of disease severity. Twenty children with JoRRP undergoing routine debridement of papillomas were recruited and followed for ≥1 year. Demographical features, clinical severity scores, and surgeries over time were tabulated. Biopsies were used to establish a tissue bank and primary cell cultures for HPV6 vs. HPV11 genotyping and evaluation of viral gene expression. We found that patients with HPV11+ disease had an earlier age at disease onset, higher frequency of surgeries, increased number of lifetime surgeries, and were more likely to progress to malignancy. However, the amplitude of viral E6/E7 gene expression did not account for increased disease severity in HPV11+ patients. Determination of HPV strain is not routinely performed in the standard of care for JoRRP patients; we demonstrate the utility and feasibility of HPV genotyping using RNA-ISH for screening of HPV11+ disease as a biomarker for disease severity and progression in JoRRP patients.

Keywords: HPV strain; JoRRP; RRP; juvenile-onset recurrent respiratory papillomatosis; laryngeal papillomas; low-risk HPV.

Figure 1

Clinical and demographical characteristics of the recruited JoRRP cohort. (A) At the time of recruitment, measures of disease severity across 20 patients in the cohort showed an average Derkay score of 8.0 (range 0–19) and 23 previous surgeries (range 0–125). (B) Summary of demographical characteristics of the cohort from Table 2. (C) Patient characteristic summary graphs indicating that patients were predominately white and male and had an early (≤5 yrs) age at disease onset. (D) The number of lifetime surgeries at the end of the study is broken down by demographical sub-categories. H = Hispanic, AA = African American.

Figure 2

HPV11+ versus HPV6+ status, and not the age of onset, is associated with worse clinical course and increased severity. (A) Results of negative binomial regression analysis on lifetime surgeries at the end of the study period shows that white patients, males, HPV11+ patients, and patients with high Derkay scores had an increased risk of high surgical interventions, whereas patients with a later age of onset had a decreased risk of the need for surgical interventions. (B) The average number of surgeries per year since diagnosis and during the first year after recruitment for HPV6+ (gray) and HPV11+ (purple) patients. HPV11+ patients had increased surgeries per year since diagnosis (p = 0.02) but not in the first year after recruitment (p = 0.58). (C) Age at disease onset distribution HPV6+ and 11+ patients show an association between HPV11 positivity and earlier age at disease onset (p = 0.04).

Figure 3

Patient clinical course categorized by low surgical frequency, sustained disease, disease regression, or disease progression (A) Clinical course per patient with the y-axis starting at the surgical number at the time of recruitment. Patients were categorized by few surgical interventions (defined as ≤2/year), disease regression (defined as a decrease to ≤1/year), sustained disease, or disease progression (diagnosis of lung SCC indicated by arrow). Across the clinical course groups, the number of surgeries in the first year following recruitment (B), the average surgeries per year since disease onset (C) and the age at disease onset (D) show statistically significant differences across the groups (p < 0.001, p = 0.04, and p = 0.02, respectively).

Figure 4

HPV viral gene expression levels vary across patients independent of HPV genotype. (A) Routine DNA-ISH on papillomas shows HPV+ positive cells predominantly in the more superficial layers of the epithelium. Scale bar, 100 µm. HPV6/11 viral E6 and E7 gene expression (white) by RNA-ISH is variable in both (B) HPV6+ and (C) HPV11+ patient samples; scale bar, 100 µm. Two patient samples are shown for both HPV6+ and HPV11+ samples. Nuclei are highlighted by DAPI stain (blue) with cell membranes stained in red by WGA (B) or ECad (C).

Figure 5

Proposed pipeline for screening HPV11. Clinical symptoms determine the need for endoscopy with biopsy being performed when masses are visualized. Pathologic diagnosis of papilloma results in routine clinical testing in most centers to determine low-risk versus high-risk HPV status (Step 1). A recommendation is proposed to supplement this initial step by testing those biopsies positive for low-risk HPV using HPV strain-selective probes (Step 2) in order to identify HPV11 positive patients that should be more aggressively monitored for disease progression and need for treatment intervention (emphasized in red).

Figure 6

A two-step approach to optimize detection of HPV infection with HPV6/HPV11 subclassification. (A) Representative H&Es of papillomas from patient 11 and patient 15 that were previously HPV genotyped as HPV6+ and HPV11+, respectively. (B) Clinically validated low-risk combined HPV6/11 DNA-ISH showing robust staining in many cells. (C) RNA-ISH using strain-selective HPV6 and HPV11 probes showing restricted HPV6 or HPV11 staining in patients 11 and 15, respectively. Note the reduced number of positive cells using the HPV6 and HPV11 RNA selective probes as compared to the combined HPV6/11 DNA probes on serial sections from the same papillomas. Scale bar, 25 µm.