Alpha, Beta, and Gamma HPV Infection in Juvenile Onset Recurrent Respiratory Papillomatosis

Purpose The aim of our study is to evaluate the prevalence of different HPV genera – alpha, beta and gamma – in Juvenile onset Recurrent Respiratory Papillomatosis (JoRRP) and examine the association of type and genus-specic viral features with the clinical outcome of disease. Methods This retrospective observational study includes consecutive patients with JoRRP who were treated in a referral centre between October 2000 and October 2020. All patients underwent cold excision and laser vaporization of papillomatous lesions. Samples were analysed for the presence of 120 viral genotypes (22 alpha-HPV, 46 beta-HPV, 52 gamma-HPV) using a highly sensitive multiplex genotyping assay. Twenty patients with JoRRP, aged 0.3–11 years old, were included, with a median follow-up of 13.5 years. All samples were HPV DNA positive: 20 (100%) for alpha-HPV DNA; 7 (35%) for beta – HPV DNA; 0 for gamma-HPV DNA. Three groups were dened according to the number of infections: seven cases (35%) with HPV mono-infection; ten cases (50%) with HPV double-infection; three cases (15%) with ≥ 3 HPV infections. At diagnosis, patients with ≥ 3 HPV infections reported higher median Derkay’s score than those with mono-infection (21 vs 14, P = 0.018). Number of HPV infections was also associated with clinical outcomes, with an average of 0.5 surgical procedures/year in patients with mono-infection, 1.2 for double-infection, 2.6 for ≥ 3 infections (P = 0.006). Despite the small sample size, these preliminary data support an association between the number of different alpha and beta HPV co-infections and the clinical severity of the disease.


Introduction
Juvenile onset Recurrent Respiratory Papillomatosis (JoRRP) is a rare and challenging HPV-induced disease, frequently associated with mucosal HPV-type infection, in particular HPV 6 and 11, and characterized by the growth of squamous papillomata in the airway epithelium [1].It is de ned by the onset before 12 years of age with the peak of incidence being commonly before 5 years [2]. Because of JoRRP's typical recurrence, patients often require multiple surgical debulking procedures to maintain an airway patency [3].
There is currently no eradicative cure for JoRRP. Furthermore, no reliable markers predicting the course of the disease, the risk of recurrence or malignant transformation exist [4]. However, the new adjuvant therapies appear to be promising in improving the control of the disease [5]. It could be, therefore, interesting and innovating having biological markers that can guide clinicians to select patients eligible for adjuvant therapies.
Many studies have investigated whether the severity and aggressivity of JoRRP maybe in uenced by the infection with mucosal HPV types, 6 and 11, but the results provided inconsistent and contrasting conclusions [1,6]. To the best of our knowledge, no study has ever investigated the prevalence of HPV genera other than mucosal alpha genus in JoRRP. Interestingly, some beta HPV types from species 3 appears to have dual tropism, being detected in nasal or anal mucosa and the skin [7,8]. Thus, in this study we have evaluated the prevalence of different HPV genera -alpha, beta and gamma, in JoRRP with a highly sensitive genotyping assays and investigated whether different pattern of HPV infection may in uence the clinical outcome of the disease.

Study population
This observational study included 20 consecutive children aged < 12 years old at onset of JoRRP, in a referral centre (Policlinico Universitario di Padova) between October 2000 and October 2018 and followed-up until October 2020. A clinical and surgical database of all patients, including age and Derkay's score [9] at onset, yearly number of surgical procedures since the diagnosis, duration of followup, surgical and controls videos, surgical and clinical outcomes have been maintained prospectively since October 2000. All patients were treated with both cold surgical and laser vaporisation with active aspiration by the same expert surgeon (CC) in the same single centre. The whole study was approved by the ethic committee for clinical experimentation of Treviso and Belluno provinces (Ethic votes: 345/AULSS9 and 421/AULSS9).
Inclusion criteria comprehended: a) diagnosis of JoRRP; b) age ≤ 12 years old at onset; c) histological and clinical data available in hospital database; d) written consent for data processing.
Depending on clinical course of the disease, considering the number of surgical procedures/year and the presence of a longitudinal progression of papillomatosis, we strati ed the patients in three groups: a. in remission; b. with persistence; c. in progression. We de ned in remission patients with no evidence of papillomata in the past 5 years; with persistence the patients submitted to less than 4 surgical procedures in the last year and/or without a longitudinal progression of papillomatosis, and in progression the patient submitted to more than or equal to 4 surgical procedures in the last year and/or with a longitudinal progression of papillomatosis.
Collection of samples and DNA extraction Para ned tissue samples for molecular analysis were obtained from surgical specimens. Micro slices of all para ned samples were shipped to the Infection and Cancer Biology Group at the International Agency for Research on Cancer in Lyon, France. DNA was obtained by an overnight incubation at 37°C of the para n tissue sections in a digestion buffer (10 mM Tris/HCl pH 7.4, 0.5 mg/ml proteinase K, and 0.4% Tween 20) [10]. To monitor the possible occurrence of cross-contamination between the different specimens during DNA extraction, tubes containing only buffer were also included (1 tube with buffer every 10 specimens).
HPV type-speci c PCR bead-based multiplex genotyping assay (TS-MPG) The identi cation of 120 viral genotypes was performed by using a high sensible type -speci c multiplex genotyping assays (TS-MPG), which combine multiplex polymerase chain reaction (PCR) and bead-based Luminex technology (Luminex Corp., Austin TX, USA) as described in the literature [11,12].
Multiplex type-speci c PCR uses speci c primers for the detection of 22 184,197,199,200,201,202). Primers for the ampli cation of beta-globin were also added to provide a positive control for the quality of the template DNA. Ampli cation of beta-globin gene showed that in all cases the samples contained good quality DNA. The results were expressed as mean uorescence intensity (MFI) [13]. The cut off was computed by adding 5 MFI to 1.1 times the median background value.

Statistical analysis
Differences in socio-demographic and clinical characteristics of the patients across strata were assessed with the Kruskal-Wallis test or Fisher's exact test. The values of P reported in the tables are considered signi cant when ≤.05. For each patient and for each year after diagnosis, the cumulative number of surgical procedures was calculated as the total number of surgical procedures from the diagnosis.

Clinical results
Patients' clinical data were included in Table 1. Twenty patients who met the inclusion criteria were included in the study. The median age at diagnosis was 3.0 years old (range 0.3-11.0 years old) and the male:female ratio was approximately 1:2. The median duration of follow up was 13.5 years (range 2-20 years) and the Derkay's score at onset (clinical and anatomical score) varied between 5 and 23 with a median value of 14. Papillomata demonstrated different localizations along the airways with different prevalence. As shown in Table 2, the larynx was the most involved site (95% of cases). The median number of procedures/year at onset was 2, ranging between 1 and 10. The total number of surgeries ranged from 1 to 45 with a median value of 9.5, while the maximum number of surgeries/year ranged from 1 and 13, with a median value of 3. All samples were HPV-DNA positive and they reported at least one infection for alpha-HPV DNA; 7 cases (35%) were also positive for beta-HPV DNA, while no gamma-HPV DNA was detected in any specimen ( Table 1).

Clinical and Virological correlations
The three groups showed similar age at onset. In all groups, the main site of papillomata was the larynx, involved in 100% of cases with co-infection and in 86% of patients with mono-infection. Although not signi cant, multiple localization was more frequent in patients with ≥3 HPV infections than those with one or two co-infections (67% vs 30%, respectively). The Derkay's score at diagnosis increased with the increasing number of co-infections, reaching 21 for ≥3 HPV co-infection compared to 9 for patients with mono-infection (P=0.018) ( Table 2). The median number of surgical interventions at onset was higher for patients with ≥3 multiple infections, though not signi cant (Table 3). However, the maximum number of surgical procedures/year (P=0.012), the average number of surgical procedures/year (P=0.022) and the total number of surgeries (P=0.006) increased with increasing number of HPV co-infections. Three patients were considered in progression and they were all co-infected patients: one positive for double coinfection and two positives for multiple co-infections. The majority of patient with HPV mono-infection (57%) was clinically in remission, whereas no one showed progression (Table 3).
Clinical outcome was also evaluated as required number of surgical procedures after diagnosis. Interestingly, the average cumulative number of surgical procedures attened after approximately 5 years from onset for patients with mono-infection or double-infections, even if at a lower level for the former than the latter (average cumulative number= 5.3 and 14.7, respectively). Conversely, patients with ≥3 HPV infections showed an upward trend over the all period of observation, reaching an average cumulative number of 43.0 surgical procedures after 20 years from diagnosis ( Figure 1). Cumulative number of surgical procedures was also calculated using information on HPV6 and HPV11 alone ( Figure  2). Patient with HPV6/HPV11 co-infection showed the worse clinical course, whereas those with HPV11 infection showed the best clinical course.

Discussion
To the best of our knowledge, this is the rst study that analysed the presence of a large number of alpha, beta and gamma HPVs types in JoRRP using a highly sensitive method. The data show that all JoRRP were associated with infections of mucosal alpha HPV types 6 and 11, which are well-known etiological factors of JoRRP. In addition, several JoRRP were also positive for some beta HPV types. Despite the small study size, the ndings highlight an association of multiple HPV infections with the severity of disease in comparison to single HPV infections.
The main problems of recurrent respiratory papillomatosis are still the lack of an eradicating treatment and the impossibility to predict its clinical outcome because of its unpredictable nature and the still limited knowledge on the pathogen-host interaction [14]. This lack of information has prompted many clinicians to try to provide a prognosis of the disease, looking for the risk factors most involved in the aggressiveness of RRP. Most of the available studies in literature found a correlation between the aggressiveness of RRP and the aetiology by HPV11 [15].
It has been assumed that HPV11 E7 may interact more effectively with the transporter associated with antigen processing subunit 1 (TAP1) than HPV6 E7, leading to reduced immune responses and the more severe disease course observed in HPV11 RRPs [16,17]. However, other studies did not provide evidence for the higher aggressive properties of HPV11 versus HPV6 1 . Indeed, some studies reached opposite conclusions, being HPV6 more associate with higher JoRRP severity in comparison to HPV11 [18,19].
Other studies highlight that age at diagnosis, rather than the genotype of HPV involved, can impact on the clinical history of the disease [1,6].
Our ndings showed that single infections have a lower prevalence than co-infections and that the latter have a much more aggressive outcome than the others which instead tend to stabilize after a follow-up of about 4 years. (Figure 1) Considering the results of prevalence of HPV genera (alpha -beta -gamma) and the clinical data, it is evident that in the present case series an increased number of co-infections was associated with increasing aggressiveness of the disease determined both by the number of surgical procedures and the longitudinal spread of the disease (Figure 1). On the other hand, by considering the clinical course of papillomatosis with respect to alpha-HPV infection only, it was observed that HPV11 is the genotype involved in a more aggressive course of the disease. However, it was clear only when HPV11 was associated with other viral genotypes (co-infections) but not in cases of mono-infection, where instead the disease has a more indolent course (Figure 2).
Multiple studies concerning the biological mechanism and the role of interactions and co-infections between different HPV genotypes were conducted on other widely spread HPV-related tumors [20]. Primarily on cervical cancer, about which several authors agree on the association between genotype-speci c HPV interactions, multiple viral co-infections and increased risk of cancer onset. 21 Concerning respiratory papillomatosis, in 2010 Donne and colleagues did not nd a certain clinical difference between HPV6 and HPV11 and recognized the possible impact of untested HPV-variants co-infections on the clinical outcome of RRP [22].
Other authors had also highlighted the presence of co-infections in respiratory papillomatosis, considering both other viruses -such as EBV, HSV and CMV, and other alpha-HPV genotypes, assuming their co-presence as an additional risk factor for the clinical outcome of the RRP [23]. In 2015 Omland and colleagues decided to stratify RRP patients depending on alpha -HPV viral load without considering HPV co-infections. They nally demonstrate the absence of a correlation between the RRP prognosis and the HPV viral load, de ned as irrelevant if < 5 copies/cell [6]. Other authors, such as Buchinsky, excluded cases of alpha-HPV-DNA co-infections from their cluster because, in their opinion, it was impossible to determine which of the different involved genotypes was responsible for the clinical phenotype of the disease [1].
However, the data of the present study do not provide any evidence of a direct role of co-infections in determining the greater aggressiveness of the disease. It has been observed that immunosuppressed patients often harbour multiple oral HPV infections [24]. Thus, the association between the aggressiveness of JoRRP and multiple HPV infections may re ect patient's immuno-suppressive status which more directly may affect the aggressive course of the disease.
Limited sample size is the major limitation of the present study, and, therefore, the results should be considered as preliminary. Nonetheless, results are strongly indicative of a new prognostic trend in JoRRP that would bene t the inclusion of a larger sample for further validation. Indeed, small sample size mainly impact on false negative results as the study is not powered to detect small differences. Further, the small sample size did not allow multivariable analyses. Conversely, the homogeneity of patients, treated and followed up by the same centre, is to be account among study strength.

Conclusions
JoRRP still remains an investigating pathology in terms of treatment and clinical course. Both alpha and beta HPV genera may be involved in JoRRP pathogenesis and the number of different HPV-type coinfections may serve as a prognostic indicator of the clinical course of the disease. The results of this study, although preliminary and based on a small number of patients, underline the importance of a highly sensitive diagnostic method in this pathology.
Declarations article and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization.
Funding: No funding was received to assist with the preparation of this manuscript Con icts of interest/Competing interests: The authors have no con ict of interests to declare that are relevant to the content of this article Availability of data and material: All data and materials used in this study are now available in clinical database Authors' contributions: All the authors have contributed actively to the study and have approved of the nal version of the manuscript   Figure 1 Cumulative number of surgeries according to number of co-infection with alpha-HPV and with beta-HPV