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 follow-up, 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 stratified the patients in three groups: a. in remission; b. with persistence; c. in progression. We defined 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
Paraffined tissue samples for molecular analysis were obtained from surgical specimens. Micro slices of all paraffined 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 paraffin 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-specific PCR bead-based multiplex genotyping assay (TS-MPG)
The identification of 120 viral genotypes was performed by using a high sensible type – specific 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-specific PCR uses specific primers for the detection of 22 alpha-papillomavirus types (HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68a and b, 70, 73 and 82), 46 beta-papillomavirus types (HPV5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, 96, 98, 99, 100, 104, 105, 107, 110, 111, 113, 115, 118, 120, 122, 124, 143, 145, 150, 151, 152, 159, 174) and 52 gamma–papillomavirus types (HPV4, 48, 50, 60, 65, 88, 95, 101, 103, 108, 109, 112, 116, 119, 121, 123, 126, 127, 128, 129, 130, 131, 132, 133, 134, 148, 149, 156, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 175, 178, 179, 180, 184, 197, 199, 200, 201, 202). Primers for the amplification of beta-globin were also added to provide a positive control for the quality of the template DNA. Amplification of beta-globin gene showed that in all cases the samples contained good quality DNA. The results were expressed as mean fluorescence 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 significant 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.