Background: The Recurrent Respiratory papillomatosis (RRP) is a laryngeal disease induced by the Human Papilloma Virus (HPV) in the respiratory or phonatory epithelium. The evolution of each patient is uncertain and despite the surgical treatment there is a high risk of recurrence.

Objective: Identify HPV types in RRP patients, point clinical outcomes and compare them between groups to assess the individual prognosis of each subtype.

Methods: Descriptive and retrospective study of 34 patients with RRP submitted to surgery for excision of papillomas. Polymerase chain reaction (PCR) tests were performed for molecular identification of HPV types with questionnaires from medical records for each patient. Correlations were made through statistical analysis.

Results: From the 34 collected samples, 4 didn’t amplified by PCR. Among the remaining 30, 15 (50%) were HPV 11, 11 (36.3%) of HPV 6 and 4 (13.7%) of HPV 18. No type 16 was found. Men were 60% of patients and women 40%. The incidence of dysplasia was 10% of cases and mean age at diagnosis was 19.73 years. There were no statistically significant differences between groups HPV 6, 11 and 18 to the outcomes dysplasia, tracheostomy, number of surgeries, age at diagnosis, duration of disease and surgeries per year of disease. Mortality was statistically higher in the group HPV 18.

Conclusion: The most prevalent HPV types in our patients were HPV 11 and HPV 6. The HPV 18 had a tendency to proportionally worse clinical outcomes and more aggressive disease, being also statistically was more related to the occurrence of death. Larger studies should be encouraged to research the statistical correlations of patient prognosis and HPV types.

In most cases RRP is presented as multiple lesions affecting the supraglottis, the commissure and the anterior one-third of vocal folds. In the presence of first symptoms patients show alteration on vocal quality, dyspnea, stridor, cough or recurrent respiratory infections. With a bimodal age pattern, the outset of symptoms varies and may start at early childhood, after 5 years, or later in young adults between 20 and 40 years old. Severe cases may occur when the entire laryngeal is compromised and more invasive cases may also extend to the lung parenchyma. Complete obstruction of the airway results on acute respiratory failure and a cricothyroidotomy or a tracheostomy may be required.

Diagnosis is carried out through the patient history and clinical suspicion associated to laryngoscopy or nasofibroscopy for visualization and confirmation through a biopsy of the lesions.

The disease progression is uncertain and it may evolve quickly to a respiratory failure, remain stable for years, or even regress spontaneously. However in most of the cases it is persistent and recurrent. Standard RRP treatment consists on the surgical removal of neoplastic buds from the airways. The most commonly used technique is the cold excision of the papillomas through micro-surgery. Other options of treatment include microdebridment and CO₂ laser ablation. Since the disease has a high recurrence index new studies point out alternatives to control the disease through the use of interferon, cidofovir, podophyllin, hormones among others. According to Kayode [1] and Avelino et al. [2,3], cidofovir and interferon have presented better results lowering the number of surgical retreatment per year; however there is still no consensus on a standardized treatment.

It is extremely important to perform a biopsy of the lesions in all patients in order to confirm the diagnosis and exclude malignancy. The genotyping of HPV is carried out using the samples collected for the biopsy. The types of the virus that causes this disease can be identified through sensitive and reliable tests such as: in situ hybridization, hybrid capture and polymerase chain reaction (PCR).

So far more than 100 subtypes of HPV have been described. HPV is still divided into three classifications: low risk (HPV 6 and HPV 11), medium risk and high risk of malignancy (that includes HPV 16 and 18). In RRP the most prevalent HPV types are 6 and 11, corresponding to something around 80% to 90% of these patients. The number of patients with HPV 16 and 18 is the lower one, ranging from 0 to 20% from patients, according to literature [1-3].

Thus knowledge on HPV types involved in RRP is important in order to determine prophylactic and therapeutic approaches for these patients, including immunization against diseases caused by HPV.

Therefore the aim of this study was to identify HPV types in patients with RRP through PCR method and report their clinical outcomes and correlate them statistically in order to assess the prognosis of each type.

METHOD

This is a retrospective descriptive study carried out from August of 2008 to November of 2015, at the Otorhinolaryngology Clinic from the Hospital de Clínicas of the Federal University of Paraná – Brazil (HC-UFPR). The material collected from 34 patients with suspected or diagnosed RRP at the hospital’s laryngology unit, establishing correlation with clinical outcomes through questionnaire and medical record.

These patients were submitted to surgery for exertion of papilloma between 2008 and 2015. The material was sent for a histopathologic evaluation at the laboratory of anatomic pathology from the same hospital.

The collected material was stored in paraffin blocks. After histological confirmation the blocks were microdissected. The obtained material was submitted to centrifugation and then an enzymatic digestion method was applied using proteinase K in order to extract viral DNA of the samples.

DNA amplification was carried out through polymerase chain reaction (PCR) in order to obtain just viral subtypes HPV 6, 11, 16 or 18 from the samples.

The questionnaire used for retrospective analysis of medical records of the studied patients presented the following variables:

•      Number of surgical interventions

•      Age of diagnosis (years)

•      Disease duration (years)

•      Surgeries per year of disease

•      Presence of dysplasia

•      Need for tracheostomy

•      Death

According to the objectives established in this research the following hypothesis were determined:

1. Is there a statistically significant difference between mean values of the variables number of surgeries; age of diagnosis (years); disease duration (years) and surgeries per year of disease and HPV types (6, 11, 16 and 18)?

2. Is there a statistically significant relation between the variables dysplasia; tracheostomy; death and HPV types (6, 11, 16 and 18)?

Hypothesis 1 was verified using the Kruskal-wallis test, because since all evaluated variables presented a continuous scale for at least one subtype of HPV data were not normally distributed. Shapiro-Wilk test was applied for normality verification with significance level of 0.05. When statistically significant difference between HPV types was identified by Kruskal-Wallis test, pairwise comparison was performed using Dunn’s multiple comparison tests. Significance level was 0.05.

According to the age of diagnosis RRP can be classified as juvenile, with onset before 6 years old and adult, and adult with onset between 20 and 40 years old. In the present study the mean age was 19.73 years indicating prevalence of adult RRP in our patients. Only 13 patients (43%) were under 10 years old when. The adult form of RRP is usually associated to a lower number of lesions and recurrence rates as well as lower risk of malignancy [12].

The transmission of HPV in RRP is not yet fully understood. There are strong evidences of vertical transmission however it is still controversial if the transmission occurs exclusively through birth canal or transplacentally as well. Studies show the presence of HPV in the amniotic fluid of mother with condylomatosis. Other studies also report the presence of HPV in the airway of newborns of mothers with no history of genital warts suggesting subclinical and latent infections. It is assumed that transmission may also occur through autoinoculation and oral sex.

There are a great number of researches on RRP focused on the prophylaxis and the use of active immunization. The developed vaccines have an important role assisting the host on fighting the infections well as to avoid vertical contamination. Bivalent vaccine is broadly commercialized and the tetravalent is already available in the Brazilians Public Health system. It is also assumed that the vaccine may also improve the immunity of a host that is already contaminated reducing the risk or relapse. 

With the implementation of prophylactic measures such as health assistance, active immunization, control of sexually transmitted diseases and proper care to avoid vertical transmission there will be a lower incidence of the disease and thus the possibility of eradicating RRP.

CONCLUSION

Recurrent Respiratory Papillomatosis is a rare and potentially fatal disease. Along the search for new diagnosis methods and treatments for our patients, this study has shown the importance of HPV genotyping for the prognosis.

Molecular identification of the types of HPV in our patients was in accordance to the literature, with a predominance of HPV 6 and 11 and few cases of HPV 18. In clinical outcomes correlation there was no statistical significant differences between subtype 6, 11 and 18 groups regarding the number of surgeries, dysplasia, tracheostomy, disease duration, age of diagnosis and surgeries per year of disease. However the variable “death” was higher with statistically significant difference in HPV 18 group.

These results stimulate the search for new ways to determine a prognosis and treat these patients. Further studies with larger samples are required in order to study the statistical correlation between HPV subtypes and clinical outcomes.

1.       Kayode AS (2012) The descriptive review, from recurrent respiratory papillomatosis of the disease, an enigmatic. Arquivos Int Otorrinolaringol 16: 108-114.

2.       Avelino MAG, Zaiden TCDT, Gomes RO (2013) Tratamento cirúrgico e terapias adjuvantes na papilomatose respiratória. Braz J Otorhinolaryngol 79: 636-642.

3.       Avelino Melissa AG, Gutzman RL, Fujita RR, Pignatari S, Weckx Luc LM, et al. (2004) Estudo do efeito do Cidofovir na papilomatose laríngea recorrente em crianças: Relato preliminar. Rev Bras Otorrinolaringol 70: 734-738.

4.       Ullmann EV (1923) On the etiology of laryngeal papilloma. Acta Otolaryngol 5: 317-374.

5.       Naren NV, Pine HS, Underbrink MP (2012) Recurrent respiratory papillomatosis. Otolaryngol Clin North Am 45: 671.

6.       Levi JE, Delcelo R, Alberti VN, Torloni H, Villa LL (1989) Human papillomavirus DNA in respiratory papillomatosis detected by in situ hybridization and the polymerase chain reaction. Am J Pathol 135: 1179-1184.

7.       Sanchez GI, Jaramillo R, Cuello G, Quintero K, Baena A, et al. (2012) Human papillomavirus genotype detection in recurrent respiratory papillomatosis (RRP) in Colombia. Head Neck 35: 229-234.

8.       Wiatrak BJ, Wiatrak DW, Broker TR, Lewis L (2004) Recurrent respiratory papillomatosis: A longitudinal study comparing severity associated with human papilloma viral types 6 and 11 and other risk factors in a large pediatric population. Laryngoscope 114: 1-23.

9.       Tjon Pian Gi RE, San Giorgi MR, Slagter-Menkema L, van Hemel, van der Laan, et al. (2015) Clinical course of recurrent respiratory papillomatosis: Comparison between aggressiveness of human papillomavirus-6 and human papillomavirus-11. Head Neck 37: 1625-1632.

10.    Aaltonen LM, Rihkanen H, Vaheri A (2002) Human papillomavirus in larynx. Laryngoscope 112: 700-707.

11.    Therezita PP, Galvão C, Bussoloti Filho I (2006) Prevalência do papilomavírus humano (HPV) na cavidade oral e na orofaringe. Rev Bras Otorrinolaringol 72: 272-282.

12.    Filho JAX, Simoceli L, Imamura R, Tsuji DH, Sennes LU (2003) Papilomatose laríngea recorrente: Experiência de 10 anos. Rev Bras Otorrinolaringol 69: 599-604.