An exploratory study of human papillomavirus detection in oral rinses from patients with recurrent respiratory papillomatosis
論文ID: 25-00026
詳細
論文ID: 25-00026
Purpose: Recurrent respiratory papillomatosis (RRP) is associated with low-risk types of human papillomavirus (HPV). With HPV DNA testing, the oral rinse of RRP patients may be a useful liquid biopsy, as previously shown in patients with oropharyngeal cancer.
Methods: Oral rinse, along with palatine and pharyngeal tonsil swabs, were collected from seven patients with persistent RRP. HPV DNA detection was performed using polymerase chain reaction, followed by genotype identification.
Results: HPV DNA was detected in five of seven oral rinse samples, but not in any palatine or pharyngeal tonsil swabs. HPV6 was identified in four of the five HPV-positive oral rinses, which was consistent with the RRP tissues.
Conclusion: HPV DNA can be detected in oral rinses from patients with RRP, suggesting the utility of the oral rinse as a liquid biopsy. In contrast, neither the palatine nor the pharyngeal tonsils were reservoirs of HPV in study patients with RRP.
Recurrent respiratory papillomatosis (RRP) is a chronic human papillomavirus (HPV)-associated disease characterized by papillomatous lesions in the upper airway, predominantly affecting the larynx. Although rare, it is intractable due to its tendency to recur and spread throughout the aerodigestive tract1-3). Low-risk types of HPV, particularly HPV6 and HPV11, have been implicated in the pathogenesis of RRP4), while high-risk types of HPV, especially HPV16, are known to cause oropharyngeal cancer (OPC). We previously reported that HPV DNA was frequently detected in both oral rinse and palatine tonsil swab samples obtained from patients with OPC who were positive for p16 immunostaining, a surrogate marker for HPV-associated OPC5). Regarding RRP, there have been reports investigating HPV DNA detection in oral rinses, but its significance in clinical settings remains unclear6-8). In the present study, we investigated HPV DNA detection in the oral rinses of RRP patients from a different perspective than those of these previous studies, by comparing it with detections made with palatine and pharyngeal tonsil swabs, aiming to clarify, at least in part, the pathophysiology of diseases caused by low-risk HPV.
Seven patients with RRP who underwent treatment at our hospital were enrolled in this study. All had undergone multiple surgeries and had persistent disease at the time of enrollment. Prior to the present study, HPV6 was detected in all patients from at least one sample of surgically removed tissue. This study was approved by the Medical Ethics Committee of Kanazawa University (Approval number:2014-054) and the Research Ethics Committee of Fukushima Medical University (Approval number:2992, 2996), and written informed consent was obtained from all enrolled patients.
Sample collectionOral rinse samples were collected after gargling with 20 mL of normal saline for 20 seconds, then centrifuged at 3,000 rpm for 10 minutes, as described previously5). Palatine tonsil swab samples were collected after brushing the palatine tonsils for cytodiagnosis, like a Pap smear, also as described previously5,9). Pharyngeal tonsil swab samples were collected endonasally using a swab with a movable cover that allowed exposure in the nasopharynx only, to prevent endonasal contamination, as previously reported9). All samples were suspended in a preservative solution for liquid-based cytology (Medical & Biological Laboratories, Nagoya, Japan) and stored at 4°C until use5,9).
HPV DNA detectionDNA extraction and HPV DNA detection were performed as previously reported5,9). Briefly, the β-globin genome fragment was first amplified by polymerase chain reaction (PCR) to confirm the presence of extracted DNA5,9). Then, an auto-nested PCR was performed on β-globin-positive samples to increase sensitivity, with 36 cycles in the first amplification and 20 cycles in the second amplification, using GP5+/6+ primers5,9,10). This process generated approximately 140 base-pair fragments from the HPV L1 structural gene of multiple mucosotrophic HPV types5,9,11-13). The amplified DNA was visualized by ultraviolet illumination with ethidium bromide after electrophoresis on a 2% agarose gel5,9). The p1203 PML2d HPV-16 plasmid, a gift from Peter Howley (Addgene plasmid # 10869), was used as the positive control, while all reagents except for DNA were included in the negative control5,9).
HPV genotypingHPV genotyping using the HPV GenoArray test kit (Hybribio, Hong Kong, China) was performed on HPV DNA-positive samples as previously described5,9). This test kit can detect 37 HPV genotypes, including 15 high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68), six low-risk types (6, 11, 42, 43, 44, and CP8304), and 16 probable low-risk types (26, 34, 40, 54, 55, 57, 61, 67, 69, 70, 72, 73, 82, 83, and 84).
The patients consisted of six males and one female. The mean and median ages were 55 and 53 years, respectively, with a range of 31-75 years. The mean and median laryngeal severity scores based on Derkay’s report were 3.9 and 3, respectively, with a range of 2-8 (Table 1)14).
β-Globin was detected in all oral rinse, palatine tonsil swab, and pharyngeal tonsil swab samples. HPV DNA was detected in oral rinse samples from five of the seven patients (71%) (Figure 1 and Table 1). HPV DNA was detected in both the first and second PCR in two patients (Patients 6 and 7) and only in the second PCR in the remaining three patients (Figure 1 and Table 1). HPV genotyping identified HPV6 in four HPV DNA-positive samples while the genotype was not identifiable in the remaining case (Table 1). In contrast, HPV DNA was not detected in either the palatine tonsil swabs or pharyngeal tonsil swabs of any patient, even with a second PCR (Figure 1 and Table 1).
Laryngeal lesions in patients and detection of HPV DNA
*Based on Derkay’s anatomical scoring system for the 13 laryngeal regions14). N, negative;P, positive.
HPV DNA detection in oral rinses, palatine tonsil swabs, and pharyngeal tonsil swabs from patients with recurrent respiratory papillomatosis (RRP). Each sample was electrophoresed on different gels because the present study was performed as a part of a larger project investigating HPV DNA detection in oral rinses, palatine tonsil swabs, and pharyngeal tonsil swabs from patients with various head and neck cancers and benign otolaryngologic diseases, including RRP5,9). Therefore, Figure 1 is a compilation of the relevant sections from the original electrophoresis images of each patient, presented without any modifications. The upper, middle, and lower bands in the “M” lane indicate 300, 200, and 100 base-pairs, respectively. M, DNA ladder marker;PC, positive control;NC, negative control;O, oral rinse;Pa, palatine tonsil swab;Ph, pharyngeal tonsil swab.
Liquid biopsy targeting viral DNA has been gaining attention in the field of oncology, particularly for Epstein-Barr virus (EBV)-associated nasopharyngeal cancer (NPC) and HPV-associated OPC15). In particular, plasma cell-free EBV DNA in NPC has been used as a diagnostic and prognostic marker in clinical settings15). Circulating plasma HPV DNA in OPC has also been suggested as a diagnostic and prognostic marker similar to NPC15).
Given that the association of HPV with cervical cancer is a clinically established reason for cervical screening with primary HPV testing, oral rinses or saliva may be useful samples for OPC screening due to their non-invasive nature and ease of collection. Currently, the sensitivity and specificity of HPV detection in the oral rinses of OPC patients are reported to be 72% and 92%, respectively15,16). Rettig and Yoshida reported in their respective studies that the detection of HPV DNA in the oral rinse of OPC patients was rare after treatment; however, when detected, it was reportedly associated with a poor prognosis5,17).
Detection of HPV DNA from oral rinse samples of RRP patients was first reported by Born et al., who reported the oral rinses of 25 of 27 patients as HPV-positive6). A linear array performed on 15 of the 25 HPV-positive samples yielded four invalid results due to insufficient DNA and 11 valid results, consisting of HPV6, HPV11 and HPV62 in one sample each, HPV81 in five samples, and negative results in three samples6). In addition, four oral rinse samples from the sexual partners of six of the HPV-positive patients were also HPV-positive, suggesting viral transmission6). Hao et al. reported that 22 of 23 RRP patients had HPV-positive oral rinse specimens7). They analyzed HPV sequences in oral rinse and RRP tissue samples from two of these HPV-positive patients and found that the sequences of HPV in the oral rinses were identical to those of RRP tissue samples from the same patients, suggesting that the HPV detected in the oral rinse originated from the RRP lesions in those patients7). Recently, Chantre-Justino et al. reported that HPV DNA was detected in five of seven salivary cellular pellets from preoperative RRP patients8). Although it was not a longitudinal study with the same set of patients, HPV DNA was not detected in oral rinses collected from RRP patients during disease-free intervals, suggesting its association with disease activity8).
In the present study, HPV6 was the only HPV genotype detected in the oral rinse samples. Although strict sequence concordance was not examined, we confirmed that HPV genotypes detected in lesion tissues and oral rinses were identical in most patients who were positive for HPV DNA. In addition, we analyzed palatine and pharyngeal tonsil swab samples to confirm whether the HPV in oral rinses was not derived from the palatine or pharyngeal tonsils, which were not investigated in previous studies. HPV infection in the palatine or pharyngeal tonsils has been reported in cases of OPC and NPC18,19) and HPV may also infect these tonsils even in individuals without cancer, as we previously reported9). Although, to the best of our knowledge, no studies have examined HPV infection in the palatine and/or pharyngeal tonsils in RRP, the results of the present study suggest that neither the palatine nor pharyngeal tonsils were likely infected with HPV in our RRP patients, and therefore did not serve as reservoirs of HPV in the oral rinses.
In conclusion, considering the results of previous reports6-8) and those of the present study, with HPV DNA testing, the oral rinse has potential as a useful liquid biopsy. However, this pilot study has several limitations. First, the sample size was small. Second, we could not find any correlations between HPV DNA detection in oral rinse samples and lesion severity, particularly with the first PCR. This may be due to the varying number of cells collected, a point to be clarified in the future. Third, although all patients had persistent disease after multiple surgeries, we did not collect any longitudinal data on these patients. Nevertheless, to the best of our knowledge, this is the first Asian study to investigate HPV DNA in oral rinses of patients with RRP, and the data in the present study provide a basis for future studies. As RRP is a rare disease and single-center studies often have a limited sample size, we are currently planning a multicenter prospective study with the aim of clarifying the significance and clinical implications of HPV DNA detection in oral rinses.
This study was supported in part by JSPS KAKENHI Grant (JP26462598, SM), a research grant from the Daiwa Securities Health Foundation (currently Daiwa Securities Foundation) (42-17, SM), and a research grant from the Support Unit for Conducting Clinically Essential Studies in St. Luke’s International University (2016, SM).
The authors have no conflict of interest to disclose.
