- Email: [email protected]
Gardasil Vaccination for Recurrent Laryngeal Papillomatosis in Adult Men Second Report: Negative Conversion of HPV in Laryngeal Secretions
ARTICLE IN PRESS Gardasil Vaccination for Recurrent Laryngeal Papillomatosis in Adult Men Second Report: Negative Conversion of HPV in Laryngeal Secretions *Ryoji Hirai, †Kiyoshi Makiyama, †Hiroumi Matsuzaki, and *Takeshi Oshima, *†Tokyo, Japan Summary: Background. In our first report on antibody levels in middle-aged and older men with recurrent laryngeal papillomatosis (RLP), we reported increases in human papillomavirus (HPV) antibody levels similar to those seen in adult women and young men. We posited that HPV antibodies produced in laryngeal mucus by Gardasil would prevent postoperative reinfection in patients with RLP. Study Design. This is a case series study. Purpose. The purpose of this study was to examine whether Gardasil injection effectively inhibits recurrence of RLP. Specifically, in this second report, whether HPV antibodies produced in laryngeal secretions by Gardasil are capable of causing negative conversion of HPV-DNA (deoxyribonucleic acid) in laryngeal mucosa was investigated. Methods. A total of 11 patients for whom antibodies were measured in the first report were studied. Before vaccination and after 1 year Post-vaccination, HPV screening tests were performed on laryngeal secretions, and whether HPV-DNA negative conversion had occurred was evaluated. At the time of collection of laryngeal secretions, the presence or absence of laryngeal papillomas was examined. Results. Before vaccination, all patients were HPV low-risk positive on laryngeal secretion screening tests. After vaccination, three patients were positive. Laryngeal papillomas remained in five patients. Conclusions. The HPV-DNA test showed negative conversion in eight of 11 (72.7%) patients after vaccination. Residual laryngeal papillomas were found in five of 11 (45.5%) patients. The serum HPV antibody titer did not differ significantly between the group in which laryngeal secretions showed HPV negative conversion and the group in which conversion did not occur. The serum antibody titer did not differ significantly as a function of whether there were residual tumors. Key Words: Recurrent laryngeal papilloma–Quadrivalent HPV vaccine–Gardasil–Laryngeal secretions–Antibody titers.
INTRODUCTION We have reported that human papillomavirus (HPV)-6/11 infection rates were higher in patients with recurrent laryngeal papillomatosis (RLP) than in patients with nonrecurrent laryngeal papillomatosis.1 We also detected HPV-DNA (deoxyribonucleic acid) from normal laryngeal mucus in the region around papillomas. This kind of HPV infection may be a factor in the recurrence of papillomas after repeated surgery. HPV infection must be treated as part of RLP treatment. We speculated that vaccination with the quadrivalent HPV vaccine Gardasil could suppress postoperative recurrence if the vaccine were able to induce HPV antibodies in laryngeal mucus.2 It has been previously reported that the serum HPV antibody levels increase in healthy adult women and young men after vaccination with Gardasil,3,4 but there are no reports on antibody levels in middle-aged and older men. In our first report, we therefore investigated antibody levels in middle-aged and older patients with RLP, and we found similar increases in HPV Accepted for publication July 21, 2017. From the *Department of Otolaryngology- Head and Neck Surgery, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi, Tokyo 173-8610, Japan; and the †Department of Otolaryngology-Head and Neck Surgery, Nihon University Hospital, 1-6 Kandasurugadai, Chiyoda, Tokyo 101-8309, Japan. Address correspondence and reprint requests to Ryoji Hirai, Department of Otolaryngology- Head and Neck Surgery, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan. E-mail: [email protected] Journal of Voice, Vol. ■■, No. ■■, pp. ■■-■■ 0892-1997 © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvoice.2017.07.017
antibody levels. At the same time, Tjon Pian Gi et al found a similar increase in HPV antibody levels after Gardasil vaccination in six male patients aged 30 years or older with recurrent respiratory papillomatosis.5 In 2009, Schwarz reported a correlation between serum and cervicovaginal secretion levels of HPV-16 and HPV-18 immunoglobulin G (IgG) after vaccination with Cervarix, indicating that HPV antibodies undergo transudation in secretions when serum antibody levels increase.6 Pinto et al tested for oral HPV antibodies in mouthwash samples from middle-aged men after vaccination with quadrivalent HPV vaccine and found that 137 of 147 (93.2%) patients had HPV-16/18 antibodies and that these had transferred to the airway mucosa.7 These findings support our posited mechanism by which Gardasil vaccination suppresses reinfection. Specifically, HPV antibodies produced in the laryngeal mucosa by Gardasil vaccination are thought to prevent postoperative reinfection at the operating site by HPV-DNA released from non-tumor regions of laryngeal mucosa in patients with RLP. If our hypothesis is correct, HPV-DNA in laryngeal mucus should become negative after Gardasil vaccination. To test this theory, whether HPV-DNA in laryngeal secretions underwent negative conversion after Gardasil vaccination was investigated. To prove our theory, relationships among the HPV-DNA results for laryngeal swabbed fluids collected before and after Gardasil vaccination, the presence or absence of residual laryngeal papilloma after vaccination, and negative conversion of the laryngeal secretions were examined.
ARTICLE IN PRESS 2
Journal of Voice, Vol. ■■, No. ■■, 2017
TABLE 1. Results for HPV Antibody Levels Before and After Vaccination, Screening Tests, and Presence or Absence of Laryngeal Papillomas Before and 1 Year After Vaccination
Patient No. 1 12 13 14 15 16 17 18 19 10 11
M7
1 Year After Vaccination HPV Screening Tests
1 Year After Vaccination Presence or Absence of Laryngeal Papillomas
1600 788 1282 141 449 258 137 895 363 1167 519
Positive Negative Negative Negative Negative Negative Negative Positive Negative Negative Positive
Presence Absence Presence Presence Absence Absence Absence Presence Absence Absence Presence
Quantitative Results (mMU/mL)
Age
Before Vaccination HPV Screening Tests
HPV Type
M0
M7
M0
32 33 39 44 45 46 46 48 49 54 65
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
6 6 6 6 6 6 6 11 6 6 6
<11 <11 <11 <11 <11 <11 21 <11 <11 13 <11
632 287 1475 144 240 2070 139 755 476 350 354
<8 <8 9 <8 <8 <8 <8 <8 <8 <8 <8
SUBJECTS Twelve HPV positive, male patients with RLP who were examined by the otorhinolaryngology department of Nihon University Hospital and gave written consent to participate were eligible for the study. One of the 12 patients, a 74-year-old, was excluded because he was treated by radiotherapy for concomitant laryngeal cancer. The remaining 11 subjects were included in the analysis. The subjects were 32 to 65 years old (mean age 45.5 years). HPV infection was determined by an HPV-DNA test using liquid-phase hybridization8–11 or a consensus primer-directed polymerase chain reaction system.12 Screening was done with liquidphase hybridization, and all subjects were HPV low-risk positive; there were no HPV high-risk cases. In a typing test by polymerase chain reaction, 10 subjects were HPV-6 positive, and one was HPV-11 positive. METHODS Combination therapy with papilloma resection and vaccine therapy was used. For surgery, the papilloma was completely resected or vaporized with the Ho:yttrium aluminum garnet laser in microlaryngeal surgery. For vaccine therapy, Gardasil (Merck Sharp and Dohme Corp., a subsidiary of Merck and Co., Inc., Kenilworth, NJ) was injected three times. The surgery was conducted either before the vaccine injection or after the first vaccine injection. The Gardasil injections were done according to the directions for use, with the second injection given 2 months after the first injection and the third injection given 6 months after the first injection. Negative conversion of HPV-DNA was investigated by performing screening tests using laryngeal secretions before vaccination and at least 1 year after vaccination. Furthermore, the presence or absence of laryngeal papillomas was examined when laryngeal secretions were collected. The presence of relationships of negative conversion and the presence or absence of papillomas with the HPV antibody level was tested using Mann-Whitney U tests.
RESULTS Table 1 shows the HPV low-risk group test results for the laryngeal mucosa swabbed fluids before and after vaccination, the presence or absence of papillomas at the time of collection of laryngeal secretions after vaccination, the results of HPV typing before vaccination, and the HPV antibody titers at the time of the first vaccination and in the seventh month. All patients were positive for low-risk−group HPV before vaccination. After vaccination, three patients remained positive, and the others were negative. Typing tests showed that two of the three positive patients were positive for HPV-6, and one was positive for HPV-11. The HPV-6−positive group (10 of 11 patients) was divided into two groups according to whether negative conversion occurred. The mean serum antibody level was 647.63 mMU/mL in the negative-conversion group (eight patients) and 493.00 mMU/mL in the non-conversion group (two patients). The Mann-Whitney U test found no significant difference in serum antibody levels between the negative-conversion group and the non-conversion group (P = 0.4334). In HPV-6−positive group, the mean serum antibody titer of four patients who had papillomas even after vaccination was 651.25 mMU/mL, whereas the mean serum antibody titer of six patients without papillomas was 593.67 mMU/mL. There appeared to be no significant relationship between the presence or absence of papillomas and the serum HPV-6 antibody titer in the Mann-Whitney U test (P = 0.8815). DISCUSSION This study was a follow-up to our first report. The same patients were examined, although one who underwent radiotherapy for concomitant laryngeal cancer was excluded. Few reports have investigated the serum antibody titers in patients with RLP who were vaccinated with Gardasil.3,4 In the present study, there were only 11 patients, but they were the same patients as in our first report on the presence or absence of laryngeal papillomas and HPV-DNA in laryngeal secretions. A literature search identified
ARTICLE IN PRESS Ryoji Hirai, et al
Secont Report: Negative Conversion of HPV in Laryngeal Secreations
no other reports regarding patients with RLP in whom laryngeal secretions and serum antibody titers were investigated. For five patients (patients 1, 3, 6, 7, and 8), multiple HPV-DNA tests were performed before vaccination, and all were positive. The finding that the HPV-DNA test became negative after vaccination is considered to be due to the effect of vaccination rather than a coincidence. The HPV-DNA test became negative after vaccination in 8 of 11 (73%) patients, but laryngeal papillomas remained in 5 of 11 (45.5%). In other words, papillomas disappeared in 6 of 11 (54.5%) patients. Presence or absence of papillomas after vaccination and laryngeal secretion HPV-DNA The results of this study showed that there were three patients in whom HPV-DNA was positive and papillomas were present after vaccination. In contrast, two patients (patients 3 and 4) were laryngeal secretion-negative despite having papillomas. If laryngeal secretions become negative for HPV-DNA, the possibility of recurrence after laser surgery is thought to be low. There may be a time lag between HPV-DNA negative conversion and the presence of papillomas. The follow-up period of about 1 year was short for examining the relationship with suppression of recurrence, and the result should be considered only as a reference finding. For a third report, we intend to investigate the effect on suppression of RLP recurrence at 5 years after Gardasil vaccination. Negative conversion of HPV-DNA in laryngeal secretions Vaccination with quadrivalent HPV vaccine causes production of anti-HPV-16 IgG in saliva,7 in the same way that antiinfluenza IgG is seen in saliva after influenza vaccination.13 As far as we know, IgG levels in saliva and laryngeal secretions have not been studied in the HPV low-risk group. The studies by Schwarz6 and Pinto7 suggest that HPV antibodies produced by vaccination can occur in body fluids via transudation from the blood. If antibodies can be induced in laryngeal mucus, it should be possible to prevent reinfection, as described previously. HPV-DNA became negative after vaccination in most of the patients in the present study. It was therefore highly probable that antibodies were present in the laryngeal mucus. This result seems to confirm our hypothesis that HPV antibodies produced in the laryngeal mucosa by Gardasil vaccination prevent postoperative reinfection at the operating site by HPVDNA released from non-tumor regions of laryngeal mucosa in patients with RLP.1 Relationship between laryngeal secretions and serum antibody levels Laryngeal swabs remained positive in three of the 11 patients. There was nothing notable about the age of these three patients compared with the negative conversion group. When we looked for differences in antibody production ability between the negative conversion group and the non-conversion group, among the 10 HPV-6−positive patients, the mean antibody level was lower in the two who remained positive than in the eight who became negative, but the difference was not significant.
3
Pinto et al reported that HPV-16/18 antibody levels in the mouth correlated with serum antibody levels,7 but they were about 500fold lower than serum levels. These results suggest that patients 4 and 7, for example, had very low HPV antibody levels in laryngeal mucus, even though the HPV-DNA test became negative after vaccination. One patient was positive for HPV-11, and the test for HPV in laryngeal secretions remained positive after vaccination. This patient had a high antibody level of 895 mMU/mL. Further study is needed to identify the factors other than antibody levels that contribute to HPV-negative conversion. Relationship between presence or absence of papillomas after vaccination and the serum antibody titer In 10 HPV-6–positive patients, the mean HPV serum antibody titer was 651.25 mMU/mL in patients with residual papillomas, whereas it was 593.67 mMU/mL in patients whose papillomas had disappeared. The presence or absence of laryngeal papillomas in these patients showed a slightly higher result in the group with papillomas, but the difference was not significant. From this, it can be thought that the serum antibody titer is not related to efficacy in suppressing papilloma recurrence. This study found that, after vaccination, many patients became negative on the screening test for HPV in laryngeal secretions. HPV-DNA can be thought to have disappeared from the laryngeal secretions of such patients. The laryngeal secretion HPVDNA test has great potential for providing useful information for evaluating acquisition of HPV immunity due to Gardasil vaccination. Future studies need to investigate a larger number of cases. CONCLUSIONS The results of HPV-DNA tests using laryngeal swabs taken before and after vaccination were compared in 11 patients with RLP who were vaccinated with the quadrivalent HPV vaccine Gardasil. The HPV-DNA test showed negative conversion in 8 of 11 (73%) patients after vaccination. Of the three patients who remained positive, two were positive for HPV-6, and one was positive for HPV-11. The serum HPV antibody level did not differ between the negative conversion group and the non-conversion group. There was also no significant difference in the relationship between the presence or absence of residual tumors and the serum antibody titer. These results seem to confirm our hypothesis that HPV antibodies produced in the laryngeal mucosa by Gardasil vaccination prevent postoperative reinfection at the operating site by HPV-DNA released from non-tumor regions of laryngeal mucosa in patients with RLP. REFERENCES 1. Makiyama K, Hirai R, Matsuzaki H, et al. Assessment of human papilloma virus infection in adult laryngeal papilloma using a screening test. J Voice. 2013;27:230–235. 2. Makiyama K, Hirai R, Matsuzaki H. Gardasil vaccination for recurrent laryngeal papillomatosis in adult men: first report: changes in HPV antibody titer. J Voice. 2017;31:104–106. 3. Munoz N, Manalastas R Jr, Pitisuttithum P, et al. Safety, immunogenicity, and efficacy of quadrivalent human papillomavirus (types 6, 11, 16, 18)
ARTICLE IN PRESS 4
4.
5.
6.
7.
8.
Journal of Voice, Vol. ■■, No. ■■, 2017 recombinant vaccine in women aged 24–45 years: a randomised, double-blind trial. Lancet. 2009;373:1949–1957. Hillman RJ, Giuliano AR, Palefsky JM, et al. Immunogenicity of the quadrivalent human papillomavirus (type 6/11/16/18) vaccine in males 16 to 26 years old. Clin Vaccine Immunol. 2012;19:261– 267. Tjon Pian Gi RE, San Giorgi MR, Pawlita M, et al. Immunological response to quadrivalent HPV vaccine in treatment of recurrent respiratory papillomatosis. Eur Arch Otorhinolaryngol. 2016;273:3231–3236. Schwarz TF, Spaczynski M, Schneider A, et al. Immunogenicity and tolerability of an HPV-16/18 AS04-adjuvanted prophylactic cervical cancer vaccine in women aged 15–55 years. Vaccine. 2009;27:581– 587. Pinto LA, Kemp TJ, Torres BN, et al. The quadrivalent HPV vaccine induces HPV-specific antibodies at the oral cavity: results from the mid-adult male vaccine trial-the MAM trial. J Infect Dis. 2016;214:1276– 1283. Nobbenhuis MA, Walboomers JM, Helmerhorst TJ, et al. Relation of human papillomavirus status to cervical lesions and consequences for
9.
10.
11.
12.
13.
cervical-cancer screening: a prospective study. Lancet. 1999;354:20– 25. Clavel C, Masure M, Bory JP, et al. Human papillomavirus testing in primary screening for the detection of high-grade cervical lesions: a study of 7932 women. Br J Cancer. 2001;84:1616–1623. Ronco G, Giorgi-Rossi P, Carozzi F, et al. Human papillomavirus testing and liquid-based cytology in primary screening of women younger than 35 years: results at recruitment for a randomised controlled trial. Lancet Oncol. 2006;7:547–555. Clavel C, Masure M, Bory JP, et al. Hybrid Capture II-based human papillomavirus detection, a sensitive test to detect in routine high-grade cervical lesions: a preliminary study on 1518 women. Br J Cancer. 1999;80:1306–1311. Yoshikawa H, Kawana T, Kitagawa K, et al. Detection and typing of multiple genital human papillomaviruses by DNA amplification with consensus primers. Jpn J Cancer Res. 1991;82:524–531. Brokstad KA, Eriksson JC, Cox RJ, et al. Parenteral vaccination against influenza does not induce a local antigen-specific immune response in the nasal mucosa. J Infect Dis. 2002;185:878–884.
INTRODUCTION We have reported that human papillomavirus (HPV)-6/11 infection rates were higher in patients with recurrent laryngeal papillomatosis (RLP) than in patients with nonrecurrent laryngeal papillomatosis.1 We also detected HPV-DNA (deoxyribonucleic acid) from normal laryngeal mucus in the region around papillomas. This kind of HPV infection may be a factor in the recurrence of papillomas after repeated surgery. HPV infection must be treated as part of RLP treatment. We speculated that vaccination with the quadrivalent HPV vaccine Gardasil could suppress postoperative recurrence if the vaccine were able to induce HPV antibodies in laryngeal mucus.2 It has been previously reported that the serum HPV antibody levels increase in healthy adult women and young men after vaccination with Gardasil,3,4 but there are no reports on antibody levels in middle-aged and older men. In our first report, we therefore investigated antibody levels in middle-aged and older patients with RLP, and we found similar increases in HPV Accepted for publication July 21, 2017. From the *Department of Otolaryngology- Head and Neck Surgery, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi, Tokyo 173-8610, Japan; and the †Department of Otolaryngology-Head and Neck Surgery, Nihon University Hospital, 1-6 Kandasurugadai, Chiyoda, Tokyo 101-8309, Japan. Address correspondence and reprint requests to Ryoji Hirai, Department of Otolaryngology- Head and Neck Surgery, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan. E-mail: [email protected] Journal of Voice, Vol. ■■, No. ■■, pp. ■■-■■ 0892-1997 © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvoice.2017.07.017
antibody levels. At the same time, Tjon Pian Gi et al found a similar increase in HPV antibody levels after Gardasil vaccination in six male patients aged 30 years or older with recurrent respiratory papillomatosis.5 In 2009, Schwarz reported a correlation between serum and cervicovaginal secretion levels of HPV-16 and HPV-18 immunoglobulin G (IgG) after vaccination with Cervarix, indicating that HPV antibodies undergo transudation in secretions when serum antibody levels increase.6 Pinto et al tested for oral HPV antibodies in mouthwash samples from middle-aged men after vaccination with quadrivalent HPV vaccine and found that 137 of 147 (93.2%) patients had HPV-16/18 antibodies and that these had transferred to the airway mucosa.7 These findings support our posited mechanism by which Gardasil vaccination suppresses reinfection. Specifically, HPV antibodies produced in the laryngeal mucosa by Gardasil vaccination are thought to prevent postoperative reinfection at the operating site by HPV-DNA released from non-tumor regions of laryngeal mucosa in patients with RLP. If our hypothesis is correct, HPV-DNA in laryngeal mucus should become negative after Gardasil vaccination. To test this theory, whether HPV-DNA in laryngeal secretions underwent negative conversion after Gardasil vaccination was investigated. To prove our theory, relationships among the HPV-DNA results for laryngeal swabbed fluids collected before and after Gardasil vaccination, the presence or absence of residual laryngeal papilloma after vaccination, and negative conversion of the laryngeal secretions were examined.
ARTICLE IN PRESS 2
Journal of Voice, Vol. ■■, No. ■■, 2017
TABLE 1. Results for HPV Antibody Levels Before and After Vaccination, Screening Tests, and Presence or Absence of Laryngeal Papillomas Before and 1 Year After Vaccination
Patient No. 1 12 13 14 15 16 17 18 19 10 11
M7
1 Year After Vaccination HPV Screening Tests
1 Year After Vaccination Presence or Absence of Laryngeal Papillomas
1600 788 1282 141 449 258 137 895 363 1167 519
Positive Negative Negative Negative Negative Negative Negative Positive Negative Negative Positive
Presence Absence Presence Presence Absence Absence Absence Presence Absence Absence Presence
Quantitative Results (mMU/mL)
Age
Before Vaccination HPV Screening Tests
HPV Type
M0
M7
M0
32 33 39 44 45 46 46 48 49 54 65
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive
6 6 6 6 6 6 6 11 6 6 6
<11 <11 <11 <11 <11 <11 21 <11 <11 13 <11
632 287 1475 144 240 2070 139 755 476 350 354
<8 <8 9 <8 <8 <8 <8 <8 <8 <8 <8
SUBJECTS Twelve HPV positive, male patients with RLP who were examined by the otorhinolaryngology department of Nihon University Hospital and gave written consent to participate were eligible for the study. One of the 12 patients, a 74-year-old, was excluded because he was treated by radiotherapy for concomitant laryngeal cancer. The remaining 11 subjects were included in the analysis. The subjects were 32 to 65 years old (mean age 45.5 years). HPV infection was determined by an HPV-DNA test using liquid-phase hybridization8–11 or a consensus primer-directed polymerase chain reaction system.12 Screening was done with liquidphase hybridization, and all subjects were HPV low-risk positive; there were no HPV high-risk cases. In a typing test by polymerase chain reaction, 10 subjects were HPV-6 positive, and one was HPV-11 positive. METHODS Combination therapy with papilloma resection and vaccine therapy was used. For surgery, the papilloma was completely resected or vaporized with the Ho:yttrium aluminum garnet laser in microlaryngeal surgery. For vaccine therapy, Gardasil (Merck Sharp and Dohme Corp., a subsidiary of Merck and Co., Inc., Kenilworth, NJ) was injected three times. The surgery was conducted either before the vaccine injection or after the first vaccine injection. The Gardasil injections were done according to the directions for use, with the second injection given 2 months after the first injection and the third injection given 6 months after the first injection. Negative conversion of HPV-DNA was investigated by performing screening tests using laryngeal secretions before vaccination and at least 1 year after vaccination. Furthermore, the presence or absence of laryngeal papillomas was examined when laryngeal secretions were collected. The presence of relationships of negative conversion and the presence or absence of papillomas with the HPV antibody level was tested using Mann-Whitney U tests.
RESULTS Table 1 shows the HPV low-risk group test results for the laryngeal mucosa swabbed fluids before and after vaccination, the presence or absence of papillomas at the time of collection of laryngeal secretions after vaccination, the results of HPV typing before vaccination, and the HPV antibody titers at the time of the first vaccination and in the seventh month. All patients were positive for low-risk−group HPV before vaccination. After vaccination, three patients remained positive, and the others were negative. Typing tests showed that two of the three positive patients were positive for HPV-6, and one was positive for HPV-11. The HPV-6−positive group (10 of 11 patients) was divided into two groups according to whether negative conversion occurred. The mean serum antibody level was 647.63 mMU/mL in the negative-conversion group (eight patients) and 493.00 mMU/mL in the non-conversion group (two patients). The Mann-Whitney U test found no significant difference in serum antibody levels between the negative-conversion group and the non-conversion group (P = 0.4334). In HPV-6−positive group, the mean serum antibody titer of four patients who had papillomas even after vaccination was 651.25 mMU/mL, whereas the mean serum antibody titer of six patients without papillomas was 593.67 mMU/mL. There appeared to be no significant relationship between the presence or absence of papillomas and the serum HPV-6 antibody titer in the Mann-Whitney U test (P = 0.8815). DISCUSSION This study was a follow-up to our first report. The same patients were examined, although one who underwent radiotherapy for concomitant laryngeal cancer was excluded. Few reports have investigated the serum antibody titers in patients with RLP who were vaccinated with Gardasil.3,4 In the present study, there were only 11 patients, but they were the same patients as in our first report on the presence or absence of laryngeal papillomas and HPV-DNA in laryngeal secretions. A literature search identified
ARTICLE IN PRESS Ryoji Hirai, et al
Secont Report: Negative Conversion of HPV in Laryngeal Secreations
no other reports regarding patients with RLP in whom laryngeal secretions and serum antibody titers were investigated. For five patients (patients 1, 3, 6, 7, and 8), multiple HPV-DNA tests were performed before vaccination, and all were positive. The finding that the HPV-DNA test became negative after vaccination is considered to be due to the effect of vaccination rather than a coincidence. The HPV-DNA test became negative after vaccination in 8 of 11 (73%) patients, but laryngeal papillomas remained in 5 of 11 (45.5%). In other words, papillomas disappeared in 6 of 11 (54.5%) patients. Presence or absence of papillomas after vaccination and laryngeal secretion HPV-DNA The results of this study showed that there were three patients in whom HPV-DNA was positive and papillomas were present after vaccination. In contrast, two patients (patients 3 and 4) were laryngeal secretion-negative despite having papillomas. If laryngeal secretions become negative for HPV-DNA, the possibility of recurrence after laser surgery is thought to be low. There may be a time lag between HPV-DNA negative conversion and the presence of papillomas. The follow-up period of about 1 year was short for examining the relationship with suppression of recurrence, and the result should be considered only as a reference finding. For a third report, we intend to investigate the effect on suppression of RLP recurrence at 5 years after Gardasil vaccination. Negative conversion of HPV-DNA in laryngeal secretions Vaccination with quadrivalent HPV vaccine causes production of anti-HPV-16 IgG in saliva,7 in the same way that antiinfluenza IgG is seen in saliva after influenza vaccination.13 As far as we know, IgG levels in saliva and laryngeal secretions have not been studied in the HPV low-risk group. The studies by Schwarz6 and Pinto7 suggest that HPV antibodies produced by vaccination can occur in body fluids via transudation from the blood. If antibodies can be induced in laryngeal mucus, it should be possible to prevent reinfection, as described previously. HPV-DNA became negative after vaccination in most of the patients in the present study. It was therefore highly probable that antibodies were present in the laryngeal mucus. This result seems to confirm our hypothesis that HPV antibodies produced in the laryngeal mucosa by Gardasil vaccination prevent postoperative reinfection at the operating site by HPVDNA released from non-tumor regions of laryngeal mucosa in patients with RLP.1 Relationship between laryngeal secretions and serum antibody levels Laryngeal swabs remained positive in three of the 11 patients. There was nothing notable about the age of these three patients compared with the negative conversion group. When we looked for differences in antibody production ability between the negative conversion group and the non-conversion group, among the 10 HPV-6−positive patients, the mean antibody level was lower in the two who remained positive than in the eight who became negative, but the difference was not significant.
3
Pinto et al reported that HPV-16/18 antibody levels in the mouth correlated with serum antibody levels,7 but they were about 500fold lower than serum levels. These results suggest that patients 4 and 7, for example, had very low HPV antibody levels in laryngeal mucus, even though the HPV-DNA test became negative after vaccination. One patient was positive for HPV-11, and the test for HPV in laryngeal secretions remained positive after vaccination. This patient had a high antibody level of 895 mMU/mL. Further study is needed to identify the factors other than antibody levels that contribute to HPV-negative conversion. Relationship between presence or absence of papillomas after vaccination and the serum antibody titer In 10 HPV-6–positive patients, the mean HPV serum antibody titer was 651.25 mMU/mL in patients with residual papillomas, whereas it was 593.67 mMU/mL in patients whose papillomas had disappeared. The presence or absence of laryngeal papillomas in these patients showed a slightly higher result in the group with papillomas, but the difference was not significant. From this, it can be thought that the serum antibody titer is not related to efficacy in suppressing papilloma recurrence. This study found that, after vaccination, many patients became negative on the screening test for HPV in laryngeal secretions. HPV-DNA can be thought to have disappeared from the laryngeal secretions of such patients. The laryngeal secretion HPVDNA test has great potential for providing useful information for evaluating acquisition of HPV immunity due to Gardasil vaccination. Future studies need to investigate a larger number of cases. CONCLUSIONS The results of HPV-DNA tests using laryngeal swabs taken before and after vaccination were compared in 11 patients with RLP who were vaccinated with the quadrivalent HPV vaccine Gardasil. The HPV-DNA test showed negative conversion in 8 of 11 (73%) patients after vaccination. Of the three patients who remained positive, two were positive for HPV-6, and one was positive for HPV-11. The serum HPV antibody level did not differ between the negative conversion group and the non-conversion group. There was also no significant difference in the relationship between the presence or absence of residual tumors and the serum antibody titer. These results seem to confirm our hypothesis that HPV antibodies produced in the laryngeal mucosa by Gardasil vaccination prevent postoperative reinfection at the operating site by HPV-DNA released from non-tumor regions of laryngeal mucosa in patients with RLP. REFERENCES 1. Makiyama K, Hirai R, Matsuzaki H, et al. Assessment of human papilloma virus infection in adult laryngeal papilloma using a screening test. J Voice. 2013;27:230–235. 2. Makiyama K, Hirai R, Matsuzaki H. Gardasil vaccination for recurrent laryngeal papillomatosis in adult men: first report: changes in HPV antibody titer. J Voice. 2017;31:104–106. 3. Munoz N, Manalastas R Jr, Pitisuttithum P, et al. Safety, immunogenicity, and efficacy of quadrivalent human papillomavirus (types 6, 11, 16, 18)
ARTICLE IN PRESS 4
4.
5.
6.
7.
8.
Journal of Voice, Vol. ■■, No. ■■, 2017 recombinant vaccine in women aged 24–45 years: a randomised, double-blind trial. Lancet. 2009;373:1949–1957. Hillman RJ, Giuliano AR, Palefsky JM, et al. Immunogenicity of the quadrivalent human papillomavirus (type 6/11/16/18) vaccine in males 16 to 26 years old. Clin Vaccine Immunol. 2012;19:261– 267. Tjon Pian Gi RE, San Giorgi MR, Pawlita M, et al. Immunological response to quadrivalent HPV vaccine in treatment of recurrent respiratory papillomatosis. Eur Arch Otorhinolaryngol. 2016;273:3231–3236. Schwarz TF, Spaczynski M, Schneider A, et al. Immunogenicity and tolerability of an HPV-16/18 AS04-adjuvanted prophylactic cervical cancer vaccine in women aged 15–55 years. Vaccine. 2009;27:581– 587. Pinto LA, Kemp TJ, Torres BN, et al. The quadrivalent HPV vaccine induces HPV-specific antibodies at the oral cavity: results from the mid-adult male vaccine trial-the MAM trial. J Infect Dis. 2016;214:1276– 1283. Nobbenhuis MA, Walboomers JM, Helmerhorst TJ, et al. Relation of human papillomavirus status to cervical lesions and consequences for
9.
10.
11.
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