Research

JAMA Otolaryngology–Head & Neck Surgery | Original Investigation

Human Papillomavirus Genotype Detection

in Oral Gargle Samples Among Men With Newly Diagnosed
Oropharyngeal Squamous Cell Carcinoma
Laura Martin-Gomez, MD, PhD; Anna R. Giuliano, PhD; William J. Fulp, MS; Jimmy Caudell, MD, PhD;
Michelle Echevarria, MD; Bradley Sirak, MPH; Martha Abrahamsen, MPH; Kimberly A. Isaacs-Soriano, MPH;
Juan C. Hernandez-Prera, MD; Bruce M. Wenig, MD; Kathryn Vorwald, MD, DDS; Caitlin P. McMullen, MD;
J. Trad Wadsworth, MD, MBA; Robbert J. Slebos, PhD; Christine H. Chung, MD

Invited Commentary
IMPORTANCE The most common cause of oropharyngeal squamous cell carcinoma is human
papillomavirus (HPV) infection, and currently the standard of care to determine the HPV
infection status in this type of carcinoma is to use p16 immunohistochemistry as a surrogate
marker of high-risk HPV infection. Although p16 immunohistochemistry is limited by the
inability to determine the specific HPV genotypes, oral gargle samples may be a readily
available source of HPV DNA for genotyping.

OBJECTIVE To determine the specific HPV genotypes present in both oral gargle samples and
tumor specimens.

DESIGN, SETTING, AND PARTICIPANTS This prospective, biomarker cohort study conducted at
a single specialized cancer hospital in Florida screened approximately 800 potentially eligible
participants from May 2014 through October 2017. To be eligible for participation, patients
had to meet all of the following criteria: 18 years of age or older, male sex, newly diagnosed as
having stage I to IV cancer of the oropharynx, a squamous cell carcinoma diagnosis,
treatment naive or at least 4 weeks after chemoradiation or surgical treatment of other
diseases, fully understand the study procedures and risks involved, and voluntarily agree to
participate by signing an informed consent statement.

MAIN OUTCOMES AND MEASURES Detection rate of HPV infection and HPV genotypes in oral
gargle samples and tumor specimens.

RESULTS A cohort of 204 male participants with newly diagnosed oropharyngeal squamous
cell carcinoma was assessed in this prospective collection of comprehensive clinical data and
oral gargle samples. Most study participants (190 [93.1%]) were white and ever smokers (114, Author Affiliations: Center for
55.9%), with a median age of 61 years (range, 35-87 years). The HPV infection status could be Immunization and Infection Research
assessed in 203 of 204 participants (99.5%) using oral gargle samples: 35 samples (17.2%) in Cancer, Tampa, Florida
(Martin-Gomez, Giuliano, Sirak,
were negative for HPV infection, whereas 168 samples (82.8%) were positive for HPV Abrahamsen, Isaacs-Soriano, Slebos,
infection. The detection rate of HPV genotypes was 93.0% in tumor specimens (160 Chung); Department of Biostatistics
specimens) and 82.8% (168 samples) in oral gargle samples. The oral gargle samples and Bioinformatics, Moffitt Cancer
Center, Tampa, Florida (Fulp);
frequently had low-risk HPV genotypes that were not detected in tumors, but these low-risk
Department of Radiation Oncology,
genotypes were always a coinfection with high-risk genotypes. Moffitt Cancer Center, Tampa, Florida
(Caudell, Echevarria); Department of
CONCLUSIONS AND RELEVANCE Oral gargle samples can be used to detect the majority of Pathology, Moffitt Cancer Center,
Tampa, Florida (Hernandez-Prera,
clinically relevant HPV genotypes found in oropharyngeal squamous cell carcinoma, but the Wenig); Department of Head and
interpretation of HPV detected in these samples should be assessed with caution for general Neck–Endocrine Oncology, Moffitt
cancer risk assessment given that sensitive assays can concomitantly detect low-risk Cancer Center, Tampa, Florida
genotypes. (Vorwald, McMullen, Wadsworth,
Slebos, Chung).
Corresponding Author: Christine H.
Chung, MD, Department of Head and
Neck–Endocrine Oncology, 12902
JAMA Otolaryngol Head Neck Surg. doi:10.1001/jamaoto.2019.0119 Magnolia Dr, Tampa, FL 33612
Published online March 28, 2019. (christine.chung@moffitt.org).

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 Research Original Investigation
T
he oropharynx, which includes the tonsils, base of the
tongue, soft palate, and oropharyngeal walls, is one of
the main anatomic sites within the head and neck re-
gion. Squamous cell carcinoma is the most common histol-
ogy of cancers arising in the oropharynx, and those oropha-
r y nge a l s q u a m o u s c e l l c a r c i n o m a s ( O P S C C s) w it h
predominantly or exclusively nonkeratinizing features are
closely linked with and are often predictive of the presence of
high-risk human papillomavirus (HPV). The main risk factors
associated with OPSCC are long-term tobacco use and HPV
infection. 1,2 For unclear reasons, the incidence of HPV-
related OPSCC is rapidly increasing, particularly in men, while
the incidence of tobacco-related OPSCC is decreasing as fewer
people smoke. 3,4 It is well established that HPV-related
OPSCC has distinct molecular and clinical characteristics lead-
ing to a favorable prognosis compared with tobacco-related
OPSCC.5-7
For accurate staging and prognosis, it is the standard of care
to determine the HPV infection status in patients with OPSCC
by using p16 immunohistochemistry (IHC) as a surrogate
marker of HPV infection.8 Surrogate HPV testing via p16 IHC
is widely recommended because of a high correlation with
other methods of detection, low cost, and ease of testing within
the current workflow in general pathology laboratories.9 How-
ever, it is limited by an inability to determine the specific HPV
genotypes and has been validated as a surrogate marker only
in the oropharynx, excluding its use in cancers arising in other
sites within the head and neck. While determination of pres-
ence or absence of HPV by p16 IHC is sufficient for the pur-
pose of prognosis, it is not sufficient for potential therapeutic
strategies targeting specific HPV genotypes, such as therapeu-
tic vaccines that often target HPV 16, the most common geno-
type associated with OPSCC.10
In addition, most HPV testing has been performed in tu-
mor tissue only, and sometimes HPV testing is not feasible with-
out the patient undergoing an invasive procedure to obtain
more tumor tissue. An alternative to tumor HPV testing is the
use of DNA obtained from oral gargle or rinse samples, which
are more accessible for collection. There is emerging data that
HPV detection in oral rinse or gargle samples is highly
sensitive.11-13 Therefore, the present study evaluated the spe-
cific HPV genotypes in both oral gargle samples and tumor
specimens derived from a cohort of patients newly diag-
nosed as having OPSCC, with a prospective collection of com-
prehensive clinical data.
Methods
Study Design and Participants
From May 2014 through October 2017, approximately 800 par-
ticipants were screened from the Head and Neck and Radia-
tion Oncology Clinics at Moffitt Cancer Center in Tampa,
Florida, as part of an ongoing biomarker study. To be eligible
for participation, patients had to meet all of the following eli-
gibility criteria: 18 years of age or older, male sex, newly diag-
nosed as having stage I to IV cancer of the oropharynx (Inter-
national Classification of Diseases for Oncology, third edition,
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Human Papillomavirus Genotype Detection in Oropharyngeal Squamous Cell Carcinoma
Key Points
Question Can oral gargle samples be used for determination of
human papillomavirus genotypes in men with oropharyngeal
squamous cell carcinoma?
Findings In this cohort study of 204 male participants, most of
the clinically relevant human papillomavirus genotypes found in
oropharyngeal squamous cell carcinoma were detected using oral
gargle samples. When low-risk human papillomavirus genotypes
were detected, they were always a coinfection with high-risk
genotypes.
Meaning The interpretation of human papillomavirus detected in
oral gargle samples should be evaluated with caution in general
cancer risk assessment given that sensitive assays can
concomitantly detect low-risk human papillomavirus genotypes.
first revision, sites: C01.9, C05.1/2, C09.0/1/8/9, or C10.1/2/3/
4/8/9), a squamous cell carcinoma histologic diagnosis, treat-
ment naive or at least 4 weeks after chemoradiation or surgi-
cal treatment of other diseases, fully understand the study
procedures and risks involved, and voluntarily agree to par-
ticipate by signing an informed consent statement. Reasons
for refusal to participate or exclusion were recorded to ac-
count for potential selection biases. The study protocol was
approved by the Chesapeake Institutional Review Board, Co-
lumbia, Maryland, and the Moffitt Cancer Center Scientific Re-
view Committee. All included participants provided signed in-
formed consent statements.
Data Collection
All participants prospectively completed an extensive health
and risk factor questionnaire at their baseline visit. Only 2 par-
ticipants (0.1%) opted to fill out a paper version of the ques-
tionnaire; the remaining used the online, computer-assisted,
self-interviewing administered survey method as previously
described.14,15 Male sex was determined by self-report. Only
men were included because the incidence of the cancer is 5
times higher among men than among women. With limited
funding, we decided to focus on men only. Baseline demo-
graphic and behavioral characteristics were determined from
the questionnaire. Electronic medical records were reviewed
and abstracted for information related to diagnosis, treat-
ment, and overall survival. The seventh edition of the Ameri-
can Joint Committee on Cancer staging manual was used to
stage participants at initial diagnosis, but the eighth edition
was used retrospectively for the analyses.
A pathology panel composed of 2 board-certified patholo-
gists (J.C.H.-P. and B.M.W.) blinded to diagnoses and patient
characteristics independently reviewed tumor specimens.
Evaluation of tumor histology and p16INK4a (p16 clone E6H4,
CINtec immunoassay) IHC was determined using a pathology
panel, with a 95% concordance of the independent readings.
Discordant results, as well as 10% of all readings, were reex-
amined by a third independent pathologist. The p16 result was
considered positive when at least 70% of the cells showed
strong and diffuse nuclear and cytoplasmic staining, as rec-
ommended by the College of American Pathologists.9
jamaotolaryngology.com
 Human Papillomavirus Genotype Detection in Oropharyngeal Squamous Cell Carcinoma
The HPV genotype was assessed for all tumor tissue speci-
mens and oral gargle samples collected at the time of recruit-
ment using the in vitro reverse hybridization assay RHA Kit HPV
SPF10-LiPA25 (DDL Diagnostic Laboratory) for the qualita-
tive identification of HPV DNA. The LiPA25 targets a 65–base
pair fragment of the L1 region of the HPV genome. This assay
has been shown to detect up to 20% more HPV types than other
marketed assays, making this the most sensitive assay for
samples with low copy number or mixed-type HPV samples
such as those obtained in oral gargle samples.16,17 This method
has reliably identified HPV types in anogenital epithelia18,19 as
well as in oral gargle samples.20,21
Statistical Analysis
Descriptive statistics, including the median and range for con-
tinuous measures and proportions and frequencies for cat-
egorical measures, were summarized for patient and clinical
characteristics. Univariate Cox proportional hazards regres-
sion models were used to compare HPV status with respect to
overall survival. Overall survival was measured from date of
biopsy to last patient contact or death. Agreement between oral
gargle sample, and tumor specimen HPV status was calcu-
lated with 95% CIs for each HPV type. Statistical analyses were
conducted using R, version 3.5.1 (The R Foundation).
Results
Patient Characteristics
In total, 502 male participants potentially eligible for this study
were screened by study coordinators (including K.A.I.-S.), and
225 provided informed consent and were enrolled. After en-
rollment, 32 participants (14.2%) were excluded for various rea-
sons, including 13 participants having initiated treatment, 6
having recurrent disease, and 2 having nonsquamous cell his-
tology. A total of 204 participants with base of tongue, tonsil,
or soft palate tumors were included in the final analyses. The
HPV genotype agreement between oral gargle samples and tu-
mor specimens was calculated for the 171 pairs available
(Figure).
The majority of the 204 study participants were white (190,
93.1%) and non-Hispanic (189, 92.6%), had a median (range)
age of 61 (35-87) years, and were ever smokers (114, 55.9%),
which include either current or former (quit ≥12 months be-
fore diagnosis) smokers with a median (range) of 20 (0-132)
pack-years (Table 1). A substantial percentage of patients (89,
43.6%) had a palatine tonsillectomy prior to diagnosis. Among
them, 67 patients (75.3%) had the operation more than or equal
to 10 years ago, 20 patients (22.5%) had the operation less than
10 years ago, and such information was missing for 2 patients
(2.2%). In addition, 58 of 92 patients (63%) with primary tu-
mors in the base of the tongue and 6 of 10 (60%) with primary
tumors in the soft palate had a palatine tonsillectomy, whereas
25 of 98 patients (26%) with tonsillar primary tumors had a
palatine tonsillectomy. These findings suggest that patients
with a history of palatine tonsillectomy had a lower chance of
having OPSCC in the tonsils compared with the base of the
tongue or soft palate. This finding also suggests a prophylac-
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Original Investigation Research
Figure. Study Participant Selection Process
Between May 2014 and October 2017
Approximately 800 participants screened
from the Head and Neck Oncology Clinic
Approximately 300 excluded for nonsquamous cell
histology, nonoropharyngeal site, prior treatment,
metastatic disease, or being a woman
502 Screened as potentially eligible
OPSCC study participants
277 Ineligible
115 Recurrent disease
53 Treated at outside facility
31 Unable to recruit before starting treatment
25 Not interested in study goals
19 Recently treated with chemoradiation
12 Language barrier
11 Multiple disease sites
7 Other (prisoner, hospice care, or dementia)
4 Reason unknown
225 Signed informed consent for study
21 Excluded from analysis
13 Participants had been treated (chemoradiation
or surgery) within the previous 4 weeks
6 Recurrent cases of OPSCC
2 Nonsquamous cell histology
204 Included in the descriptive analysis,
of whom 171 had both an oral gargle
sample and tumor specimen available
for calculation of HPV agreement
HPV indicates human papillomavirus; OPSCC, oropharyngeal squamous cell
carcinoma.
tic tonsillectomy does not protect from development of
OPSCC.
Histologic Characteristics, Treatments, and Outcomes
Primary tumors were found in the base of the tongue (95,
46.6%), tonsil (99, 48.5%), and soft palate (10, 4.9%) in the oro-
pharynx (Table 2). Overall, 175 tumor specimens (85.8%) were
positive for p16 as determined by IHC. The remaining tumor
specimens were either p16 negative (19, 9.3%) or there was in-
sufficient tumor tissue for p16 IHC determination (10, 4.9%).
The majority of participants (139, 68.1%) received concurrent
chemoradiation as the primary treatment, whereas 44 partici-
pants (21.6%) received radiotherapy alone. Surgery was in-
cluded as part of the treatment in the rest of the patients. The
median (range) follow-up was 12.7 (0.5-40.9) months. Over-
all survival was evaluated based on the HPV status deter-
mined by HPV SPF10-LiPA25 using DNA from the oral gargle
samples and tumor specimens and by p16 IHC using the tu-
mor specimens. Patients with HPV positivity determined by
all 3 methods had better overall survival, with lower hazard
ratios compared with patients with HPV negativity (oral gargle
high-risk HPV: hazard ratio, 0.44; 95% CI, 0.18-1.10; tumor p16
by IHC: hazard ratio, 0.44; 95% CI, 0.14-1.34; tumor high-risk
HPV: hazard ratio, 0.69; 95% CI, 0.16-3.03) (Table 3).
E3
 Research Original Investigation Human Papillomavirus Genotype Detection in Oropharyngeal Squamous Cell Carcinoma

Table 1. Demographic, Behavioral, and Oral Health Characteristics Table 2. Histologic Characteristics, Treatment, and Outcomes
of 204 Study Participants of 204 Study Participants

Characteristic No. (%) of Participants Characteristic No. (%) of Participants

Race Tumor location
White 190 (93.1) Base of tongue 95 (46.6)
Black 8 (3.9) Tonsil 99 (48.5)
Other 6 (2.9) Soft palate 10 (4.9)
Ethnicity Stage at presentation (AJCC, eighth edition)
Hispanic 15 (7.4) p16-Positive stage I 100 (49.0)
Non-Hispanic 189 (92.6) p16-Positive stage II/III 75 (36.8)
Age at diagnosis, y p16-Negative stage I/II 4 (2.0)
Median (range) 61 (35-87) p16-Negative stage III/IV 15 (7.4)
35-49 24 (11.8) Not available 10 (4.9)
50-59 63 (30.9) p16INK4a (by IHC)
60-69 78 (38.2) Positive 175 (85.8)
≥70 39 (19.1) Negative 19 (9.3)
Smoking status Not available 10 (4.9)
Never smoker 88 (43.1) First course of treatment
Former smoker 102 (50.0) Radiotherapy alone 44 (21.6)
Current smoker 12 (5.9) Surgery alone 1 (0.5)
Not available 2 (1.0) Radiotherapy plus surgery 5 (2.5)
Cigarettes among 113 smokers, pack-years Chemotherapy plus radiotherapy 139 (68.1)
Median (range) 20 (0-132) Chemotherapy plus radiotherapy plus surgery 12 (5.9)
≤5 23 (11.3) Not available 3 (1.5)
6-29 47 (23.0) Vital status
≥30 43 (21.1) Alive 185 (90.7)
Not available 91 (44.6) Deceased 19 (9.3)
Alcohol drinks per drinking occasion in the past Abbreviations: AJCC, American Joint Committee on Cancer;
month
IHC, immunohistochemistry.
No alcohol 80 (39.2)
1-4 drinks 94 (46.1)
≥5 drinks 26 (12.7) (3.9%). Within the tumor specimens, 3 had low-risk HPV geno-
Not available 4 (2.0)
types detected (1 specimen had HPV 11 and 2 specimens had
HPV 6); however, all 3 of these specimens had HPV 16 con-
Tonsillectomy
comitantly detected. Similarly, of the few oral gargle samples
Yes 89 (43.6)
that had a low-risk HPV genotype detected, it was always a coin-
No 111 (54.4)
fection with a high-risk genotype. The agreement for each HPV
Not available 4 (2.0)
type was calculated in all 171 participants with oral gargle
Time since tonsillectomy, years ago
sample-tumor specimen pairs (Table 4). The agreement was
<10 20 (9.8)
73.7% for HPV 16 (95% CI, 66.4%-80.1%) and 94.2% (95% CI,
≥10 67 (32.8)
89.5%-97.2%) for HPV 18.
No tonsillectomy or not available 117 (57.4)

HPV Genotypes in Oral Gargle and Tumor Specimens

The HPV infection status could be determined in 203 of 204
(99.5%) of the oral gargle samples: 168 samples (82.8%) were
positive for HPV, and 35 samples (17.2%) were negative for HPV
as determined by HPV SPF10-LiPA25. Only 1 oral gargle sample
failed DNA quality assessment. Among the 204 cases, 172 cases
also had available tumor specimens: 160 (93.0%) were posi-
tive for HPV, and 12 (7.0%) were negative for HPV (Table 4).
Furthermore, multiple HPV types were present in 13 of the tu-
mor specimens (7.6%) and 20 of the oral gargle samples (9.9%).
The most commonly found genotype was HPV 16 in both tu-
mor specimens (143 of 172, 83.1%) and oral gargle samples (128
of 203, 63.1%), followed in frequency by HPV 18, which was
present in 8 tumor specimens (4.7%) and 8 oral gargle samples
Discussion
Determination of HPV status using p16 expression in OPSCC
at the time of diagnosis is current standard practice.9 In the
present study, we evaluated demographic, clinical, and his-
topathologic characteristics of a cohort of patients with newly
diagnosed OPSCC.
We determined specific HPV genotypes in both oral gargle
samples and tumor specimens at the time of diagnosis. The
most frequent HPV genotype detected in both the oral gargle
samples and the tumor specimens was HPV 16 followed by HPV
18. The detection rate of the HPV genotypes in oral gargle
samples was high at 82.8%. The agreement between oral gargle
samples and tumor specimens was 73.7% for HPV 16 and 94.0%

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 Human Papillomavirus Genotype Detection in Oropharyngeal Squamous Cell Carcinoma Original Investigation Research

Table 3. Univariate Cox Proportional Hazards Regression Models for Overall Survival
No. of Participants (No. of
Variable Deaths) Level HR (95% CI)
Oral gargle high-risk HPV 202 (19) Negative 1 [Reference]
Positive 0.44 (0.18-1.10)
Tumor p16 (by IHC) 193 (18) Negative 1 [Reference]
Positive 0.44 (0.14-1.34)
Tumor high-risk HPV 171 (18) Negative 1 [Reference] Abbreviations: IHC,
immunohistochemistry; HPV, human
Positive 0.69 (0.16-3.03)
papillomavirus; HR, hazard ratio.

Table 4. HPV Type Distribution and Agreement Between Oral Gargle Samples
and Tumor Specimens of Study Participants

No. (%) of Specimens Agreement (n = 171)a

Oral Gargle Sample
HPV Type (n = 203) Tumor Specimen (n = 172) No. % (95% CI)
HPV HR typesb
HPV 16 128 (63.1) 143 (83.1) 126 73.7 (66.4-80.1)
HPV 18 8 (3.9) 8 (4.7) 161 94.2 (89.5-97.2)
HPV 31 1 (0.5) 1 (0.6) 169 98.8 (95.8-99.9)
HPV 33 9 (4.4) 12 (7.0) 165 96.5 (92.5-98.7)
HPV 35 5 (2.5) 7 (4.1) 165 96.5 (92.5-98.7)
HPV 39 1 (0.5) 0 170 99.4 (96.8-100)
HPV 45 1 (0.5) 1 (0.6) 169 98.8 (95.8-99.9)
HPV 51 3 (1.5) 1 (0.6) 168 98.2 (95.0-99.6) Abbreviations: HPV, human
papillomavirus; HR, high risk;
HPV 52 1 (0.5) 3 (1.7) 167 97.7 (94.1-99.4) LR, low risk.
HPV 56 1 (0.5) 0 170 99.4 (96.8-100) a
Oral gargle sample-tumor specimen
HPV 58 1 (0.5) 1 (0.6) 171 100 pairs were available for 171 patients.
b
HPV 68 1 (0.5) 0 170 99.4 (96.8-100) Includes the following HR HPV
c types: 2, 3, 4, 5, 6, 7, 8, 11, 13, 14, 16,
HPV LR types
18, 20, 26, 27, 28, 30, 21, 32, 33, 34,
HPV 6 5 (2.5) 2 (1.2) 165 96.5 (92.5-98.7) 35, 37, 39, 40, 42, 43, 44, 45, 55, 56,
HPV 11 1 (0.5) 1 (0.6) 170 99.4 (96.8-100) 57, 58, 59, 61, 62, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 81, 82,
HPV 44 2 (1.0) 0 170 99.4 (96.8-100)
83, 84, 85, 86, 87, 89, 90, 91, 95,
HPV 53 1 (0.5) 0 170 99.4 (96.8-100) 97, 102, 106, 114, and 115.
HPV 66 3 (1.5) 0 169 98.8 (95.8-99.9) c
Includes the following LR HPV
HPV 70 2 (1.0) 0 170 99.4 (96.8-100) types: 6, 11, 16, 18, 31, 33, 34, 35, 39,
40, 42, 43, 44, 45, 51, 52, 53, 54, 56,
HPV 74 3 (1.5) 0 168 98.2 (95.0-99.6)
58, 59, 66, 68, 70, 73, and 74.

or higher for all other HPV types. In addition, low-risk HPV
genotypes were frequently detected in the oral gargle samples;
when low-risk HPV genotypes were detected, they were al-
ways a coinfection with high-risk genotypes in our OPSCC co-
hort. The interpretation of HPV detection in oral gargle samples
in a cancer-free population for cancer risk assessment, how-
ever, should be evaluated with caution given that sensitive as-
says can concomitantly detect low-risk genotypes, and only a
small fraction of the positive tests will be high-risk HPV geno-
types. For example, when 1626 men in Brazil, Mexico, and the
United States were tested for HPV using oral rinse-and-gargle
samples, the prevalence of any HPV genotype was 4%, with
the prevalence of high-risk genotypes at 1% and low-risk geno-
types at 3%.20 In addition, most individuals clear an HPV in-
fection within 1 year. Lifetime risk of developing OPSCC re-
mains very low.
Currently the preferred method of HPV status determina-
tion is p16 IHC, which does not provide specific HPV geno-
type information. It is a very pragmatic approach to HPV in-
fection status determination. However, determination of HPV
genotypes may be more relevant as HPV-targeted cancer pre-
vention and therapies move forward in development. For pre-
vention of HPV infection, there are several US Food and Drug
Administration–approved prophylactic HPV vaccines com-
posed of HPV L1-containing virus-like particles that have been
successful in preventing the acquisition of the virus in healthy
populations as well as in preventing reinfections.22 For ex-
ample, the HPV 9-valent vaccine prevents infection by high-
risk HPV 16, 18, 31, 33, 45, 52, and 58 as well as low-risk HPV 6
and 11.23 Based on our data, it is reassuring that the current pre-
ventive vaccine adequately covers up to 95.7% of the high-
risk HPV genotypes seen in OPSCC. However, there is no spe-
cific US Food and Drug Administration indication at this time
for using these vaccines to prevent OPSCC. Meanwhile, their
effect on OPSCC incidence remains unknown, and it will still
take several decades to be fully elucidated.
By contrast, most therapeutic HPV vaccines currently being
studied target the most common genotype, HPV 16, and are

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 Research Original Investigation Human Papillomavirus Genotype Detection in Oropharyngeal Squamous Cell Carcinoma

based on the premise that cellular immunity can be attained
by targeting HPV-infected cells that express viral oncopro-
teins, including E6 and E7, by generating cytotoxic T-
lymphocyte responses against these viral proteins.24,25 De-
velopment of these vaccines has been under way for the last
decade, and some vaccines are currently being tested in clini-
cal trials as monotherapy or in combination with other first-
line treatments, such as chemotherapy, surgery, or check-
point inhibitors.26,27 Determination of the HPV 16 genotype in
OPSCC is required before enrolling in clinical trials evaluating
these therapeutic vaccines. Based on our study, HPV genotyp-
ing could be easily obtained using HPV DNA from readily ac-
cessible oral gargle samples.
Strengths and Limitations
A major strength of our study is that demographic, behav-
ioral, clinical, pathologic, and outcome data were prospec-
tively collected. However, there are several limitations. All par-
ticipants were enrolled at the Moffitt Cancer Center, a
specialized cancer hospital. This may have led to a bias in the
population of cancer cases included to study because per-
haps more advanced, harder-to-treat cases are often referred
for treatment to specialized centers of cancer care. This is a
single-center study, and the results might not be applicable to
other populations. We enrolled only men, and most patients
were non-Hispanic, white individuals. We also had only a small
number of HPV-negative cases in this cohort of patients for a
robust comparison against the HPV-positive cases, and the fol-
low-up time was relatively short. In addition, the sample size
of HPV detection using tumor specimens (n = 172) was smaller
than that using oral gargle samples (n = 203) owing to insuf-
ficient tumor cells in the tissue specimens or lack of available
tumors for testing.
Conclusions
Our study showed that oral gargle samples can be used to de-
tect the majority of the clinically relevant HPV genotypes found
in men with OPSCC. When low-risk HPV genotypes were de-
tected, they were always a coinfection with high-risk geno-
types. However, the interpretation of HPV detected in oral
gargle samples should be assessed with caution in cancer risk
assessment of a cancer-free population given that sensitive as-
says can concomitantly detect low-risk genotypes. Further-
more, as development of HPV-targeted treatment ap-
proaches, such as genotype-specific therapeutic vaccines,
moves forward, it may be important to adapt an HPV detec-
tion method that can determine the specific HPV genotypes
for each patient.

ARTICLE INFORMATION
Accepted for Publication: January 22, 2019.
Published Online: March 28, 2019.
doi:10.1001/jamaoto.2019.0119
Author Contributions: Drs Giuliano and Chung had
full access to all of the data in the study and take
responsibility for the integrity of the data and the
accuracy of the data analysis.
Concept and design: Giuliano, Abrahamsen, Chung.
Acquisition, analysis, or interpretation of data:
Martin-Gomez, Giuliano, Fulp, Caudell, Echevarria,
Sirak, Isaacs-Soriano, Hernandez-Prera, Wenig,
Vorwald, McMullen, Wadsworth, Slebos, Chung.
Drafting of the manuscript: Martin-Gomez, Giuliano,
Slebos, Chung.
Critical revision of the manuscript for important
intellectual content: Giuliano, Fulp, Caudell,
Echevarria, Sirak, Abrahamsen, Isaacs-Soriano,
Hernandez-Prera, Wenig, Vorwald, McMullen,
Wadsworth, Slebos, Chung.
Statistical analysis: Fulp.
Obtained funding: Giuliano.
Administrative, technical, or material support:
Martin-Gomez, Giuliano, Echevarria, Abrahamsen,
Isaacs-Soriano, Vorwald, Wadsworth, Slebos,
Chung.
Supervision: Giuliano, Abrahamsen, Chung.
Conflict of Interest Disclosures: Dr Martin-Gomez
reported receiving grant support from the Alfonso
Martin Escudero Foundation during the conduct of
the study; she is currently employed at
GlaxoSmithKline. Dr Giuliano is a member of Merck
& Co Inc Global and Scientific Advisory Boards. Her
institution has received funding for
investigator-initiated studies from Merck & Co Inc.
No other disclosures were reported.
Funding/Support: This study was funded by
exploratory/developmental grant R21DE024816
from the National Institute of Dental and
Craniofacial Research to Dr Giuliano.
Role of the Funder/Sponsor: The funder had no
role in the design and conduct of the study;
collection, management, analysis, and
interpretation of the data; preparation, review, or
approval of the manuscript; and decision to submit
the manuscript for publication.
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