ORIGINAL PAPER

Human papillomavirus (HPV) 16 and the prognosis of headand neck cancer in a geographical region with a low prevalenceof HPV infection

Rossana Veronica Mendoza Lopez • Jose Eduardo Levi • Jose Eluf-Neto •

Rosalina Jorge Koifman • Sergio Koifman • Maria Paula Curado •

Pedro Michaluart-Junior • David Livingstone Alves Figueiredo • Fabiano Pinto Saggioro •

Marcos Brasilino de Carvalho • Luiz Paulo Kowalski • Marcio Abrahao •

Francisco de Gois-Filho • Eloiza Helena Tajara • Tim Waterboer •

Paolo Boffetta • Paul Brennan • Victor Wunsch-Filho

Received: 27 June 2013 / Accepted: 15 January 2014 / Published online: 29 January 2014

� Springer International Publishing Switzerland 2014

Abstract

Background The role of human papillomavirus (HPV) on

head and neck squamous cell carcinoma (HNSCC) survival

in regions with low HPV prevalence is not yet clear. We

evaluated the HPV16 infection on survival of HNSCC

Brazilian patient series.

Methods This cohort comprised 1,093 HNSCC cases

recruited from 1998 to 2008 in four Brazilian cities and

followed up until June 2009. HPV16 antibodies were

analyzed by multiplex Luminex assay. In a subset of 398

fresh frozen or paraffin blocks of HNSCC specimens, we

analyzed for HPV16 DNA by L1 generic primer poly-

merase chain reaction. HNSCC survival according to

HPV16 antibodies was evaluated through Kaplan–Meier

method and Cox regression.

Results Prevalence of HPV16 E6 and E6/E7 antibodies

was higher in oropharyngeal cancer than in other head and

neck tumor sites. HPV16 DNA positive in tumor tissue was

also higher in the oropharynx. Seropositivity for HPV16 E6

antibodies was correlated with improved HNSCC survival

and oropharyngeal cancer. The presence of HPV16 E6/E7

R. V. M. Lopez (&) � V. Wunsch-Filho

Faculdade de Saude Publica, Universidade de Sao Paulo,

Av. Dr. Arnaldo, 715, Sao Paulo CEP 01246-904, Brazil

e-mail: rossana@usp.br

R. V. M. Lopez

Instituto de Ensino e Pesquisa, Hospital de Cancer de Barretos,

Barretos, Brazil

J. E. Levi

Instituto de Medicina Tropical, Universidade de Sao Paulo,

Sao Paulo, Brazil

J. Eluf-Neto

Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo,

Brazil

R. J. Koifman � S. Koifman

Escola Nacional de Saude Publica, Fundacao Oswaldo Cruz,

Rio de Janeiro, Brazil

M. P. Curado

Hospital Araujo Jorge, Goiania, Brazil

M. P. Curado � P. Boffetta

International Prevention Research Institute, Lyon, France

P. Michaluart-Junior

Hospital das Clınicas, Faculdade de Medicina, Universidade de

Sao Paulo, Sao Paulo, Brazil

D. L. A. Figueiredo � F. P. Saggioro

Hospital das Clınicas, Faculdade de Medicina de Ribeirao Preto,

Universidade de Sao Paulo, Ribeirao Preto, Brazil

M. B. de Carvalho

Hospital Heliopolis, Sao Paulo, Brazil

L. P. Kowalski

Hospital do Cancer A.C. Camargo, Sao Paulo, Brazil

M. Abrahao

Hospital Sao Paulo, Universidade Federal de Sao Paulo,

Sao Paulo, Brazil

F. de Gois-Filho

Instituto do Cancer Arnaldo Viera de Carvalho, Sao Paulo,

Brazil

E. H. Tajara

Faculdade de Medicina de Sao Jose do Rio Preto, Sao Paulo,

Brazil

123

Cancer Causes Control (2014) 25:461–471

DOI 10.1007/s10552-014-0348-8

antibodies was correlated with improved HNSCC survival

and oropharyngeal cancer survival. The death risk of oro-

pharyngeal squamous cell carcinoma patients HPV16 E6/

E7 antibodies positive was 78 % lower than to those who

test negative.

Conclusion Oropharyngeal squamous cell carcinoma is

less aggressive in the HPV16 E6/E7 positive serology

patients. HPV16 E6/E7 antibody is a clinically sensible

surrogate prognostic marker of oropharyngeal squamous

cell carcinoma.

Keywords Human papillomavirus � Serology �Prognosis � Head and neck cancer � Prevalence

Introduction

Head and neck squamous cell carcinomas (HNSCC)

include tumors of the oral cavity, oropharynx, and larynx,

which vary with respect to their etiology and prognosis.

Worldwide, these tumors account for 650,000 new cases

and 350,000 deaths every year [1]. Latin America, partic-

ularly Brazil, has a relatively high incidence of HNSCC.

Among both sexes, the rates of oral cavity and larynx

cancer were 2.8 and 2.2/100,000, respectively, in 2008 [2].

The relative survival rate for HNSCC is approximately

50–70 %, including all sites, clinical stages, and forms of

treatment. In Brazil, the 5-year survival rate for oral and

oropharyngeal cancer is \50 % [3].

Tobacco smoking and alcohol consumption are the most

important risk factors for HNSCC [4]. The International

Agency for Research on Cancer recognizes human papil-

lomavirus (HPV) type 16 as the only type of HPV that is

carcinogenic to organs other than the cervix uteri, includ-

ing the anus, penis, vagina, vulva, oral cavity, oropharynx,

and tonsils [5]. HPV16 has been associated with increased

risk of head and neck cancer, particularly oropharynx and

tonsil tumors in high as well as low HPV prevalence

regions [6–10]. In regions with high HPV prevalence,

studies have shown that HPV16-positive patients have a

better HNSCC prognosis than those who are HPV16-neg-

ative [11–14]. However, the impact of HPV16 on HNSCC

survival in regions with low HPV prevalence is not known.

We analyzed the relationship between HPV16 infection

and the overall survival of patients with HNSCC and

cancer in specific sites (oral cavity, oropharynx, hypo-

pharynx, and larynx) in Brazil, a country with a low

prevalence of HPV infection.

Materials and methods

Study subjects

This study includes incident HNSCC cases confirmed by

histology and diagnosed between 1998 and 2008. The cases

originated from two multicentre studies in Brazil. The

Latin American study was conducted from 1998 to 2002 in

seven cities; for the present study, we only included cases

from the cities of Goiania, Rio de Janeiro, and Sao Paulo.

The Gencapo study was conducted from 2003 to 2008 in

the cities of Ribeirao Preto and Sao Paulo. Both studies

were approved by the clinical centres’ ethics committees

and by the National Commission on Ethics in Research.

Written consent was given by each patient participating in

the studies.

HNSCC was classified into one of four categories

according to anatomical subsites, each of which has a

distinct prognosis: oral cavity, oropharynx, hypopharynx,

and larynx. The International Classification of Diseases,

version 10 [15], was used to classify the tumor subsite

according to the method used by Hashibe and colleagues

[16]: oral cavity (C00.3–C00.9, C02.0–C02.3, C02.8,

C02.9, C03.0, C03.1, C03.9, C04.0, C04.1, C04.8, C04.9,

C05.0, C05.8, C05.9, C06.0–C06.2, C06.8, C06.9, C14.0,

C14.2, and C14.8), oropharynx (C01.9, C02.4, C05.1,

C05.2, C09.0, C09.1, C09.8, C09.9, C10.0–C10.4, C10.8,

and C10.9), hypopharynx (C12.9, C13.0–C13.2, C13.8, and

C13.9), and larynx (C32.0–C32.3, C32.8, and C32.9).

All patients underwent face-to-face interviews immedi-

ately after diagnosis with trained interviewers who used a

structured questionnaire to obtain information about vari-

ables that could affect HNSCC survival, including tobacco

smoking, alcohol consumption, and education. Patient

hospital records were reviewed to obtain additional infor-

mation on tumor clinical stage, treatment, and time since

diagnosis until dead or last information. The tumor’s

clinical stage was classified as CS I–IV according to the

TNM classification system, 6th edition [17]. Additional

information on cause of death was validated through death

certificates obtained from the Sao Paulo State Death Reg-

istry (for patients from Ribeirao Preto and Sao Paulo) and

from the Goiania Population Cancer Registry (for patients

from Goiania). In Rio de Janeiro, death certificates were

included in the hospital medical records.

T. Waterboer

German Cancer Research Center (DKFZ), Heidelberg, Germany

P. Boffetta

The Tisch Cancer Institute, Mount Sinai School of Medicine,

New York, NY, USA

P. Brennan

International Agency for Research on Cancer, Lyon, France

462 Cancer Causes Control (2014) 25:461–471

123

Patients were recruited into the study from November

1998 to December 2008, and they were followed until 30

June 2009. Of the 1,275 eligible cases at the start of the

study, 182 were excluded because no information was

available on tobacco smoking, alcohol consumption, edu-

cation, tumor clinical stage, or treatment. Ultimately, 1,093

cases with complete follow-up records were included in the

analysis.

Biological samples

In both studies, blood was obtained from each patient at the

time of the interview. Tumor tissue samples from biopsies

or surgical procedures were obtained for a subset of 398

cases (36 %), including 198 fresh-frozen samples and 200

paraffin-embedded samples.

DNA extraction and detection of HPV DNA

For patient samples from the Gencapo study, DNA was

extracted from paraffin slices using the Nucleon HT kit

(GE Life Sciences, Sao Paulo, Brazil) and examined using

the Inno-LiPA HPV Genotyping kit (Innogenetics, Gent,

Belgium) [18]. For patient samples from the Latin Amer-

ican study, DNA was extracted from fresh tumor tissue

using the QIAamp DNA Mini kit (Qiagen, Valencia, CA,

Table 1 Baseline characteristics of HNSCC study patients, Brazil, 1998–2008

All patients Oral cavity Oropharynx Hypopharynx Larynx

n = 1,093 n = 321 n = 252 n = 115 n = 405

n (%) n (%) n (%) n (%) n (%)

Study

Latin America 727 (66.5) 181 (56.4) 200 (79.4) 84 (73.0) 262 (64.7)

GENCAPO 366 (33.5) 140 (43.6) 52 (20.6) 31 (27.0) 143 (35.3)

Sex

Female 148 (13.5) 56 (17.4) 39 (15.5) 7 (6.1) 46 (11.4)

Male 945 (86.5) 265 (82.6) 213 (84.5) 108 (93.9) 359 (88.6)

Age (years)

\55 435 (39.8) 145 (45.2) 115 (45.6) 43 (37.4) 132 (32.6)

55–64 373 (34.1) 101 (31.5) 91 (36.1) 39 (33.9) 142 (35.1)

65–75 225 (20.6) 55 (17.1) 37 (14.7) 24 (20.9) 109 (26.9)

C75 60 (5.5) 20 (6.2) 9 (3.6) 9 (7.8) 22 (5.4)

Education (years)

0 160 (14.6) 41 (12.8) 37 (14.7) 18 (15.7) 64 (15.8)

1–7 589 (53.9) 163 (50.8) 144 (57.1) 63 (54.8) 219 (54.1)

8–10 226 (20.7) 73 (22.7) 39 (15.5) 25 (21.7) 89 (22.0)

C11 118 (10.8) 44 (13.7) 32 (12.7) 9 (7.8) 33 (8.1)

Tobacco smoking

Nonsmoker 53 (4.8) 21 (6.5) 15 (6.0) 3 (2.6) 14 (3.5)

Former smoker 273 (25.0) 61 (19.0) 50 (19.8) 30 (26.1) 132 (32.6)

Current smoker 767 (70.2) 239 (74.5) 187 (74.2) 82 (71.3) 259 (64.0)

Alcohol consumption

Nondrinker 97 (8.9) 35 (10.9) 13 (5.2) 5 (4.3) 44 (10.9)

Former drinker 390 (35.7) 82 (25.5) 101 (40.1) 50 (43.5) 157 (38.8)

Current drinker 606 (55.4) 204 (63.6) 138 (54.8) 60 (52.2) 204 (50.4)

Tumor stage

T1/T2 366 (33.5) 133 (41.4) 83 (32.9) 24 (20.9) 126 (31.1)

T3 325 (29.7) 60 (18.7) 84 (33.3) 44 (38.3) 137 (33.8)

T4 402 (36.8) 128 (39.9) 85 (33.7) 47 (40.9) 142 (35.1)

Treatment

Surgery 347 (31.7) 129 (40.2) 39 (15.5) 20 (17.4) 159 (39.3)

Radiotherapy 214 (19.6) 36 (11.2) 73 (29.0) 35 (30.4) 70 (17.3)

Surgery ? radiotherapy 335 (30.6) 114 (35.5) 66 (26.2) 31 (27.0) 124 (30.6)

Other 197 (18.0) 42 (13.1) 74 (29.4) 29 (25.2) 52 (12.8)

Cancer Causes Control (2014) 25:461–471 463

123

USA). HPV DNA detection was performed by polymerase

chain reaction (PCR) with the generic primers PGMY09/

11, which amplify a fragment spanning *450 bp of the L1

region of most mucosal HPV types [19], in the presence of

human b-globin primers, which amplify a fragment of

268 bp as a positive control. The reaction conditions were

as follows: 200 lM dNTPs, 4 mM magnesium chloride,

80 nM PGMY09/11, and 20 nM PCO4/GH20 (b-globin)

oligonucleotides, 250 ng of template DNA, and 1 U of Taq

polymerase (Invitrogen, Sao Paulo, Brazil). The thermo-

cycling profile consisted of an initial incubation of 5 min at

94 �C, followed by 40 cycles of 94 �C (1 min), 55 �C

(1 min), and 72 �C (1 min) and a final elongation step of

5 min at 72 �C in a PE 2400 (Applied Biosystems, Foster

City, CA, USA) or a MasterCycler gradient thermal cycler

(Eppendorf AG, Hamburg, Germany). The PCR products

were analyzed by electrophoresis on a 2 % agarose gel

stained with ethidium bromide and observed under

ultraviolet light. Further genotyping of PGMY09/11-posi-

tive samples was conducted by restriction fragment length

polymorphism using the enzymes and patterns described by

Bernard et al. [20]. These procedures and analyses were

conducted at the Institute of Tropical Medicine at the

University of Sao Paulo in Brazil.

Detection of HPV antibodies

Antibodies against HPV were analyzed by multiplex

Luminex serology assay. This antibody detection method is

based on a glutathione S-transferase capture ELISA [21] in

combination with fluorescent bead technology [22]. To

determine a positive serological response, mean fluores-

cence intensity values were dichotomised as antibody

positive or negative for the serological response to onco-

proteins E1, E2, E4, E6, E7, and L1 of HPV16; oncopro-

teins E6, E7, and L1 of HPV 18, 31, 33, and 35;

Table 2 Characteristics of HNSCC patients by HVP DNA and HPV16 DNA status, Brazil, 1998–2008

Total n = 398 HPV DNA p HPV16 DNA p

Negative Positive Negative Positive

n = 363 n = 35 n = 384 n = 14

n (%) n (%) n (%) n (%)

Sex 0.0631 0.0851

Female 50 42 (84.0) 8 (16.0) 46 (92.0) 4 (8.0)

Male 348 321 (92.2) 27 (7.8) 338 (97.1) 10 (2.9)

Age (years) 0.0782 0.9411

\55 162 141 (87.0) 21 (13.0) 155 (95.7) 7 (4.3)

55–64 141 132 (93.6) 9 (6.4) 136 (96.5) 5 (3.5)

65–75 76 71 (93.4) 5 (6.6) 74 (97.4) 2 (2.6)

C75 19 19 (100.0) 0 19 (100.0) 0

Tobacco smoking 0.4382 0.1061

Nonsmoker 15 13 (86.7) 2 (13.3) 13 (86.7) 2 (13.3)

Former smoker 107 95 (88.8) 12 (11.2) 103 (96.3) 4 (3.7)

Current smoker 276 255 (92.4) 21 (7.6) 268 (97.1) 8 (2.9)

Alcohol consumption 0.0102 0.0751

Nondrinker 38 32 (84.2) 6 (15.8) 34 (89.5) 4 (10.5)

Former drinker 140 122 (87.1) 18 (12.9) 136 (97.1) 4 (2.9)

Drinker 220 209 (95.0) 11 (5.0) 214 (97.3) 6 (2.7)

Tumor Site 0.2462 0.9591

Oral cavity 121 113 (93.4) 8 (6.6) 117 (96.7) 4 (3.3)

Oropharynx 91 85 (93.4) 6 (6.6) 87 (95.6) 4 (4.4)

Hypopharynx 44 41 (93.2) 3 (6.8) 43 (97.7) 1 (2.3)

Larynx 142 124 (87.3) 18 (12.7) 137 (96.5) 5 (3.5)

Treatment 0.0262 0.1861

Surgery 143 128 (89.5) 15 (10.5) 136 (95.1) 7 (4.9)

Radiotherapy 76 76 (100) 0 76 (100) 0

Surgery ? radiotherapy 122 109 (89.3) 13 (10.7) 118 (96.7) 4 (3.3)

Other 57 50 (87.7) 7 (12.3) 54 (94.7) 3 (5.3)

1 Fisher’s exact test. 2 chi-square test

464 Cancer Causes Control (2014) 25:461–471

123

oncoproteins E6 and L1 of 45, 52, and 58; and oncoprotein

L1 of HPV 6, 11, and 77. Means plus three standard

deviations were calculated to define cutoffs, excluding

outliers [23]. These procedures and analyses were con-

ducted at the German Cancer Research Center in Heidel-

berg, Germany.

Statistical analysis

Frequencies and percentages were calculated for categori-

cal variables, and the associations between variables were

tested using Fisher’s exact test or chi-square tests. The

concordance between HPV DNA and serological response

was calculated using the Kappa statistic. Cox model

regression was performed to calculate the hazard ratios

(HR) and 95 % confidence intervals (95 % CI) for the

association of HPV on HNSCC overall survival, adjusting

for gender, age (years\55, 55–64, 65–74, and C75), study

group (Latin America or Gencapo), education (years of

schooling: 0, 1–7, 8–10, and C11), tobacco smoking

(nonsmoker, former smoker, current smoker), alcohol

consumption (nondrinker, former drinker, current drinker),

Table 3 Characteristics of HNSCC patients by HPV16 E6, E7, L1, E6/E7 serology antibodies, Brazil, 1998–2008

Total HPV16 E6 HPV16 E7 HPV16 L1 HPV16 E6/E7

Negative Positive Negative Positive Negative Positive Negative Positive

n = 1,013 n = 80 n = 833 n = 260 n = 1,018 n = 75 n = 1,066 n = 27

n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%)

Sex

Female 148 136 (91.9) 12 (8.1) 116 (78.4) 32 (21.6) 132 (89.2) 16 (10.8) 141 (95.3) 7 (4.7)

Male 945 877 (92.8) 68 (7.2) 717 (75.9) 228 (24.1) 886 (93.8) 59 (6.2) 925 (97.9) 20 (2.1)

p1 0.692 0.506 0.041 0.080

Age (years)

\55 435 395 (90.8) 40 (9.2) 337 (77.5) 98 (22.5) 403 (92.6) 32 (7.4) 420 (96.6) 15 (3.4)

55–64 373 345 (92.5) 28 (7.5) 285 (76.4) 88 (23.6) 349 (93.6) 24 (6.4) 365 (97.9) 8 (2.1)

65–75 225 216 (96.0) 9 (4.0) 164 (72.9) 61 (27.1) 208 (92.4) 17 (7.6) 221 (98.2) 4 (1.8)

C75 60 57 (95.0) 3 (5.0) 47 (78.3) 13 (21.7) 58 (96.7) 2 (3.3) 60 (100.0) 0

p1 0.093 0.592 0.657 0.277

Tobacco smoking

Nonsmoker 53 43 (81.1) 10 (18.9) 36 (67.9) 17 (32.1) 47 (88.7) 6 (11.3) 47 (88.7) 6 (11.3)

Former smoker 273 253 (92.7) 20 (7.3) 208 (76.2) 65 (23.8) 255 (93.4) 18 (6.6) 265 (97.1) 8 (2.9)

Current smoker 767 717 (93.5) 50 (6.5) 589 (76.8) 178 (23.2) 716 (93.4) 51 (6.6) 754 (98.3) 13 (1.7)

p1 0.004 0.341 0.420 \0.001

Alcohol

Nondrinker 97 90 (92.8) 7 (7.2) 77 (79.4) 20 (20.6) 88 (90.7) 9 (9.3) 95 (97.9) 2 (2.1)

Former drinker 390 370 (94.9) 20 (5.1) 302 (77.4) 88 (22.6) 365 (93.6) 25(6.4) 384 (98.5) 6 (1.5)

Drinker 606 553 (91.3) 53 (8.7) 454 (74.9) 152 (25.1) 565 (93.2) 41 (6.8) 587 (96.9) 19 (3.1)

p1 0.101 0.492 0.601 0.275

Tumor Site

Oral cavity 321 301 (93.8) 20 (6.2) 246 (76.6) 75 (23.4) 302 (94.1) 19 (5.9) 316 (98.4) 5 (1.6)

Oropharynx 252 225 (89.3) 27 (10.7) 191 (75.8) 61 (24.2) 237 (94.0) 15 (6.0) 238 (94.4) 14 (5.6)

Hypopharynx 115 113 (98.3) 2 (1.7) 86 (74.8) 29 (25.2) 105 (91.3) 10 (8.7) 115 (100.0) 0

Larynx 405 374 (92.3) 31 (7.7) 310 (76.5) 95 (23.5.) 374 (92.3) 31 (7.7) 397 (98.0) 8 (2.0)

p1 0.017 0.976 0.620 0.002

Treatment

Surgery 347 319 (91.9) 28 (8.1) 254 (73.2) 93 (26.8) 319 (91.9) 28 (8.1) 339 (97.7) 8 (2.3)

Radiotherapy 214 200 (93.5) 14 (6.5) 166 (77.6) 48 (22.4) 199 (93.0) 15 (7.0) 210 (98.1) 4 (1.9)

Surgery ? radiotherapy 335 310 (92.5) 25 (7.5) 259 (77.3) 76 (22.7) 313 (93.4) 22 (6.6) 324 (96.7) 11 (3.3)

Other 197 184 (93.4) 13 (6.6) 154 (78.2) 43 (21.8) 187 (94.9) 10 (5.1) 193 (98.0) 4 (2.0)

p1 0.887 0.458 0.609 0.697

1 chi-square test or Fisher’s exact test

Cancer Causes Control (2014) 25:461–471 465

123

tumor clinical stage (T1/T2, T3, and T4), and treatment

(surgery, radiotherapy, surgery plus radiotherapy, another

treatment). The dependence between oral cavity, hypo-

pharynx, and larynx cancer (combined) survival and oro-

pharynx cancer (separately) survival on the presence of

HPV16 antibodies was evaluated using the Kaplan–Meier

method, and the differences between the curves were

assessed using log-rank tests. For the descriptive analysis

and multiple survival analyses, we used IBM� SPSS�

Statistics version 18 (IBM Corp, Somers, NY, USA) and

Stata/SE 11.0 for Windows (StataCorp LP, College Station,

TX, USA). All reported p values are two-sided with a

significance level of 0.05.

Results

At the end of the follow-up period, 356 of the study

patients were alive, 626 had died, and 111 were lost to

follow-up. The median survival of the cohort members was

32 months (95 % CI 27–37 months). HNSCC 5-year

overall survival after diagnosis was 38 %, and disease-

specific survival was 43 %.

The description of study group, demographic charac-

teristics, tobacco and alcohol consumption, clinical stage

and treatment for all patients with serological response to

HPV is presented in Table 1 and corresponding to all

patients with serological samples. Of the HNSCC cases,

321 (29.4 %) affected the oral cavity, 252 (23.1 %)

affected the oropharynx, 115 (10.5 %) affected the

hypopharynx, and 405 (37.1 %) affected the larynx. The

majority of the patients were male, and 37 % were between

51 and 60 years old. The education level was low; 68.5 %

had \8 years of schooling. Only 4.8 % of the HNSCC

patients reported that they did not smoke, and 8.9 % did

not report alcohol consumption at the time of the interview.

An advanced clinical tumor stage (T3 or T4) at diagnosis

was observed in many of the HNSCC cases, and the most

common treatment approach was surgery or a combination

of surgery plus radiotherapy.

The overall prevalence of HPV DNA was 8.8 % and for

HPV16 DNA was 3.5 % in tumor tissue. Table 2 shows the

prevalence of HPV DNA and HPV16 DNA according to

gender, age, tobacco smoking, alcohol consumption, ana-

tomical site, and treatment. Association statistically sig-

nificant was observed in alcohol consumption and

treatment with HPV DNA status. The HPV16 DNA prev-

alence in tumor tissue was higher in women, patients

50 years old or younger, nonsmokers, and nondrinkers. In

addition, the prevalence of HPV16 DNA was higher in

patients with oropharyngeal cancer than in patients with

cancers of other head and neck sites. No association was

observed between treatment and HPV16 DNA status;

however, we observed that all patients who received

radiotherapy were negative to HPV16 DNA. A total of

8.8 % of HNSCC patients tested positive for HPV DNA in

tumor tissue (35/398), of which 40 % had HPV16 (14/35).

Table 3 displays the prevalence of HPV16 antibodies

by gender, age, tobacco smoking, alcohol consumption,

anatomical site, and treatment. The overall prevalence of

Table 4 Concordance between HPV16 DNA and HPV16 serological antibodies by HNSCC subsite, Brazil, 1998–2008

Oral cavity Kappap1

Oropharynx Kappap1

Hypopharynx Kappap1

Larynx Kappap1

HPV16 DNA HPV16 DNA HPV16 DNA HPV16 DNA

Negative Positive Negative Positive Negative Positive Negative Positiven = 117 n = 4 n = 87 n = 4 n = 43 n = 1 n = 137 n = 5n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%)

HPV16 E6 -0.044 0.578 NA 0.145

Negative 110 (94.0) 4 (100.0) 0.614 84 (96.6) 1 (25.0) \0.001 43(100.0)

1 (100.0) 123 (89.8) 3 (60.0) 0.039

Positive 7 (6.0) 0 3 (3.4) 3 (75.0) 0 0 14 (10.2) 2 (40.0)

HPV16 E7 0.040 0.062 -0.044 0.033

Negative 82 (70.1) 2 (50.0) 0.391 63 (72.4) 2 (50.0) 0.332 30 (69.8) 1 (100.0) 0.512 101 (73.7) 3 (60.0) 0.496

Positive 35 (29.9) 2 (50.0) 24 (27.6) 2 (50.0) 13 (30.2) 0 36 (26.3) 2 (40.0)

HPV16 L1 -0.048 0.416 -0.039 0.063

Negative 108 (92.3) 4 (100.0) 0.564 84 (96.6) 2 (50.0) \0.001 38 (88.4) 1 (100.0) 0.717 125 (91.2) 4 (80.0) 0.392

Positive 9 (7.7) 0 3 (3.4) 2 (50.0) 5 (11.6) 0 12 (8.8) 1 (20.0)

HPV16E6/E7

-0.029 0.477 NA 0.171

Negative 114 (97.4) 4 (100.0) 0.746 85 (97.7) 2 (50.0) \0.001 43(100.0)

1 (100.0) 133 (97.1) 4 (80.0) 0.042

Positive 3 (2.6) 0 2 (2.3) 2 (50.0) 0 0 4 (2.9) 1 (20.0)

1 p for Kappa statistic

NA not available

466 Cancer Causes Control (2014) 25:461–471

123

HPV16 antibodies among HNSCC patients was as fol-

lows: E6 (7.3 %), E7 (23.8 %), L1 (6.9 %), and E6/E7

(2.5 %). HPV16 E6 and E6/E7 antibodies had the lowest

prevalence overall among the HNSCC patients, but the

prevalence of these antibodies was higher among younger

patients, nonsmokers, and those with oropharyngeal can-

cer. According to treatment, the data showed no associ-

ation with serological response to HPV. Relative

frequencies were distributed similarly in each treatment

and antibodies.

In general, the concordance of HPV16 DNA and HPV16

serological antibodies was low. However, in cases of oro-

pharyngeal cancer, the concordance between HPV16 DNA

and HPV16 E6, L1, and E6/E7 was moderate (0.578, 0.416,

and 0.477, respectively) (Table 4).

Seropositivity for antibodies against HPV16 E6 and E6/

E7 was significantly associated with a better HNSCC

prognosis. Oropharyngeal cancer patients with HPV16 E6/

E7 seropositivity had a 78 % lower risk of death compared

with those with negative serology (HR = 0.22; 95 % CI

0.06–0.76). No association between HPV16 DNA in tumor

tissue and HNSCC survival was observed (HR = 0.74;

95 % CI 0.27–2.07). Greater survival rates were observed

among patients with oropharyngeal tumors who were

HPV16 DNA-positive, but this result was not precise

(HR = 0.04; 95 % CI 0.001–1.75) (Table 5). We observed

a poorer prognosis among men, among patients with

advanced clinical tumor stage (T3 and T4), and among

those treated with surgery and radiotherapy. Considering

only oropharyngeal cancer, older patients and smokers

(former and current) had lower overall survival (data no

showed).

Based on the results in Table 5, Kaplan–Meier survival

curves were calculated for oral cavity, hypopharyngeal,

and laryngeal cancers combined (Fig. 1a, b) and for oro-

pharyngeal cancer cases separately (Fig. 1c, d), consider-

ing seropositivity for HPV16 E6 (Fig. 1a, c) and E6/E7

(Fig. 1b, d), respectively. The prognosis was clearly better

for oropharyngeal cancer patients with a positive serolog-

ical response to HPV16 E6/E7 (Log-rank test; p = 0.016)

(Fig. 1d).

Seropositivity for antibodies against HPV16 E6 and E6/

E7 was significantly associated with a better HNSCC

prognosis. Oropharyngeal cancer patients with HPV16 E6/

E7 seropositivity had a 78 % lower risk of death compared

with those with negative serology.

Table 6 shows the characteristics of HNSCC patients by

availability of biological samples for HPV DNA status. No

differences were observed in patients according to gender,

age, tobacco and alcohol consumption, anatomical site, and

tumor stage. Tumor tissue samples were only available for

surgery cases and association was observed for HPV16

DNA (p = 0.041).Ta

ble

5O

ver

all

surv

ival

of

HN

SC

Cp

atie

nts

by

HP

V1

6se

rolo

gic

alan

tib

od

ies

and

HP

V1

6D

NA

,B

razi

l,1

99

8–

20

08

nH

NS

CC

pn

Ora

lca

vit

yp

nO

rop

har

yn

xp

nH

yp

op

har

yn

xp

nL

ary

nx

pH

R1

(95

%C

I)H

R1

(95

%C

I)H

R1

(95

%C

I)H

R1

(95

%C

I)H

R1

(95

%C

I)

HP

V1

6E

6

Neg

ativ

e1

,01

31

30

11

22

51

11

31

37

41

Po

siti

ve

80

0.6

3(0

.44

–0

.89

)0

.01

02

00

.98

(0.5

1–

1.8

9)

0.9

47

27

0.7

0(0

.37

–1

.32

)0

.26

82

0.7

7(0

.08

–7

.96

)0

.82

93

10

.58

(0.3

0–

1.1

5)

0.1

19

HP

V1

6E

7

Neg

ativ

e8

33

12

46

11

91

18

61

31

01

Po

siti

ve

26

01

.00

(0.8

3–

1.2

2)

0.9

75

75

1.0

7(0

.74

–1

.56

)0

.71

56

10

.73

(0.5

0–

1.0

9)

0.1

21

29

1.2

3(0

.73

–2

.08

)0

.43

19

51

.02

(0.7

1–

1.4

7)

0.9

24

HP

V1

6L

1

Neg

ativ

e1

,01

81

30

21

23

71

10

51

37

41

Po

siti

ve

75

0.9

3(0

.67

–1

.30

)0

.66

81

90

.98

(0.5

1–

1.8

9)

0.9

62

15

0.7

5(0

.34

–1

.65

)0

.47

61

00

.91

(0.4

1–

2.0

3)

0.8

10

31

1.0

3(0

.57

–1

.85

)0

.92

6

HP

V1

6E

6/E

7

Neg

ativ

e1

,06

61

31

61

23

81

11

5N

A3

97

1

Po

siti

ve

27

0.3

4(0

.16

–0

.73

)0

.00

65

1.8

5(0

.56

–6

.13

)0

.31

41

40

.22

(0.0

6–

0.7

5)

0.0

16

0N

A8

0.2

2(0

.03

–1

.58

)0

.13

2

HP

V1

6D

NA

Neg

ativ

e3

84

11

17

18

71

43

NA

13

71

Po

siti

ve

14

0.7

4(0

.27

–2

.07

)0

.56

64

2.0

2(0

.39

–1

0.5

7)

0.4

07

40

.05

(0.0

01

–1

.77

)0

.09

81

NA

50

.76

(0.0

9–

6.2

3)

0.7

96

1H

azar

dra

tio

adju

sted

for

stu

dy

,g

end

er,

age,

edu

cati

on

,to

bac

cosm

ok

ing

,al

coh

ol

con

sum

pti

on

,cl

inic

altu

mo

rst

age,

and

trea

tmen

t.N

A:

no

tav

aila

ble

Cancer Causes Control (2014) 25:461–471 467

123

Discussion

This is the largest study conducted in Brazil to assess the

prevalence of HPV in a sample of HNSCC patients and to

determine the corresponding survival rates.

Although the prevalence of HPV16 DNA in this cohort

of HNSCC patients was only 3.5 % (14/398), the groups

with the highest prevalence of positivity were females

(8 %), those 50 years old or younger (4.9 %), nonsmokers

(13.3 %), nondrinkers (10.5 %), and patients with oro-

pharynx tumors (4.4 %). These prevalence levels of

HPV16 DNA are lower than those observed in other

regions of the world [24]. Regarding HPV antibodies in

serum, we observed a high prevalence of HPV16 E7

(positive in about 24 % of all HNSCC cases), regardless of

tumor site. Oropharyngeal tumors had a higher prevalence

of HPV16 E6 (10.7 %) and HPV16 E6/E7 (5.6 %) than

tumors in other anatomical sites of the head and neck.

Notably, these levels of HPV16 E6 and E6/E7 antibodies

are much lower than those previously reported in the

United States [7, 14].

The presence of HPV16 DNA alone does not seem to be

a good indicator of prognosis for HNSCC patients. We

observed that HPV16 DNA had different prognostic value

depending on the tumor site: poorer for oral cavity cancer

and better for oropharynx and larynx cancers, although the

estimates lacked precision.

For oropharyngeal cancer, we observed a moderate

concordance between HPV16 DNA and HPV16 E6 and E6/

E7 serology. Previous studies have detected significantly

higher oropharyngeal cancer death rates for patients who

are seronegative for HPV16 E6 or E6/E7 [14, 25–29]. In

our study, HPV16 E6 and E6/E7 seropositivity were

associated with a reduction in death from HNSCC. How-

ever, when we examined the Kaplan–Meier survival curves

for the combined tumors of the oral cavity, hypopharynx

Fig. 1 Kaplan–Meier curves of overall survival according to sero-

logical response to HPV16 E6 and HPV16 E6/E7 in patients with oral

cavity, hypopharynx, and larynx cancers (combined) and in patients

with oropharynx cancer. A HPV16 E6 in patients with oral cavity,

hypopharynx, and larynx cancers. B HPV16 E6/E7 in patients with

oral cavity, hypopharynx, and larynx cancers. C HPV16 E6 in

patients with oropharyngeal cancer. D HPV16 E6/E7 in patients with

oropharyngeal cancer. Overall survival of 841 patients with oral

cavity, hypopharynx, and larynx cancers (A and B). Overall survival

in 252 patients with oropharynx cancer (C and D)

468 Cancer Causes Control (2014) 25:461–471

123

and larynx and, separately, oropharynx tumors, the strong

association of HPV16 E6/E7 with oropharyngeal cancer

prognosis became clear.

There are some limitations to our study. We were not

able to obtain information on crucial covariates for 14.3 %

(182/1,275) of the initial cases, and we were also unable to

follow up with 10.2 % (111/1,093) of the patients included

in the study. The patients who were lost might have

affected the study’s precision. Another limitation is that

tissue samples, from which HPV16 DNA was extracted,

were only available for a portion of the cases (36.4 %),

which could compromise the evaluation of the association

of HPV16 DNA and HNSCC survival. Tumor tissue

availability depends of the tumor size and also if the patient

was submitted to surgery or not. However, the subset for

which no tissue samples were available was comparable to

the entire sample with respect to demographics, lifestyle,

and clinical characteristics (Table 6).

We observed that patients who are positive for HPV16

E6/E7 antibodies represent a group with a higher percent-

age of oropharyngeal cancer, females and nonsmokers.

This profile explains the better prognosis of this group.

However, the survival advantage associated with HPV16

infection in patients with oropharyngeal squamous cell

Table 6 Characteristics of HNSCC patients by availability of HPV16 DNA, Brazil, 1998–2008

Total

n = 1,093

n (%)

No samples

n = 695

n (%)

Samples available n = 398 p1

HPV16 DNA

Negative Positive

n = 384 n = 14

n (%) n (%)

Sex 0.475

Female 148 (13.5) 98 (14.1) 46 (12.0) 4 (28.6)

Male 945 (86.5) 597 (85.9) 338 (88.0) 10 (71.4)

Age (years) 0.637

\55 435 (39.8) 273 (39.3) 155(40.4) 7 (50.0)

55–64 373 (34.1) 232 (33.4) 136 (35.4) 5 (35.7)

65–75 225 (20.6) 149 (21.4) 74 (19.3) 2 (14.3)

C75 60 (5.5) 41 (5.9) 19 (4.9) 0

Tobacco smoking 0.290

Nonsmoker 53 (4.8) 38 (5.5) 13 (3.4) 2 (14.3)

Former smoker 273 (25.0) 166 (23.9) 103 (26.8) 4 (28.6)

Current smoker 767 (70.2) 491 (70.6) 268 (69.8) 8 (57.1)

Alcohol consumption 0.832

Nondrinker 97 (8.9) 59 (8.5) 34 (8.9) 4 (28.6)

Former drinker 390 (35.7) 250 (36.0) 136 (35.4) 4 (28.6)

Drinker 606 (55.4) 386 (55.5) 214 (55.7) 6 (42.9)

Site 0.867

Oral cavity 321 (29.4) 200 (28.8) 117 (30.5) 4 (28.6)

Oropharynx 252 (23.1) 161 (23.2) 87 (22.7) 4 (28.6)

Hypopharynx 115 (10.5) 71 (10.2) 43 (11.2) 1 (7.1)

Larynx 405 (37.1) 263 (37.8) 137 (35.7) 5 (35.7)

Tumor stage 0.233

T1/T2 366 (33.5) 228 (32.8) 133 (34.6) 5 (35.7)

T3 325 (29.7) 219 (31.5) 102 (26.6) 4 (28.6)

T4 402 (36.8) 248 (35.7) 149 (38.8) 5 (35.7)

Treatment 0.041

Surgery 347 (31.7) 204 (29.4) 136 (35.4) 7 (50.0)

Radiotherapy 214 (19.6) 138 (19.9) 76 (19.8) 0

Surgery ? radiotherapy 335 (30.7) 213 (30.6) 118 (30.7) 4 (28.6)

Other 197 (18.0) 140 (20.1) 54 (14.1) 3 (21.4)

1 chi-square test for association between availability of tumor tissues (with samples or not) and characteristic

Cancer Causes Control (2014) 25:461–471 469

123

carcinoma likely reflects the interplay between the virus

and lymphoid tissue, accounting for the differences in the

biology of the disease and the treatment response [30].

In conclusion, patients with oropharyngeal cancer who

test positive for HPV16 E6/E7 antibodies survived longer

than those who test negative, then HPV16 E6/E7 antibodies

are a clinically relevant indicator of prognosis for oro-

pharyngeal squamous cell carcinoma. This result, which

was obtained in a geographical region with a low preva-

lence of HPV infection, is similar to results reported in

regions with a high prevalence of HPV infection. Test for

HPV 16 E6/E7 antibodies may highlight to clinicians those

patients who could be treated for a shorter duration and

lowering the risk of unnecessary toxic effects.

Acknowledgments This work was supported by Fundacao de

Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [10/50733-6;

09/52031-1 and 04/12054-9] and by the European Commission

[IC18-CT97-0222].

Conflict of interest None declared.

References

1. Argiris A, Karamouzis MV, Raben D, Ferris RL (2008) Head and

neck cancer. Lancet 371:1695–1709

2. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM

(2010) GLOBOCAN 2008 v2.0, Cancer Incidence and Mortality

Worldwide: IARC CancerBase No 10 [Internet]. Lyon, France:

International Agency for Research on Cancer; [cited 2012 Aug

30]. http://globocan.iarc.fr

3. Carvalho AL, Ikeda MK, Magrin J, Kowalski LP (2004) Trends

of oral and oropharyngeal cancer survival over five decades in

3267 patients treated in a single institution. Oral Oncol 40:71–76

4. Szymanska K, Hung RJ, Wunsch Filho V, Eluf Neto J, Curado

MP, Koifman S et al (2011) Alcohol and tobacco, and the risk of

cancers of the upper aerodigestive tract in Latin America: a case–

control study. Cancer Causes Control 22:1037–1046

5. Human papillomaviruses (2007) Lyon, France: International

Agency for Research on Cancer- IARC Monograph on the

Evaluation of Carcinogenic Risks to Humans

6. Herrero R, Castellsague X, Pawlita M, Lissowska J, Kee F,

Balaram P et al (2003) Human papillomavirus and oral cancer:

The International Agency for Research on Cancer Multicenter

Study. J Natl Cancer Inst 95:1772–1783

7. Smith EM, Ritchie JM, Pawlita M, Rubenstein LM, Haugen TH,

Turek LP et al (2006) Human papillomavirus seropositivity and

risks of head and neck cancer. Int J Cancer 120:825–832

8. D’Souza G, Kreimer AR, Viscidi R, Pawlita M, Fakhry C, Koch

WM et al (2007) Case-control study of human papillomavirus and

oropharyngeal cancer. N Engl J Med 356:1944–1956

9. Nasman A, Attner P, Hammarstedt L, Du J, Eriksson M, Giraud

G et al (2009) Incidence of human papillomavirus (HPV) positive

tonsillar carcinoma in Stockholm, Sweden: an epidemic of viral-

induced carcinoma? Int J Cancer 125:362–366

10. Ribeiro KB, Levi JE, Pawlita M, Koifman S, Matos E, Eluf-Neto

J et al (2011) Low human papillomavirus prevalence in head and

neck cancer: results from two large case–control studies in high-

incidence regions. Int J Epidemiol 40:489–502

11. Fakhry C, Westra WH, Li S, Cmelak A, Ridge JA, Pinto H et al

(2008) Improved survival of patients with human papillomavi-

rus–positive head and neck squamous cell carcinoma in a pro-

spective clinical trial. J Natl Cancer Inst 100:261–269

12. Smith EM, Rubenstein LM, Ritchie JM, Lee JH, Haugen TH,

Hamsikova E et al (2008) Does treatment seropositivity to human

papillomavirus have prognostic significance for head and neck

cancers? Cancer Epidemiol Biomarkers Prev 17:2087–2096

13. Ang KK, Harris J, Wheeler R, Weber R, Rosenthal DI, Nguyen-

Tan PF et al (2010) Human papillomavirus and survival of

patients with oropharyngeal cancer. N Engl J Med 363:24–35

14. Liang C, Marsit CJ, McClean MD, Nelson HH, Christensen BC,

Haddad RI et al (2012) Biomarkers of HPV in head and neck

squamous cell carcinoma. Cancer Res 72:5004–5013

15. World Health Organization (1992) International statistical clas-

sification of diseases and related health problems. 10th rev. World

Health Organization, Washington, DC

16. Hashibe M, Brennan P, Benhamou S, Castellsague X, Chen C,

Curado MP et al (2007) Alcohol drinking in never users of

tobacco, cigarette smoking in never drinkers, and the risk of head

and neck cancer: pooled analysis in the International Head and

Neck Cancer Epidemiology Consortium. J Natl Cancer Inst

99:777–789

17. Sobin LH, Wittekind C (2002) TNM classification of malignant

tumours. International Union Against Cancer, 6th edn. Wiley-

Liss, New York

18. Kleter B, Van Doorn LJ, Schrauwen L, Molijn A, Sastrowijoto S,

Ter Schegget J et al (1999) Development and clinical evaluation

of a highly sensitive PCR-reverse hybridization line probe assay

for detection and identification of anogenital human papilloma-

virus. J Clin Microbiol 37:2508–2517

19. Coutlee F, Gravitt P, Kornegay J, Hankins C, Richardson H,

Lapointe N et al (2002) Use of PGMY primers in L1 consensus

PCR improves detection of human papillomavirus DNA in gen-

ital samples. J Clin Microbiol 40:902–907

20. Bernard HU, Chan SY, Manos MM, Ong CK, Villa LL, Delius H

et al (1994) Identification and assessment of known and novel

human papillomaviruses by polymerase chain reaction amplifica-

tion, restriction fragment length polymorphisms, nucleotide

sequence, and phylogenetic algorithms. J Infec Dis 170:1077–1085

21. Sehr P, Zumbach K, Pawlita M (2001) A generic capture ELISA

for recombinant proteins fused to glutathione-S-transferase: val-

idation for HPV serology. J Immunol Methods 253:153–162

22. Waterboer T, Sehr P, Michael KM, Franceschi S, Nieland JD,

Joos TO et al (2005) Multiplex human papillomavirus serology

based on in situ-purified glutathione s-transferase fusion proteins.

Clin Chem 51:1845–1853

23. Clifford GM, Shin HR, Oh JK, Waterboer T, Ju YH, Vaccarella S

et al (2007) Serologic response to oncogenic human papilloma-

virus types in male and female university students in Busan,

South Korea. Cancer Epidemiol Biomarkers Prev 16:1874–1879

24. Kreimer AR, Clifford GM, Boyle P, Franceschi S (2005) Human

papillomavirus types in head and neck squamous cell carcinomas

worldwide: a systematic review. Cancer Epidemiol Biomarkers

Prev 14:467–475

25. Dayyani F, Etzel CJ, Liu M, Ho CH, Lippman SM, Tsao AS

(2010) Meta-analysis of the impact of human papillomavirus

(HPV) on risk and overall survival in head and neck squamous

cell carcinomas (HNSCC). Head Neck Oncol 2:15. doi:10.1186/

1758-3284-2-15

26. Smith EM, Pawlita M, Rubenstein LM, Haugen TH, Hamsikova

E, Turek LP (2010) Risk factors and survival by HPV-16 E6 and

E7 antibody status in human papillomavirus positive head and

neck cancer. Int J Cancer 127:111–117

27. Rotnaglova E, Tachezy R, Salakova M, Prochazka B, Kosl’abova

E, Vesela E et al (2011) HPV involvement in tonsillar cancer:

470 Cancer Causes Control (2014) 25:461–471

123

prognostic significance and clinically relevant markers. Int J

Cancer 129:101–110

28. Rubenstein LM, Smith EM, Pawlita M, Haugen TH, Hamsikova

E, Turek LP (2011) Human papilomavırus serologic follow-up

response and relationship to survival in head and neck cancer: a

case-comparison study. Infec Agent Cancer 6:9. doi:10.1186/

1750-9378-6-9

29. Smith EM, Rubenstein LM, Haugen TH, Pawlita M, Turek LP

(2012) Complex etiology underlies risk and survival in head and

neck cancer human papillomavirus, tobacco, and alcohol: a case

for multifactor disease. J Oncol. doi:10.1155./2012/571862

30. Mendez E (2012) Biomarkers of HPV infection in oropharyngeal

carcinomas: can we find simplicity in the puzzle of complexity?

Cancer Res 72:4896–4898

Cancer Causes Control (2014) 25:461–471 471

123