SEARCH

SEARCH BY CITATION

Keywords:

  • human papillomavirus;
  • HPV16 viral load;
  • oral cancer;
  • oropharyngeal cancer

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Case selection
  5. Results
  6. Discussion
  7. References

A considerable subset of oropharyngeal squamous cell carcinomas (SCCs) are positive for human papillomavirus (HPV); however, delineating etiologically-associated HPV infections from SCCs with concurrent HPV infection unrelated to tumorigenesis is challenging. Viral load assessment in biopsy specimens may help facilitate such differentiation. HPV16 viral load and serologic markers were assessed among oral and oropharyngeal cases from a multinational study conducted by the International Agency for Research on Cancer (IARC). HPV16 viral load, measured semiquantitatively by PCR-enzyme immunoassay, was dichotomized as high or low based on the median optical density value. Serologic antibodies to HPV16 virus-like particles (VLPs) and to HPV16 E6 and E7 proteins were measured by ELISA. Compared to HPV DNA-negative cases (n = 852), HPV16 DNA-positive cases with high viral load (n = 26) were significantly more likely to originate in the oropharynx (odds ratio [OR], 12.0; 95% confidence interval [CI], 5.2–27.5) and, after adjustment for tumor site (AdjOR), have antibodies against HPV16 VLPs (AdjOR, 14.6; 95% CI, 6.0–35.6), E6 (AdjOR, 57.6; 95% CI, 21.4–155.3) and E7 (AdjOR, 25.6; 95% CI, 9.3–70.8). HPV16 DNA-positive cases with low viral load (n = 27) were more commonly oropharyngeal (OR, 2.7; 95% CI, 1.1–6.2) and seropositive for HPV16 VLPs (AdjOR, 2.7; 95% CI, 1.1–6.9), E6 (AdjOR, 3.0; 95% CI, 0.7–14.0) and E7 (AdjOR, 3.5; 95% CI, 0.7–16.3), compared to HPV DNA-negative cases; the associations, however, were neither as strong nor as significant as the associations for high viral load. As there appears to be a strong association between HPV16 serologic markers and viral load, in the absence of data on serologic markers, HPV16 viral load may be used to help delineate the subset of HPV16 DNA-positive oral and oropharyngeal cancers that may be the consequence of HPV infection. © 2005 Wiley-Liss, Inc.

Numerous studies have provided consistent evidence that human papillomavirus (HPV), the necessary cause of cervical cancer, is present in tumor biopsies from approximately 20-50% of oropharyngeal squamous cell carcinomas (SCCs) and a smaller subset of oral SCCs.1, 2, 3, 4 Among HPV DNA-positive oropharyngeal SCCs, 90% are positive for HPV16.1, 2, 3, 4 Nonetheless, HPV DNA detection in tumor biopsies may not be sufficient evidence of causation. HPV16 DNA from tumor specimens analyzed jointly with markers of expression of the viral oncogene E6, mutational patterns of the cancer suppressor gene TP53 and levels of allelic loss, have helped identify a subset of these cancers that may be the consequence of HPV infection.1, 2, 5, 6, 7, 8

HPV viral load, a measure of the amount of viral DNA in biopsy specimens, alone or in conjunction with well-characterized HPV serologic assays, may clarify the role of HPV among oral and oropharyngeal cases. Antibodies against HPV E6 and E7 are markers of an invasive HPV-associated malignancy9, 10 and are rarely present among individuals with HPV DNA-negative oral and oropharyngeal tumors.1 Antibodies against HPV virus-like particles (VLPs) are considered a marker of cumulative, lifetime HPV infection,11, 12, 13, 14, 15 and are associated with HPV-related disease, but not as strongly as E6 and E7 antibodies. While these markers do not allow for inferences on causality, evaluation of the associations between high and low viral load with HPV16 serologic markers among HPV16 DNA-positive and -negative oral and oropharyngeal SCCs may delineate the subset more likely the result of HPV infection. Viral load assessment may also compensate for the less than optimal sensitivity in each of the HPV serologic markers currently available.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Case selection
  5. Results
  6. Discussion
  7. References

Oral and oropharyngeal SCC cases from the International Agency for Research on Cancer (IARC) multinational case-control study of HPV and oral cancer were selected for this analysis. The methods and main results of the parent study have been previously reported.1 Briefly, incident cases with oral and oropharyngeal SCC were recruited from referral centers and hospitals in 9 countries (Australia, Canada, Cuba, Italy, India, Northern Ireland, Poland, Spain and Sudan) from 1996 to 1999. Following informed consent, biologic samples were collected prior to cancer treatment and included cancer biopsies and serologic samples. Specimens were stored at −70 or −40°C (depending on study site) and shipped to designated laboratories for analysis.

Case selection

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Case selection
  5. Results
  6. Discussion
  7. References

Only cases with HPV DNA results from beta-globin positive tumor biopsy specimens were included (n = 908); cancer-free controls were not used in the present analysis. Cases positive for high-risk HPV types other than HPV16 were excluded (n = 3) because only antibodies against HPV16-specific VLP, E6 and E7 were available. Our final analytic population consisted of 53 HPV16 DNA-positive oral (n = 28) and oropharyngeal (n = 25) SCCs and 852 HPV DNA-negative cases.

HPV DNA and viral load analysis

The general primer-mediated GP5+/6+ polymerase chain reaction enzyme immunoassay (PCR-EIA) was used to detect and type 20 HPV types6 from biopsy material.1 Of the 53 HPV16-positive SCCs included in this analysis, the PCR-EIA optical density (OD) values obtained after a 1-hr substrate incubation were used to assess the relative amount of HPV16 DNA.16 A previous study demonstrated that, within the range of 1 × 10 to 1 × 106 genome equivalents, the EIA OD value obtained after 1 hr of substrate incubation shows a linear relation with the amount of input DNA.17 Consequently, EIA ODs can semiquantitatively assess the relative viral load.

Detection of antibodies against HPV16 VLPs

Plasma samples were tested for antibodies against HPV16 VLPs by ELISA18 and results were dichotomized as seropositive or seronegative as described previously.1

Detection of antibodies against HPV16 E6 and E7

Antibodies against HPV16 E6 and E7 proteins were detected in an ELISA that utilizes the glutathione S-transferase (GST) capture method with bacterially-expressed full-length E6 and E7 fused to GST as the antigens.19 The assay cutoff point for seropositivity was set and samples were dichotomized as seropositive or seronegative, as described previously.1

Statistical analysis

The minimum, maximum and median values of HPV16 viral load were described for the 53 HPV16 DNA-positive cases. The median values of viral load for oral and oropharyngeal cases were compared by use of the nonparametric Mann-Whitney test.

HPV16 DNA-positive cases were dichotomized as “high” and “low” viral load based on the median viral load value (OD = 1.92) from the 53 HPV16 DNA-positive cases. Three categories were therefore established for analysis: (i) HPV DNA-negative cases (n = 852), (ii) HPV16 DNA-positive cases with low viral load (n = 27) and (iii) HPV16 DNA-positive cases with high viral load (n = 26).

Tumor site and stage,20 seropositivity to HPV16 VLPs, E6 and E7, and a combined measure of seropositivity to E6 and/or E7, were evaluated by use of maximum-likelihood multinomial (polytomous) logistic regression to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs). HPV DNA-negative cases served as the reference category and the high and low viral load groups were each compared to the reference category. Age (in 10-year categories), tumor site, tobacco use (cigarettes per day), alcohol use (drinks per day) and seropositivity to HPV16 serologic markers beyond the 1 in question, were each investigated as possible confounders in adjusted multinomial logistic regression models.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Case selection
  5. Results
  6. Discussion
  7. References

Among the HPV16 DNA-positive cases, viral load OD values ranged from 0.27 to 2.53; the median value was 1.92 (Fig. 1). The median viral load was significantly higher among cases with oropharyngeal SCC (OD = 2.40) compared to oral SCC (OD = 1.59; p = 0.006). Compared to oral cancers, cancers arising in the oropharynx, the head and neck cancer site most commonly associated with HPV infection, were more than twice (95% CI, 1.1–6.2) as likely to have low viral load and 12 times (95% CI, 5.2–27.5) as likely to have high viral load (Table I). Large tumor size,20 a possible confounding factor for viral load in tumor specimens, was not associated with HPV DNA-positivity or viral load (Table I).

thumbnail image

Figure 1. Distribution of HPV16 viral load optical density values among the 53 HPV16 DNA-positive cancer cases, by tumor site. Oral SCC cases are represented by closed circles, oropharyngeal SCC cases by the letter “x.” HPV = human papillomavirus; SCC = squamous cell carcinoma.

Download figure to PowerPoint

Table I. Tumor-Related Classifications and HPV16 Serologic Markers Among HPV16 DNA-Positive Cases with Low and High Viral Load Compared to HPV DNA-Negative Cases*
 HPV DNA-negative casesHPV16 DNA-positive cases, low viral loadHPV16 DNA-positive cases, high viral load
 no. (%)ano. (%)ano. (%)a
  •  Odds ratios (OR) 95% confidence intervals (95%CI).–

  • *

    Viral load dichotomized at the median value, (optical density=1.92).–

  • a

    Columns are not equal to the total population due to missing data.–

  • b

    ORadjusted for tumor site (oral cavity versus oropharynx). HPV = human papillomavirus.

Tumor site   
 Oral cavity736 (86.4)19 (70.4)9 (34.6)
 Oropharynx116 (13.6)8 (29.6)17 (65.4)
 OR (95%CI)1.02.7 (1.1–6.2)12.0 (5.2–27.5)
Tumor size20   
 T0-T2416 (53.3)8 (36.4)15 (62.5)
 T3-T4365 (46.7)14 (63.6)9 (37.5)
 OR (95%CI)1.02.0 (0.8–4.8)0.7 (0.3–1.6)
 Adjusted OR (95%CI)b1.01.8 (0.7–4.3)0.5 (0.2–1.2)
HPV16 VLP antibodies   
 Seronegative717 (90.3)21 (77.8)10 (40.0)
 Seropositive77 (9.7)6 (22.2)15 (60.0)
 OR (95%CI)1.02.7 (1.0–6.8)14.0 (6.1–32.2)
 Adjusted OR (95%CI)b1.02.7 (1.1–6.9)14.6 (6.0–35.6)
HPV16 E6 antibodies   
 Seronegative792 (97.8)25 (92.6)9 (34.6)
 Seropositive18 (2.2)2 (7.4)17 (65.4)
 OR (95%CI)1.03.5 (0.8–16.0)83.1 (32.7–211.3)
 Adjusted OR (95%CI)b1.03.0 (0.7–14.0)57.6 (21.4–155.3)
HPV16 E7 antibodies   
 Seronegative794 (98.0)25 (92.6)15 (57.7)
 Seropositive16 (2.0)2 (7.4)11 (42.3)
 OR (95%CI)1.04.0 (0.9–18.2)36.4 (14.5–91.5)
 Adjusted OR (95%CI)b1.03.5 (0.7–16.3)25.6 (9.3–70.8)
HPV16 E6 and/or E7 antibodies   
 Seronegative779 (96.2)24 (88.9)9 (34.6)
 Seropositive31 (3.8)3 (11.1)17 (65.4)
 OR (95%CI)1.03.1 (0.9–11.0)47.5 (19.6–114.9)
 Adjusted OR (95%CI)b1.02.8 (0.8–10.0)36.0 (14.1–92.0)

Serologic biomarkers of HPV16 were assessed among HPV DNA-negative, HPV16 DNA-positive cases with low viral load, and HPV16 DNA-positive cases with high viral load. HPV16 VLP seroprevalence significantly increased from 9.7% of HPV DNA-negative cases to 22.2% of low viral load HPV16 DNA-positive cases (OR adjusted for tumor site [AdjOR], 2.7; 95% CI, 1.1–6.9) to 60.0% of high viral load HPV16 DNA-positive cases (AdjOR, 14.6; 95% CI, 6.0–35.6; p for trend <0.001). Compared to HPV DNA-negative cases, low viral load was not significantly associated with E6 seropositivity. However, high HPV viral load significantly increased the odds of HPV16 E6 seropositivity 57-fold (AdjOR, 57.6; 95% CI, 21.4–155.3) (Table I). High viral load was also significantly associated with HPV16 E7 seropositivity (AdjOR, 25.6; 95% CI, 9.3–70.8) compared to HPV DNA-negative cases (Table I). Adjustment for age (in 10-year categories) did not affect the magnitude or significance of the associations of viral load with tumor site, grade, or HPV16 serologic markers (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Case selection
  5. Results
  6. Discussion
  7. References

To determine if viral load assessment may contribute to defining the subset of HPV-positive oral and oropharyngeal cancers likely due to HPV infection, we investigated associations among HPV16 viral load and established viral markers, namely, HPV16 VLP, E6 and E7 seropositivity. High viral load among HPV16 DNA-positive oral and oropharyngeal cancers was both highly and significantly associated with these serologic biomarkers compared to HPV DNA-negative cases. In contrast, low viral load was only modestly associated with serologic markers, and these associations often did not attain statistical significance.

HPV16 VLP seropositivity was elevated 14-fold among cases with high viral load, but only 3-fold among cases with low viral load, compared to HPV DNA-negative cases. These associations suggest that VLP seropositivity, a marker of past HPV infection,11, 12, 13, 14, 15 rather than HPV-associated tumorigenesis, may actually be the result of HPV exposure that preceded, and possibly contributed to, HPV-related carcinogenesis. More importantly, seropositivities for HPV16 E6 or E7, recognized markers of an HPV-associated malignancy,9, 10 were remarkably higher among cases with high viral load compared to HPV DNA-negative cases. Again, the corresponding associations for low viral load cases with E6 or E7 seropositivities were modest. While the serologic assays are not site-specific and may be the result of HPV infection and cancer at other mucosal sites, the magnitude of the associations are unlikely to be a result of unidentified tumors rather than tumors of the head and neck. The marked difference between the associations of serologic markers with low compared to high viral load provides evidence that high viral load in biopsy specimens, in conjunction with seropositivity to markers of an HPV-associated malignancy, may identify a subset of these cancers in which HPV is biologically active.

Large studies on cervical smears suggested that persistent HPV infection,21 cytological progression,21, 22, 23, 24 and the risk of a high-grade cervical lesion21, 22, 23, 24, 25 are each associated with high HPV viral load in the cervix. As a consequence, viral load assessment was proposed to distinguish clinically relevant HPV infections in the cervix,26 yet has only been assessed in a few studies of sites beyond the cervix. Indeed, 1 study showed a markedly higher viral load in HPV DNA containing oral and oropharyngeal SCCs with E6 mRNA expression than in those without.6 Another study revealed that viral load was significantly higher among HPV DNA-positive cancers occurring in the tonsil, a subsite of the oropharynx, compared to HPV DNA-positive nonoropharyngeal cancers.27

Most of these studies employed the gold-standard for HPV viral load assessment: real-time PCR.27, 28 However, based on this study, it seems that the less costly, more feasible semiquantitative method may also have utility in epidemiologic studies in which laborious real-time PCR assays may not be possible. Important limitations may exist for both semiquantitative and quantitative viral load assessment. The amount of cancer cells in a biopsy specimen may impact viral load quantitation, and substantial variability may exist in viral load measurements within a single tumor.29 Additionally, it remains uncertain whether the measurement of viral load is the result of a few cells with greater number of HPV DNA copies, or many cells with few copies. Nonetheless, it seems that estimated viral load might contribute to defining the subset of HPV-positive oral and oropharyngeal cancers that are more likely the result of HPV infection. Varying the cutoff point for high vs. low viral load did not appreciably affect the results (for receiver operating characteristic [ROC] curve analysis of viral load OD predicting E6/E7 seropositivity, the area under the curve was 0.81).

While we would not advise the use of a semiquantitative viral load assay for predictive value on an individual basis, in the absence of a quantitative assay, this estimate of the actual viral load may importantly contribute to population-based studies on the etiology of HPV-associated oral and oropharyngeal cancer.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Case selection
  5. Results
  6. Discussion
  7. References
  • 1
    Herrero R, Castellsagué X, Pawlita M, Lissowska J, Kee F, Balaram P, Rajkumar T, Sridhar H, Rose B, Pintos J, Fernández L, Idris A, et al. Human papillomavirus and oral cancer: the International Agency for Research on Cancer multicenter study. J Natl Cancer Inst 2003; 95: 177283.
  • 2
    Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, Zahurak ML, Daniel RW, Viglione M, Symer DE, Shah KV, Sidransky D. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000; 92: 70920.
  • 3
    Schwartz SM, Daling JR, Doody DR, Wipf GC, Carter JJ, Madeleine MM, Mao EJ, Fitzgibbons ED, Huang S, Beckmann AM, McDougall JK, Galloway DA. Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection. J Natl Cancer Inst 1998; 90: 162636.
  • 4
    Smith EM, Ritchie JM, Summersgill KF, Klussmann JP, Lee JH, Wang D, Haugen TH, Turek LP. Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers. Int J Cancer 2004; 108: 76672.
  • 5
    Dai M, Clifford GM, le Calvez F, Castellsagué X, Snijders PJ, Pawlita M, Herrero R, Hainaut P, Franceschi S. Human papillomavirus type 16 and TP53 mutation in oral cancer: matched analysis of the IARC multicenter study. Cancer Res 2004; 64: 46871.
  • 6
    van Houten VM, Snijders PJF, van den Brekel MW, Kummer JA, Meijer CJLM, van Leeuwen B, Denkers F, Smeele LE, Snow GB, Brakenhoff RH. Biological evidence that human papillomaviruses are etiologically involved in a subgroup of head and neck squamous cell carcinomas. Int J Cancer 2001; 93: 2325.
  • 7
    Wiest T, Schwarz E, Enders C, Flechtenmacher C, Bosch FX. Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control. Oncogene 2002; 21: 15107.
  • 8
    Braakhuis BJM, Snijders PJF, Keune WJ, Meijer CJLM, Ruijter-Schippers HJ, Leemans CR, Brakenhoff RH. Genetic patterns in head and neck cancers that contain or lack transcriptionally active human papillomavirus. J Natl Cancer Inst 2004; 96: 9981006.
  • 9
    Sun Y, Eluf-Neto J, Bosch FX, Muñoz N, Booth M, Walboomers JM, Shah KV, Viscidi RP. Human papillomavirus-related serological markers of invasive cervical carcinoma in Brazil. Cancer Epidemiol Biomarkers Prev 1994; 3: 3417.
  • 10
    Meschede W, Zumbach K, Braspenning J, Scheffner M, Benitez-Bribiesca L, Luande J, Gissmann L, Pawlita M. Antibodies against early proteins of human papillomaviruses as diagnostic markers for invasive cervical cancer. J Clin Microbiol 1998; 36: 47580.
  • 11
    Kirnbauer R, Hubbert NL, Wheeler CM, Becker TM, Lowy DR, Schiller JT. A virus-like particle enzyme-linked immunosorbent assay detects serum antibodies in a majority of women infected with human papillomavirus type 16. J Natl Cancer Inst 1994; 86: 4949.
  • 12
    Wideroff L, Schiffman MH, Nonnenmacher B, Hubbert N, Kirnbauer R, Greer CE, Lowy D, Lorincz AT, Manos MM, Glass AG, Scott DR, Sherman ME, et al. Evaluation of seroreactivity to human papillomavirus type 16 virus-like particles in an incident case-control study of cervical neoplasia. J Infect Dis 1995; 172: 142530.
  • 13
    Carter JJ, Koutsky LA, Wipf GC, Christensen ND, Lee SK, Kuypers J, Kiviat N, Galloway DA. The natural history of human papillomavirus type 16 capsid antibodies among a cohort of university women. J Infect Dis 1996; 174: 92736.
  • 14
    Dillner J, Kallings I, Brihmer C, Sikstrom B, Koskela P, Lehtinen M, Schiller JT, Sapp M, Mardh PA. Seropositivities to human papillomavirus types 16, 18, or 33 capsids and to Chlamydia trachomatis are markers of sexual behavior. J Infect Dis 1996; 173: 13948.
  • 15
    Kjaer SK, Chackerian B, van den Brule AJ, Svare EI, Paull G, Walbomers JM, Schiller JT, Bock JE, Sherman ME, Lowy DR, Meijer CJLM. High-risk human papillomavirus is sexually transmitted: evidence from a follow-up study of virgins starting sexual activity (intercourse). Cancer Epidemiol Biomarkers Prev 2001; 10: 1016.
  • 16
    Molano M, Van den Brule A, Plummer M, Weiderpass E, Posso H, Arslan A, Meijer CJLM, Munoz N, Franceschi S. Determinants of clearance of human papillomavirus infections in Colombian women with normal cytology: a population-based, 5-year follow-up study. Am J Epidemiol 2003; 158: 48694.
  • 17
    Jacobs MV, van den Brule AJ, Snijders PJF, Helmerhorst TJ, Meijer CJLM, Walboomers JM. A non-radioactive PCR enzyme-immunoassay enables a rapid identification of HPV 16 and 18 in cervical scrapes after GP5+/6+ PCR. J Med Virol 1996; 49: 2239.
  • 18
    Viscidi RP, Ahdieh-Grant L, Clayman B, Fox K, Massad LS, Cu-Uvin S, Shah KV, Anastos KM, Squires KE, Duerr A, Jamieson DJ, Burk RD, et al. Serum immunoglobulin G response to human papillomavirus type 16 virus-like particles in human immunodeficiency virus (HIV)-positive and risk-matched HIV-negative women. J Infect Dis 2003; 187: 194205.
  • 19
    Sehr P, Zumbach K, Pawlita M. A generic capture ELISA for recombinant proteins fused to glutathione S-transferase: validation for HPV serology. J Immunol Methods 2001; 253: 15362.
  • 20
    Fleming ID, Cooper JS, Henson DE, Hutter RVP, Kennedy BJ, Murphy GP, O'Sullivan B, Yarbro JW. AJCC cancer staging manual, 5th. ed. Philadelphia: Lippincott-Raven, 1997.
  • 21
    van Duin M, Snijders PJF, Schrijnemakers HF, Voorhorst FJ, Rozendaal L, Nobbenhuis MA, van den Brule AJ, Verheijen RH, Helmerhorst TJ, Meijer CJLM. Human papillomavirus 16 load in normal and abnormal cervical scrapes: an indicator of CIN II/III and viral clearance. Int J Cancer 2002; 98: 5905.
  • 22
    Josefsson AM, Magnusson PK, Ylitalo N, Sorensen P, Qwarforth-Tubbin P, Andersen PK, Melbye M, Adami HO, Gyllensten UB. Viral load of human papilloma virus 16 as a determinant for development of cervical carcinoma in situ: a nested case-control study. Lancet 2000; 355: 218993.
  • 23
    Ylitalo N, Josefsson A, Melbye M, Sorensen P, Frisch M, Andersen PK, Sparen P, Gustafsson M, Magnusson P, Ponten J, Gyllensten U, Adami HO. A prospective study showing long-term infection with human papillomavirus 16 before the development of cervical carcinoma in situ. Cancer Res 2000; 60: 602732.
  • 24
    Ho GY, Studentsov YY, Bierman R, Burk RD. Natural history of human papillomavirus type 16 virus-like particle antibodies in young women. Cancer Epidemiol Biomarkers Prev 2004; 13: 1106.
  • 25
    Cuzick J, Terry G, Ho L, Hollingworth T, Anderson M. Type-specific human papillomavirus DNA in abnormal smears as a predictor of high-grade cervical intraepithelial neoplasia. Br J Cancer 1994; 69: 16771.
  • 26
    Snijders PJF, van den Brule AJ, Meijer CJLM. The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity. J Pathol 2003; 201: 16.
  • 27
    Koskinen WJ, Chen RW, Leivo I, Makitie A, Back L, Kontio R, Suuronen R, Lindqvist C, Auvinen E, Molijn A, Quint WG, Vaheri A, et al. Prevalence and physical status of human papillomavirus in squamous cell carcinomas of the head and neck. Int J Cancer 2003; 107: 4016.
  • 28
    Mellin H, Dahlgren L, Munck-Wikland E, Lindholm J, Rabbani H, Kalantari M, Dalianis T. Human papillomavirus type 16 is episomal and a high viral load may be correlated to better prognosis in tonsillar cancer. Int J Cancer 2002; 102: 1528.
  • 29
    Klussmann JP, Weissenborn SJ, Wieland U, Dries V, Kolligs J, Jungehuelsing M, Eckel HE, Dienes HP, Pfister HJ, Fuchs PG. Prevalence, distribution, and viral load of human papillomavirus 16 DNA in tonsillar carcinomas. Cancer 2001; 92: 287584.