Affiliations of authors: M. L. Gillison (Department of Medical Oncology), W. M. Koch, R. B. Capone, M. Spafford, L. Wu, D. Sidransky (Department of OtolaryngologyHead and Neck Surgery), W. H. Westra, M. Viglione (Department of Pathology), D. E. Symer (Department of Molecular Biology and Genetics), The Johns Hopkins University School of Medicine, Baltimore, MD; M. L. Zahurak, Department of Biostatistics, The Johns Hopkins Oncology Center, Baltimore; K. V. Shah, R. W. Daniel, Department of Molecular Microbiology and Immunology, The Johns Hopkins School of Hygiene and Public Health, Baltimore.
Correspondence to: Keerti V. Shah, M.D., Dr.P.H., The Johns Hopkins School of Hygiene and Public Health, 615 N. Wolfe St., Rm. E-5012, Baltimore, MD 21205 (e-mail: kvshah{at}jhsph.edu).
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ABSTRACT |
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INTRODUCTION |
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High-risk HPVs (e.g., HPV16 and HPV18) are known to be tumorigenic in human epithelial tissues. These viruses are a necessary but insufficient cause of cervical squamous cell carcinoma and have been implicated in the development of other anogenital squamous cell cancers. Two viral oncoproteins of high-risk HPVs, E6 and E7, promote tumor progression by inactivating the TP53 gene (also known as p53) and retinoblastoma tumor suppressor gene products, respectively (58). Accordingly, these viral oncoproteins are capable of transforming primary human keratinocytes from either genital or upper respiratory tract epithelia (9) and disrupting cell-cycle regulatory pathways in the genetic progression of HNSCC (10,11). The TP53 gene is mutated in approximately 45% of HNSCC (11) and, although pRb mutations are rare, expression of upstream regulators of pRb function, such as TP16 (12) and cyclin D (11,13), is commonly altered in HNSCC. Therefore, HPV infection may represent an alternative, but functionally comparable, molecular pathway for HNSCC tumorigenesis.
Mucosal HPVs are known to infect the upper respiratory tract (4). Low-risk HPV6 and HPV11 cause both benign genital condylomata and respiratory papillomas (14). HPV genomic sequences have also been identified in HNSCC, but markedly varied estimates of viral prevalence (range, 8%100%) (15) have impeded clarification of the relationship between HPV presence and head and neck cancer development. Despite this variability, studies (16,17) have suggested an association of HPVs with cancers in the oropharynx and, especially, with tonsillar carcinomas (1820).
In a recent casecontrol study (21), HPV presence in the oral cavity was associated with increased risk of oral cavity or oropharyngeal cancer (odds ratio [OR] = 3.7; 95% confidence interval [CI] = 1.59.3), independent of alcohol and tobacco exposure. Men with cancers at these sites also had sexual risk factors similar to those for women with cervical cancer and known to be associated with HPV exposure (22). These include young age at first intercourse, a history of multiple sexual partners, and a history of genital warts (22). Seropositivity to HPV16 capsid protein was significantly greater in oral cancer patients than in control subjects, providing further evidence of prior HPV16 exposure in cancer cases (22).
We sought to clarify the role that HPV plays in HNSCC development by performing a detailed virologic analysis of 253 fresh-frozen HNSCC tumor specimens. In addition, patient risk factor profiles, clinical and pathologic data, and tumor p53 status were used to establish epidemiologic, pathologic, and molecular correlates of HPV positivity.
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SUBJECTS AND METHODS |
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Patients with histologically confirmed, newly diagnosed, or recurrent HNSCC were studied. All patients participated in research protocols in the Division of Head and Neck Cancer Research approved by the Institutional Review Board of The Johns Hopkins Hospital, Baltimore, MD, and affiliated institutions and gave written informed consent for banking of tumor tissue. Demographic data, including age at diagnosis, sex, race, and alcohol and tobacco exposures, were obtained from medical records. Tumor site, stage, and grade were determined from review of operative, radiology, and pathology reports. Tumor stage was assigned on the basis of best available stagingclinical or pathologicby use of the 1997 American Joint Committee on Cancer (AJCC) staging criteria (23). The patient's average weekly consumption of alcohol at diagnosis was recorded in whiskey equivalents consumed per week (one whiskey equivalent = 10 g of alcohol 1 oz of 86-proof liquor
one 3.6-oz glass of wine containing 12% alcohol
one beer) (2). Patients were classified as light or nondrinkers (<10 whiskey equivalents per week), moderate drinkers (1020 whiskey equivalents per week), or heavy drinkers (>20 whiskey equivalents per week). Patients were also classified as never, current, or former (former smokers are those who have quit for >12 months before diagnosis) daily tobacco smokers (cigarette, pipe, and/or cigar). All patients with newly diagnosed or recurrent disease were followed prospectively from the date of original diagnosis by the head and neck surgeon. The date and clinical status of patients were recorded at each follow-up appointment (median of every 3 months from diagnosis). Primary treatment modality (radiation therapy, chemoradiation therapy, surgery, etc.) as well as the date and cause of death were documented.
Tumor Specimens
Fresh tumor specimens were frozen within 30 minutes of resection and stored at -80°C until processing. Microdissection was used to ensure that more than 70% of isolated DNA was from tumor tissue. DNA was purified from samples as described (24). DNA purification and HPV-detection assays were performed in separate laboratories to reduce contaminations.
Review of hematoxylineosin-stained slides of available tumor specimens was performed independently by two pathologists (W. H. Westra and M. Viglione) who did not know the results of the HPV testing, and differences were resolved by joint review. Tumor grade was recorded as well, moderate, or poor according to the criteria of the World Health Organization (25). Tumors were further characterized according to the absence or presence of "basaloid" features as defined by small, dark, crowded cells with scant cytoplasm, hyperchromatic nuclei, marked mitotic activity, a predominant lobular pattern of growth, and the absence of prominent keratinization (26).
Cell Lines
SiHa (HPV16-positive), CaSki (HPV16-positive), HeLa (HPV18-positive), C 4II (HPV18-positive), C-33A (HPV-negative), and K562 (HPV-negative) cell lines were used as controls in polymerase chain reaction (PCR), Southern blot, and in situ hybridization assays (2730). High-molecular-weight genomic DNA was isolated from cell lines for Southern blot analysis (31), and cell blocks were prepared from control cell lines for in situ hybridization as described previously (32).
Consensus L1 PCR
HPV genomic sequences were detected by PCR amplification by use of consensus degenerate primers (MY09/MY11/HMB01) complementary to the conserved L1 region of HPV as described (3335). Amplification of a ß-globin gene fragment was performed by use of PCO4 and GH20 primers (34) to control for target DNA integrity. ß-Globin-negative samples were excluded from further analysis. PCR products dot blotted onto Biotrans nylon membranes (ICN Pharmaceuticals, Inc., Costa Mesa, CA) were hybridized with biotin end-labeled oligonucleotide probes for ß-globin. A "generic" mixture of HPV probes (HPV16, HPV18, HPV51, and HPV66) and 33 HPV type-specific probes (synthesized at DNA Synthesis Core Facility, The Johns Hopkins School of Hygiene and Public Health, Baltimore, MD) were used for the identification of HPV type. Hybridization signals were detected by use of a chemiluminescence system on Hyperfilm ECL (Amersham Life Science Inc., Arlington Heights, IL).
To ensure the specificity of results, PCR products from all specimens positive for HPV in the dot-blot format were confirmed by Southern blot hybridization. Specimens with amplification products of the expected size were considered to be positive. Specimens positive for HPV by generic probe on Southern blot analysis that could not be further type specified underwent further analysis. PCR products from the L1 region amplification were cloned by use of the TA cloning kit from Invitrogen Corp. (Carlsbad, CA) and sequenced by use of the fluorescent dideoxy terminator method of cycle sequencing following ABD protocols (The Perkin-Elmer Corp., Applied Biosystems Division [ABD], Foster City, CA) (36). A BLAST search was performed to assign L1 sequences to known HPV types.
E7 Type-Specific PCR
Because rare false-negative L1 region amplifications could occur as a result of integration events in that region, all tumor DNAs were also tested for HPV16 and HPV18 by amplification of the viral E7 region by use of type-specific primers. Tumors positive for HPV33 or HPV31 by the L1 primers were confirmed by PCR amplification with type-specific primers for the E7 region as described (37) with the following modifications: a 5' oligonucleotide primer for HPV16 amplification, HPV16 E7.642, 5'-ATTAAATGACAGCTCAGAGGA-3', was substituted for the HPV16 E7.671 primer described; all reactions contained 3 mM MgCl2; and the annealing temperature for the HPV33 type-specific amplifications was reduced to 45°C. Amplification was followed by dot-blot hybridization (37) and confirmed by Southern blot hybridization.
E6 Open Reading Frame Sequencing
Tumor specimens positive for HPV16 were further subclassified into HPV16 variants by amplification and sequencing of the HPV16 E6 coding region (nucleotides 104559) by use of nested PCR (38). The PCR product was purified for direct sequencing with the use of the QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA). DNA templates were sequenced by use of the fluorescent dideoxy terminator method of cycle sequencing (36). Coding sequences, including all identified base-pair (bp) substitutions, were confirmed by sequencing both strands of DNA and by repeat amplification and sequencing. Analysis was performed by use of Sequencher Software 3.1 (Gene Codes Corp., Ann Arbor, MI). HPV16 nucleotide variations were compared with reference HPV DNA sequence (38). Placement of HPV16 variants into the major phylogenetic groups was performed by inspection (39).
Southern Blot Hybridization
Tumors positive for HPV16 by PCR, for which at least 25 µg of tumor DNA were available, were examined for HPV16 sequences by Southern blot hybridization of unamplified tumor DNAs. Full-length HPV16 genomic DNA (7905 bp) in pGEM II (Promega Corp., Madison, WI) was gel purified and labeled to a high specific activity with [-32P]deoxycytidine triphosphate by the random primer method (Random Primers DNA Labeling System; Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD). Nonamplified tumor DNA (25 µg) was digested with PstI (New England Biolabs, Inc., Beverly, MA), ethanol precipitated, and separated on a 1.25% agarose gel. The DNA was denatured and transferred to a nylon membrane (GeneScreen Plus; Du Pont NEN, Boston, MA). After hybridization to radiolabeled HPV16 probe, the membrane was exposed to film (Kodak, Rochester, NY) for 2472 hours at -70°C.
In Situ Hybridization
In situ hybridization was performed by use of the catalyzed reporter deposition system for the detection of HPV16 DNA in formalin-fixed, paraffin-embedded tissues also prepared at the time of resection as described (32).
TP53 Sequencing
A 1.8-kilobase TP53 gene fragment, encompassing exons 59, was amplified from purified tumor DNA by PCR (40) and sequenced directly (41). This region was chosen because somatic mutations outside this DNA-binding region are rare in human malignancies (42,43). Mutations were confirmed by repeated amplification and sequencing of the tumor DNA.
Statistical Analysis
Factors associated with HPV status were selected on cross-tabulations and logistic regression modeling. Cross-tabulations were analyzed by use of the chi-square test or Fisher's exact test, where appropriate. A logistic regression model was used to determine the effects of multiple factors on HPV status. Results are summarized as ORs and corresponding 95% CIs.
In survival analysis, the primary statistical end points were overall survival (death from all causes) and disease-specific survival (death from HNSCC). Event time distributions for these end points were estimated by use of the method of Kaplan and Meier (44) and compared by use of the log-rank statistic (45) or the proportional hazards regression model (46). The assumption of proportional hazards for primary and recurrent cancers was not appropriate; therefore, survival models were stratified for this factor. The simultaneous effect of two or more factors was studied by use of stratified multivariate proportional hazards models. Factors tested for prognostic value included sex, age at diagnosis, race, tobacco and alcohol exposures, tumor stage, lymph node status, HPV presence, tumor location, and tumor grade. In the subset of patients for whom TP53 sequence data were available (n = 166), presence of a mutation in the TP53 gene was also evaluated as a prognostic factor for survival. Estimates of relative risk are presented as hazard ratios (HRs) and corresponding 95% CIs. All P values reported are two-sided. Computations were performed by use of STATA statistical software (StataCorp, College Station, TX), and KaplanMeier curves were created in SAS (47).
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RESULTS |
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The study population consisted of 259 patients with a histologically confirmed diagnosis of HNSCC from June 1987 through October 1998. Six of the 259 patients were excluded from further analysis because ß-globin DNA could not be amplified from purified tumor DNA. The majority of tumor specimens (from 200 patients) were obtained from the primary tumor at diagnosis. The remaining 53 tumor specimens were obtained by biopsy of recurrent, local disease.
The characteristics of the study population (n = 253) largely reflect the demographics of head and neck cancer patients in the United States (Table 1). Patient ages ranged from 17 to 91 years (median, 63 years; interquartile range, 5471 years). The primary tumor was located in the nasopharynx (n = 2), oral cavity (n = 84), oropharynx (n = 60 ), hypopharynx (n = 21), or larynx (n = 86). Seventy percent of the patients (177 of 253) presented with locally advanced, stage III or IV disease. With regard to combined alcohol and tobacco exposures, the study population consisted of 33 (13%) nonsmokers/light or nondrinkers, 100 (39%) current smokers/light or nondrinkers, 64 (25%) current smokers/moderateheavy drinkers, 40 (16%) former smokers/light or nondrinkers, and nine (4%) former smokers/moderateheavy drinkers. Exposure information was insufficient to classify seven (3%) patients. No patients in this study were nonsmokers/moderateheavy drinkers.
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HPV genomic DNA was detected in 55 (22%) of 253 tumor specimens by use of the MY09/MY11/HMBO1 primers to amplify the L1 region of the viral genome. HPV type was identified as HPV16 in 49 tumors, HPV33 in three tumors, and one tumor was positive for HPV16 and HPV31. Two additional tumors positive for HPV by the generic probe could not be type specified. After cloning and sequencing of amplified DNA, one of these two tumors was identified as HPV11. The other tumor was not successfully cloned but was considered to be positive for analysis because the amplified DNA was of the appropriate size on Southern blot when hybridized to the generic probe.
With the use of type-specific primers for the E7 region, seven additional tumors were positive for HPV. Six of seven were positive for HPV16, and one was positive for HPV18 (an oral cavity tumor). All four specimens positive for HPV33 and HPV31 on amplification of the L1 region were confirmed by use of type-specific primers. Therefore, with the use of the combination of the L1 and E7 region primers, 62 (25%) of 253 (95% CI = 19%30%) tumor specimens were positive for HPV genomic DNA. HPV16 accounted for the majority of the HPVs identified, 90% (56 of 62). Five of six of the remaining viral isolates were high-risk types: HPV31, 2% (one of 62, also HPV16 positive); HPV33, 5% (three of 62); and HPV18, 2% (one of 62). One (2%) of the 62 cases (a laryngeal tumor) remained untyped. HPV11, a low-risk HPV type, was identified by cloning as described above from one oropharyngeal tumor. The HPV positivity at different anatomic sites was as follows: oral cavity, 12% (10 of 84 specimens); oropharynx, 57% (34 of 60); hypopharynx, 10% (two of 21); larynx, 19% (16 of 86); and nasopharynx (none of two).
HPV16 Variant Sequence Analysis
To confirm that the different HPV16 isolates were independent and tumor specific, the E6 open reading frame of HPV16-positive tumors was sequenced. DNA sequencing of the E6 region allows identification of unique viral variants and categorization of these variants within one of six major phylogenetic groups (38). Sequence data were attainable for 52 (93%) of 56 HPV16-positive tumors because DNA from the other four samples had been exhausted. Observed sequence variations were compared with a reference sequence of a European prototype, HPV16 "E-P-350T" (Table 2) (38). Seventeen distinct HPV16 variants were identified in the 52 isolates sequenced; of these, seven were novel variants not previously reported (E-G315T, E-G315G, E-C395G, E-A478T, E-A132T, Af1-C311, and Af1-A389). The majority of isolates could be classified into the same phylogenetic group as the European prototype, 75% (39 of 52) (Table 2
). Asian (17% [nine of 52]), North American (4.0% [two of 52]), and African 1 (4.0% [two of 52]) variants were also identified.
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HPV16 Identification by Southern Blot and In Situ Hybridization
The presence of HPV16 in tumors found by PCR amplification was further examined by use of two highly specific methods, Southern blot analysis of unamplified tumor DNA with an HPV16 probe and in situ hybridization for HPV16 sequences on paraffin-imbedded tumor samples. We verified that a single copy of integrated HPV16 could be detected by Southern blot, with an appropriate band pattern change, using 10 µg of genomic DNA isolated from SiHa cells, known to contain a single copy of integrated virus (data not shown). Fifty-seven percent (12 of 21) of HPV16 PCR-positive (12 of 18 in oropharyngeal and none of three in nonoropharyngeal) tumors tested were positive for HPV16 by Southern blot by the use of 25 µg of unamplified tumor DNA (Fig. 1). Banding patterns observed on Southern blot hybridization may help distinguish episomal and integrated forms of the virus. Because HPV16 DNA has six PstI restriction sites, episomal virus has six distinct bands, and viral integration alters this pattern. Banding patterns consistent with episomal (lane 12), both episomal and integrated (lane 7), and integrated (lane 4) forms of the virus were seen (Fig. 1
). These results do not exclude the possibility that altered banding patterns were due to unusual multimeric episomal forms with minor rearrangements or deletions (48). Formal confirmation of viral integration in these tumors would require digestion with other informative enzymes (HindIII and BamHI), two-dimensional gel electrophoresis, and/or cloning of cell genomeviral junctions (49). These procedures could not be performed because of limited quantities of tumor DNA.
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There were significant associations between HPV presence and both location and grade of the primary tumor (Table 1). Oropharyngeal tumors were significantly more likely to be HPV positive (34 [57%] of 60; OR = 9.7; 95% CI = 4.222) than oral cavity tumors (10 [12%] of 84) or all tumors arising from nonoropharyngeal sites (28 [14%] of 193; OR = 7.7; 95% CI = 4.015). HPV-positive oropharyngeal tumors arose predominantly from the palatine or lingual tonsils (32 [94%] of 34): HPV positivity was 62% (32 of 52) for tumors on the tonsil/base of tongue and 25% (two of eight) for tumors at other oropharyngeal sites. The association of HPV presence with tonsillar location was very strong (OR = 9.1; 95% CI = 4.618) when compared with all nontonsillar tumors. Poorly differentiated tumors were more likely to be HPV positive (OR = 3.5; 95% CI = 1.48.8) than well-differentiated and moderately differentiated tumors (Table 1
). For the subset of cases reviewed for basaloid characteristics (n = 148) (see "Subjects and Methods" section), HPV-positive tumors were significantly more likely to have a characteristic basaloid morphology (OR = 19.8; 95% CI = 5.374) than HPV-negative tumors (Table 1
).
The median age of patients with HPV-negative tumors was 64 years and was not significantly different from that of patients with HPV-positive tumors, 60.5 years (Wilcoxon rank sum; P = .42). There was a trend for patients with HPV-positive tumors to be nondrinkers or light drinkers, but this difference was not statistically significant (Table 1). Other factors unrelated to HPV status on univariate logistic regression analysis included sex, race, presence of lymph node disease, primary therapy received, and tobacco exposure (Table 1
). Neither the tumor specimen tested (primary or recurrent) nor the AJCC stage was associated with the presence of HPV (data not shown).
Direct sequencing of exons 59 of the TP53 gene has been completed for an unselected subset (166 [66%] of 253) of tumors in this study. TP53 mutations were identified in 39% (95% CI = 32%47%) of sequenced tumors. HPV-positive tumors appeared less likely to harbor a TP53 mutation, but this difference was not statistically significant (Table 1). Although specimens taken from the primary tumor were more likely to have undergone TP53 sequencing than recurrent tumors (chi-squared(1 df); P = .04), the subset of patients with TP53 sequence data did not differ significantly from those without with regard to HPV status, location of the primary tumor, sex, race, stage, lymph node status, age, smoking status, or alcohol consumption (chi-squared(1 df); P>.10).
A logistic regression model of factors related to the presence of HPV in HNSCC was constructed. Both poor tumor grade (OR = 2.4; 95% CI = 1.24.9) and oropharyngeal tumor site (OR = 6.2; 95% CI = 3.112.1) independently increased the probability of HPV presence (Table 1). The magnitude and direction of these associations were not altered appreciably by the addition of other nonsignificant factors to the model, including age, race, sex, tumor site, tumor grade, lymph node status, and alcohol or tobacco exposure.
Because the majority of HPV-positive tumors arose from the oropharynx, we compared risk factors for HPV positivity for HNSCC located in the oropharynx (n = 60) versus other sites (n = 193) (Table 3). Regardless of location, there were no differences between HPV-positive and HPV-negative tumors with respect to sex, age at diagnosis, race, and lymph node status. However, in the oropharynx, poor tumor grade, basaloid morphology, and wild-type TP53 increased the probability of HPV positivity, while moderate to heavy alcohol intake decreased the probability of HPV positivity (Table 3
). In the oropharyngeal subset, TP53 mutations were inversely associated with HPV; 67% of HPV-negative but only 10% of HPV-positive tumors harbored TP53 mutations (Fisher's exact test; P = .001) (Table 3
). Nonsmokers were more frequent in the HPV-positive group (seven [21%] of 34) than in the HPV-negative oropharynx group (one [4%] of 25), but this difference was not statistically significant. By contrast, at nonoropharyngeal sites, only basaloid morphology significantly increased the probability of HPV positivity (Table 3
).
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Survival Analysis
To determine whether the presence of HPV in head and neck tumors had prognostic significance, we analyzed both overall survival and disease-specific survival. Survival data were available for 252 of the 253 patients in this study. One hundred deaths, including 71 from head and neck cancer and 29 from unrelated causes, have occurred in the study population. Patients were followed for a median of 31 months (range, 5 days to 241 months). The status of eight patients (3%) who transferred their care to another institution after being followed at Johns Hopkins Hospital (Baltimore, MD) for 7 weeks to 96 months (median, 16 months) was unknown. Therefore, these patients were considered lost to follow-up. The estimated median overall survival of the 252 patients was 85 months. Seventy percent of all patients survived for 2 years and 58% survived for 5 years. The estimated median survival of the HPV-negative group was 76 months, while that of the HPV-positive group was estimated to be greater than 91 months.
For the entire group of 252 subjects, patients with HPV-positive tumors had significantly improved overall survival when compared with patients with HPV-negative tumors (HR = 0.57; 95% CI = 0.341.0). Factors found to be predictive of poor overall survival by univariate proportional hazards regression analysis included presence of lymph node disease (HR = 1.8; 95% CI = 1.22.8) or advanced locoregional disease (stage 3 or 4 versus stage 1 or 2) at diagnosis (HR = 1.7; 95% CI = 1.12.7), age greater than 60 years (HR = 1.9; 95% CI = 1.22.9), and heavy weekly consumption of alcohol (HR = 2.1; 95% CI = 1.23.6) (Table 4). Sex, race, and tumor location were not prognostically significant. After adjustment for lymph node status, age, and alcohol consumption, patients with HPV-positive tumors were estimated to have approximately a 40% reduction in risk of death from all causes (HR = 0.60; 95% CI = 0.351.0) when compared with patients with HPV-negative tumors (Table 4
).
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Survival data were also compared separately for oropharyngeal and nonoropharyngeal HNSCC patients. There were three cancer deaths in 34 patients with HPV-positive and 10 cancer deaths in 26 patients with HPV-negative oropharyngeal cancers. Disease-specific survival was significantly improved in the HPV-positive oropharyngeal group (HR = 0.26; 95% CI = 0.070.98; P = .05). Among patients with nonoropharyngeal cancers, there were six cancer deaths in 28 patients with HPV-positive and 52 cancer deaths in 165 patients with HPV-negative cancers. Disease-specific survival was similar in the two groups (HR = 0.62; 95% CI = 0.261.5; P = .28).
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DISCUSSION |
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A predisposition of the oropharyngeal mucosa to malignant transformation by HPV was first suggested when HPV16 was detected in tumors of the tongue, tonsil, and pharynx but not in control tissues (51). Another study (50) found HPV in oropharyngeal cancers three times as often as other HNSCC primaries (18.6% versus 6.1%; P = .02). In two case series, 50%60% of tonsillar carcinomas (a subset of oropharyngeal tumors) were HPV positive in comparison to 6%10% of tumors at other sites (P<.001) (16,17). Consistently high prevalence of HPV in tonsillar carcinomas has been found by use of various methods of HPV detection (1620,52,53), and active viral oncogene transcription and genomic integration have been observed in case studies of these cancers (19,53,54).
We have now confirmed and extended previous studies showing a strong association between HPV and oropharyngeal cancer. Our findings suggest that HPV-positive oropharyngeal cancers arising from the lingual and palatine tonsils are a distinct molecularpathologic entity etiologically linked to infection by high-risk HPVs, especially HPV16. Moreover, a clonal association of HPV with cancer cells is strongly supported by the specific localization of HPV in tumor cells at all cancer stages (preinvasive, invasive, and lymph node) and its probable integration into the genome of some tumors. In contrast to HPV-negative oropharyngeal cancers, these cancers have distinct pathology (more frequently basaloid), tumor biology (fewer p53 mutations), risk factors (less associated with alcohol consumption and perhaps smoking), and clinical course (improved survival) (Table 3).
An etiologic link between HPV and nonoropharyngeal tumors is less firmly established. Nonoropharyngeal HPV-positive tumors were not statistically significantly different from HPV-negative tumors with regard to alcohol consumption and tobacco exposure and survival. Also, nonoropharyngeal cancers that were HPV positive by PCR were rarely HPV positive by Southern blot or in situ hybridization. We have no reason to suspect that HPV presence by PCR in these tumors represents false-positive findings. However, because of PCR's exquisite sensitivity, latent infections pathologically unrelated to the tumor could be detected. Nevertheless, single case reports of nonoropharyngeal tumors have provided incontrovertible evidence that the HPV16 genome was integrated into cancer cells and that the viral genome was transcriptionally active (55).
HPV-associated HNSCCs have a morphologic appearance that deviates from conventional keratinizing squamous cell carcinoma (17,18). In our detailed histopathologic evaluation, HPV-positive tumors not only were poorly differentiated and nonkeratinizing but also were strongly associated with a "basaloid" morphology (OR = 18.7; 95% CI = 2.1167). A basaloid subtype is a well-recognized morphologic variant of HNSCC (26) and, like the subset of HPV-positive HNSCC tumors, basaloid tumors predominantly occur in the oropharynx (19,52,5658). In this study, basaloid features were statistically significantly associated with HPV-positive tumors, both in and outside the oropharynx. The association between HPV and basaloid differentiation is substantiated further by the basaloid phenotype of HPV-associated squamous cell carcinomas of the anus (59), penis (60,61), and vulva (62,63).
The role of p53 mutations in the pathogenesis of certain head and neck cancers (64,65) may be substituted by HPV infection because viral E6 protein can inactivate p53 by targeting the protein for ubiquitination and degradation (7). Although p53 mutations were found in about one third of all tumors, there was a marked difference in p53 mutation frequency between HPV-positive and HPV-negative tumors in the oropharynx. The inverse relationship (67% versus 10%) between p53 mutation and HPV strengthens the etiologic role of HPV in oropharyngeal cancers. The overall frequency of p53 inactivation in HNSCC (p53 mutation and/or high-risk HPV infection) was 55% in this entire series and 85% in the oropharynx, emphasizing the importance of p53 abrogation in HNSCC progression.
The coexistence of both HPV and p53 mutations in a single tumor, observed in 11 cases, may be explained by the variable sensitivity of certain p53 mutants to E6 degradation (66,67) and/or by tumor promotion via p53-independent viral mechanisms (68,69) such as disruption of pRb function by HPV E7 (70). Evidence for activity of high-risk HPV E7 was previously observed in a subset of HNSCCs found to have reduced pRb expression by immunohistochemistry. These tumors tended to arise from the tonsil, were poorly differentiated, were more likely to occur in nonsmokers, and were HPV16 or HPV33 positive (52). Similar findings were reported in another study of tonsillar cancers (19).
The inverse association between p53 mutations and HPV presence in the oropharynx further suggests two parallel or overlapping pathways of HNSCC development: one driven by environmental toxins (e.g., tobacco and alcohol) and another driven by an infectious agent (e.g., high-risk HPVs). Although in previous studies (7,50) nonsmokers were more likely to have HPV-positive head and neck tumors than smokers, viral infections may act synergistically with tobacco and alcohol exposures. Exposure of HPV16- or HPV18-immortalized human keratinocyte cell lines to tobacco-related carcinogens resulted in substantially more genetic alterations leading to cellular transformation not seen in keratinocytes transfected with low-risk HPV or without HPV and exposed to the same carcinogens (7174). This synergy was also supported by a recent casecontrol study of oral cavity and oropharyngeal cancer patients. HPV16-seropositive nonsmokers had a twofold and HPV16-seronegative current smokers had a 5.8-fold risk of HNSCC compared with seronegative nonsmokerslight drinkers; HPV16-seropositive current smokers had an approximately 15-fold increase in HNSCC risk (22).
The "casecase" or "case-only" format of this study was used specifically to investigate the differences between HPV-positive and HPV-negative head and neck cancers to enhance the specificity of the association between HPV and head and neck cancer (75). Multivariate analysis of the data shown in Table 1 indicates that the two populations of patients may be more similar than different with respect to the major environmental risk factors of alcohol and tobacco exposure. However, a healthy control group would be required to further investigate possible synergistic interactions between tobacco, alcohol, and HPV exposures. Alcohol and tobacco exposure histories in this study were limited to qualitative categories because of the dependence on medical record data. Indeed, our qualitative data may underestimate differences between HPV-positive and HPV-negative patients that may become more evident with measurements of lifetime cumulative exposures.
We found that HPV-positive HNSCC patients had significantly improved disease-specific survival when compared with patients with HPV-negative tumors, even after adjustment for age, lymph node status, and heavy alcohol consumption (Table 4). Because of retrospective data collection, we were unable to adjust for possible confounding factors, such as nutrition and performance status or the presence of comorbid illness. Prior survival analyses led to contradictory conclusions but were limited by small sample size, short follow-up, and/or lack of disease-specific survival analysis (17,52,7678).
The improved disease-specific survival in patients with HPV-positive HNSCC is somewhat surprising and remains unexplained. Because HPV-positive tumors may be less associated with alcohol and tobacco exposure and HPV infections tend to be focal, field cancerization (in which the upper respiratory epithelium is repeatedly exposed to carcinogens) (10) may be less applicable. Patients with these tumors may be less susceptible to the development of synchronous or metasynchronous tumors in the lungs, esophagus, and elsewhere in the head and neck that could adversely affect long-term survival. Because the majority of HNSCC recurrences occur within approximately 18 months of diagnosis, this hypothesis is supported by the separation of disease-specific survival curves at 18 months (Fig. 3) and the rarity of cancer deaths beyond 18 months in the HPV-positive group. Because of the retrospective nature of the study, we were unable to evaluate the effect of HPV presence on response to primary therapy, including commonly employed radiation therapy. Although there is no evidence that the presence of HPV alters the radiosensitivity of tumors (7981), radiation therapy has been shown to increase both major histocompatibility complex class I and E6 and E7 expression, and an increased immune surveillance could contribute to improved survival (82).
The predominance of oncogenic, high-risk viral types (HPV16, HPV18, HPV31, and HPV33) in HNSCC (16,17,19, 52,78,83,84) previously identified as the major HPV types in cervical carcinomas worldwide (85,86) argues for a potentially analogous role for these viruses in the development of malignancy in the upper airway. However, these results need to be interpreted within the context of the study's limitations. As a retrospective case series at a tertiary referral center, it is unclear how these results extend to other populations with different environmental exposures and genetic backgrounds. However, similar HPV prevalence estimates have been reported in a population-based study (22) and in case series in the United States (17) and in Europe (50).
Discrete, tumor-specific HPV16 E6 variants were identified in 52 of 56 HPV16-positive tumors after repeated amplification and sequencing, arguing against artifacts, such as laboratory contamination or PCR-induced mutations. Infection by particular HPV16 variants may increase both the risk of progression to high-grade cervical (87) and anal (88) intraepithelial neoplasia and invasive cervical cancer (89). The striking similarity between the frequency distribution of HPV16 variants in HNSCC and that previously reported in cervical cancers in North America (39) is consistent with a predisposition of certain HPV16 variants to increase the risk of invasive cancer (87,90). Alternatively, this similarity may merely reflect the overall frequency distribution of HPV infections in the populations studied. Future evaluation of HPV variants in the upper airway of noncancer subjects, estimated to have a prevalence in adults of 5%11% (4), may help to distinguish between these possibilities.
The means by which HPV is transmitted to the upper airway is unclear. Although oral HPV infection is rare in newborn children of infected mothers (91) and in children prior to sexual activity (92), infections increase after onset of sexual activity (93). Epidemiologic studies [reviewed in (94)] of cervical cancer have clearly demonstrated that high-risk mucosatropic HPVs are transmitted by sexual contact. Although HPV presence in head and neck cancers has not yet been convincingly linked to specific sexual practices such as oral sex (21,22,95), HPV positivity has been linked to the number of sexual partners in three casecontrol studies (21,22,95). The presence of HPV in preinvasive and invasive disease as well as lymph node metastases suggests that viral presence precedes disease progression. However, a prospective, seroepidemiologic study of HPV exposure and subsequent development of head and neck cancer is needed to determine that exposure precedes disease (96).
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We thank C. Wheeler for her comments on the HPV16 variant analysis, A. Alberg for his review and comments on the manuscript, T.-C. Wu for his advice concerning the in situ hybridization, and J. Rocca for her assistance with medical record retrieval.
Presented in part at the 1999 International Human Papillomavirus Conference, Charleston (SC), and at the Thirty-fifth Annual Meeting of the American Society of Clinical Oncology, Atlanta (GA).
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Manuscript received September 22, 1999; revised February 22, 2000; accepted February 24, 2000.
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