Advances in Anatomic Pathology:
Human Papillomavirus Detection in Head and Neck Squamous Cell Carcinomas
Snow, Anthony N. MD; Laudadio, Jennifer MD
Department of Pathology, Wake Forest University Baptist Medical Center, Winston-Salem, NC
Financial Support to Disclose: None.
Reprints: Jennifer Laudadio, MD, Department of Pathology, Wake Forest University Baptist Medical Center, Medical Center Boulevard, Winston-Salem, NC 27157 (e-mail: firstname.lastname@example.org).
Squamous cell carcinomas of the head and neck (HNSCC) are a frequent diagnosis in anatomic pathology practice. Tobacco use and heavy alcohol consumption are known risk factors for HNSCC but in other cases human papillomavirus (HPV) is linked to carcinogenesis. HPV proteins E6 and E7 promote oncogenesis by blocking the action of p53 and pRB, respectively. An absence of p53 mutations in addition to expression of p16 are part of the distinct molecular profile identified in the subset of HNSCCs because of HPV. Various methods are available for HPV detection but polymerase chain reaction and in situ hybridization techniques are commonly used. Both methods are amenable for testing formalin-fixed paraffin-embedded tissue that is a sample type readily available to the pathologist. HPV is detectable in approximately a quarter of all HNSCCs, and is particularly prevalent in the oropharynx in which the positivity rates approach 40%. A vast majority of HPV-related HNSCCs are owing to HPV type 16 with types 18, 31, and 33 accounting for almost all of the remaining cases. HPV-related HNSCCs are associated with better prognosis for both recurrence and survival. This group of tumors has also been shown to respond well to radiation treatment. As the clinical relevance of HPV in HNSCCs continues to emerge, anatomic pathologists are likely to receive increasing requests for testing. Herein, the authors review the biological and clinical aspects of HPV-associated HNSCC and review techniques for HPV detection.
Worldwide, more than 640,000 cases of head and neck cancer are diagnosed every year and account for more than 350,000 deaths.1 Annually, approximately 50,000 cases are diagnosed in the United States alone and cause approximately 12,000 deaths.2 Tobacco and alcohol use are established risk factors for squamous cell carcinomas of the head and neck (HNSCCs).3–5 Human papillomavirus (HPV) has also been identified as an etiologic agent in a subset of HNSCCs. HPV infection may even act synergistically with carcinogenic agents such as tobacco and alcohol. Even though smoking rates are steady or even slightly decreasing, the incidence of oropharyngeal squamous cell carcinomas (SCC) is increasing in the United States, specifically in males and young adults.6,7 HPV positivity rates in oropharyngeal SCCs are also increasing.6
Many different technologies are available for HPV detection in HNSCCs. Both in situ hybridization (ISH) and amplification by polymerase chain reaction (PCR) are commonly used and are applicable for formalin-fixed paraffin-embedded (FFPE) tissue. The clinical use of testing HNSCCs for HPV is growing as HPV status has prognostic significance and predicts response to radiation treatment. However, there continues to be lack of uniformity in testing methodology and uncertainty remains regarding the use for HPV typing.
HPV GENOME AND ROLE IN ONCOGENESIS
More than 100 types of human papillomavirus, a member of the papillomaviridae family, have been identified.8 Different HPV types share between 71% and 89% of their genetic sequence but vary with regards to their tissue tropism and association with carcinogenesis.8 Alpha papillomaviruses are implicated in mucosal and genital infections, whereas β papillomaviruses cause cutaneous infections. HPV types are assigned as high risk or low risk for carcinogenesis, based upon epidemiologic data of HPV association with cervical carcinoma.
The HPV genome consists of circular double-stranded DNA, approximately 7.9 kilobases in size. The genome consists of a noncoding long-control region, 6 early genes (E1, E2, E4-E7), and 2 late genes that encode the viral capsid (L1 and L2).9 Two early genes, E1 and E2, control gene transcription, and replication.10 Other early genes, E6 and E7, have pivotal roles in oncogenesis. After infection of a squamous epithelial cell, the HPV genome may remain episomal or become integrated into the host genome. When the HPV genome integrates into the host cell DNA, E2 is disrupted allowing for overexpression of E6 and E7 oncogenes and immortalization of the infected cell. E6 protein disrupts normal apoptosis by binding and inactivating the tumor suppressor p53 and promoting its degradation.11,12 E6 also activates telomerase allowing the regenesis of the ends of chromosomes after cell division.13 The ability to regenerate telomeres contributes to cellular immortality. E7 oncoprotein functions by binding and degrading pRB.14,15 Blocking the function of pRB allows for unchecked progression through the cell cycle and unlimited tumor cell proliferation.
The action of HPV E6 and E7 is necessary for malignant transformation. Blockage of these 2 oncoproteins through the use of short hairpin RNAs results in loss of cell viability and apoptosis.16 Furthermore, disrupting E6 and E7 in oropharyngeal cell lines results in increased levels of p53 and pRB and increased levels of p53-activated genes.16 These findings indicate that in HPV-induced carcinomas the p53 and pRB pathways remain intact. Active transcription of HPV with generation of E6 and E7 oncoproteins is necessary for tumor cell proliferation, suppression of apoptosis, and ultimately carcinogenesis.
MOLECULAR PROFILE OF HPV-POSITIVE HNSCCs
HPV-positive HNSCCs are genetically and molecularly distinct from HPV-negative tumors providing further evidence for a casual role of HPV in oncogenesis. The different genetic profile also suggests that the HPV infection is an early event in oncogenesis. Frequent molecular abnormalities in HNSCCs include TP53 (encoding p53) mutations, deletion or silencing of CDKN2A (encoding p16), and loss of heterozygosity (LOH) at 3p and 17p.17–19 In contrast, carcinomas in which HPV is transcriptionally active are less likely to harbor LOH at 3p, 9p, and 17p.20 Loss of 16q is associated with HPV-positive HNSCCs, but losses of 9p21 and 3p are rare as is amplification of 11q13.21
At the molecular level, HPV-associated tumors are less likely to harbor TP53 mutations than HPV-negative cases.22,23 In particular, HPV transcriptional activity as measured by E6 or E7 mRNA positivity is associated with an absence of TP53 mutations.20,24,25 The absence of p53 mutation in this subset of SCCs is consistent with the degradation of p53 by E6 and consistent with the finding that apoptosis is possible when the actions of E6 and E7 are blocked. HPV type 16-positive carcinomas also differ from other HNSCCs in that they have higher levels of nuclear β-catenin contributing to cellular proliferation.26 As compared with HPV-negative SCCs, cases positive for HPV have strong expression of p16, a component of the retinoblastoma tumor suppressor pathway. Expression of p16 can be detected by immunohistochemistry (IHC) and is strongly and diffusely expressed in HPV-associated tumors but absent in HPV-negative carcinomas.21,23,27–30 One study found 82% of HPV type 16-positive carcinomas to be p16 positive by IHC as compared with 7% of HPV-negative cases.28 Cases that are HPV positive but negative for p16 expression are molecularly more similar to HPV-negative cases suggesting that in these instances HPV is not directly involved in carcinogenesis.31 Expression of E6 and/or E7 mRNA is specifically associated with p16 expression.21,25 As carcinomas with detectable HPV DNA but no detectable mRNA are less likely to express p16 and cases that do not express p16 are molecularly similar to HPV-negative tumors, IHC for p16 may be a reasonable surrogate for detecting transcriptionally active HPV infection. On the basis of these molecular findings, an immunohistochemical profile of p53 negativity, p16 diffuse positivity, and β-catenin nuclear positivity is consistent with HPV-associated carcinoma.
Detection methods relating to HPV infection of HNSCCs vary widely in scope and type. The method choice depends greatly upon the desired information, available tissue type, and resources. Current methods target protein, DNA, and mRNA. Specimen types evaluated may be frozen, fresh, or FFPE tissue. Cytologic preparations and saliva have also been examined.
The southern blot has long been considered the gold standard for detection of specific DNA sequence and has been used to validate a majority of the currently used HPV detection techniques. However, with its technical demand, necessity for large quantities of DNA, and lack of significant advantage over other techniques, its use in clinical applications for HPV detection is rare.
The use of p16 IHC as a surrogate for HPV is intriguing. This method has been shown to have 100% sensitivity in screening for transcriptionally active infection.32 However, HPV independent pathways of oncogenesis can lead to increased expression of p16 and the specificity is only 79%.32 When targeting HPV-type 16 DNA, 1 study found 93% correlation between p16 expression and HPV status.33 Some of the discordant results could be explained by the presence of HPV other than type 16. Begum et al34 found similar results when comparing p16 IHC with HPV type 16 detection by ISH. A total of 92% of HPV 16-positive tumors showed p16 expression versus 6% of HPV-negative tumors.
Although evidence for the use of p16 is growing, clinical and research studies apply amplification techniques most commonly.35 The inherent plasticity of PCR lends itself to the creation of many iterations of the technique aimed at multiple pieces of clinical information. Manipulation of sequence specificity combined with chemical and thermal stringency in single or multiplex reactions achieve this level of diversity. Downstream detection of the PCR products adds yet another layer of variation.
The most straight forward amplification assay is a PCR reaction directed at a single HPV type using a specific primer set.36,37 The amplification product can then be detected by simple gel electrophoresis using intercalation with ethidium bromide for visualization under ultraviolet light (Fig. 1A). Using type-specific PCR is a reasonable and cost-effective approach for testing HNSCCs, especially oropharyngeal carcinomas, as HPV type 16 accounts for the vast majority of HPV found. Type-specific PCR can also be used as a confirmatory step after wide-spectrum HPV detection.
Several amplification techniques have been developed for wide-spectrum HPV detection. If desired, type discrimination can then be pursued by subsequent manipulation of the PCR product. The use of several specific primer pairs combined in a multiplex reaction is one option for wide-spectrum HPV detection. Alternatively, consensus primers can be used. The earliest developed consensus primer sets (MY09/11, GP5/6, and their derivatives) target a highly conserved portion of the L1 gene.38,39 The general primer (GP) 5/6 set was originally designed to target HPV 6, 11, 16, 18, 31, 33 but was later found to amplify at least 27 types.40,41 In an attempt to limit nonspecific amplification products, a modified consensus primer set dubbed GP5+/6+ was later developed.41 Another consensus primer set, MY09/11, can amplify at least 30 HPV types including 6, 11, 16, 18, 31, and 33.42 The GP5+/6+ and MY09/11 primer sets amplify segments of 150 and 450 base pairs, respectively.43,44 Even smaller fragments of target DNA can be amplified using short-fragment PCR. This technique has the potential to be especially useful for FFPE tissue, which generally yields fragmented nucleic acid. One such primer set known as SPF10 has been successfully used for identification of broad spectrum HPV in laryngeal tissue.44 Additional gains in sensitivity have been sought by using nested PCR. This method involves an initial amplification step followed by a second amplification of the initial PCR products. For specificity, the second amplification is carried out using primers that sit internal to the first primer pair. One example of this method uses an initial set of primers directed at a 441 base pair sequence within the L1 gene followed by a nested primer set to amplify a secondary 335 base pair product.45 In comparison with this protocol, use of a single step amplification detected the presence of HPV in only 29% of the nested PCR-positive samples.45 However, most clinical data have been generated from methods without this level of sensitivity, and the high sensitivity of nested PCR may lead to false positives.
After broad spectrum HPV amplification, several techniques are available for target detection and type discrimination. If multiplex primer sets directed at high-risk HPV types prevalent in HNSCC are used, type discrimination may not be necessary. If consensus primers are used and include low-risk HPV types such as 6 and 11, typing will be necessary to determine the clinical relevance of infection. Restriction fragment length polymorphism (RFLP) is a common high throughput type discrimination method that can be applied to consensus HPV amplification products. As of the genetic sequence variation between HPV types, unique restriction patterns will be detected on gel electrophoresis after enzymatic digestion. One of the most commonly used enzyme combinations is PstI, HaeIII, and RsaI with or without DdeI (Fig. 1B).46 If the RFLP method is chosen for clinical testing, interpretation can be challenging given the numerous possible restriction patterns. Other detection techniques for HPV type identification include the dot blot, reverse line blot, and DNA enzyme immunoassay. These procedures all involve hybridization of amplified target DNA to HPV type-specific probes. Either the probe or the target is labeled and can be detected using enzymatic means. Dot blotting is rarely used but reverse line blot and DNA enzyme immunoassays are applied more commonly. The dot blot and reverse line blot techniques show nearly identical type discrimination when compared, but reverse line blot requires less DNA and less hands-on time.39
All of the amplification and type discrimination techniques described above have been developed and validated for both anogenital and head and neck applications. They adequately and equivalently amplify the target of interest, in this case L1. However, concern has arisen regarding the clinical utility of targeting L1. Multiple portions of the HPV genome, including L1, may be deleted in the process of integration into the host genome contributing to false negative results.47 The number of false negatives secondary to genome integration when using PCR alone is estimated at 7% and can be overcome by targeting E7 for amplification.48 For this reason, assays have been developed, which amplify portions of E6 and E7. Both genes contain highly conserved regions (preintegration and postintegration) and are good functional targets because of their role in tumorigenesis.47,49–52 Both the L1 and E6 or E7 targeted primer sets are acceptable strategies and are prevalent within current literature.
The use of real-time PCR or real-time RT-PCR offer advantages as compared with the end-point PCR methods already described. Real-time PCR requires less hands-on time and eliminates the need for post-PCR manipulation, thereby reducing the risk of contamination. However, it does require a capital equipment investment in a real-time PCR instrument. Real-time PCR and RT-PCR techniques have been used to further elucidate the clinical significance of HPV infections in HNSCCs. These platforms allow assays to be designed for either wide spectrum or type-specific HPV detection. Real-time PCR provides a method of determining viral load in a clinical sample. Higher viral loads may be more clinically relevant then low levels of detectable virus. Real-time RT-PCR also generates quantitative information and by targeting mRNA adds evidence for active gene transcription. HNSCCs that are HPV DNA positive but negative for E6 or E7 mRNA expression have been shown to be molecularly similar to HPV DNA-negative cases.32 Furthermore, expression of E6 and E7 mRNA is associated with higher viral loads as compared with mRNA-negative cases.21
Many studies have shown reproducible results with RNA-based assays when using frozen tissue as a substrate but this material is not always available for testing. Although RNA assays based on FFPE tissue offer more of a challenge, it provides the opportunity for retrospective testing and is often the only sample type available to the pathologist. Multiple studies have compared RNA extraction from fresh or frozen tissue with that from FFPE tissue. The greatest decrease in RNA quality occurs immediately after fixation and processing. Comparative data between real-time RT-PCR of RNA extracted from fresh tissue and from tissue immediately after formalin fixation and paraffin embedding shows a 10-fold greater expression in the fresh specimens.53 Other variables such as prefixation time (up to 12 h) and storage time of FFPE blocks (up to 15 y) did not show a comparable decrease in the ability to amplify RNA as the fixation process itself.53 Denaturing gel electrophoresis of extracted RNA from FFPE tissue shows an increase in fragmentation of RNA strands relative to that which was extracted from fresh tissue.53 A logical supposition is that a decrease in amplicon target length would increase the success rate of RNA amplification from FFPE samples. This concept was confirmed when the amplicon size was decreased from 291 base pairs to 99 base pairs and compared between fresh and immediately extracted FFPE tissue.53 The FFPE tissue showed 90 times greater expression levels with the smaller amplicon. This study found that the optimal amplicon length is approximately 135 base pairs, and that targets above 175 base pairs show a significant increase in the false negative results.53 As RNA degradation is potentially nonrandom, the exploration for preserved targets within E6 or E7 should be further investigated. Coupled with a variety of tested extraction methods, this information may lead to improved techniques for RNA analysis from FFPE tissues.54
In Situ Hybridization
In situ hybridization using chromogen or fluorophore labeled probes is a reproducible technique applicable to detection of a wide range of HPV types particularly from FFPE tissues. Interpretation of chromogenic ISH is relatively straight forward for an anatomic pathologist and does not require the use of a fluorescent microscope. Using ISH allows the localization of detected HPV DNA in the nucleus of carcinoma cells, and dot-like positivity indicates viral integration into the cell genome (Fig. 2A). Type discrimination, in most cases, requires multiple slides with specific probes. Probe pools targeting low-risk and high-risk types provide a useful screening technique that can limit subsequent reactions. Signal enhancement techniques such as tyramide signal amplification have increased the sensitivity of ISH reactions 10-fold to 100-fold. This technique requires the use of a biotinylated probe to which an avidin-conjugated horseradish peroxidase is added. The biotin-conjugated tyramide reagent is activated by the horseradish peroxidase at which point it covalently links to protein substrate in the region. This step leaves an increased amount of biotin substrate localized to the region of probe binding which supplies binding sites for another round of avidin-conjugated horseradish peroxidase. At this point, additional amplification steps can be carried out, or the addition of a chromogen or fluorophore can be initiated.55–57
The hybrid capture technique (Qiagen Valencia, CA) is used extensively among pathology labs to detect 13 high-risk HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) in cervical cytology specimens. Currently, the use of this method is limited for HNSCC HPV testing, but the technique has potential for screening oral brushings. In this assay, sample DNA is chemically denatured into single-stranded DNA and hybridized to specific RNA probes. This solution is added to a microwell plate coated with anti-DNA: RNA hybrid antibody. A second anti-DNA: RNA hybrid antibody conjugated to multiple equivalents of alkaline phosphatase is applied to provide signal amplification and a platform for a chemiluminescent substrate. Signal is reported as relative light units after normalization with a control signal. Two studies have used the hybrid capture technique to screen cytologic brushings from grossly normal oropharyngeal tissue for high-risk HPV. In 1 study of 50 healthy volunteers, zero individuals proved to be positive for high-risk HPV.58 In another study, brushings from 4 of 70 women with a history of cervical carcinoma tested positive for high-risk HPV using the hybrid capture method.59 At this time, oral brush cytology has not achieved a sensitivity or specificity sufficiently competitive with surgical biopsy for diagnosis and prospective studies are necessary to determine the clinical use of such screening. With advancement in technique and potential increase in the utilization of oral brush cytology, the use of hybrid capture for HNSCC testing may increase.60,61
Detection Method Comparison
There are nearly innumerable options for HPV detection in HNSCCs and no standardization of procedures. PCR has the advantage of sensitivity with the ability to detect less than 1 viral genome copy per cell. The use of ISH and IHC offer advantages in terms of cost, access, and potentially turnaround time. Several studies have compared assays in an attempt to optimize clinically relevant HPV detection. Smeets et al32 used FFPE tumor tissue (oropharynx and oral cavity) and serum from 48 patients and calculated the sensitivity and specificity for various detection techniques. The techniques for comparison included p16 protein expression by IHC, PCR using GP5+/6+consensus primers and enzyme immunoassay for genotyping, real-time RT-PCR targeting HPV type 16 E6 mRNA, real-time PCR targeting HPV type 16 E7, and detection of serum antibodies against HPV type 16 L1, E6, and E7 proteins. This study focused on identification of transcriptionally active species and used real-time RT-PCR carried out on frozen tissue targeting E6 and E7 as the standard for positivity. Using this approach, a tumor positive for HPV DNA but negative for RNA is considered a false positive. The sensitivities and specificities of each method are shown in Table 1. On the basis of their findings, the authors propose a testing algorithm of first screening for HPV using p16 IHC, which is highly sensitive but lacks specificity. After positive p16 results confirmatory testing with consensus GP5+/6+ PCR is carried out. This approach has 100% sensitivity and specificity and would reduce the number of cases needing PCR testing.32 Singhi and Westra33 noted a similar sensitivity with p16 IHC (100%), but advocate the use of ISH to confirm a positive IHC result. In this algorithm, chromogenic ISH specifically for HPV type 16 follows a positive p16 IHC test. If the type-specific ISH is negative, chromogenic ISH using consensus probes for other HPV types is carried out.33 Both algorithms exploit the high sensitivity of p16 IHC and bolster it with a second more specific confirmatory method. Both are practical approaches with supportive data and provide a step toward defining a clinically relevant standardized testing protocol.
HPV PREVALENCE IN HNSCCs
In determining which HNSCC cases to test for HPV, the association between HPV positivity and clinical characteristics is not clear-cut. The use of demographic data to predict HPV status performs only moderately.62 The strongest correlation is between HPV positivity and a history of nonsmoking.3,6,21,27,31,62,63 A correlation between nondrinking and HPV-positive status has also been found3,22,31,62,63 but some studies have failed to show this association.20,21,64 Patients with HPV-positive HNSCCs tend to be younger in age.6,21,62,65,66 There is also data indicating that HPV-positive HNSCCs are associated with a higher number of sexual partners.3,67,68 Other factors shown to be associated with HPV-positive oropharyngeal carcinoma include positive serology for HPV type 16 L1 capsid protein and HPV oral infection.3 No consistent link between HPV status and sex has been identified.
Aside from clinical characteristics, certain pathologic features are associated with HPV status. HPV-positive tumors are more likely to be poorly differentiated and have basaloid morphology (Fig. 2B).22,27 However, it may be that only a subgroup of HPV-positive tumors, those with evidence of transcriptional activity, display this morphology. Expression of E6/E7 mRNA or p16 has specifically been shown to be associated with poorly differentiated basaloid squamous cell carcinomas.21,24,69 Oropharyngeal SCCs that are HPV positive and express p16 are more likely to be higher stage and poorly differentiated than p16-negative tumors.31
Reported rates of HPV positivity in HNSCCs widely vary. This inconsistency can be attributed to differences in methodology of HPV detection and differences in sample types, populations, and anatomic sites tested. Furthermore, some studies have tested for HPV type 16 only whereas others have included wide-spectrum HPV testing. HPV positivity rates range from 3% to 40% in HNSCCs (Table 2).20–22,24,25,27,28,33,64,70 In a study by Singhi and Westra,33 69% of HNSCCs were HPV positive. This prevalence is higher than those reported in other studies and is likely owing to over-representation of oropharyngeal tumors. In 1 meta-analysis including PCR and ISH detection methods, HPV prevalence was 34.5% in HNSCCs.35 This prevalence rate is fairly similar to the 26% reported in another meta-analysis that included only studies using PCR detection.38 It is interesting to note that, the type of sample tested (FFPE versus fresh-frozen tissue) did not impact the detected positivity rate.38
The prevalence of HPV is higher in the oropharynx than any other head and neck site.22,27,28,33,62 Studies of oropharyngeal tumors alone have revealed anywhere from 14% to 72% of these carcinomas to be HPV positive (Table 3).3,6,30,31,62,63,65,66 In Termine's meta-analysis 38% of oropharyngeal carcinomas were HPV positive.35 Kreimer's meta-analysis found an almost identical HPV prevalence, 35.6%, in oropharyngeal squamous cell carcinomas.38 This prevalence is higher than the 23.5% and 24% positivity rates in oral cavity and laryngeal squamous cell carcinomas, respectively.38 In studies that compared anatomic sites, 57% to 82% of oropharyngeal versus 0.8% to 9% of nonoropharyngeal squamous cell carcinomas were HPV positive.22,28,33,62
On the basis of molecular signatures that suggest transcriptionally active HPVs are a distinct entity, it may be more relevant to assess the presence of E6/E7 mRNA or p16 expression to determine the number of HNSCCs due to HPV. It is interesting to note that, only 40% to 50% of HPV-positive HNSCC cases have detectable E6/E7 mRNA.20,21,24,25 Furthermore, tumors that express E6 and/or E7 mRNA or are positive for p16 expression are more likely to be from the oropharynx than any other site.21,24,25,33
HPV type 16 is by far the most common type detected in HNSCCs. Type 16 accounts for 78.6% to 100% of HPV-positive oropharyngeal cases.6,20,21,27,63,66 In meta-analysis, 87% of oropharyngeal HPV-positive cases were determined to be type 16.38 Although a majority of oropharyngeal squamous cell carcinomas are related to HPV16, the expansion of HPV type testing can more accurately determine the prevalence. On the basis of analysis of Kreimer's review data, types 16, 18, 31, and 33 account for 99% of HPV-positive HNSCCs.38 Other head and neck sites are more likely than oropharyngeal tumors to be related to types other than HPV 16. About 32% of oral cavity and 31% of laryngeal HPV carcinomas are owing to HPV types other than 16.38 HPV type 18 accounts for 1% of oropharyngeal, 8% of oral cavity, and 4% of laryngeal HPV-positive SCCs.38 Coinfections are possible and most frequently include HPV16.38 HPV33 is often reported and has been found in up to 10% of HPV-positive HNSCCs.21 Numerous other HPV types have been rarely detected in HNSCCs and include types 6, 11, 35, 45, 51, 52, 56, 58, 59, and 68.3,22,27,38,63 It is notable that HPV types 6 and 11, considered low-risk types, have been reported. In these cases, HPV may not be the etiologic agent driving carcinogenesis but rather a transient infection.
HPV DNA can be detected through all stages of carcinogenesis, from dysplasia to invasive carcinoma.28 HPV DNA can also be detected in premalignant lesions such as leukoplakia and lichen planus.71,72 In a review by McKaig et al,70 20% of benign and premalignant head and neck lesions were HPV positive with a higher positivity rate (35.9%) detected by Southern Blot than by ISH (18.5%). Using ISH, HPV can also be seen in normal mucosa adjacent to carcinoma.21 Furthermore, HPV DNA can be detected in specimens from patients without a history of HNSCC. A total of 3.1% of tonsillar biopsies and 12.2% of oral rinse samples tested positive for HPV DNA.73 HPV type 16 was most frequently detected and coinfections were found.73 Prospective studies are necessary to clarify the clinical significance of such findings.
HPV IN HNSCC METASTASES
The identical HPV type as found in primary tumors can also be detected in metastatic foci from HNSCCs.21 In those primary tumors with detectable E6 mRNA and lymph node metastases, the metastatic carcinoma is also mRNA positive.24 A total of 22.5% to 27% of metastatic HNSCC lesions are HPV positive consistent with positivity rates in primary head and neck carcinomas.69,74 Many patients with HNSCC first come to medical attention because of a neck mass. Fine needle aspiration (FNA) is a commonly used technique in such instances to establish a diagnosis. If squamous cell carcinoma is diagnosed, patients then undergo extensive imaging and possibly nonselective biopsies to identify the primary tumor location. If a primary site remains unidentified, the patient may be subjected to wide field radiation as opposed to localized treatment. Unfortunately, no histologic feature can reliably indicate the primary site but HPV testing may be useful as the detection of HPV suggests an oropharyngeal primary.
In cervical node metastases, specifically from unknown primaries 37.5% and 38% have been found to be HPV positive.33,64 Using HPV 16 ISH, 53% of metastatic lesions from oropharyngeal tumors were positive but no metastases from other head and neck sites had detectable HPV.34 As HPV positivity is most often seen in oropharyngeal tumors, detection of HPV in a metastatic focus can help localize the primary tumor and allow for site-specific biopsies. In 1 study, 78% of HPV-positive cervical node metastases were found to be of oropharyngeal origin.74
Cystic lymph node metastases may be encountered in patients with HNSCC. This type of metastatic lesion is especially common in patients with oropharyngeal SCC. A total of 41% of patients with primary oropharyngeal carcinoma have been found to have cystic lymph node metastases,75 and 85% of cystic lymph node metastases are of oropharyngeal origin and in the remaining 15%, the primary tumor site remains unidentified.75 FNA of such metastases may result in false negative diagnoses because of the hypocellularity, presence of inflammation, and associated debris. In such instances, HPV testing on FNA material can be useful in distinguishing benign cystic lesions and cystic squamous cell metastases. HPV detection can also help differentiate a recurrent or metastatic HNSCC from a second non-HPV-related primary tumor.
HPV STATUS IMPACTS PROGNOSIS
Key factors impacting prognosis in HNSCC cases are tumor size and nodal status, but the importance of HPV status in prognosis is emerging. HPV-positive tumors tend to be of higher stage, often owing to positive lymph node status.65 However, even though higher stage, no overall survival difference is observed between HPV-positive and negative cases.64 HPV positivity is an independent prognostic factor for improved survival in HNSCC patients.22,27,29,66 Prospective analysis confirms HPV DNA positivity as a prognosticator for improved overall and progression-free survival.27 In addition to being a favorable prognostic factor in all HNSCCs, HPV-positive status is associated with improved survival and local control in specifically oropharyngeal SCCs.6,63,65 Use of quantitative PCR may have prognostic relevance as patients with higher HPV type 16 viral loads have better overall and recurrence-free survival as compared with patients negative for HPV or with low-viral loads (less than 50 viral copies per tumor cell).76 Mellin et al77 also observed improved disease-specific survival for patients with higher viral loads (greater than 190 E6 viral copies per control gene).
Some studies have shown that improved prognosis is only true for carcinomas with transcriptionally active HPV infections. In oropharyngeal tumors, detectable E6/E7 mRNA is associated with significantly better prognosis.21 In contrast, SCCs positive for HPV but negative for E6/E7 mRNA have similar prognosis to HPV-negative tumors. The expression of p16 has also been associated with better overall survival, local recurrence-free survival, and disease-free survival.31 Five year overall survival was 79% for HPV-positive p16-positive tumors versus 20% for HPV-negative and 18% for HPV-positive p16-negative cases.31 Results were similar for 5-year disease-free survival rates; 75% for HPV-positive p16 expressing tumors versus 15% for HPV-negative and 13% for HPV-positive and p16-negative cases.31 In patients treated with radiotherapy, p16 expression as a surrogate of HPV positivity was associated with improved locoregional tumor control, disease-free survival, and overall survival.78 The benefit was greatest in patients with poorly differentiated, oropharyngeal, or higher-stage tumors.78 Complete clinical response to initial therapy was achieved in all patients with HPV-positive p16 expressing SCCs.31
Independent of primary tumor site, HPV-positive HNSCCs have a higher response rate to treatment with either chemotherapy or chemoradiation.27 In patients treated with radiation therapy, HPV positivity is associated with improved relapse-free survival and improved disease-specific survival independent of tumor stage.66 Multivariate analysis shows that HPV positivity is prognostic for lower risk of recurrence and improved overall survival in patients with oropharyngeal SCCs treated with chemoradiation.65 Improved prognosis and response to therapy in HPV-related tumors may be attributable to the absence of TP53 mutations. In the absence of mutation, the apoptotic pathway can respond to radiation treatment. An alternate theory is that patients with HPV-positive tumors lack field cancerization attributable to tobacco use and alcohol consumption. In HNSCCs negative for HPV, the adjacent epithelium may harbor early oncogenic mutations limiting the response to therapy.
HPV-associated carcinomas are a distinct subset of HNSCCs driven by the actions of E6 and E7 oncoproteins. These tumors have a molecularly distinct profile as compared with HPV-negative SCCs. Although correlations are not perfect, patients with a social history negative for tobacco use and alcohol consumption are more likely to have HPV-associated tumors. Meta-analysis data shows that HPV is detectable in 26% to 35% of HNSCCs.35,38 The prevalence of HPV-associated SCCs is higher in the oropharynx than any other anatomic site. The reason why HPV prevalence is higher in oropharyngeal SCCs remains unclear but may be owing to the distinct reticulated crypt epithelium in tonsillar tissue. Most HPV-positive HNSCCs are owing to HPV type 16 and the inclusion of 3 additional types (18, 31, and 33) will account for almost all HPV-associated HNSCCs. In patients with HPV-positive primary HNSCCs, HPV can also be detected in metastatic foci.
Many questions remain regarding the best testing strategies for detecting HPV in HNSCCs, and there is currently no single recommended method. FFPE has been shown in multiple studies to be an acceptable sample type for testing. Both PCR and ISH-based methods are well-studied validated techniques. IHC for p16 expression may also be used as a surrogate for HPV positivity. The decision of which method to use will be laboratory dependent and determined on the basis of cost, expertise, necessary equipment, sensitivity, and specificity. Testing for HPV type 16 only is an option but expansion to include other high-risk types will help avoid false negatives, especially in tumors from sites other than the oropharynx. Importantly, studies generating data showing HPV as a favorable prognostic factor were not limited to only testing for HPV type 16.
Data are conflicting whether the detection of transcriptionally active HPV is important. Although some studies have indicated that the favorable prognosis of HPV positivity is applicable only to cases with detectable mRNA or p16, the 1 prospective study available used ISH and multiplex PCR to detect HPV DNA. This study confirms HPV DNA positivity has prognostic significance and one may conclude that RNA detection is unnecessary. Two testing algorithms have been proposed and need to be further validated. Both strategies include screening for HPV with p16 IHC followed by more specific confirmatory testing using either PCR or ISH.
HPV-associated HNSCCs have been shown to have a better prognosis both in terms of recurrence and survival. This group of carcinomas also responds favorably to radiation treatment. Many centers are routinely testing HNSCCs for HPV given the prognostic implications. However, treatment strategies do not currently differ on the basis of HPV status. In the future, determination of HPV status may be used to guide treatment decisions and posttreatment surveillance. Detection of oral HPV in patients without HNSCC may also take on a screening role for early detection. Clinical trials will provide further guidance.
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human papillomavirus; head and neck squamous cell carcinoma; oropharyngeal carcinoma; HPV testing
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