long regarded as a uniform group of tumors that differed only as a function of anatomic site , ongoing studies indicate that hnsccs may not be as homogenous as previously supposed . recognition of distinct molecular genetic profiles now permits finer resolution of hnscc into distinct subtypes that differ with respect to risk factors , pathogenesis , and clinical behavior . hpv - associated oropharyngeal carcinoma has recently been recognized as a unique subtype of hnscc . hpv status has a profound effect on patient prognosis , and it may soon guide therapy . accordingly , hpv status will become a standard component in the diagnostic reporting of all oropharyngeal carcinomas . this review covers recent advances toward defining hpv - related oropharyngeal carcinoma , and draws specific attention to pathology concerns that impact on the diagnosis and reporting of these cancers . hnscc is the eighth most common cancer worldwide with approximately 650,000 new cases reported annually . beginning in the early 1980s , the incidence of hnscc in the united states and elsewhere has been in retreat . accordingly , hnscc is often perceived as a preventable disease , and its ultimate demise simply a matter of implementing behavior modification strategies . this great optimism has recently been shaken by a sobering escalation of a specific type of cancer that is asserting its dominance on the head and neck oncology landscape . this anomalous rise bucks the prevailing notion that hnscc incident rates can be controlled through smoking prevention and cessation . indeed , the escalation of oropharyngeal carcinoma points to the participation of non - traditional behavioral and environmental factors driving this disturbing epidemiologic trend . the prototypic patient with oral cancer is an older man who has smoked cigarettes and drank alcohol for many years . this prototype , however , no longer fits the patient with oral cancer who now enters the doors of our clinics and hospitals . patients now tend to be younger ( between 40 and 60  years ) white men who have never smoked cigarettes or drank alcohol . traditional risk factors have been supplanted by other powerful risk factors relating to sexual practices , the most important of these being high number of sexual partners , history of oral - genital sex , and history of oral - anal sex [ 5 , 6 ] . oral anogenital contact is an important route for the transmission of hpv to the oral cavity . certain conditions and behaviors that alter antitumor immunity may be important in transforming an hpv oral infection towards hpv - related malignancy . as one example , marijuana use has recently been identified as an independent risk factor for hpv - positive hnscc , and the strength of this association increases with intensity , duration , and cumulative years of marijuana smoking . long scrutinized as a potential source of dna - damaging carcinogens , marijuana smoke may be more relevant for its immunomodulatory effects . binding , in turn , can suppress immune responses , diminish host responses to viral pathogens , and attenuate antitumor activity . these hpv - positive cancers are increasingly recognized as a distinct subgroup of hnscc with a biological and clinical profile that diverges from that of their hpv - negative counterparts ( table  1 ) . at the molecular genetic level , hpv - positive hnsccs express the viral oncoproteins e6 and e7 , overexpress the p16 gene product , and only infrequently harbor p53 gene mutations . regarding clinical behavior , the mechanisms underlying this favorable prognosis may involve the combined effects of immune surveillance to viral - specific tumor antigens , an intact apoptotic response to radiation , and the absence of widespread genetic alterations associated with smoking ( i.e. , field cancerization).table  1comparison of hpv - positive and hpv - negative head and neck cancershpv - positivehpv - negativeincidenceincreasingdecreasingageyoungeroldergender3:1 men3:1 menrisk factorssexual behaviortobacco , alcoholcofactorsmarijuana , immunosuppressiondiet , oral hygienemolecular genetics findingsp16 p16 rb rb p53 wild - typep53 mutatedanatomic sitelingual and palatine tonsilsall sitespathologic findings  primarybasaloidkeratinized  lymph node metastasiscysticsolidsurvivalbetterworse comparison of hpv - positive and hpv - negative head and neck cancers the pathologic features of hpv - positive hnscs also deviate from the moderately differentiated keratinizing morphology that typifies most hnsccs . hpv - positive hnsccs consistently : ( 1 ) arise from the tonsillar crypts ; ( 2 ) are unassociated with dysplasia of the surface epithelium ; ( 3 ) exhibit lobular growth ; ( 4 ) are permeated by infiltrating lymphocytes ; ( 5 ) lack significant keratinization ; and ( 6 ) demonstrate a prominent  basaloid  morphology ( fig .  1 ) . two microscopic features of hpv - related oropharyngeal cancers are likely to cause diagnostic ambiguity . first , hpv - related hnscc is customarily misperceived as a poorly differentiated carcinoma based on the immature appearance of the tumor cells . in point of fact , the appearance of the tumor cells closely emulates the appearance of the reticulated epithelium  the specialized epithelium lining the tonsillar crypts from which hpv - related cancers arise . in other words , hpv - related oropharyngeal cancers are in fact highly differentiated , not poorly differentiated as so widely assumed . second , use of the term  basaloid  as a diagnostic descriptor is confusing for the way it invites an erroneous connection with basaloid squamous cell carcinoma  a subtype of hnscc notorious for its aggressive clinical behavior . within the basaloid subtype , detection of hpv is a highly favorable prognostic factor that helps identify a subset of cancers that departs from the highly aggressive behavior associated with this variant . until consensus panels put forward a classification scheme for oropharyngeal carcinomas that underscores their relationship with hpv while avoiding confusion with the aggressive basaloid variant , it is our practice to : ( 1 ) classify these tumors as non - keratinizing squamous cell carcinomas , ( 2 ) suspend the use of the descriptors such as  poorly differentiated  and  basaloid  , and ( 3 ) routinely report on the hpv status of all hnsccs arising in the oropharynx.fig .  1typical histopathologic appearance of an hpv - related cancer of the head and neck . a the carcinomas tend to arise from the tonsillar crypts and infiltrate the lymphoid stroma as expanding tumor lobules . b the lobules of tumor cells are permeated by lymphocytes , lack keratinization , and have a basaloid appearance typical histopathologic appearance of an hpv - related cancer of the head and neck . a the carcinomas tend to arise from the tonsillar crypts and infiltrate the lymphoid stroma as expanding tumor lobules . b the lobules of tumor cells are permeated by lymphocytes , lack keratinization , and have a basaloid appearance determination of hpv status will certainly become standard practice in the pathologic evaluation of oropharyngeal cancers . as a biomarker , hpv detection is emerging as a valid method of discerning the presence and progress of disease encompassing all aspects of patient care from early cancer detection , to tumor localization   ( fig . 2 ) , to selection of patients most likely to benefit from specific therapies , to post - treatment tumor surveillance . a variety of detection methods are in current use including pcr - based strategies , type - specific in situ hybridization ( ish ) techniques , and immunohistochemical detection of surrogate biomarkers ( e.g. p16 protein ) . standardization of hpv detection in the clinical arena must begin with selection of the best detection platform for universal application.fig . the presence of hpv as evident by p16 immunohistochemical staining ( b ) and hpv-16 in situ hybridization ( hybridization dots within tumor cell nuclei , inset ) point to the oropharynx as the site of tumor origin metastatic squamous cell carcinoma of unknown primary origin . the presence of hpv as evident by p16 immunohistochemical staining ( b ) and hpv-16 in situ hybridization ( hybridization dots within tumor cell nuclei , inset ) point to the oropharynx as the site of tumor origin the preferential use of ish methods over pcr - based methods is supported both by biological and practical considerations . the reported large variation of hpv prevalence in hnscc is a largely a reflection of the inability of non - quantitative pcr methods to discern virus that is biologically meaningful from virus that is biologically irrelevant . in contrast , punctuate hybridization signals within the nuclei of tumor cells is a pattern of staining only seen following hpv dna integration into the host genome , and thus is more closely linked with relevant viral infections . importantly , the improved specificity of hpv detection by ish does not come at the expense of sensitivity . the introduction of various signal amplification steps has significantly improved the sensitivity of this technique , even to the point of viral detection down to one viral copy per cell . the development of nonfluorescent chromogens now allows visualization of dna hybridization using conventional light microscope . adaptation of ish to formalin - fixed and paraffin - embedded tissues has made this technique compatible with standard tissue - processing procedures and amendable to retrospective analysis of archival tissue blocks while most pcr - based methods are optimized to fresh frozen samples . ish is a feasible and cost - effective test for most diagnostic laboratories that routinely process formalin - fixed and paraffin - embedded tissue blocks . in hpv - positive oropharyngeal carcinomas , functional inactivation of rb by the viral oncoprotein e7 is known to induce an up regulation of p16 expression , reaching levels that can be readily detected by routine immunohistochemistry . accordingly , p16 immunohistochemistry is often advocated as a reliable surrogate marker of hpv - induced neoplasia of oropharynx . direct comparison of p16 immunohistochemical staining and hpv-16 ish for large numbers of hnsccs reveals a discrepancy rate of about 25% . in a subset of discrepant cases , high p16 expression is due to the presence of some other ( non-16 ) hpv type as confirmed by wide spectrum ish . the remaining discrepancies likely reflect the imperfection of p16 as a surrogate marker . using e6/e7 mrna levels as conclusive evidence of hpv involvement . the limitations of any single detection assay may be offset using algorithms that combine the strengths of complementary assays . given a sensitivity that approaches 100% , p16 immunostaining is a good first line assay for eliminating hpv - negative cases from additional analysis . ish can be run concurrently with p16 immunostaining or as a second - line assay following a positive p16 result . given a specificity approaching 100% , a positive hpv-16 ish reduces the numbers of false positive cases by p16 staining alone . a p16-positive / hpv-16-negative result singles out a subset of tumors that qualify for rigorous analysis for other ( i.e. non-16 ) oncogenic hpv types . for this third - line assay , we use a consensus ish probe that that detects an extended panel of hpv types . p16 immunohistochemistry and hpv ish are standardized techniques that are easily applied to formalin - fixed and paraffin - embedded tissues . as automated ish technologies are brought on - line , turnaround time will be further shortened and standardization across various diagnostic laboratories will be enhanced . the escalating incidence of oropharyngeal carcinoma in the absence of a parallel rise in smoking and alcohol consumption suggests that nontraditional behavioral and environmental factors are driving this aberration . hpv , particularly type 16 , has been established as a causative agent in up to 70% of oropharyngeal cancers . these hpv - positive hnsccs differ in important respects from hpv - negative hnscc including risk factors , molecular genetic alterations , microscopic appearance , and clinical behavior . diagnostic pathologists are now faced with the challenge of accurately discerning hpv status of oropharyngeal cancers .