cervical infection by one of approximately 15 high - risk hpv types is generally accepted as the necessary causative agent of cervical cancer [ 1 , 2 ] .
the upper aerodigestive tract , including the oral cavity , the pharynx , and the larynx , is also lined by a squamous mucous membrane and because of the morphological similarities and epitheliotropic nature of hpv , a link between head and neck squamous cell carcinoma ( hnscc ) and hpv seems logical .
hnscc is usually etiologically linked to tobacco and/or alcohol or other lifestyle habits , but a minority of patients develop hnscc in the absence of exposure to these factors or any other obvious predisposing genetic defect .
several investigators have reported detection of hpv infection in healthy oral mucosa as well as in squamous cell carcinoma of the oral cavity ( oscc ) and oropharynx ( opscc ) [ 4 , 5 ] , which are related to sexual behaviour and younger age .
hpv has shown to have a predilection for certain , especially nonkeratinized , anatomical sites of the oropharynx .
high - risk hpv types , such as hpv 16 , have been detected even in clinically normal oropharyngeal mucosa . in the cervix , the persistence of hpv infection may increase the likelihood of viral integration and concomitant deregulation of viral protein expression , leading to overexpression of e6/e7 oncoproteins [ 7 , 8 ] .
in oral cancers the integration of hpv dna into the host 's cell genome is probably a less common event .
various hpv types have been identified in patients with oral cancer , but clinical and case control studies have not proved a causal relationship between the virus and oral carcinomas beyond any reasonable doubt [ 8 , 9 , 11 ] .
hpv dna detection alone is regarded as insufficient evidence for a causal role in oral cells transformation and is more like a secondary invader [ 7 , 10 ] .
hpv integration into the host cell genome however , is a causal factor in head and neck carcinogenesis [ 1113 ] . in many reports ,
in which the hpv - positivity varies from 40 to 57% , there is no reference to the viral integration of the hpv positive samples [ 5 , 8 , 10 ] .
the purpose of our study is to define hpv detection in patients with osccs and to evaluate the detection of e6/e7 high - risk hpv mrna as a possible biomarker for posttreatment surveillance .
sixty - four ( 64 ) cytological samples were obtained from patients with hnscc of the oral cavity , ( 35 oral tongue , 8 maxilla , 4 mandible , 8 floor of mouth , 6 retromolar trigone , and 3 buccal mucosa ) who visited the outpatient clinic of the radiotherapy department of the regional anticancer oncology hospital  saint savvas  , athens , greece , between november 2008 and november 2011 .
ethical approval was granted by the ethics committee of regional anticancer oncology hospital of athens  st .
the samples were collected using a brush and swabbing the mouth , maxilla and mandible , oral tongue , and buccal mucosa and were preserved in thin prep ( preservcyt solution , hologic uk ) .
cytological samples were transferred in lysis buffer ( nuclisens lysis buffer , biomrieux hellas s.a , cat no .
200292 ) for 30 minutes , then total nucleic acid was extracted by the offboard protocol with the nuclisens easymag platform ( biomrieux hellas s.a ) , according to the manufacturer 's instructions .
dna quality test was carried out using human globin , beta , primer set kit ( maxim biotech , inc . , south san francisco , ca , usa ) according to manufacturer 's instructions . to assess rna integrity , 5  g of rna per sample
then , the papillocheck hpv - screening ( greiner , germany ) was used for the type - specific identification of 24 types of hpv ( 15 high - risk types : 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , 68 , 73 , and 82 , probable high - risk types : 53 , 66 and 7 low - risk types : 6 , 11 , 40 , 42 , 43 , 44/55 , and 70 ) .
this technology is based on a dna chip for the type - specific identification of 24 types of hpv .
e1-based pcr was performed according to the manufacturer 's guidelines . for each sample , we mixed 19.8  l papillocheck mastermix , 0.2  l hotstartaq plus dna polymerase ( 5u/l , qiagen , cat no .
hybridization is followed by mixing 30  l of the papillocheck hybridization buffer in a new reaction tube with 5  l of the pcr product at room temperature and transferring 25  l of the hybridization mix into each compartment of the chip .
the chip was washed in 3 washing solutions , centrifuged for 3 minutes at 5000  rpm , and scanned on the checkscannertm . a commercial real - time nuclisens easyq assay ( nuclisens easyq hpv 1.1 , biomrieux hellas s.a , cat no .
290003 ) was performed for the qualitative detection of hpv e6/e7 mrna of five high - risk hpv types ( 16 , 18 , 31 , 33 , and 45 ) according to the manufacturer 's instructions .
firstly , three premixes were made by adding reagent sphere diluent ( tris - hcl , 45% dmso ) into reagent spheres ( nucleotides , dithiotreitol , and mgcl2 ) . in each premix
, we added u1a / hpv 16 , hpv 33/45 , or hpv 18/31 primer and molecular beacon mixes , kcl stock solution , and nasba water .
secondly , 10  l of this premix was distributed to each well in a reaction plate , and the addition of 5  l rna followed .
the plates were incubated for 4 minutes at 65c to destabilize secondary structures of rna , followed by cooling down to 41c .
the reaction was started by addition of enzymes ( amv - rt , rnase h , t7 rna polymerase , and bovine serum albumin ) and measured in real time using the lambda fl 600 fluorescence reader ( bio - tek , winooski , vt ) at 41c for 2 hours and 30 minutes . following diagnosis and molecular analysis , patients with positive histology of hnscc of the oral cavity , with a karnofsky index ( ki )  70% , and with normal renal , hepatic and bone marrow function were included .
baseline laboratories studies requirements included neutrophils greater than 1.5  10/l and platelets > 100  10/l .
exclusion criteria were distant metastatic disease , a life expectancy of < 3 months , pregnancy , previous cancer disease within 5 years of study entry , existence of second primary tumor , and karnofsky index ( ki ) of < 70% .
furthermore , patients with severe renal , liver , cardiovascular , or pulmonary diseases and patients with hematological malignancies were excluded .
all participants underwent an extensive presurgical evaluation that included clinical examination , panendoscopy , complete blood count and biochemical profile , clearance creatinine , cardiovascular examination , chest x - ray or ct , and ct and/or mri of head and neck .  
patients with early stage resectable disease ( stage i - ii ) and selected patients of stage iii ( t2n1m0 ) were treated surgically .
patients with locally advanced disease ( unresectable or marginally resectable ) , stage iii - iva , were treated either with induction chemotherapy or radical chemoradiation .
depending on response after induction chemotherapy , patients underwent either surgery followed by postoperative chemoradiation or radical combined radiation therapy with chemotherapy . before induction chemotherapy
the palpable edges of the primary lesion ( both the longest and the shortest axes ) were marked by at least four points , which were 0.5  cm away .  
tumor response was evaluated after the end of induction chemotherapy using a clinical examination and ct or mri of the head and neck with tumor volumetry .
complete response ( cr ) was defined as a reduction of the tumor volume  70% up to the complete disappearance .
partial response ( pr ) was defined as at least a 30% decrease in the sum of diameters of tumor , taking as reference the baseline sum diameters .
progressive disease ( pd ) was defined as enlargement of tumor volume  20% or new tumor manifestations . stable disease ( sd ) was defined as insufficient shrinkage to qualify for pr and insufficient increase to qualify for pd .
the median follow - up interval was 18 months ( range 4 to 36 months ) .
patients are monitored every two months in the first two years , every three to four months in the next 3 to 5 years , and once a year thereafter until death .
disease - free survival was estimated from the end of treatment to tumor recurrence , distant metastasis , or death from any cause .
chi - squared tests were performed to assess statistical significance of any differences in prevalence .
2  2 contingency tables fisher 's exact test was performed along with odds ratio and 95% confidence intervals calculation .
we collected 64 samples from the oral cavity , 19 of which were unsatisfactory for molecular analysis due to insufficient quantity of dna , thus 45 samples were finally analysed ( table 1 ) .
the patients ' ages ranged from 19 to 82 years ( mean age : 51.9 years ) .
clinical , pathological , and treatment characteristics of the patients are described in table 1 .
papillocheck assay detected high risk hpv dna in 3/45 of the osccs ( 2 sccs of the oral tongue and 1 of the floor of the mouth ) .
the different hpv types revealed by the dna and rna test are shown in table 2 . in the 3 high - risk hpv dna positive oral cavity cancers , hpv 16
was detected in 2 samples ( 1 case of scc of the oral tongue and 1 of the floor of the mouth ) and hpv 18 in 1 sample ( 1 case of oral tongue scc ) .
in addition , the low - risk hpv 6 was found in 2 oral tongue samples .
further analysis using the nuclisens easyq assay detected e6/e7 mrna expression from the five high - risk hpv types ( 16 , 18 , 31 , 33 , and 45 ) in 4/45 of the total samples ( 8.9% ) ( 3 cases of oral tongue scc and 1 of the floor of the mouth ) .
hpv 16 was the commonest type revealed by the rna test as shown in table 2 .
as far as the site - specific oscc is concerned , hpv 16 was detected in 3 out of 5 hpv dna positive oral cavity sccs ( 2 cases of oral tongue scc and 1 of floor of the mouth ) , while hpv 18 was found in 1 out of 5 hpv - infected oral cavity sccs ( 1 case of oral tongue scc ) , by the rna detection method .
8 of 45 patients ( 1 stage i ( t1n0m0 ) , 5 stage ii ( t2n0m0 ) , 2 stage iii ( t2n1m0 ) ) underwent surgical excision ( 7 oral tongue , 1 maxilla ) .
4 of them had no adverse features , and they did not undergo any further treatment . the rest of the 4 patients with adverse features ( > 2 positive ln , resection margins < 5  mm , perineural tumor invasion , and vascular space invasion ) received external beam radiation therapy ( ebrt ) 5460  gy . all patients who were treated surgically are so far disease - free ( 7 hpv dna negative and 1 hpv dna positive ( defined as absence of recurrence at the primary site , assessed by physical examination at every clinic visit and imaging studies at specified intervals ) .
however , one of them developed a second primary tumor of the lung and so far is disease - free from both primaries ( table 2 ) . from the 37 patients with locally advanced ( unresectable or marginally resectable ) disease ( stage iii - iva ) ,
28 patients underwent induction chemotherapy with 2 cycles of tpf ( docetaxel 75  mg / m , ( d1 ) cisplatin 75  mg / m ( d1 ) , and 5-fu 500  mg / m ( d1-d5 ) ( referred as treatment protocol a ) and 9 patients underwent radical external beam radiation therapy ( ebrt : 6872  gy ) with concurrent chemotherapy ( cisplatin ) ( d1d22 ) and weekly cetuximab due to medical contraindications for induction chemotherapy with tpf ( referred as treatment protocol b ) .   from 28 patients treated with protocol a , 18 had complete response .
10 of them underwent radical surgery and 8 of them underwent radical radiotherapy with concurrent chemotherapy .
depending on histological findings , patients underwent ebrt 5460  gy with or without concurrent chemotherapy ( cisplatin 75  mg / m d1 , d22 ) and weekly cetuximab .
for the patients with high - risk features , such as r1 resection , extra capsular nodal spread , and vascular space invasion , perineural invasion total dose of 66 gy was granted . from 18 patients with complete response , 14 patients
are today free of disease ( 3 hpv dna positive ( hpv 18 , 16 , 6 ) , 11 hpv dna negative ) , 2 died from lung metastasis ( 1 hpv dna positive ( hpv 16 ) , 1 hpv dna negative ) , and 2 are still alive with local recurrence ( 2 hpv dna negative ) ( table 2 ) .
the rest of the 10 patients who were treated with protocol a had partial response ( table 1 ) .
all of them underwent radical ebrt ( 6872  gy ) with concurrent chemotherapy ( cisplatin ) ( d1d22 ) and weekly cetuximab .
1 of them is free of disease , 6 died from metastatic disease , 1 died from other causes , and 2 of them are still alive with local recurrence . from 9 patients with stage iii - iva who were treated with protocol b ,
5 of them are free of disease , 1 died from metastatic disease , and 3 are still alive with local recurrence .
of the approximately 120  hpv types so far known to infect the mucosal surfaces of the genital tract , 14 are considered to be  high risk  or  oncogenic 
[ 1 , 2 ] . some of these high - risk types have been found in the oral cavity and oropharynx of both cancer - free adult individuals and in patients suffering from hnscc [ 4 , 7 , 8 , 12 ] .
hpv 16 , the hpv type most prevalent in cervical sccs , is also the most common type present in hpv - positive hnsccs [ 10 , 1315 ]
. the great variation in hpv prevalence found in osccs in different studies may be due to differences among the analyzed population , but also due to differences in the samples tested ( i.e. , formalin - fixed or fresh biopsies , exfoliated fresh cells ) , the methods of dna extraction , and , most importantly , the hpv detection methods used [ 8 , 16 ] . several studies using a variety of techniques , including immunohistochemistry , in situ hybridization , dot blot and southern blot hybridization , and polymerase chain reaction ( pcr ) , have been used to demonstrate the presence of hpv genome in hnscc [ 4 , 5 , 7 , 15 ] .
our study shows that the use of dna and rna detection methods confirms the prevalence of hpv infection among patients with osccs .
hpv 16 infection has been found in 2 cases . in both cases infected by hpv 16 ,
what is interesting is that the rna test revealed hpv 16  e6/e7  mrna .
this may be explained by the fact that total viral dna of hpv 16 has been integrated into the host genome , and , therefore , it can not be detected by the dna test . in 75% of the hpv - positive sccs of the oral tongue ,
hpv implication in oral tongue cancer has already been stated by many authors [ 5 , 17 ] .  
on the other hand , clinical results indicated that the association between patient mortality and hpv detection was not statistically significant ( p = 0.4215 ) .
the prevalence of hpv among currently disease - free patients was also not statically significant ( p = 1.000 ) .
as already stated , e6/e7 mrna expression from high - risk hpv types is probably a less common event in hnscc patients [ 13 , 14 ] .
nevertheless different investigators ' results suggest that hpv - positive carcinomas represent a different tumor entity [ 14 , 18 ] .
high - risk hpv types demonstrate an integration tendency , as can be assumed in our study by the mrna oncoproteins ' expression .
we should also mention , as many investigators indicate , that low - risk hpv types ( hpv 6 in our case ) have been found in some head and neck carcinomas and might be implicated in the carcinogenesis process [ 14 , 18 , 19 ] .
although authors support better prognosis for hpv - positive oral cancer cases [ 3 , 14 , 16 , 18 ] , hpv - positive tumors are usually diagnosed in higher stage than hpv - negative tumors .
therefore there is a small subgroup of hpv oral cancers that demonstrate a worse response to treatment and have a lower rate of survival .
sexual transmission of oncogenic hpv genotypes is universally accepted for the anogenital region , whereas the route of infection for the oropharyngeal region is unclear [ 2 , 18 ] .
although several studies have recently identified hpv , particularly type 16 , in a subset of squamous cell carcinoma of the tonsils and base of the tongue [ 9 , 14 , 16 , 21 ] , the possibility of sexual or other transmission of hpv in oral cancer patients also needs to be explored [ 5 , 6 , 18 ]
. the immune response might also affect the survival , but it seems to depend on whether hpv is present in the tumor cells in an episomal form or as an integrated virus [ 14 , 18 ] .
although our data showed a relatively low hpv and oral cancer association , people from different geographical regions often demonstrate higher or lower hpv prevalence in oscc , which could also be the reason for our results . despite the heterogeneity between different studies , hpv appears to play an important role in some types of oropharyngeal carcinomas and possibly a small subgroup of cancers in the oral cavity   and may represent an alternative pathway in carcinogenesis to the known and established factors of tobacco and alcohol [ 9 , 18 ]
the ability to control head and neck cancer will therefore depend on three basic cornerstones : prevention , detection , and early diagnosis .
the recent development of an hpv vaccine might offer hope for the prevention of cervical and anogenital carcinomas and possibly also represent an additional prevention option for a substantial number of patients with hnscc .
this study described the detection rates and attribution of hpv genotypes as well as the e6/e7 mrna expression of site - specific osccs in 45 greek patients .
dna and rna assays detected the same genotypes in all high - risk hpv infected samples .
this study confirms the prevalence of hpv infection among patients with osccs . future analysis and followup of more osccs
 the relation between hpv and head and neck cancer has recently and extensively been investigated . 
 the purpose of this study was to indentify hpv genotypes , as well as e6/e7 mrna expression of high - risk hpvs ( 16 , 18 , 31 , 33 and 45 ) in oral squamous cell carcinomas ( osccs ) from 45 greek patients . 
 the overall prevalence of hpv dna positive osccs was 11.1% ( 5/45 ) , while high - risk hpv dna was found in 6.7% ( 3/45 ) of osccs . 
 e6/e7 mrna expression was detected in 8.9% ( 4/45 ) of the oral cavity samples . 
 our data indicated that hpv 16 was the commonest genotype identified in hpv - positive osccs by both dna and rna tests . 
 this study confirms the prevalence of hpv infections among patients with osccs . 
 future analysis and followup of more osccs will enable us to correlate hpv detection and clinical outcome . 