head and neck squamous cell carcinoma ( hnscc ) is the sixth most common cancer worldwide , with an average 5-year survival rate of around 50% ( 1 ) . despite improvements in the therapeutic modalities ,
the prognosis of these patients has not been significantly improved in the last three decades .
alcohol and both smoked and smokeless tobacco use are associated with increased risk of developing malignancy of the oral cavity and pharynx ( 2 ) .
recently , the role of oncogenic viruses especially hpv in the pathogenesis of scc has attracted the researchers attention .
hpv viral proteins , e6 and e7 , play major roles in the cervical as well as orophararyngeal carcinogenesis .
this proteins act through inactivation of tumor suppressor genes , p53 and retinoblastoma ( rb ) genes , resulting in cell immortalization and transformation ( 4 )
. perhaps one of the most important developments in the head and neck oncology over the past decade is the recognition of hpv status in oropharyngeal sccs ( opsccs ) .
it is obvious that hpv - ralated opscc has unique epidemiological and clinical entity and it had better distinguishes from the ocsccs because of unknown role of hpv in non - oropharyngeal head and neck cancers ( 5 - 7 ) .
( 9 ) , and lingen et al . in 2013 ( 10 ) considered low etiologic role for hpv status in ocsccs .
mentioned that probably other risk factors that are still unknown play roles in the pathogenesis of ocsccs . in recent years , much attention has been paid to the prognostic role of hpv status in hnscc .
some researchers have found that patients with hpv detected in their cancers , have better prognosis compared to those without hpv ( 11 - 15 ) .
nevertheless there are studies that have found no difference or poorer prognosis for hpv related tumors ( 16 - 22 ) .
in this study , we have investigated the prevalence rate of hpv status in patients with ocscc , and its significance in the clinicopathological characteristics of tumors and patients prognosis .
132 patients with diagnosis of ocscc were included in this study between 2001 and 2013 .
their records were collected from the files of department of pathology , faculty of dentistry and omid hospital , mashhad university of medical sciences , iran .
age , gender , anatomical region , tnm stage , histological grade , therapeutic interventions , events including local , distant relapse and death were extracted from the medical records database .
the haematoxylin and eosin stained sections from all specimens were reviewed by two pathologists to confirm the histology and grading .
histological grading was assessed according to the who guidelines and tumor staging was reported according to the american joint committee on cancer staging criteria ( ajcc sixth edition , 2002 ) .
cleaning off the microtome blade with xylene and ethanol after cutting each paraffin block was taken for preventing and monitoring contamination of samples .
the tumor samples were deparaffinized using a series of xylene and ethanol washes . in this study ,
the tissue sample was mixed with 400 l digestion buffer which consisted of tris - cl ( 100 mm , ph 7.5 ) and tween-20 ( 0.055 ) and proteinase k 3l ( 20 mg / ml ; frementase , germany ) , followed by incubation at 56c for 3 hour .
after the complete digestion of tissue , the mixture was incubated at 100c in hotblock ( accublock digital dry baths ; usa ) to inactivate proteinase k. the samples were then centrifuged at 5000 rpm , and the supernatant containing dna was used for pcr amplification ( 96-well thermal cycler ; applied biosystem , usa ) . the quality of dna was determined by amplification of housekeeping gene ( -globin ) using the gh20/pc04 primers ( cinnagen , iran ) .
a consensus sequence from the l1 region of the hpv genome was amplified using gp5+/gp6 + primers ( cinnagen , iran ) ( 2 ) .
gp5:5-ttt gtt act gtg gta gat act ac-3 and gp6:5-gaa aaa taa act gta aat cat att c-3 were used as the primer set .
hpv typing ( hpv 6 , 11 , 16 , 18 and 31 ) was also performed using hpv type specific primers ( cinnagen , iran ) ( 19 ) .
pcr products were separated in 2% agarose gels and visualized by green veiwer staining ( parstous , iran ) .
all analyses were performed using data processing program spss / pc version 16.0 for windows ( spss inc . , chicago , il , usa ) .
chi square , mann -whitney , student s t test and fisher s exact tests were used to assess the statistical significant differences between hpv status and clinicopathological parameters .
the os and dfs rates were estimated by the kaplan - meier method , and differences in survival rates were analyzed with the log - rank test .
5 to 10 m sections were cut from each paraffin block . cleaning off the microtome blade with xylene and ethanol after cutting each paraffin block
the tumor samples were deparaffinized using a series of xylene and ethanol washes . in this study ,
the tissue sample was mixed with 400 l digestion buffer which consisted of tris - cl ( 100 mm , ph 7.5 ) and tween-20 ( 0.055 ) and proteinase k 3l ( 20 mg / ml ; frementase , germany ) , followed by incubation at 56c for 3 hour . after the complete digestion of tissue ,
the mixture was incubated at 100c in hotblock ( accublock digital dry baths ; usa ) to inactivate proteinase k. the samples were then centrifuged at 5000 rpm , and the supernatant containing dna was used for pcr amplification ( 96-well thermal cycler ; applied biosystem , usa ) . the quality of dna was determined by amplification of housekeeping gene ( -globin ) using the gh20/pc04 primers ( cinnagen , iran ) .
a consensus sequence from the l1 region of the hpv genome was amplified using gp5+/gp6 + primers ( cinnagen , iran ) ( 2 ) .
gp5:5-ttt gtt act gtg gta gat act ac-3 and gp6:5-gaa aaa taa act gta aat cat att c-3 were used as the primer set .
hpv typing ( hpv 6 , 11 , 16 , 18 and 31 ) was also performed using hpv type specific primers ( cinnagen , iran ) ( 19 ) .
pcr products were separated in 2% agarose gels and visualized by green veiwer staining ( parstous , iran ) .
all analyses were performed using data processing program spss / pc version 16.0 for windows ( spss inc . , chicago , il , usa ) .
chi square , mann -whitney , student s t test and fisher s exact tests were used to assess the statistical significant differences between hpv status and clinicopathological parameters .
the os and dfs rates were estimated by the kaplan - meier method , and differences in survival rates were analyzed with the log - rank test .
moreover , eight patients were excluded because their clinicopathological databases were not complete . remaining 114 ocscc cases comprised of 58 men and 56 women .
the mean age of patients was 58.6 years ( range 19 - 85 years).the follow - up period ranged from 6 months to 8 years .
the hpv sequences were detected in 15 patients ( 13.16% ) : 5 cases with hpv11 ( 33.3% ) , one case with hpv6 ( 6.7% ) and two cases with both hpv6 and 11 ( 13.3% ) . in 7 patients ( 46.7% )
patients characteristics and tumor features of the hpv positive and negative ocscc cases are compared in table 1 .
no significant differences were observed between the two groups in the variables of age , gender , tumor site , size and histological grading .
fifty five hpv - negative patients ( 55.6% ) were n0 , while five hpv - positive patients ( 33.3% ) were n0 .
in addition , while twenty two hpv - negatives ( 22.2% ) were n2 , eight hpv - positive patients ( 53.3% ) were n2 .
the median lymph node involvement was significantly higher in hpv positive group compared to hpv negative group ( p = 0.045 ) . as a result , significantly more stage iva was seen among the hpv - positive tumors compared to the hpv - negative ones ( 66.7% vs. 35.4% , p = 0.04 ) .
most of the hpv positive and negative cases were treated with surgery and adjuvant radiation therapy ( table 2 ) .
thirty four out of 99 hpv - negative patients ( 34.3% ) died from their disease compared with 11 out of 15 in the hpv - positive cases ( 73.3% ) .
chi square test revealed a significant relation between the rate of death and hpv positive status ( p = 0.004 ) .
survival and hpv dna status : the median dfs was 13.9 months among the hpv - positive and 49.9 months among the hpv - negative patients with a significant difference ( p = 0.029 ) .
also the median os of 15.7 months in the hpv - positive group compared to 49.6 months in the hpv - negative group was significantly better ( p = 0.017 ) .
the kaplan - meier survival curves for the hpv - positive and negative cases are shown in figure 1 .
we used multivariate analysis involving cox proportional hazards model to determine which factors were jointly predictive of os and dfs .
the hpv status ( hr = 2.10 , ci = 1.02 to 4.28 , p = 0.041 ) and stage ( hr = 1.92 , ci = 1.36 to 2.70 , p <
in addition , both hpv status ( hr = 2.80 , ci = 1.35 to 5.79 , p = 0.005 ) and stage of disease ( hr = 1.95 , ci = 1.37 to 2.79 , p <
moreover , eight patients were excluded because their clinicopathological databases were not complete . remaining 114 ocscc cases comprised of 58 men and 56 women .
the mean age of patients was 58.6 years ( range 19 - 85 years).the follow - up period ranged from 6 months to 8 years .
the hpv sequences were detected in 15 patients ( 13.16% ) : 5 cases with hpv11 ( 33.3% ) , one case with hpv6 ( 6.7% ) and two cases with both hpv6 and 11 ( 13.3% ) . in 7 patients ( 46.7% )
patients characteristics and tumor features of the hpv positive and negative ocscc cases are compared in table 1 .
no significant differences were observed between the two groups in the variables of age , gender , tumor site , size and histological grading .
fifty five hpv - negative patients ( 55.6% ) were n0 , while five hpv - positive patients ( 33.3% ) were n0 .
in addition , while twenty two hpv - negatives ( 22.2% ) were n2 , eight hpv - positive patients ( 53.3% ) were n2 .
the median lymph node involvement was significantly higher in hpv positive group compared to hpv negative group ( p = 0.045 ) . as a result , significantly more stage iva was seen among the hpv - positive tumors compared to the hpv - negative ones ( 66.7% vs. 35.4% , p = 0.04 ) .
most of the hpv positive and negative cases were treated with surgery and adjuvant radiation therapy ( table 2 ) .
thirty four out of 99 hpv - negative patients ( 34.3% ) died from their disease compared with 11 out of 15 in the hpv - positive cases ( 73.3% ) .
chi square test revealed a significant relation between the rate of death and hpv positive status ( p = 0.004 ) .
survival and hpv dna status : the median dfs was 13.9 months among the hpv - positive and 49.9 months among the hpv - negative patients with a significant difference ( p = 0.029 ) .
also the median os of 15.7 months in the hpv - positive group compared to 49.6 months in the hpv - negative group was significantly better ( p = 0.017 ) .
the kaplan - meier survival curves for the hpv - positive and negative cases are shown in figure 1 .
we used multivariate analysis involving cox proportional hazards model to determine which factors were jointly predictive of os and dfs .
the hpv status ( hr = 2.10 , ci = 1.02 to 4.28 , p = 0.041 ) and stage ( hr = 1.92 , ci = 1.36 to 2.70 , p <
in addition , both hpv status ( hr = 2.80 , ci = 1.35 to 5.79 , p = 0.005 ) and stage of disease ( hr = 1.95 , ci = 1.37 to 2.79 , p <
the participation of hpv in the oral and oropharyngeal carcinogenesis was first proposed in 1983 by syrjanen et al . ( 23 ) and then supported by several other studies . according to many studies
, the hpv prevalence rate in oral cancer is varied between 0% - 100% ( 6 , 19 , 24 - 26 ) .
this widespread variability can be in part due to differences in methodology of hpv detection ways and differences in sample types , populations , and anatomic sites tested ( 27 , 28 ) .
moreover , various studies have reported conflicting results for the probable role of hpv on the patient 's prognosis ( 15 , 16 , 29 ) . in the current study ,
regarding the sample size , the majority of small to medium sized studies ( < 100 patients ) have revealed a wide variation in hpv prevalence rates ( varying between 0% and 100% ) ; however , larger studies generally tend to show lower hpv prevalence rates ( varying between 1.4% and 48.8% ) .
( 27 ) have mentioned the sample size as an important factor in the reportedly heterogeneous prevalence rate of hpv , which is in accordance with our findings .
the hpv positivity rate of 13.6% in our study is almost similar to those other reports of oral cavity cancers which are ranged from 12% to 70% for opscc ( 2 , 15 , 22 , 30 ) versus 3% to 15% for ocscc ( 2 , 20 , 31 ) . while there is another report that mentions a 25% incidence rate of hpv among tongue cancers in iran and yet all of them subtypes of 18 and 16 ( 32 ) , in the current study , 15 cases ( 13.16% )
were identified as hpv positive and the detected viral subtypes were subtypes 6 and 11 .
however , it is not still clear whether these subtypes result in an infection driven carcinogenesis or they are just a transient infection ( 5 , 33 ) .
pannone et al . mentioned that hpv 6 , which was known as  low risk  or  non - oncogenic  subtype to the cervix , was present in a larger number of head and neck cancers .
hpv 6 and 11 have been found in some tonsillar and laryngeal carcinomas and in the malignant transformation of benign laryngeal papillomas , hpv 11 has been most commonly implicated . hpv 6 and
11 have also been on suspicion of malignancies such as verrucous carcinoma of the oral cavity ( 34 , 35 ) .
hpv 6 and 11 were also observed in other studies on cancers of the head and neck ( 36 , 37 ) .
studies on opscc often suggested that the incidence of hpv in this region is more prevalent ( 5 , 33 , 38 ) .
it is also mentioned that hpv - mediated opsccs are more likely late stage , poorly differentiated , and with large lymph nodes in the neck , and in spite of that they have less risk of recurrence and death ( 2 , 5 , 24 , 33 , 39 - 41 ) . some studies indicate that such hpv - positive tumors are more radio - sensitive and have better overall survival rate ( 33 , 42 , 43 ) .
these patients tend to be younger and often male with less consumption of alcohol and smoking ( 33 , 41 , 44 ) .
there is also data indicating that patients with hpv - positive opscc are associated with a higher number of sexual partners ( 33 , 34 ) . in sccs
limited to the oral cavity , usually the hpv prevalence rate is lower and hpv16 is also the most common type detected ( 2 , 28 ) .
some studies have suggested that hpv does not play a significant role in the etiology of ocscc ( 8 - 10 ) and also does not play a role in the progression toward malignancy , even though some studies have demonstrated the role of hpv16 and 18 in oral carcinogenesis ( 45 - 47 ) .
notably , because of relatively low frequency of hpv in ocsccs , only few studies have attamped to correlate hpv status with clinical outcome ( 5 , 24 , 46 , 47 ) . in most of the studies of hnsccs , the demonstrated clinicopatological features about hpv positive cases are usually related to the oropharyngeal samples ; therefore , little is known about the influence of hpv on clinical course of patients with the oral cavity cancer because of the lower hpv prevalence .
no data currently supports the idea that hpv is significantly associated with improved outcome for patients with oral cancer .
only few published studies on patients with oral cavity carcinoma specifically examined the impact of hpv on outcome .
kaminagakura studied 114 patients and found a nonsignficant trend towards improved survival for 22 hpv - positive patients ( 47 ) .
sugiyama et al . demonstrated a nonsignficant trend towards improved overall survival for hpvpositive oral cavity cancer patients ( 48 ) .
smith found no association with hpv and outcome for patients with oral carcinoma , based on either serology or tumor hpv detection ( 49 ) .
also in isayeva s cohort study of 89 patients with oral cavity carcinoma , no significant association was found for patients with either hpv16/18 e6e7 rna and time to disease progression or disease specific survival ( 5).therefore , future studies on oral cavity scc should be powered to address the important clinical issue of hpv status . in the present study , the mean age of the cases was 58.6 years , and there was no statistical difference between the hpv - positive and the hpv - negative groups based on age .
however , in some studies , the hpv - associated patients who suffer from oropharyngeal cancers tend to be younger ( 33 , 39 , 42 , 44 ) . in our study ,
53.3% of hpv - positive patients were males and 46.7% were females , and there was no meaningful relation between gender and the hpv status .
such findings have been observed in some other studies ( 29 ) , although some studies on oropharynx lesions have reported that the hpv - positive cases are predominantly male ( 33 , 41 , 42 , 44 ) . in the present study , the most common tumor site was tongue , and there was no meaningful statistical relation between tumor location and the hpv status .
similar findings have been reported in other studies ( 19 , 29 ) . according to the tumor grade , although 60 % of the hpv - positive patients were grade 2 and 59.6% of hpv - negative patients were grade1 , no meaningful statistical difference was found between hpv status and histological grading of the tumor ( 16 , 19 ) .
although in most of the oropharyngeal cancer studies advanced tumor stages have been observed in the hpv - positive cases ( 18 , 33 , 39 , 41 , 44 , 50 ) , there are some studies which have not seen any difference ( 16 , 19 , 32 ) . in this study 55.6 % of the hpv - negative patients and only 33.3% of the hpv - positive patients were n0 .
moreover , 66.7% of the hpv positive groups and only 35.4% of the hpv negative ones were stage iva .
( 51 ) have reported similar findings , although in some studies no difference have been observed ( 13 ) and in most of the hpv - related opscc cases higher lymph node involvement have been observed ( 18 , 33 , 39 ) . in the current study
local recurrence was the most commen type of relapse , and there was no statistically significant difference according to hpv status . in most of the hpv - related opscc studies low reccurence rate have been observed ( 2 , 50 ) , although weinberger et al .
have noted that hpv status has no prognostic value for local recurrence alone ( 52 ) . in our study ,
34.4% of the hpv - negative patients and 73.3% of the hpv - positive patients died of disease .
that statistically meaningful relation was observed between hpv status and patients death . less mortality risk in hpv - positive cases
but as far as we know , no study on oral cavity cancers has especially dealt with the mortality rate of hpv - positive patients .
we found that hpv - negative patients survived significantly longer than hpv - positive patients when both the os and the dfs were measured .
similar findings have been reported in other studies based on tumors with higher stages of disease ( 16 , 22 ) .
while the majority of oropharyngeal studies have reported an improved prognosis of hpv positive tumors ( 2 , 5 , 18 , 19 , 33 , 39 - 41 , 44 , 50 , 53 ) , there are still other studies which have reported no difference ( 2 , 5 , 18 , 19 ) . in our study
there are other studies which indicate hpv - related opscc patients , have a lower intake of tobacco and alcohol ( 33 , 39 , 44 , 50 ) .
currently , little is known about the influence of hpv on the clinical course and survival of patients with ocscc .
although the findings of this study revealed relatively low prevalance of hpv dna in ocsccs and also the well known high risk subtypes were not observed , the carcinogenic role of this virus and its less common variant of hpv 6 and 11 , which we did see in the current study , can not be totally ignored in the oral cavity cancers . regarding high mortality rate of these variants of hpv - positive tumors , further investigations with higher sample volume using more developed methods
currently , little is known about the influence of hpv on the clinical course and survival of patients with ocscc .
although the findings of this study revealed relatively low prevalance of hpv dna in ocsccs and also the well known high risk subtypes were not observed , the carcinogenic role of this virus and its less common variant of hpv 6 and 11 , which we did see in the current study , can not be totally ignored in the oral cavity cancers . regarding high mortality rate of these variants of hpv - positive tumors , further investigations with higher sample volume using more developed methods are still required .
 backgroundsquamous cell carcinoma ( scc ) is the most common malignancy of the oral cavity . 
 a relationship between the human papilloma virus ( hpv ) infection and the prognosis of oral cavity scc ( ocscc ) has been discussed before.objectiveswe investigated the prevalence rate of hpv status in patients with ocscc , and its effects on clinicopathological characteristics of tumors and patients prognosis.patients and methodssections of formalin - fixed , paraffin - embedded tissue blocks from 114 histopathologically confirmed ocscc cases were investigated in this study . polymerase chain reaction ( pcr ) was applied to evaluate the hpv status in the samples.resultsfifteen ( 13.16% ) cases were identified as hpv positive . 
 the detected viral subtypes in this study were the subtypes 6 and 11 . 
 the stage and especially lymph node stage was significantly higher in the hpv positive group compared to the hpv negative group ( p = 0.04 ) . 
 disease free survival ( dfs ) was remarkably lower in the hpv positive group compared to the hpv negative group ( 13.9 vs. 49.9 months , p = 0.02 ) . 
 overall survival ( os ) was also significantly inferior in the hpv positive group ( 15.7 vs. 49.6 months , p = 0.01 ) . in the current study 
 , no significant differences were observed between two groups in relation to the variables of age , gender , tumors site , tumor size , tumor grading and also the recurrence rate.conclusionsthe observed higher mortality rate among the hpv positive group indicates the poorer prognosis of this group in comparison with the hpv negative patients . 
 the incidence rate of hpv infection was low in the studied samples ; however , interaction of subtypes 6 and 11 of hpv in poorer prognosis of the patients and a carcinogenic role of hpv in ocscc can not be ruled out . 
1. Background
2. Objectives
3. Patients and Methods
3.1. Detection of HPV DNA Was Performed by PCR
3.2. Statistical Analysis
4. Results
4.1. Characteristics of Cases
5. Discussion
5.1. Conclusion