lung cancer remains the leading cause of cancer - related deaths worldwide , causing over a million deaths per year worldwide , with an estimated 221,130 new cases and 156,940 deaths in 2011 in the united states . ten to 20 percent of patients with nsclc have never smoked . besides active smoking other etiologic risk factors for nsclc are known ( e.g. , toxins , radon , and air pollution ) ; human papilloma virus ( hpv ) also has been proposed as a potential cause for nsclc . in his meta - analysis of studies that have looked for detection of hpv in lung cancer , it was noted that hpv detection rates in bronchial carcinomas were found to be highly variable , ranging from 0 percent to 100 percent . on average , of 2,468 bronchial carcinomas , hpv dna has been reported in 536 ( 21.7% ) of squamous and nonsquamous nsclc histologies . the genome of hpv is a circular double - stranded dna molecule of approximately 8000  bp . the open reading frames ( orf ) include e1 , e2 , e4 , e5 , e6 , and e7 , which are expressed at different stages during epithelial differentiation . the high - risk hpv e6 and e7 orf encode the viral oncoproteins that are invariably expressed in hpv - positive human cancers . e6 and e7 joint expression is necessary and sufficient for the immortalization of primary human keratinocytes in vitro . previous studies showed equal expression of e6 and e7 in all cases . e6 and e7 function mainly through decreasing the activities of the retinoblastoma tumor suppression gene and p53 pathway . low - risk hpv e6 and e7 exhibit much weaker activity than the high - risk hpv e6 and e7 proteins . levels of these igs against hpv e7 correlate with the clinical course of cervical cancer . e6 and e7 oncoprotein activity also explains the difference in potential carcinogenesis after infection with high - risk hpv versus low - risk hpv . muller et al . showed an association with seroreactivity to hpv-16 e6-e7 peptides in patients with invasive cervical cancer . antibodies to hpv-16 e6 and e7 proteins appear to be virus - specific and disease - state - specific markers of hpv - associated cervical cancer . detection of immunoglobulin ( igg ) reactivity with the e7 oncoproteins of hpv reflects the effectiveness of the immune response against cervical cancers and their precursor lesions . the reactivity of antibody with the virus oncoprotein appears to be conformationally dependent and specific for hpv type [ 4 , 9 ] .   in cervical cancer patients that ghim and jenson have followed with this serological test , a predominant igg response against e7 was associated with periods of tumor stability ( dormancy ) , especially when the cancer was confined to the lymph nodes . an igg response was usually associated with various rates of progression of tumors , particularly in extranodal sites . a recent study using pcr confirmed the presence of 16.7 percent hpv dna integrated in the dna of nsclc . considering hpv 's established causative role in upper airway malignancies and the detection of hpv dna in a significant number of lung cancers , the question of hpvs etiologic effect on this cancer deserves further study . since e6/e7 is essential to hpv 's carcinogenic effect , e6/7 is expected to be present in non - small - cell lung cancer with a pathogenesis due to hpv . this study examines the presence of antibodies ( igg ) in serum against e7 for hpv . establishing an immune response to hpv in nsclc would add further evidence to support hpv 's etiological role for nsclc . the study utilized 100 unidentified serum samples of nsclc patients and controls obtained from the cancer database and specimen repository ( cdsr ) at the james graham brown cancer center . sera from hpv-16 and -18 positive cervical cancer patients were collected as the controls at 4 and 6 different consecutive phases of clinical progression , respectively . the sera were used to measure the titration of total igg against e7 proteins of hpv-16 and -18 by elisa using recombinant e7 proteins as antigen . previously , we successfully generated e7 fusion proteins of hpv16 and 18 using molecular biological techniques . e7 genes of hpv16 or 18 were cloned into prokaryotic expression vectors , pmalc2 ( new england biolabs ) or pqe30 vector ( qiagen ) by pcr cloning technology . the e7 proteins were expressed in e. coli and purified as recommended by the manufacturers ( neb and qiagen , resp . ) . the expression and quality of these recombinant proteins were examined by sds - page , immunoblot , and elisa using standardized molecular biological techniques . the concentration of produced proteins was determined by protein titration using bio - rad protein assay ( biorad ) . direct elisa method was used to measure igg titration against e7 of hpv16 and 18 . briefly , approximately 100  ng protein / well in 50  l bicarbonate buffer ( ph 9.6 ) ( biofluid , gaithersburg , md ) was coated onto elisa microplates ( dynatech , va ) for 1  hr at 37c . after three washings with 200  l of pbs , 200  ml pbs containing 5% nonfat skimmed milk ( 5% pbs - sm ) was added for 1  hr at 37c . then sera diluted to 1/100 in pbs containing 1% bovine serum albumin ( 1% pbsa ) was added for 1  hr at rt followed by the alkaline - phosphatase-(ap- ) conjugated goat anti - igg ( h&l chains ) of human at a 1/1000 dilution in 1% pbsa for an incubation of 1  hr at 37c . the reaction was revealed with ap - chromogenic substrate ( sigma-104 p - nitrophenyl phosphate ; sigma , st louis , mo ) . positive control human sera , which had been previously tested as positive for these hpv oncoproteins , were employed for each experiment .   according to the relationship of anti - e7 igg levels of hpv16 and hpv18 , the cut - off points for the positives of hpv16 and/or 18 were selected at the slope changes using the nonlinear least square method . based on the distribution of elisa values , the cut - off values of e7 igg were found at 0.59 ( 0.045 standard deviation ) and 0.48 ( 0.036 standard deviation ) in hpv-16 and -18 , respectively . the cut - off points were used to group the patients into the positive and the negative based on whether their immune responses are beyond the cut - off points or not . the samples that showed higher values for both hpv-16 and 18 e7 proteins were selected , and the sample , which showed same distance to both cut - off lines , was decided as the case of double infections of hpv-16 and 18 . statistically significant association of the hpv positive patients with gender , disease stages , smoking status , and age were evaluated by the binary logistic regression method . as shown on table 1 , of the 100 samples , 53% came from males and 47% from females . additionally , 88% had a history of smoking ; 7% never smoked ; and in 5% the history of smoking was unknown . of the samples , 43% were adenocarcinoma ( ac ) , 39% squamous cell carcinoma ( scc ) , 10% nsclc not otherwise specified ( nos ) , 4% large cell carcinoma , and 2% neuroendocrine type ( nec ) . the control sera from hpv-16 and -18 positive cervical cancer patients were tested to see if the igg response to the e7 reflects the prognosis of the cancer . both sera showed low igg titrations in the early phase of the illness but showed instant and high immune responses to the oncoprotein in the course of the disease . the titrations increased over time with disease progression except during a short period within the hpv-18 positive group ( figure 1 ) . because of the unavailability of clinical information of the treatment modalities , the prognosis could not be correlated with the igg levels , although both patients succumbed to their disease within two to three years . low titrations appear to be due to the serum collections at early periods of the cancer progression and possibly due to chemotherapeutic treatments that cause immunosuppression .   with the elisa values of lung cancer patients , the cut - off values of the e7 at 0.59 with 0.045 standard deviations and 0.48 with 0.036 standard deviations in hpv-16 and -18 , the given cut - off , 6 and 11 patients were seropositive for the oncogenic e7 proteins of hpv-16 and -18 , respectively . out of the positives , 5 showed the cross - reactions for both e7s , but of those , 4 tended to get close to a cut - off line of one type . only one case was defined as the positive for both with the same distance to both cut - off lines . consequently , 16 nsclc patients were found to have igg titration for pv oncogenic e7 protein ( table 2 ) . 23.5% and 15.4% of ac and scc were positive for hpv e7 , respectively ( table 2 ) . of the 6 positive cases for hpv-16 , 3 were ac , 2 scc , and 1 nos . the only case with high igg levels to both types of hpvs , 16 and 18 , was ac . two ( 28% ) of 7 patients who reported never smoking were positive for hpv , and 12 ( 13.6% ) of 88 smokers were hpv positive . twenty - three of the patients had a previous history of at least one other cancer ( hpv status at time of recent lung cancer diagnosis in parentheses ) : uterine ( hpv16 ) , rectal , two nsclc , three bladder ( one hpv16 ) , three cervical , laryngeal ( hpv16 ) , endometrial ( hpv 16 ) palate , basal cell ( hpv16 ) , skin , trigone molar , renal cell ( hpv16 ) , prostate ( hpv18 ) , tongue ( hpv 16/18 ) , melanoma ( hpv16 ) , vulva , cll , and three breast cancers . the seropositivity against e7-igg was not correlated with other hpv associated cancers . using binary logistic regression any association of hpv infection with gender , disease stages , smoking status , and age of lung cancer patients the age of the patients showed significant effect on the anti - e7 levels ( figure 3 ) . both , hpv16 and 18 e7 levels , increased as the patients ' ages increased with p values of 0.02 and 0.08 . however , the other did not show any highly statistically significant association at under   =  0.05 with the sample group . this study detected igg against e7 for high risk hpv ( type 16 and 18 ) in 16% of patients with nsclc . the presence of antibodies against e7 for hpv 16/18 complements previous studies detecting dna of hpv within nsclc 's genome . the present study found that the age of the patients had a significant effect on the anti - e7 levels . the reason for this is not obvious , but may be due to reinfection or prolonged exposure to the hpv . never smoking did not correlate clearly statistically with the presence of igg directed towards hpv - e7 , although the number of never smokers was low in this study . igg against e7 was detected in squamous and nonsquamous histology of nsclc , which complements the fact that hpv dna is found in the dna of both histologies [ 3 , 10 ] . patients with a previous history of other cancers , which may be associated with hpv , did not have a higher incidence of igg levels against e7 . it is therefore unlikely that this confounded the results in this study of nsclc patients . the presence of hpv dna alone is not enough to support an active role of the virus in the development of lung cancer , but synthesis of e6 and e7 oncoproteins is a mandatory condition for cell transformation and development of cancer . the early oncoprotein e6 binds and degrades the p53 protein , while the e7 protein binds and inactivates the protein rb , resulting in abnormal cell proliferation and tumor growth [ 5 , 11 , 12 ] .   the prevalence of high igg levels against e7 in lung cancer patients , as well as the fact that the titers increased with age and during the course of the disease , may point to a role of hpv in the pathogenesis of this epidemic disease , that affects also a significant number of never smokers . in conclusion , the immune response to hpv 's oncogene e7 found in nsclc patients adds to the evidence linking hpv to nsclc carcinogenesis . in the last years the use of these vaccines may also have impact on the incidence of lung cancer . further studies should be pursued in the future to effects of hpv and vaccines against hpv on nsclc .
background . 
 a significant number of non - small - cell lung cancers ( nsclc ) have human papillomavirus ( hpv ) dna integrated in their genome . 
 this study sought to further establish hpv 's possible etiologic link to nsclc by evaluating an immune response to hpv 's oncogene , e7 , in patients with nsclc . 
 patients and methods . antibodies ( igg ) in serum against e7 for hpv 16 and 18 in 100 patients with nsclc were examined by enzyme - linked immunosorbent assay ( elisa ) . results . sixteen nsclc patients were found to have a high titration of igg for hpv oncogenic e7 protein . 
 23.5% of adenocarcinomas ( ac , ) and 15.4% of squamous cell carcinomas ( scc ) were positive for igg against hpv e7 . 
 hpv-18 ( 11% ) had a slightly higher frequency than hpv-16 ( 6% ) . 
 of the six positive cases for hpv-16 , 3 were ac , 2 scc , and 1 nos ( not otherwise specified ) . 
 for the 11 hpv-18 positives , 7 were ac , and 4 scc . 
 the one case with igg against hpv 16 and 18 was ac . 
 one case had high cross - reactive levels against e7 of hpv 16 and 18 . 
 two ( 28% ) of 7 patients who reported never smoking were positive for hpv , and 12 ( 13.6% ) of 88 smokers were hpv positive . 
 conclusions . 
 the study detected high levels of igg against e7 in 16% of nsclc patients . 
 this adds evidence to a potential role of hpv in the pathogenesis of nsclc .