a role for hpv in lung cancer has been proposed , although previous reports of hpv in lung tumor tissue have been inconclusive .
paraffin - embedded tissue specimens from 450 lung cancer patients who were smokers or never - smokers enrolled in the environment and genetics in lung cancer etiology study were evaluated for hpv dna by real - time polymerase chain reaction to determine the hpv prevalence in tissues from lung cancer patients in a representative western population .
two specimens were found to be hpv positive at low copy number but were negative on additional genotype - specific testing and on testing for a broad spectrum of hpv types .
hpv is not associated with lung carcinogenesis in lung cancer patients from a representative western population .
multiple tissue specimens were used from each patient for analysis and were not necessarily adjacent to or from the same paraffin block . also , the possible contamination of specimens during collection could not be determined . because of geographic variances in the prevalence of hpv reported in lung tumors , further studies in non - western populations should be completed .
a role for hpv in lung cancer has been proposed , although previous reports of hpv in lung tumor tissue have been inconclusive .
paraffin - embedded tissue specimens from 450 lung cancer patients who were smokers or never - smokers enrolled in the environment and genetics in lung cancer etiology study were evaluated for hpv dna by real - time polymerase chain reaction to determine the hpv prevalence in tissues from lung cancer patients in a representative western population .
two specimens were found to be hpv positive at low copy number but were negative on additional genotype - specific testing and on testing for a broad spectrum of hpv types .
hpv is not associated with lung carcinogenesis in lung cancer patients from a representative western population .
multiple tissue specimens were used from each patient for analysis and were not necessarily adjacent to or from the same paraffin block . also , the possible contamination of specimens during collection could not be determined .
because of geographic variances in the prevalence of hpv reported in lung tumors , further studies in non - western populations should be completed .
eagle is a large population - based case  control study with 2100 lung cancer patients that was designed to investigate the genetic and environmental determinants of lung cancer and smoking persistence ( 22 ) . as previously described (
22 ) , individuals with newly diagnosed lung cancer were recruited from april 22 , 2002 , to february 28 , 2005 , from 13 hospitals in the lombardy region of italy ( see notes ) .
the study was approved by the institutional review board of each participating hospital and university in italy and by the national cancer institute , bethesda , md .
lung cancer was diagnosed by standard clinical criteria and confirmed by pathology reports from surgery , biopsy , or cytology samples ( approximately 95% of patients ) or through clinical history and imaging ( approximately 5% of patients ) ( 22 ) .
tumor histology was classified by the 1999 world health organization histological typing of lung and pleural tumors ( 23 ) .
the 13 italian hospitals treat approximately 80% of all patients with newly diagnosed lung cancer in the area , which includes more than 1.3 million people aged 3579 years from five cities and surrounding towns and villages .
thus , participants in the eagle study should be broadly representative of lung cancer patients in western populations .
we decided to test 450 adult male and female lung cancer patients , including all 30 never - smokers and a random sample of 420 smokers who had an adequate number of unstained tissue slides and at least one slide available that was stained with hematoxylin and eosin .
these stained slides were reviewed by h. song to establish the presence and estimate proportion of tumor tissue in the sample , but these slides were not necessarily adjacent to the tissue that was tested for hpv dna .
all 450 patients were tested for hpv16 and hpv18 dna because these are the most common types found in cervical cancer ( 24 ) and account for the overwhelming majority of noncervical hpv  associated cancers ( 25 ) .
type - specific pcr for hpv16 and hpv18 was conducted in a laboratory at the ohio state university , as described below . for a subset of 100 patients including all never - smokers and a random selection of smokers ,
a second tissue specimen was sent to ddl diagnostic laboratory ( voorburg , the netherlands ) for broad - spectrum hpv typing .
all specimen receipt , processing , and pre - amplification analysis procedures were performed in ultraviolet light 
all equipment and reagents were specific to the pre - amplification analysis laboratory with strict one - way transport of specimens from this laboratory to a physically separate postamplification analysis laboratory .
additional protocols to prevent specimen contamination included sample processing in small batches , change of gloves after dna extraction of each sample , exclusive use of barrier pipette tips , weekly cleaning of all laboratory surfaces and equipment with 70% ethanol , and soaking of all racks and pcr plate holders in 10% bleach for a minimum of 10 minutes between each experiment .
additionally , one negative control per seven experimental samples was included in each column of each 96-well pcr plate .
dna was extracted from 450 formalin- or ethanol - fixed paraffin - embedded tissue samples by means of paraffin removal with octane , proteinase k digestion , phenol  chloroform extraction , and ethanol precipitation . briefly , after octane exposure , the sample was washed with 100% ethanol , resuspended in 200 l of digestion buffer ( 50 mm tris 
hcl at ph 8.5 , 1 mm edta , and proteinase k at 20 g / ml ) , and incubated for 48 hours at 55c .
if tissue remained , 10 l of proteinase k ( 400 g / ml ) was added , and digestion was continued for an additional 2448 hours .
chloroform by use of qiagen maxtract tubes ( qiagen , germantown , md ) and ethanol precipitated in the presence of glycogen ( 0.02 mg / ml ) .
pellets were washed once in 70% ethanol , dried at 37c in a dry incubator , and resuspended in 50 l of diethylene pyrocarbonate - treated water . dna quantity and purity ( calculated by use of the ratio of the absorbance at 260 nm to that at 280 nm [ 260/280 ratio ] )
were measured with the nanodrop spectrophotometer ( thermo fisher scientific , inc , wilmington , de ) .
an estimate of the number of cells analyzed in each pcr was made by custom taqman real - time pcr assays targeting a single - copy human gene on chromosome 7 , human endogenous retrovirus 3 ( erv3 ) ( 26 ) . in a pilot study of 19 specimens ,
dna integrity was evaluated through amplification of erv3 sequences with primers generating an amplicon of 58 , 135 , or 354 base pairs ( bp ) in size ( supplementary table 1 , available online ) .
primer and probe sequences for the 58-bp amplicon were as follows : forward primer , 5-gataatttcacactaaccgcc-3 ; reverse primer , 5-agatgctctgacttgatggt-3 ; and the probe sequence , 5-56-fam - ctcttccctcgaacctgc-3bhq1 - 3 ( where 56-fam is 56-carboxy - fluorescein and 3bhq1 is 3-black hole quencher-1 ) .
primer and probe sequences for the 135-bp amplicon were as follows : forward primer , 5-catgggaagcaagggaacta-3 ; reverse primer , 5-cccagcgagcaatacagaattt-3 ; and probe , 5-56-fam - tcttccctcgaacctgcaccatcaagtca-3bhq1 - 3. primer and probe sequences for the 354-bp amplicon were as follows : forward primer , 5-catgggaagcaagggaactaatg-3 ; reverse primer , 5-gcccacagatccagtagagg-3 ; and probe were the same as for the 135-bp amplicon above .
briefly , purified genomic dna ( 2 l ) from lung cancer specimens was analyzed .
a standard curve was generated in duplicate from a fivefold dilution series ( from 150  000 to 1.92 cells ) of a diploid human cell line , ccd-18lu ( catalog number ccl-205 , atcc [ american type culture collection ] , manassas , va ) .
results were reported as the number of human diploid genome equivalents of purified genomic dna from tumor samples that were evaluated for hpv16 and hpv18 dna by real - time pcr .
all samples were tested once , and positive samples were retested in duplicate . although dna was degraded because of fixation , the mean yield was 54.5 ng / ml . this subset analysis indicated that dna integrity was sufficient to support amplification of targets with amplicon size between approximately 58 and 135 bp .
dna specimens with at least 100 evaluable cell equivalents and a 260/280 ratio between 1.5 and 2.2 ( 27 ) were considered to have adequate dna quantity and quality for further analysis .
type - specific taqman real - time pcr targeted to the oncogenic e6 region of the genome for hpv16 ( amplicon size = 201 bp ) and the e7 region of the genome for hpv18 ( amplicon size = 137 bp ) were performed in a 96-well plate format as previously described ( 28 ) . to account for dna degradation in formalin - fixed and paraffin - embedded tissues ,
all samples were also evaluated by means of a taqman real - time pcr assay designed to amplify an 81-bp amplicon in the e6 region of the genome for hpv16 .
primers and probes were manufactured at idt technologies ( coralville , ia ) and sequences were : forward , 5-gagaactgcaatgtttcaggacc-3 ; reverse , 5-tgtatagttgtttgcagctctgtgc-3 ; and probe , 5-56-famcaggagcgacccagaaagttaccacagtt-3bhq1 - 3. standard curves were generated in duplicate by use of a fivefold dilution series from 2.5  10 copies of plasmid vector containing full - length viral genome ( pgem hpv16 ) in a background of human placental dna ( 5 ng/l ) .
reactions that were performed in an applied biosystems 7300 pcr system contained 2x taqman universal pcr master mix ( applied biosystems , foster city , ca ) , 0.1 mol of probe , 0.2 mol of each primer , and 2 l of purified tumor dna .
amplification conditions included a 12-minute incubation at 95c , followed by 50 cycles of 15 seconds at 95c and 60 seconds at 60c .
the cycle threshold ( ct ) of unknown samples was determined from an equation derived from a linear regression through the log ct of the standard curve according to the manufacturer s recommendations . for positive samples ,
hpv viral load was normalized to erv3 copy number by real - time pcr , as a measure of viral copy number per cell analyzed .
samples with any detectable hpv16 or hpv18 dna were tested in duplicate , with a mean viral load of greater than one viral copy per sample classified as real - time pcr positive .
a mean viral load of greater than or equal to one viral copy per cell was considered indicative of a clonal viral 
specimens from all 30 never - smokers and 70 randomly sampled current and former smokers were selected to test for broad - spectrum hpv typing at ddl diagnostic laboratory .
hpv typing used pcr with short pcr fragment ( spf10 ) primers , which targets the hpv l1 gene , and a mixture of conservative probes that recognize at least 54 mucosal hpv genotypes .
we used the dna enzyme immunoassay ( deia ) to determine hpv dna positivity by detecting amplimers , synthesized by biotinylated pcr primers , through hybridization to a mixture of hpv - specific probes , as described previously ( 29 ) .
stringent pcr contamination precautions that were similar to the type - specific testing for hpv16 and hpv18 conducted at the ohio state university were taken .
irradiated laminar flow hoods that were separate from a postamplification analysis laboratory . during dna isolation ,
pcr amplification , and post - pcr analyses , specific negative ( water blank ) and positive ( hpv dna extracted from the hpv18-positive hela cell line ) controls were included .
we monitored potential contamination of samples with negative controls and the sensitivity of the procedure with low - concentration positive controls .
all reagents had been prepared in a separate reagent laboratory and subjected to strict quality control to confirm efficacy and the absence of contaminants .
dna amplification by pcr with spf10 primers was followed by a reverse hybridization line - probe assay ( lipa25 , version 1 ; labo bio - medical products , rijswijk , the netherlands ) for genotyping of 25 hpv types in spf10- and deia - positive samples .
these primers produce very short pcr fragments of approximately 65 bp , making them ideal for amplification in formalin - fixed specimens ( 2931 ) .
eight specimens did not appear to have tumors in the tissue specimen received for testing .
dna was extracted for 92 specimens ( from 65 ever - smokers and 27 never - smokers ) with identifiable tumor in the tissue specimen . for each specimen ,
half of the tissue section was removed from the slide with a swab after confirming the presence of tumor cells in that part of the section .
this part of the tissue was transferred to a microtube ( sarstedt , etten - leur , the netherlands ) and digested with proteinase k buffer ( 45 mm tris 
hcl at ph 8 , 0.9 mm edta , 0.45% tween 20 , and proteinase k at 1 mg / ml ) .
proteinase k solution ( 100 l ) was added to each microtube and incubated for 1624 hours at 56c .
resulting dna preparations were stored at 20c . during this process , negative and positive dna isolation controls were included . in these samples ,
dna quality and quantity were adequate as measured by real - time pcr for the human -actin gene in the same dna specimens as used for the spf10 pcr .
for one patient ( a current smoker ) where additional investigation was warranted due to weak hpv16 positivity in the sample tested at the ohio state university , type - specific pcr for hpv16 was performed with a 92-bp amplicon as previously described ( 32 ) . our goal in determining sample size was to be able to rule out a true hpv prevalence greater than 1.0% .
using cii in stata ( statacorp lp , college station , tx ) , we determined that zero hpv positives among 450 lung cancer patients would generate a one - sided 97.5% confidence interval ( ci ) of 0% to 0.82% and that one observed hpv - positive patient among 450 lung cancer patients would provide a two - sided 95% confidence interval of 0.0056% to 1.2% ; zero positives among 400 lung cancer patients would provide a one - sided 97.5% confidence interval of 0% to 0.92% , and one observed hpv - positive patient would provide a two - sided 95% confidence interval of 0.0063% to 1.4% .
the chance of finding at least one positive when the true prevalence is 1.0% or more was 98.9% with a sample size of 450 patients ; thus , finding no positives with a sensitive assay rules out even a low prevalence of infection with high confidence .
in addition to the sample size calculations described above , we used the cii command in stata ( statacorp lp ) , version 9.0 , to estimate hpv prevalence and corresponding confidence intervals .
eagle is a large population - based case  control study with 2100 lung cancer patients that was designed to investigate the genetic and environmental determinants of lung cancer and smoking persistence ( 22 ) . as previously described (
22 ) , individuals with newly diagnosed lung cancer were recruited from april 22 , 2002 , to february 28 , 2005 , from 13 hospitals in the lombardy region of italy ( see notes ) .
the study was approved by the institutional review board of each participating hospital and university in italy and by the national cancer institute , bethesda , md .
lung cancer was diagnosed by standard clinical criteria and confirmed by pathology reports from surgery , biopsy , or cytology samples ( approximately 95% of patients ) or through clinical history and imaging ( approximately 5% of patients ) ( 22 ) .
tumor histology was classified by the 1999 world health organization histological typing of lung and pleural tumors ( 23 ) .
the 13 italian hospitals treat approximately 80% of all patients with newly diagnosed lung cancer in the area , which includes more than 1.3 million people aged 3579 years from five cities and surrounding towns and villages .
thus , participants in the eagle study should be broadly representative of lung cancer patients in western populations .
we decided to test 450 adult male and female lung cancer patients , including all 30 never - smokers and a random sample of 420 smokers who had an adequate number of unstained tissue slides and at least one slide available that was stained with hematoxylin and eosin .
these stained slides were reviewed by h. song to establish the presence and estimate proportion of tumor tissue in the sample , but these slides were not necessarily adjacent to the tissue that was tested for hpv dna .
all 450 patients were tested for hpv16 and hpv18 dna because these are the most common types found in cervical cancer ( 24 ) and account for the overwhelming majority of noncervical hpv  associated cancers ( 25 ) .
type - specific pcr for hpv16 and hpv18 was conducted in a laboratory at the ohio state university , as described below . for a subset of 100 patients including all never - smokers and a random selection of smokers ,
a second tissue specimen was sent to ddl diagnostic laboratory ( voorburg , the netherlands ) for broad - spectrum hpv typing .
all specimen receipt , processing , and pre - amplification analysis procedures were performed in ultraviolet light 
all equipment and reagents were specific to the pre - amplification analysis laboratory with strict one - way transport of specimens from this laboratory to a physically separate postamplification analysis laboratory .
additional protocols to prevent specimen contamination included sample processing in small batches , change of gloves after dna extraction of each sample , exclusive use of barrier pipette tips , weekly cleaning of all laboratory surfaces and equipment with 70% ethanol , and soaking of all racks and pcr plate holders in 10% bleach for a minimum of 10 minutes between each experiment .
additionally , one negative control per seven experimental samples was included in each column of each 96-well pcr plate .
dna was extracted from 450 formalin- or ethanol - fixed paraffin - embedded tissue samples by means of paraffin removal with octane , proteinase k digestion , phenol  chloroform extraction , and ethanol precipitation .
briefly , after octane exposure , the sample was washed with 100% ethanol , resuspended in 200 l of digestion buffer ( 50 mm tris 
hcl at ph 8.5 , 1 mm edta , and proteinase k at 20 g / ml ) , and incubated for 48 hours at 55c
. if tissue remained , 10 l of proteinase k ( 400 g / ml ) was added , and digestion was continued for an additional 2448 hours .
chloroform by use of qiagen maxtract tubes ( qiagen , germantown , md ) and ethanol precipitated in the presence of glycogen ( 0.02 mg / ml ) .
pellets were washed once in 70% ethanol , dried at 37c in a dry incubator , and resuspended in 50 l of diethylene pyrocarbonate - treated water . dna quantity and purity ( calculated by use of the ratio of the absorbance at 260 nm to that at 280 nm [ 260/280 ratio ] )
were measured with the nanodrop spectrophotometer ( thermo fisher scientific , inc , wilmington , de ) .
an estimate of the number of cells analyzed in each pcr was made by custom taqman real - time pcr assays targeting a single - copy human gene on chromosome 7 , human endogenous retrovirus 3 ( erv3 ) ( 26 ) . in a pilot study of 19 specimens , dna integrity was evaluated through amplification of erv3 sequences with primers generating an amplicon of 58 , 135 , or 354 base pairs ( bp ) in size ( supplementary table 1 , available online ) .
primer and probe sequences for the 58-bp amplicon were as follows : forward primer , 5-gataatttcacactaaccgcc-3 ; reverse primer , 5-agatgctctgacttgatggt-3 ; and the probe sequence , 5-56-fam - ctcttccctcgaacctgc-3bhq1 - 3 ( where 56-fam is 56-carboxy - fluorescein and 3bhq1 is 3-black hole quencher-1 ) .
primer and probe sequences for the 135-bp amplicon were as follows : forward primer , 5-catgggaagcaagggaacta-3 ; reverse primer , 5-cccagcgagcaatacagaattt-3 ; and probe , 5-56-fam - tcttccctcgaacctgcaccatcaagtca-3bhq1 - 3. primer and probe sequences for the 354-bp amplicon were as follows : forward primer , 5-catgggaagcaagggaactaatg-3 ; reverse primer , 5-gcccacagatccagtagagg-3 ; and probe were the same as for the 135-bp amplicon above .
briefly , purified genomic dna ( 2 l ) from lung cancer specimens was analyzed .
a standard curve was generated in duplicate from a fivefold dilution series ( from 150  000 to 1.92 cells ) of a diploid human cell line , ccd-18lu ( catalog number ccl-205 , atcc [ american type culture collection ] , manassas , va ) .
results were reported as the number of human diploid genome equivalents of purified genomic dna from tumor samples that were evaluated for hpv16 and hpv18 dna by real - time pcr .
all samples were tested once , and positive samples were retested in duplicate . although dna was degraded because of fixation , the mean yield was 54.5 ng / ml . this subset analysis indicated that dna integrity was sufficient to support amplification of targets with amplicon size between approximately 58 and 135 bp .
dna specimens with at least 100 evaluable cell equivalents and a 260/280 ratio between 1.5 and 2.2 ( 27 ) were considered to have adequate dna quantity and quality for further analysis .
type - specific taqman real - time pcr targeted to the oncogenic e6 region of the genome for hpv16 ( amplicon size = 201 bp ) and the e7 region of the genome for hpv18 ( amplicon size = 137 bp ) were performed in a 96-well plate format as previously described ( 28 ) . to account for dna degradation in formalin - fixed and paraffin - embedded tissues ,
all samples were also evaluated by means of a taqman real - time pcr assay designed to amplify an 81-bp amplicon in the e6 region of the genome for hpv16 .
primers and probes were manufactured at idt technologies ( coralville , ia ) and sequences were : forward , 5-gagaactgcaatgtttcaggacc-3 ; reverse , 5-tgtatagttgtttgcagctctgtgc-3 ; and probe , 5-56-famcaggagcgacccagaaagttaccacagtt-3bhq1 - 3. standard curves were generated in duplicate by use of a fivefold dilution series from 2.5  10 copies of plasmid vector containing full - length viral genome ( pgem hpv16 ) in a background of human placental dna ( 5 ng/l ) .
reactions that were performed in an applied biosystems 7300 pcr system contained 2x taqman universal pcr master mix ( applied biosystems , foster city , ca ) , 0.1 mol of probe , 0.2 mol of each primer , and 2 l of purified tumor dna .
amplification conditions included a 12-minute incubation at 95c , followed by 50 cycles of 15 seconds at 95c and 60 seconds at 60c .
the cycle threshold ( ct ) of unknown samples was determined from an equation derived from a linear regression through the log ct of the standard curve according to the manufacturer s recommendations . for positive samples ,
hpv viral load was normalized to erv3 copy number by real - time pcr , as a measure of viral copy number per cell analyzed .
samples with any detectable hpv16 or hpv18 dna were tested in duplicate , with a mean viral load of greater than one viral copy per sample classified as real - time pcr positive .
a mean viral load of greater than or equal to one viral copy per cell was considered indicative of a clonal viral  tumor relationship .
specimens from all 30 never - smokers and 70 randomly sampled current and former smokers were selected to test for broad - spectrum hpv typing at ddl diagnostic laboratory .
hpv typing used pcr with short pcr fragment ( spf10 ) primers , which targets the hpv l1 gene , and a mixture of conservative probes that recognize at least 54 mucosal hpv genotypes .
we used the dna enzyme immunoassay ( deia ) to determine hpv dna positivity by detecting amplimers , synthesized by biotinylated pcr primers , through hybridization to a mixture of hpv - specific probes , as described previously ( 29 ) .
stringent pcr contamination precautions that were similar to the type - specific testing for hpv16 and hpv18 conducted at the ohio state university were taken .
irradiated laminar flow hoods that were separate from a postamplification analysis laboratory . during dna isolation , pcr amplification , and post - pcr analyses ,
specific negative ( water blank ) and positive ( hpv dna extracted from the hpv18-positive hela cell line ) controls were included .
we monitored potential contamination of samples with negative controls and the sensitivity of the procedure with low - concentration positive controls .
all reagents had been prepared in a separate reagent laboratory and subjected to strict quality control to confirm efficacy and the absence of contaminants .
dna amplification by pcr with spf10 primers was followed by a reverse hybridization line - probe assay ( lipa25 , version 1 ; labo bio - medical products , rijswijk , the netherlands ) for genotyping of 25 hpv types in spf10- and deia - positive samples .
these primers produce very short pcr fragments of approximately 65 bp , making them ideal for amplification in formalin - fixed specimens ( 2931 ) .
eight specimens did not appear to have tumors in the tissue specimen received for testing .
dna was extracted for 92 specimens ( from 65 ever - smokers and 27 never - smokers ) with identifiable tumor in the tissue specimen . for each specimen ,
half of the tissue section was removed from the slide with a swab after confirming the presence of tumor cells in that part of the section .
this part of the tissue was transferred to a microtube ( sarstedt , etten - leur , the netherlands ) and digested with proteinase k buffer ( 45 mm tris 
hcl at ph 8 , 0.9 mm edta , 0.45% tween 20 , and proteinase k at 1 mg / ml ) .
proteinase k solution ( 100 l ) was added to each microtube and incubated for 1624 hours at 56c .
resulting dna preparations were stored at 20c . during this process , negative and positive dna isolation controls were included . in these samples , dna quality and quantity
were adequate as measured by real - time pcr for the human -actin gene in the same dna specimens as used for the spf10 pcr .
for one patient ( a current smoker ) where additional investigation was warranted due to weak hpv16 positivity in the sample tested at the ohio state university , type - specific pcr for hpv16 was performed with a 92-bp amplicon as previously described ( 32 ) .
all specimen receipt , processing , and pre - amplification analysis procedures were performed in ultraviolet light 
all equipment and reagents were specific to the pre - amplification analysis laboratory with strict one - way transport of specimens from this laboratory to a physically separate postamplification analysis laboratory .
additional protocols to prevent specimen contamination included sample processing in small batches , change of gloves after dna extraction of each sample , exclusive use of barrier pipette tips , weekly cleaning of all laboratory surfaces and equipment with 70% ethanol , and soaking of all racks and pcr plate holders in 10% bleach for a minimum of 10 minutes between each experiment .
additionally , one negative control per seven experimental samples was included in each column of each 96-well pcr plate .
dna was extracted from 450 formalin- or ethanol - fixed paraffin - embedded tissue samples by means of paraffin removal with octane , proteinase k digestion , phenol  chloroform extraction , and ethanol precipitation . briefly , after octane exposure , the sample was washed with 100% ethanol , resuspended in 200 l of digestion buffer ( 50 mm tris 
hcl at ph 8.5 , 1 mm edta , and proteinase k at 20 g / ml ) , and incubated for 48 hours at 55c .
if tissue remained , 10 l of proteinase k ( 400 g / ml ) was added , and digestion was continued for an additional 2448 hours .
chloroform by use of qiagen maxtract tubes ( qiagen , germantown , md ) and ethanol precipitated in the presence of glycogen ( 0.02 mg / ml ) .
pellets were washed once in 70% ethanol , dried at 37c in a dry incubator , and resuspended in 50 l of diethylene pyrocarbonate - treated water . dna quantity and purity ( calculated by use of the ratio of the absorbance at 260 nm to that at 280 nm [ 260/280 ratio ] )
were measured with the nanodrop spectrophotometer ( thermo fisher scientific , inc , wilmington , de ) .
an estimate of the number of cells analyzed in each pcr was made by custom taqman real - time pcr assays targeting a single - copy human gene on chromosome 7 , human endogenous retrovirus 3 ( erv3 ) ( 26 ) . in a pilot study of 19 specimens ,
dna integrity was evaluated through amplification of erv3 sequences with primers generating an amplicon of 58 , 135 , or 354 base pairs ( bp ) in size ( supplementary table 1 , available online ) .
primer and probe sequences for the 58-bp amplicon were as follows : forward primer , 5-gataatttcacactaaccgcc-3 ; reverse primer , 5-agatgctctgacttgatggt-3 ; and the probe sequence , 5-56-fam - ctcttccctcgaacctgc-3bhq1 - 3 ( where 56-fam is 56-carboxy - fluorescein and 3bhq1 is 3-black hole quencher-1 ) .
primer and probe sequences for the 135-bp amplicon were as follows : forward primer , 5-catgggaagcaagggaacta-3 ; reverse primer , 5-cccagcgagcaatacagaattt-3 ; and probe , 5-56-fam - tcttccctcgaacctgcaccatcaagtca-3bhq1 - 3. primer and probe sequences for the 354-bp amplicon were as follows : forward primer , 5-catgggaagcaagggaactaatg-3 ; reverse primer , 5-gcccacagatccagtagagg-3 ; and probe were the same as for the 135-bp amplicon above .
briefly , purified genomic dna ( 2 l ) from lung cancer specimens was analyzed .
a standard curve was generated in duplicate from a fivefold dilution series ( from 150  000 to 1.92 cells ) of a diploid human cell line , ccd-18lu ( catalog number ccl-205 , atcc [ american type culture collection ] , manassas , va ) .
results were reported as the number of human diploid genome equivalents of purified genomic dna from tumor samples that were evaluated for hpv16 and hpv18 dna by real - time pcr .
all samples were tested once , and positive samples were retested in duplicate . although dna was degraded because of fixation , the mean yield was 54.5 ng / ml . this subset analysis indicated that dna integrity was sufficient to support amplification of targets with amplicon size between approximately 58 and 135 bp .
dna specimens with at least 100 evaluable cell equivalents and a 260/280 ratio between 1.5 and 2.2 ( 27 ) were considered to have adequate dna quantity and quality for further analysis .
type - specific taqman real - time pcr targeted to the oncogenic e6 region of the genome for hpv16 ( amplicon size = 201 bp ) and the e7 region of the genome for hpv18 ( amplicon size = 137 bp ) were performed in a 96-well plate format as previously described ( 28 ) . to account for dna degradation in formalin - fixed and paraffin - embedded tissues ,
all samples were also evaluated by means of a taqman real - time pcr assay designed to amplify an 81-bp amplicon in the e6 region of the genome for hpv16 .
primers and probes were manufactured at idt technologies ( coralville , ia ) and sequences were : forward , 5-gagaactgcaatgtttcaggacc-3 ; reverse , 5-tgtatagttgtttgcagctctgtgc-3 ; and probe , 5-56-famcaggagcgacccagaaagttaccacagtt-3bhq1 - 3. standard curves were generated in duplicate by use of a fivefold dilution series from 2.5  10 copies of plasmid vector containing full - length viral genome ( pgem hpv16 ) in a background of human placental dna ( 5 ng/l ) .
reactions that were performed in an applied biosystems 7300 pcr system contained 2x taqman universal pcr master mix ( applied biosystems , foster city , ca ) , 0.1 mol of probe , 0.2 mol of each primer , and 2 l of purified tumor dna .
amplification conditions included a 12-minute incubation at 95c , followed by 50 cycles of 15 seconds at 95c and 60 seconds at 60c .
the cycle threshold ( ct ) of unknown samples was determined from an equation derived from a linear regression through the log ct of the standard curve according to the manufacturer s recommendations . for positive samples ,
hpv viral load was normalized to erv3 copy number by real - time pcr , as a measure of viral copy number per cell analyzed .
samples with any detectable hpv16 or hpv18 dna were tested in duplicate , with a mean viral load of greater than one viral copy per sample classified as real - time pcr positive .
a mean viral load of greater than or equal to one viral copy per cell was considered indicative of a clonal viral  tumor relationship .
specimens from all 30 never - smokers and 70 randomly sampled current and former smokers were selected to test for broad - spectrum hpv typing at ddl diagnostic laboratory .
hpv typing used pcr with short pcr fragment ( spf10 ) primers , which targets the hpv l1 gene , and a mixture of conservative probes that recognize at least 54 mucosal hpv genotypes .
we used the dna enzyme immunoassay ( deia ) to determine hpv dna positivity by detecting amplimers , synthesized by biotinylated pcr primers , through hybridization to a mixture of hpv - specific probes , as described previously ( 29 ) .
stringent pcr contamination precautions that were similar to the type - specific testing for hpv16 and hpv18 conducted at the ohio state university were taken .
irradiated laminar flow hoods that were separate from a postamplification analysis laboratory . during dna isolation , pcr amplification , and post - pcr analyses ,
specific negative ( water blank ) and positive ( hpv dna extracted from the hpv18-positive hela cell line ) controls were included .
we monitored potential contamination of samples with negative controls and the sensitivity of the procedure with low - concentration positive controls .
all reagents had been prepared in a separate reagent laboratory and subjected to strict quality control to confirm efficacy and the absence of contaminants .
dna amplification by pcr with spf10 primers was followed by a reverse hybridization line - probe assay ( lipa25 , version 1 ; labo bio - medical products , rijswijk , the netherlands ) for genotyping of 25 hpv types in spf10- and deia - positive samples .
these primers produce very short pcr fragments of approximately 65 bp , making them ideal for amplification in formalin - fixed specimens ( 2931 ) .
eight specimens did not appear to have tumors in the tissue specimen received for testing .
dna was extracted for 92 specimens ( from 65 ever - smokers and 27 never - smokers ) with identifiable tumor in the tissue specimen . for each specimen ,
half of the tissue section was removed from the slide with a swab after confirming the presence of tumor cells in that part of the section .
this part of the tissue was transferred to a microtube ( sarstedt , etten - leur , the netherlands ) and digested with proteinase k buffer ( 45 mm tris 
hcl at ph 8 , 0.9 mm edta , 0.45% tween 20 , and proteinase k at 1 mg / ml ) .
proteinase k solution ( 100 l ) was added to each microtube and incubated for 1624 hours at 56c .
resulting dna preparations were stored at 20c . during this process , negative and positive dna isolation controls were included . in these samples ,
dna quality and quantity were adequate as measured by real - time pcr for the human -actin gene in the same dna specimens as used for the spf10 pcr .
for one patient ( a current smoker ) where additional investigation was warranted due to weak hpv16 positivity in the sample tested at the ohio state university , type - specific pcr for hpv16 was performed with a 92-bp amplicon as previously described ( 32 ) .
our goal in determining sample size was to be able to rule out a true hpv prevalence greater than 1.0% .
using cii in stata ( statacorp lp , college station , tx ) , we determined that zero hpv positives among 450 lung cancer patients would generate a one - sided 97.5% confidence interval ( ci ) of 0% to 0.82% and that one observed hpv - positive patient among 450 lung cancer patients would provide a two - sided 95% confidence interval of 0.0056% to 1.2% ; zero positives among 400 lung cancer patients would provide a one - sided 97.5% confidence interval of 0% to 0.92% , and one observed hpv - positive patient would provide a two - sided 95% confidence interval of 0.0063% to 1.4% .
the chance of finding at least one positive when the true prevalence is 1.0% or more was 98.9% with a sample size of 450 patients ; thus , finding no positives with a sensitive assay rules out even a low prevalence of infection with high confidence . in addition to the sample size calculations described above , we used the cii command in stata ( statacorp lp ) , version 9.0 , to estimate hpv prevalence and corresponding confidence intervals .
among the 450 lung cancer patients , 246 ( 54.7% ) had adenocarcinoma and 137 ( 30.4% ) had squamous cell carcinoma ( table 1 ) .
most patients had a history of tobacco smoking : 220 patients ( 48.9% ) were current smokers , 198 patients ( 44.0% ) were former smokers , and 30 patients ( 6.7% ) were never - smokers .
the median age at diagnosis of lung cancer was 67.6 years ( range = 35.479.9 years ) , and the median body mass index was 25.3 kg / m ( range = 15.960.8 kg / m ) . among smokers ,
the median duration of smoking was 45 years ( range = 170 years ) , the median average smoking intensity was one pack per day ( range = 03 packs per day ) , the median pack - years was 43.8 ( range = 0.1192 pack - years ) , and the median age at initiation of smoking was 16 years of age ( range = 645 years ) .
the majority of patients had less than a high school education ( 72.5% or 319 patients ) , drank alcohol ( 84.3% or 365 patients ) , were married or cohabitating ( 82.2% or 356 patients ) , had abnormal respiratory function ( 51.7% or 186 patients ) , and had stage iib disease or less ( 71.3% or 321 patients ) ( table 2 ) .
characteristics of 450 lung cancer patients from the population - based case  control environment and genetics in lung cancer etiology study , including histology , smoking status , and human papillomavirus ( hpv ) status pcr = polymerase chain reaction . results from 399 specimens with adequate dna of 450 selected for testing . results from 92 specimens with tumor present in the sample of 100 selected for testing . includes mixed type ( adenosquamous , n = 10 ) , synchronous ( two different lung cancers , n = 6 ) , non - small cells ( n = 2 ) , and other ( including poorly differentiated , epithelial , neuroendocrine , mucoepidermoid , non - small cell , pleomorphic , sarcomatoid , n = 6 ) . descriptive characteristics of 450 lung cancer patients from the population - based case  control environment and genetics in lung cancer etiology study * copd = chronic obstructive pulmonary disease .
the median number of cells evaluable for the hpv16 and hpv18 pcr as measured by erv3 real - time pcr was 3680 cells ( range = 1.9399  750 cells ) .
combining these data , 399 ( 88.7% ) of 450 specimens had adequate dna quantity and quality for analysis ( ie , 100 or more cell equivalents and a 260/280 ratio between 1.5 and 2.2 ) . on review of the 450 slides that were stained with hematoxylin and eosin ,
412 specimens contained tumor cells . of those tumor - positive specimens , 73.8% ( n = 304 specimens ) contained greater than 60% tumor .
we included all specimens with adequate dna ( n = 399 specimens ) in the prevalence estimate . among the 399 specimens with adequate dna , one female former smoker with adenocarcinoma was positive for hpv16 on initial testing .
given the very low viral load in this tumor ( average = 0.4 per 1000 cell equivalents across three tests of the same sample ) , we tested another tumor tissue specimen from this patient .
eosin slide from this specimen consisted of greater than 80% cancer cells , indicating there was adequate tumor available to detect any hpv dna if it were present .
this patient was diagnosed with stage ib lung cancer at 72 years of age and had smoked for 48 years at an average intensity of 0.75 packs per day ( 36 pack - years ) .
pcr analysis targeting an 81-bp amplicon in the hpv16 e6 region confirmed the prior positive test ( average of two viral copies per 1000 cell equivalents ) in the first sample , and the second tumor tissue specimen from this patient was again negative .
another patient who was initially negative for hpv16 tested positive with this assay but at low copy number ( 131 per 1000 cell equivalents ) .
this second patient was a male with squamous cell carcinoma diagnosed at 64 years of age who had smoked for 43 years at an average intensity of one pack per day ( 43 pack - years ) .
eosin slide from this patient also contained greater than 80% cancer cells . in the subset of 92 specimens tested with the broad - spectrum assay spf10-deia - lipa25 ( including those from the two patients who tested positive with low viral load , as described above ) , no specimens were positive for hpv16 dna or for any hpv type .
ddl diagnostic laboratory conducted type - specific testing for hpv16 as a follow - up test for the male patient identified as being hpv positive by pcr analysis targeting an 81-bp amplicon in the hpv16 e6 region .
these results indicate that the prevalence of hpv dna is 0.0% ( table 1 ) with an upper 97.5% confidence interval of 0.92% for the 399 specimens with adequate dna , including 370 ever - smokers ( upper 97.5% confidence bound = 0.99% ) and 27 never - smokers ( upper 97.5% confidence bound = 12.8% ) .
in this study , which is to our knowledge the largest study of hpv in lung tumor tissue from a western country , we found no evidence that hpv is associated with lung carcinogenesis .
extensive laboratory efforts to avoid dna contamination and state - of - the - art , highly sensitive hpv dna detection assays were performed .
the two tumors that tested positive had a very low viral load of less than one copy of hpv16 per cell , despite being composed of greater than 80% tumor cells .
one of these patients tested negative in a separate tissue specimen , and the other was negative on additional type - specific hpv16 testing in a separate sample .
none of the subset of 92 patients was positive for any other hpv type by the very sensitive spf10-deia - lipa25 testing method .
hpv - related carcinogenesis at other anatomical sites indicates that hpv should be present in every tumor cell if it truly contributed to the development of that tumor ( 25 ) .
thus , whereas low - level hpv positivity in one tissue sample and complete lack of hpv in a separate sample may be possible , such tumor heterogeneity is unlikely to reflect a truly causal association .
on the basis of these results , the prevalence of hpv in lung tumor tissue from this population was essentially 0% .
a limitation of this study was that the tissue specimens were not necessarily adjacent or from the same block , although as described above , lack of uniform hpv results throughout the tumor would suggest that hpv was not associated with tumorigenesis . because tissue was collected for diverse purposes , it was not possible to address contamination in specimen collection .
we also used viral load to assess whether detected hpv dna was present at a meaningful level , as described above .
although dna degradation may occur in paraffin - embedded tissues , dna quality was confirmed by satisfactory 260/280 ratio and real - time pcr for the human actin gene and/or erv-3 in 399 of the 450 specimens .
these results indicate that the contamination precautions used during pcr analysis were largely sufficient to avoid contamination and that the dna from the paraffin - embedded tissues was adequate for hpv dna detection .
a particular strength of this study was that two independent laboratories extracted dna ( at the ohio state university using phenol  chloroform extraction and at ddl diagnostic laboratories using crude extraction methods , as described in  materials and methods  ) from different tissue specimens from the same patient and conducted separate pcr assays for hpv dna .
all 450 specimens were tested for the e6 and e7 oncogenes of hpv16 and hpv18 , the two types most strongly associated with cancer outside the cervix ( 25 ) , which circumvented concerns about false - negative results because of loss of the l1 gene through integration . a substantial proportion ( 92 specimens from 450 lung cancer patients )
was tested for a broad range of hpv types with the l1-based spf10-deia - lipa25 system .
the spf10-deia - lipa25 system is the gold standard for hpv dna testing in paraffin - embedded tissue because of its short pcr product ( 31 ) and ensured that specimens were tested for all carcinogenic and many noncarcinogenic hpv types .
geographic differences in the prevalence of hpv in lung tumor tissue may be associated with variation in smoking habits , sexual behaviors , or other factors related to environmental exposures , culture , topography , or genetics ( 10 ) .
asian studies typically report higher pcr - based hpv prevalences of hpv in lung tumors than european studies , with a summary meta - estimate of 11.6% ( 95% ci = 9.5% to 14.2% ) for hpv16 and 8.8% ( 95% ci = 6.0% to 12.8% ) for hpv18 in lung tumor tissues from asia compared with 3.5% ( 95% ci = 2.3% to 5.3% ) for hpv16 and
3.6% ( 95% ci = 2.3% to 5.7% ) for hpv18 in lung tumor tissues from europe ( 10 ) .
the largest pcr - based asian studies found hpv dna in 42.0% ( n = 92 specimens , 95% ci = 35.6% to 48.6% ) of 219 paraffin - embedded lung tumor tissues ( 33,34 ) and 44.1% ( n = 138 specimens , 95% ci = 38.5% to 49.8% ) of 313 fresh - frozen lung tumor tissues ( 10,35 ) .
similar to our study , the largest previous european pcr - based study from france found a very low prevalence of hpv dna in 218 fresh - frozen lung tumor tissues ( 1.8% prevalence , 95% ci = 0.7% to 4.8% , n = 4 positive samples ) ( 10,36 ) .
a study of lung cancer in taiwan found that nonsmoking female lung cancer patients were more likely to have hpv - positive lung tumor tissue than male lung cancer patients , who were more likely to smoke ( 37 ) .
environmental tobacco exposure is unlikely to account entirely for the increased risk in never - smokers .
non  smoking - related factors must therefore contribute to lung cancer among never - smokers .
given the high upper confidence limit of our prevalence estimate in never - smokers ( 12.8% ) , further study of hpv in lung tumor tissue from never - smokers may be warranted .
for example , the pcr - based prevalence of hpv in lung tumor tissues from japan ranges from 0.0% to 78.3% ( 10 ) .
although we found essentially no hpv in lung tumor tissues from italy , others have reported 12.8%21.1% hpv dna positivity in italian lung cancer patients ( 10 ) .
such disparities within the same geographic region emphasize the importance of taking precautions to avoid pcr contamination and prevent false - positive results ( 38 ) when using sensitive hpv dna detection assays to avoid false - negative results .
our study used such precautions in two independent laboratories , verified dna quality before hpv detection , and used multiple sensitive methods for hpv dna detection .
although a few previous asian and european studies have found 0% prevalence of hpv in lung tumor tissues , these studies included less than 100 patients .
with nearly 400 patients evaluated , we had a sufficient sample size to detect any true prevalence of hpv greater than 1% .
our study was twice as large as the next largest study in europe ( n = 218 specimens ) . in conclusion , using multiple state - of - the - art methods to evaluate the presence of hpv dna in resected lung cancer tumors from a representative western study population , we found no evidence that hpv is associated with the development of lung cancer .
although we detected essentially no hpv in specimens from the eagle study , evaluation in a larger population of never - smokers , in which the attributable risk of non  smoking - related risk factors is necessarily higher , may be informative .
differences in smoking habits could potentially account for the higher prevalence of hpv dna detected in lung tumor tissue from asian countries . for western populations , however , this study found no data to support that hpv is associated with lung carcinogenesis .
this work was supported by an intramural research award from the division of cancer epidemiology and genetics , national cancer institute at the national institutes of health , and a national cancer institute director s innovation award , national cancer institute at the national institutes of health to j.k . ;
general funds from the intramural research program of the national cancer institute at the national institutes of health ; and the cancer prevention fellowship program , office of preventive oncology , national cancer institute at the national institutes of health , bethesda , md .
control study and cleared the article for publication but had no role in the analysis , decision to publish , or preparation of the article .
 backgroundlung cancer kills more than 1 million people worldwide each year . whereas 
 several human papillomavirus ( hpv)associated cancers have been identified , the role of hpv in lung carcinogenesis remains controversial.methodswe selected 450 lung cancer patients from an italian population  based case  control study , the environment and genetics in lung cancer etiology . 
 these patients were selected from those with an adequate number of unstained tissue sections and included all those who had never smoked and a random sample of the remaining patients . 
 we used real - time polymerase chain reaction ( pcr ) to test specimens from these patients for hpv dna , specifically for e6 gene sequences from hpv16 and e7 gene sequences from hpv18 . 
 we also tested a subset of 92 specimens from all never - smokers and a random selection of smokers for additional hpv types by a pcr - based test for at least 54 mucosal hpv genotypes . 
 dna was extracted from ethanol- or formalin - fixed paraffin - embedded tumor tissue under strict pcr clean conditions . 
 the prevalence of hpv in tumor tissue was investigated.resultsspecimens from 399 of 450 patients had adequate dna for analysis . 
 most patients were current ( 220 patients or 48.9% ) smokers , and 92 patients ( 20.4% ) were women . when hpv16 and hpv18 type  
 specific primers were used , two specimens were positive for hpv16 at low copy number but were negative on additional type - specific hpv16 testing . 
 neither these specimens nor the others examined for a broad range of hpv types were positive for any hpv type.conclusionswhen dna contamination was avoided and state - of - the - art highly sensitive hpv dna detection assays were used , we found no evidence that hpv was associated with lung cancer in a representative western population . 
 our results provide the strongest evidence to date to rule out a role for hpv in lung carcinogenesis in western populations . 
CONTEXT AND CAVEATS
Prior knowledge
Study design
Contribution
Implications
Limitations
Materials and Methods
Study Population
HPV DNA Testing
Type-Specific PCR for HPV16 and HPV18.
Broad-Spectrum HPV Typing.
Statistical Analysis
Results
Discussion
Funding
Supplementary Material