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Assessment of Human Papillomavirus in Lung Tumor Tissue

Assessment of Human Papillomavirus in Lung Tumor Tissue DOI: 10.1093/jnci/djr003 Published by Oxford University Press 2011. Advance Access publication on February 3, 2011. This is an Open Access article distributed under the terms of the Creative Com mons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ARTICLE Jill Koshiol, Melissa Rotunno, Maura L. Gillison, Leen-Jan Van Doorn, Anil K. Chaturvedi, Letizia Tarantini, Hebin Song, Wim G. V. Quint, Linda Struijk, Alisa M. Goldstein, Allan Hildesheim, Philip R. Taylor, Sholom Wacholder, Pietro Alberto Bertazzi, Maria Teresa Landi, Neil E. Caporaso Manuscript received July 6, 2010; revised December 13, 2010; accepted December 30, 2010. Correspondence to: Jill Koshiol, PhD, Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Executive Blvd, Rm 7070, Bethesda, MD 20892-7248 (e-mail: koshiolj@mail.nih.gov). Background Lung 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. Methods We 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. Results Specimens 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. Conclusions When 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. J Natl Cancer Inst 2011;103:501–507 More than 1 million people die of lung cancer each year (1). integration into the cellular host DNA. Although geographic dif- Although smoking is the major etiologic factor, most smokers will ferences in the prevalence of HPV could potentially explain some not develop lung cancer (2), indicating that additional cofactors are variability in study results, interlaboratory variability and inade- needed for lung carcinogenesis. Lung cancer is also a major cause quate sample collection and handling leading to contamination may of cancer deaths even among never-smokers (3). also contribute to the variability in reported results (10). Therefore, Certain types of human papillomavirus (HPV) are r fi mly estab - the role of HPV in lung carcinogenesis remains unclear. lished as human carcinogens. These HPV types cause essentially all Part of the reason why HPV is hypothesized to be associated human cervical cancers (4,5) and are strongly implicated in the with lung carcinogenesis is because the respiratory tract is in close etiology of a substantial proportion of other anogenital cancers (6) proximity to the oropharynx, where HPV is known to cause a and oropharyngeal cancers (7,8). HPV has been hypothesized to substantial proportion of cancers (7,8). HPV has been detected in play a role in lung cancer pathogenesis, largely on the basis of more respiratory papillomas, which are sometimes found in the bronchi than 50 studies (9,10) in which HPV genomic DNA was detected and occasionally progress to malignancy, and also has been in 0%–100% (median = 16.7%) of lung tumor tissues. However, detected in respiratory tumors, as noted above (9,10). In addition, these studies are not den fi itive because of power constraints, re - HPV can transform bronchial cells in vitro (11). stricted study populations, use of nonspecic fi and/or insensitive Furthermore, cervical cancer survivors and immunosuppressed laboratory approaches, and use of HPV detection techniques that populations are at increased risk for lung cancer and established are limited to regions of the viral genome that may be lost with viral HPV-related malignancies (12–14). Although smoking is an jnci.oxfordjournals.org JNCI | Articles 501 The goal of our study was to complete a more definitive evalu - C O N T E X T A N D C A V E A T S ation of HPV in lung cancer. We tested for the presence of HPV DNA with highly sensitive polymerase chain reaction (PCR) and Prior knowledge took rigorous precautions against PCR contamination in tumor Human papillomavirus (HPV) is associated with cervical and oro- tissues from 450 lung cancer patients from the well-characterized pharyngeal cancers. A role for HPV in lung cancer has been pro- posed, although previous reports of HPV in lung tumor tissue have population–based case–control study, Environment and Genetics been inconclusive. in Lung Cancer Etiology (EAGLE). Study design Paraffin-embedded tissue specimens from 450 lung cancer patients Materials and Methods who were smokers or never-smokers enrolled in the Environment Study Population and Genetics in Lung Cancer Etiology study were evaluated for EAGLE is a large population-based case–control study with 2100 HPV DNA by real-time polymerase chain reaction to determine the HPV prevalence in tissues from lung cancer patients in a represen- lung cancer patients that was designed to investigate the genetic tative Western population. and environmental determinants of lung cancer and smoking per- sistence (22). As previously described (22), individuals with newly Contribution diagnosed lung cancer were recruited from April 22, 2002, to Two specimens were found to be HPV positive at low copy number February 28, 2005, from 13 hospitals in the Lombardy region of but were negative on additional genotype-specific testing and on Italy (see Notes). Each participant provided written informed con- testing for a broad spectrum of HPV types. The study found no evidence to support an association between HPV and lung cancer. sent. The study was approved by the institutional review board of each participating hospital and university in Italy and by the Implications National Cancer Institute, Bethesda, MD. Lung cancer was diag- HPV is not associated with lung carcinogenesis in lung cancer nosed by standard clinical criteria and confirmed by pathology patients from a representative Western population. reports from surgery, biopsy, or cytology samples (approximately Limitations 95% of patients) or through clinical history and imaging (approx- Multiple tissue specimens were used from each patient for analysis imately 5% of patients) (22). Tumor histology was classified by the and were not necessarily adjacent to or from the same paraffin 1999 World Health Organization Histological Typing of Lung block. Also, the possible contamination of specimens during col- and Pleural Tumors (23). The 13 Italian hospitals treat approxi- lection could not be determined. Because of geographic variances mately 80% of all patients with newly diagnosed lung cancer in the in the prevalence of HPV reported in lung tumors, further studies in area, which includes more than 1.3 million people aged 35–79 non-Western populations should be completed. years from five cities and surrounding towns and villages. EAGLE From the Editors had a participation rate of 86.6% among lung cancer patients. Thus, participants in the EAGLE study should be broadly repre- sentative of lung cancer patients in Western populations. We decided to test 450 adult male and female lung cancer established cofactor for cervical squamous cell carcinoma, it is not patients, including all 30 never-smokers and a random sample of associated with an increased risk of cervical adenocarcinoma (15). 420 smokers who had an adequate number of unstained tissue However, cervical adenocarcinoma survivors have approximately slides and at least one slide available that was stained with hema- twofold increased risk of lung cancer (16). In addition, HIV– toxylin and eosin. These stained slides were reviewed by H. Song AIDS patients, who have an increased risk of HPV and HPV- to establish the presence and estimate proportion of tumor tissue related malignancies (17), also have an increased risk of lung in the sample, but these slides were not necessarily adjacent to the cancer that has not been completely accounted for by increased tissue that was tested for HPV DNA. The samples were formalin smoking (13). These epidemiological data are consistent with a fixed for 399 patients and ethanol fixed for 51 patients. hypothesis that HPV contributes to respiratory carcinogenesis in a smoking-independent manner. HPV DNA Testing A difficulty with the hypothesis that HPV is a lung carcinogen All 450 patients were tested for HPV16 and HPV18 DNA because is the fact that HPV is not generally transmitted as an aerosol, these are the most common types found in cervical cancer (24) and making transmission to the lung difficult (18). However, the exis - account for the overwhelming majority of noncervical HPV– tence of rare recurrent respiratory papillomatosis demonstrates associated cancers (25). Type-specific PCR for HPV16 and that HPV can reach the respiratory tract. In addition, recurrent HPV18 was conducted in a laboratory at the Ohio State University, respiratory papillomatosis shows a second peak in young adults as described below. For a subset of 100 patients including all aged approximately 20 years (19) that is associated with oral sex never-smokers and a random selection of smokers, a second tissue and number of lifetime sex partners (20), consistent with sexual specimen was sent to DDL Diagnostic Laboratory (Voorburg, the transmission. The oral cavity, in which HPV infections are estab- Netherlands) for broad-spectrum HPV typing. lished largely through sexual transmission (21), may act as a reser- voir for transmission of HPV to the respiratory tract, possibly Type-Specific PCR for HPV16 and HPV18. All specimen receipt, through aspiration or mucosal transfer. The probability of such processing, and pre-amplification analysis procedures were transmission may be exceedingly low, however, considering the performed in ultraviolet light–irradiated laminar flow hoods in a rarity of recurrent respiratory papillomatosis. 502 Articles | JNCI Vol. 103, Issue 6 | March 16, 2011 laboratory with positive-pressure ventilation. All equipment and diploid genome equivalents of purified genomic DNA from tumor reagents were specific to the pre-amplification analysis laboratory samples that were evaluated for HPV16 and HPV18 DNA by real- with strict one-way transport of specimens from this laboratory to time PCR. All samples were tested once, and positive samples were a physically separate postamplification analysis laboratory. retested in duplicate. Although DNA was degraded because of Additional protocols to prevent specimen contamination included fixation, the mean yield was 54.5 ng/ mL. This subset analysis indi- sample processing in small batches, change of gloves after DNA cated that DNA integrity was sufficient to support amplification of extraction of each sample, exclusive use of barrier pipette tips, targets with amplicon size between approximately 58 and 135 bp. weekly cleaning of all laboratory surfaces and equipment with 70% The 58-bp ERV3 fragment was amplie fi d for the 431 remaining ethanol, and soaking of all racks and PCR plate holders in 10% specimens. DNA specimens with at least 100 evaluable cell equiva- bleach for a minimum of 10 minutes between each experiment. lents and a 260/280 ratio between 1.5 and 2.2 (27) were considered Additionally, one negative control per seven experimental samples to have adequate DNA quantity and quality for further analysis. was included in each column of each 96-well PCR plate. Type-specific TaqMan real-time PCR targeted to the DNA was extracted from 450 formalin- or ethanol-fixed paraffin- oncogenic E6 region of the genome for HPV16 (amplicon size = embedded tissue samples by means of paraffin removal with 201 bp) and the E7 region of the genome for HPV18 (amplicon octane, proteinase K digestion, phenol–chloroform extraction, and size = 137 bp) were performed in a 96-well plate format as previously ethanol precipitation. Briefly, after octane exposure, the sample described (28). To account for DNA degradation in formalin-fixed was washed with 100% ethanol, resuspended in 200 µL of diges- and paraffin-embedded tissues, all samples were also evaluated by tion buffer (50 mM Tris–HCl at pH 8.5, 1 mM EDTA, and pro- means of a TaqMan real-time PCR assay designed to amplify an teinase K at 20 µg/mL), and incubated for 48 hours at 55°C. If 81-bp amplicon in the E6 region of the genome for HPV16. tissue remained, 10 µL of proteinase K (400 µg/mL) was added, Primers and probes were manufactured at IDT Technologies and digestion was continued for an additional 24–48 hours. DNA (Coralville, IA) and sequences were: forward, 5′-GAGAAC was extracted in phenol–chloroform by use of Qiagen MaXtract TGCAATGTTTCAGGACC-3′; reverse, 5′-TGTATAGTTGT tubes (Qiagen, Germantown, MD) and ethanol precipitated in the TTGCAGCTCTGTGC-3′; and probe, 5′-56-FAMCAGGAGC presence of glycogen (0.02 mg/mL). Pellets were washed once in GACCCAGAAAGTTACCACAGTT-3BHQ1-3′. Standard curves 70% ethanol, dried at 37°C in a dry incubator, and resuspended in were generated in duplicate by use of a fivefold dilution series from 50 µL of diethylene pyrocarbonate-treated water. DNA quantity 2.5 × 10 copies of plasmid vector containing full-length viral and purity (calculated by use of the ratio of the absorbance at genome (pGEM HPV16) in a background of human placental 260 nm to that at 280 nm [260/280 ratio]) were measured with the DNA (5 ng/µL). Reactions that were performed in an Applied Nanodrop spectrophotometer (Thermo Fisher Scientific, Inc , Biosystems 7300 PCR system contained 2X TaqMan universal Wilmington, DE). PCR master mix (Applied Biosystems, Foster City, CA), 0.1 µmol An estimate of the number of cells analyzed in each PCR was of probe, 0.2 µmol of each primer, and 2 µL of purified tumor made by custom TaqMan real-time PCR assays targeting a single- DNA. Amplification conditions included a 12-minute incubation copy human gene on chromosome 7, human endogenous retrovi- at 95°C, followed by 50 cycles of 15 seconds at 95°C and rus 3 (ERV3) (26). In a pilot study of 19 specimens, DNA integrity 60 seconds at 60°C. The cycle threshold (CT) of unknown samples was evaluated through amplification of ERV3 sequences with was determined from an equation derived from a linear regression primers generating an amplicon of 58 , 135, or 354 base pairs (bp) through the log CT of the standard curve according to the manu- in size (Supplementary Table 1, available online). Primer and facturer’s recommendations. For positive samples, HPV viral load probe sequences for the 58-bp amplicon were as follows: forward was normalized to ERV3 copy number by real-time PCR, as a primer, 5′-GATAATTTCACACTAACCGCC-3′; reverse primer, measure of viral copy number per cell analyzed. Samples with any 5′-AGATGCTCTGACTTGATGGT-3′; and the probe sequence, detectable HPV16 or HPV18 DNA were tested in duplicate, 5′-56-FAM-CTCTTCCCTCGAACCTGC-3BHQ1-3′ (where with a mean viral load of greater than one viral copy per sample 56-FAM is 5′6-carboxy-fluorescein and 3BHQ1 is 3-Black Hole classified as real-time PCR positive. A mean viral load of greater Quencher-1). Primer and probe sequences for the 135-bp ampli- than or equal to one viral copy per cell was considered indicative con were as follows: forward primer, 5′-CATGGGAAGCAAG of a clonal viral–tumor relationship. GGAACTA-3′; reverse primer, 5′-CCCAGCGAGCAATACAG AATTT-3′; and probe, 5′-56-FAM-TCTTCCCTCGAACCTG Broad-Spectrum HPV Typing. Specimens from all 30 never- CACCATCAAGTCA-3BHQ1-3′. Primer and probe sequences smokers and 70 randomly sampled current and former smokers for the 354-bp amplicon were as follows: forward primer, were selected to test for broad-spectrum HPV typing at DDL 5′-CATGGGAAGCAAGGGAACTAATG-3′; reverse primer, Diagnostic Laboratory. HPV typing used PCR with short PCR 5′-GCCCACAGATCCAGTAGAGG-3; and probe were the same fragment (SPF ) primers, which targets the HPV L1 gene, and a as for the 135-bp amplicon above. Reaction conditions were as mixture of conservative probes that recognize at least 54 mucosal previously published (26). Briefly, purified genomic DNA (2 µL) HPV genotypes. We used the DNA enzyme immunoassay (DEIA) from lung cancer specimens was analyzed. A standard curve was to determine HPV DNA positivity by detecting amplimers, syn- generated in duplicate from a fivefold dilution series (from 150 000 thesized by biotinylated PCR primers, through hybridization to a to 1.92 cells) of a diploid human cell line, CCD-18LU (catalog mixture of HPV-specific probes, as described previously (29). number CCL-205, ATCC [American Type Culture Collection], Stringent PCR contamination precautions that were similar to the Manassas, VA). Results were reported as the number of human type-specific testing for HPV16 and HPV18 conducted at the jnci.oxfordjournals.org JNCI | Articles 503 Statistical Analysis Ohio State University were taken. All pre-amplification proce- Our goal in determining sample size was to be able to rule out a dures were performed in ultraviolet light–irradiated laminar flow true HPV prevalence greater than 1.0%. Using cii in Stata hoods that were separate from a postamplification analysis labora- (StataCorp LP, College Station, TX), we determined that zero tory. During DNA isolation, PCR amplification, and post-PCR HPV positives among 450 lung cancer patients would generate a analyses, specific negative (water blank) and positive (HPV DNA one-sided 97.5% confidence interval (CI) of 0% to 0.82% and that extracted from the HPV18-positive HeLa cell line) controls were one observed HPV-positive patient among 450 lung cancer included. We monitored potential contamination of samples with patients would provide a two-sided 95% confidence interval of negative controls and the sensitivity of the procedure with low- 0.0056% to 1.2%; zero positives among 400 lung cancer patients concentration positive controls. All reagents had been prepared in would provide a one-sided 97.5% confidence interval of 0% to a separate reagent laboratory and subjected to strict quality control 0.92%, and one observed HPV-positive patient would provide a to confirm efficacy and the absence of contaminants. two-sided 95% confidence interval of 0.0063% to 1.4%. The DNA amplification by PCR with SPF primers was followed chance of finding at least one positive when the true prevalence is by a reverse hybridization line-probe assay (LiPA , version 1; 1.0% or more was 98.9% with a sample size of 450 patients; thus, Labo Bio-Medical Products, Rijswijk, the Netherlands) for geno- finding no positives with a sensitive assay rules out even a low typing of 25 HPV types in SPF - and DEIA-positive samples. prevalence of infection with high confidence. These primers produce very short PCR fragments of approxi- In addition to the sample size calculations described above, we mately 65 bp, making them ideal for amplification in formalin- used the cii command in Stata (StataCorp LP ), version 9.0, to fixed specimens (29–31). Eight specimens did not appear to have estimate HPV prevalence and corresponding confidence tumors in the tissue specimen received for testing. DNA was intervals. 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 Results with a swab after confirming the presence of tumor cells in that Among the 450 lung cancer patients, 246 (54.7%) had adenocarci- part of the section. This part of the tissue was transferred to a noma and 137 (30.4%) had squamous cell carcinoma (Table 1). microtube (Sarstedt, Etten-Leur, the Netherlands) and digested Most patients had a history of tobacco smoking: 220 patients with proteinase K buffer (45 mM Tris–HCl at pH 8, 0.9 mM (48.9%) were current smokers, 198 patients (44.0%) were former EDTA, 0.45% Tween 20, and proteinase K at 1 mg/mL). smokers, and 30 patients (6.7%) were never-smokers. The median Proteinase K solution (100 µL) was added to each microtube and age at diagnosis of lung cancer was 67.6 years (range = 35.4–79.9 incubated for 16–24 hours at 56°C. Proteinase K solution was years), and the median body mass index was 25.3 kg/m (range = inactivated by incubating the tubes at 95°C for 10 minutes. 15.9–60.8 kg/m ). Among smokers, the median duration of smoking Resulting DNA preparations were stored at 220°C. During this was 45 years (range = 1–70 years), the median average smoking process, negative and positive DNA isolation controls were in- intensity was one pack per day (range = 0–3 packs per day), the cluded. In these samples, DNA quality and quantity were adequate median pack-years was 43.8 (range = 0.1–192 pack-years), and the as measured by real-time PCR for the human b-actin gene in the median age at initiation of smoking was 16 years of age (range = same DNA specimens as used for the SPF PCR. For one patient 6–45 years). Ninety-two patients (20.4%) were women. The ma- (a current smoker) where additional investigation was warranted jority of patients had less than a high school education (72.5% or due to weak HPV16 positivity in the sample tested at the Ohio 319 patients), drank alcohol (84.3% or 365 patients), were married State University, type-specific PCR for HPV16 was performed or cohabitating (82.2% or 356 patients), had abnormal respiratory with a 92-bp amplicon as previously described (32). Table 1. 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 HPV DNA status by PCR* testing, Smoking status, No. (%) No. (% HPV positive) Type-specific Broad-spectrum Lung cancer histology Never Former Current Unknown HPV16 and HPV18† HPV typing‡ Adenocarcinoma (n = 246) 25 (83.3) 103 (52.0) 117 (53.2) 1 (50.0) 221 (0.0) 63 (0.0) Squamous cell (n = 137) 2 (6.7) 68 (34.3) 66 (30.0) 1 (50.0) 123 (0.0) 27 (0.0) Large cell (n = 30) 1 (3.3) 10 (5.1) 19 (8.6) 0 (0.0) 24 (0.0) 0 (0.0) Small cell (n = 13) 0 (0.0) 5 (2.5) 8 (3.6) 0 (0.0) 11 (0.0) 0 (0.0) Other (n = 24)§ 2 (6.7) 12 (6.1) 10 (4.5) 0 (0.0) 20 (0.0) 2 (0.0) * 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). 504 Articles | JNCI Vol. 103, Issue 6 | March 16, 2011 function (51.7% or 186 patients), and had stage IIB disease or less Among the 399 specimens with adequate DNA, one female (71.3% or 321 patients) (Table 2). former smoker with adenocarcinoma was positive for HPV16 on The median number of cells evaluable for the HPV16 and initial testing. Given the very low viral load in this tumor (average HPV18 PCR as measured by ERV3 real-time PCR was 3680 cells = 0.4 per 1000 cell equivalents across three tests of the same (range = 1.9–399 750 cells). One specimen was negative for ERV3. sample), we tested another tumor tissue specimen from this The median 260/280 ratio was 1.63 (range = 1.23–1.94). Combining patient. We found no evidence of HPV16 or HPV18 DNA in this these data, 399 (88.7%) of 450 specimens had adequate DNA second specimen. The hematoxylin–eosin slide from this specimen quantity and quality for analysis (ie, 100 or more cell equivalents consisted of greater than 80% cancer cells, indicating there was and a 260/280 ratio between 1.5 and 2.2). On review of the 450 adequate tumor available to detect any HPV DNA if it were slides that were stained with hematoxylin and eosin, 412 specimens present. This patient was diagnosed with stage IB lung cancer at contained tumor cells. Of those tumor-positive specimens, 73.8% 72 years of age and had smoked for 48 years at an average intensity (n = 304 specimens) contained greater than 60% tumor. We in- of 0.75 packs per day (36 pack-years). cluded all specimens with adequate DNA (n = 399 specimens) in PCR analysis targeting an 81-bp amplicon in the HPV16 E6 the prevalence estimate. 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 Table 2. Descriptive characteristics of 450 lung cancer patients patient who was initially negative for HPV16 tested positive with from the population-based case–control Environment and this assay but at low copy number (131 per 1000 cell equivalents). Genetics in Lung cancer Etiology study* This second patient was a male with squamous cell carcinoma No. of patients diagnosed at 64 years of age who had smoked for 43 years at an Characteristic (% of total) average intensity of one pack per day (43 pack-years). The hema- Age, y toxylin–eosin slide from this patient also contained greater than <60 93 (20.7) 80% cancer cells. 60–69 181 (40.2) In the subset of 92 specimens tested with the broad-spectrum ≥70 176 (39.1) assay SPF -DEIA-LiPA (including those from the two patients 10 25 Sex Male 358 (79.6) who tested positive with low viral load, as described above), no Female 92 (20.4) specimens were positive for HPV16 DNA or for any HPV type. Area of Italy DDL Diagnostic Laboratory conducted type-specific testing for Brescia 72 (16.0) HPV16 as a follow-up test for the male patient identified as being Milano 268 (59.6) HPV positive by PCR analysis targeting an 81-bp amplicon in the Monza 54 (12.0) Pavia 22 (4.9) HPV16 E6 region. The specimen tested negative for HPV DNA Varese 34 (7.6) with this type-specific test. These results indicate that the preva - Education† lence of HPV DNA is 0.0% (Table 1) with an upper 97.5% confi - None 27 (6.1) dence interval of 0.92% for the 399 specimens with adequate Elementary school 161 (36.6) DNA, including 370 ever-smokers (upper 97.5% confidence Middle school 131 (29.8) High school (teaching training, job training, 105 (23.9) bound = 0.99%) and 27 never-smokers (upper 97.5% confidence college training) bound = 12.8%). University degree (bachelor, master, PhD) 16 (3.6) Alcohol consumption† No 68 (15.7) Discussion Yes 365 (84.3) Marital status† In this study, which is to our knowledge the largest study of HPV Married or cohabitating 356 (82.2) in lung tumor tissue from a Western country, we found no evi- Neither married nor cohabitating 77 (17.8) dence that HPV is associated with lung carcinogenesis. Extensive Spirometry-based respiratory function† laboratory efforts to avoid DNA contamination and state-of-the- Normal 174 (48.3) art, highly sensitive HPV DNA detection assays were performed. Mild COPD 51 (14.2) Moderate COPD 68 (18.9) The two tumors that tested positive had a very low viral load of less Severe and very severe COPD 17 (4.7) than one copy of HPV16 per cell, despite being composed of Potentially restrictive disease 50 (13.9) greater than 80% tumor cells. One of these patients tested nega- Stage tive in a separate tissue specimen, and the other was negative on IA 84 (18.7) additional type-specific HPV16 testing in a separate sample. None IB 126 (28.0) IIA 14 (3.1) of the subset of 92 patients was positive for any other HPV type by IIB 97 (21.6) the very sensitive SPF -DEIA-LiPA testing method. HPV- 10 25 IIIA 58 (12.9) related carcinogenesis at other anatomical sites indicates that HPV IIIB 43 (9.6) should be present in every tumor cell if it truly contributed to the IV 28 (6.2) development of that tumor (25). Thus, whereas low-level HPV * COPD = chronic obstructive pulmonary disease. positivity in one tissue sample and complete lack of HPV in a sep- † Numbers do not sum to total because of missing values. arate sample may be possible, such tumor heterogeneity is unlikely jnci.oxfordjournals.org JNCI | Articles 505 to reflect a truly causal association. On the basis of these results, found that nonsmoking female lung cancer patients were more the prevalence of HPV in lung tumor tissue from this population likely to have HPV-positive lung tumor tissue than male lung was essentially 0%. cancer patients, who were more likely to smoke (37). Environmental A limitation of this study was that the tissue specimens were not tobacco exposure is unlikely to account entirely for the increased necessarily adjacent or from the same block, although as described risk in never-smokers. Non–smoking-related factors must there- above, lack of uniform HPV results throughout the tumor would fore contribute to lung cancer among never-smokers. Given the suggest that HPV was not associated with tumorigenesis. Because high upper confidence limit of our prevalence estimate in never- tissue was collected for diverse purposes, it was not possible to smokers (12.8%), further study of HPV in lung tumor tissue from address contamination in specimen collection. However, all labo- never-smokers may be warranted. ratory assays were performed under stringent precautions to avoid Nevertheless, the prevalence of HPV can vary markedly within contamination. We also used viral load to assess whether detected the same country. For example, the PCR-based prevalence of HPV DNA was present at a meaningful level, as described above. HPV in lung tumor tissues from Japan ranges from 0.0% to 78.3% Although DNA degradation may occur in paraffin-embedded tis - (10). Although we found essentially no HPV in lung tumor tissues sues, DNA quality was confirmed by satisfactory 260/280 ratio and from Italy, others have reported 12.8%–21.1% HPV DNA posi- real-time PCR for the human b–actin gene and/or ERV-3 in 399 tivity in Italian lung cancer patients (10). Such disparities within of the 450 specimens. These results indicate that the contamina- the same geographic region emphasize the importance of taking tion precautions used during PCR analysis were largely sufficient precautions to avoid PCR contamination and prevent false-posi- to avoid contamination and that the DNA from the paraffin- tive results (38) when using sensitive HPV DNA detection assays embedded tissues was adequate for HPV DNA detection. to avoid false-negative results. Our study used such precautions in A particular strength of this study was that two independent two independent laboratories, verified DNA quality before HPV laboratories extracted DNA (at the Ohio State University using detection, and used multiple sensitive methods for HPV DNA phenol–chloroform extraction and at DDL Diagnostic Laboratories detection. Although a few previous Asian and European studies using crude extraction methods, as described in “Materials and have found 0% prevalence of HPV in lung tumor tissues, these Methods”) from different tissue specimens from the same patient studies included less than 100 patients. With nearly 400 patients and conducted separate PCR assays for HPV DNA. All 450 spec- evaluated, we had a sufficient sample size to detect any true preva - imens were tested for the E6 and E7 oncogenes of HPV16 and lence of HPV greater than 1%. Our study was twice as large as the HPV18, the two types most strongly associated with cancer out- next largest study in Europe (n = 218 specimens). side the cervix (25), which circumvented concerns about false- In conclusion, using multiple state-of-the-art methods to eval- negative results because of loss of the L1 gene through integration. uate the presence of HPV DNA in resected lung cancer tumors A substantial proportion (92 specimens from 450 lung cancer from a representative Western study population, we found no evi- patients) was tested for a broad range of HPV types with the dence that HPV is associated with the development of lung cancer. L1-based SPF -DEIA-LiPA system. The SPF -DEIA-LiPA 10 25 10 25 Although we detected essentially no HPV in specimens from the system is the gold standard for HPV DNA testing in paraffin- EAGLE study, evaluation in a larger population of never-smokers, embedded tissue because of its short PCR product (31) and in which the attributable risk of non–smoking-related risk factors ensured that specimens were tested for all carcinogenic and many is necessarily higher, may be informative. Differences in smoking noncarcinogenic HPV types. habits could potentially account for the higher prevalence of HPV Geographic differences in the prevalence of HPV in lung DNA detected in lung tumor tissue from Asian countries. For tumor tissue may be associated with variation in smoking habits, Western populations, however, this study found no data to support sexual behaviors, or other factors related to environmental expo- that HPV is associated with lung carcinogenesis. sures, culture, topography, or genetics (10). Asian studies typically References report higher PCR-based HPV prevalences of HPV in lung 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA tumors than European studies, with a summary meta-estimate of Cancer J Clin. 2005;55(2):74–108. 11.6% (95% CI = 9.5% to 14.2%) for HPV16 and 8.8% (95% 2. Thun MJ, Henley SJ, Calle EE. Tobacco use and cancer: an epidemiologic CI = 6.0% to 12.8%) for HPV18 in lung tumor tissues from Asia perspective for geneticists. Oncogene. 2002;21(48):7307–7325. compared with 3.5% (95% CI = 2.3% to 5.3%) for HPV16 and 3. Sun S, Schiller JH, Gazdar AF. Lung cancer in never smokers—a different 3.6% (95% CI = 2.3% to 5.7%) for HPV18 in lung tumor tissues disease. Nat Rev Cancer. 2007;7(10):778–790. 4. Bosch FX, de Sanjose S. The epidemiology of human papillomavirus in- from Europe (10). The largest PCR-based Asian studies found fection and cervical cancer. Dis Markers. 2007;23(4):213–227. HPV DNA in 42.0% (n = 92 specimens, 95% CI = 35.6% to 5. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is 48.6%) of 219 paraffin-embedded lung tumor tissues (33,34) and a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999; 44.1% (n = 138 specimens, 95% CI = 38.5% to 49.8%) of 313 189(1):12–19. fresh-frozen lung tumor tissues (10,35). Similar to our study, the 6. Munoz N, Castellsague X, de Gonzalez AB, Gissmann L. Chapter 1: HPV in the etiology of human cancer. Vaccine. 2006;24S3(23):S1–S10. largest previous European PCR-based study from France found a 7. Kreimer AR, Clifford GM, Boyle P, Franceschi S. Human papillomavirus very low prevalence of HPV DNA in 218 fresh-frozen lung tumor types in head and neck squamous cell carcinomas worldwide: a systematic tissues (1.8% prevalence, 95% CI = 0.7% to 4.8%, n = 4 positive review. Cancer Epidemiol Biomarkers Prev. 2005;14(2):467–475. samples) (10,36). Part of this geographic discrepancy may be 8. Marur S, D’Souza G, Westra WH, Forastiere AA. HPV-associated head because of differences in smoking habits. For example, Asian and neck cancer: a virus-related cancer epidemic. Lancet Oncol. women typically do not smoke. A study of lung cancer in Taiwan 2010;11(8):781–789. 506 Articles | JNCI Vol. 103, Issue 6 | March 16, 2011 9. Giuliani L, Favalli C, Syrjanen K, Ciotti M. Human papillomavirus infec- 31. Gravitt PE, Viscidi R. Chapter 5: measurement of exposure to human tions in lung cancer. Detection of E6 and E7 transcripts and review of the papillomaviruses. In: Rohan TE, Shah KV, eds. Cervical Cancer: From literature. Anticancer Res. 2007;27(4C):2697–2704. Etiology to Prevention (Cancer Prevention-Cancer Causes). Boston, MA: 10. Srinivasan M, Taioli E, Ragin CC. Human papillomavirus type 16 and 18 Kluwer Academic Publishers; 2004:119–141. in primary lung cancers—a meta-analysis. Carcinogenesis. 2009;30(10): 32. van Doorn LJ, Molijn A, Kleter B, Quint W, Colau B. Highly effective 1722–1728. detection of human papillomavirus 16 and 18 DNA by a testing algorithm 11. Willey JC, Broussoud A, Sleemi A, et al. Immortalization of normal combining broad-spectrum and type-specific PCR. J Clin Microbiol. human bronchial epithelial cells by human papillomaviruses 16 or 18. 2006;44(9):3292–3298. Cancer Res. 1991;51(19):5370–5377. 33. Miyagi J, Kinjo T, Tsuhako K, et al. Extremely high Langerhans cell 12. Caporaso NE, Dodd KW, Tucker MA, et al. Chapter 6: new malignancies infiltration contributes to the favourable prognosis of HPV-infected following cancer of the respiratory tract. In: Curtis RE, Freedman DM, squamous cell carcinoma and adenocarcinoma of the lung. Histopathology. Ron E, eds. New Malignancies Among Cancer Survivors: SEER Cancer 2001;38(4):355–367. Registries, 1973-2000. Bethesda, MD: National Cancer Institute; 34. Miyagi J, Tsuhako K, Kinjo T, Iwamasa T, Hirayasu T. Recent striking 2006:145–179. NIH Publication 05-5302. changes in histological differentiation and rate of human papillomavirus 13. Chaturvedi AK, Pfeiffer RM, Chang L, et al. Elevated risk of lung cancer infection in squamous cell carcinoma of the lung in Okinawa, a subtropical among people with AIDS. AIDS. 2007;21(2):207–213. island in southern Japan. J Clin Pathol. 2000;53(9):676–684. 14. Kalliala I, Dyba T, Nieminen P, Hakulinen T, Anttila A. Mortality in a 35. Wang Y, Wang A, Jiang R, et al. Human papillomavirus type 16 and 18 long-term follow-up after treatment of CIN. Int J Cancer. 2010;126(1): infection is associated with lung cancer patients from the central part of 224–231. China. Oncol Rep. 2008;20(2):333–339. 15. International Collaboration of Epidemiological Studies of Cervical 36. Coissard CJ, Besson G, Polette MC, et al. Prevalence of human papillo- Cancer. Comparison of risk factors for invasive squamous cell carcinoma maviruses in lung carcinomas: a study of 218 cases. Mod Pathol. 2005; and adenocarcinoma of the cervix: collaborative reanalysis of individual 18(12):1606–1609. data on 8,097 women with squamous cell carcinoma and 1,374 women 37. Cheng YW, Chiou HL, Sheu GT, et al. The association of human papil- with adenocarcinoma from 12 epidemiological studies. Int J Cancer. 2007; lomavirus 16/18 infection with lung cancer among nonsmoking Taiwanese 120(4):885–891. women. Cancer Res. 2001;61(7):2799–2803. 16. Chaturvedi AK, Kleinerman RA, Hildesheim A, et al. Second cancers after 38. Koshiol J, Kreimer AR. Lessons from Australia: HPV is not a major risk squamous cell carcinoma and adenocarcinoma of the cervix. J Clin Oncol. factor for esophageal squamous cell carcinoma. Cancer Epidemiol Biomarkers 2009;27(6):967–973. Prev. 2010;19(8):1889–1892. 17. Heard I. Prevention of cervical cancer in women with HIV. Curr Opin HIV AIDS. 2009;4(1):68–73. Funding 18. Gillison ML, Shah KV. Chapter 9: role of mucosal human papillomavirus This work was supported by an Intramural Research Award from the Division of in nongenital cancers. J Natl Cancer Inst Monogr. 2003;31):57–65. Cancer Epidemiology and Genetics, National Cancer Institute at the National 19. Shykhon M, Kuo M, Pearman K. Recurrent respiratory papillomatosis. Institutes of Health, and a National Cancer Institute Director’s Innovation Clin Otolaryngol Allied Sci. 2002;27(4):237–243. Award, National Cancer Institute at the National Institutes of Health to J.K.; 20. Kashima HK, Shah F, Lyles A, et al. A comparison of risk factors in General Funds from the Intramural Research Program of the National Cancer juvenile-onset and adult-onset recurrent respiratory papillomatosis. Institute at the National Institutes of Health; and the Cancer Prevention Laryngoscope. 1992;102(1):9–13. Fellowship Program, Office of Preventive Oncology, National Cancer Institute 21. Gillison ML. Current topics in the epidemiology of oral cavity and oro- at the National Institutes of Health, Bethesda, MD. The Division of Cancer pharyngeal cancers. Head Neck. 2007;29(8):779–792. Epidemiology and Genetics reviewed and approved the EAGLE case–control 22. Landi MT, Consonni D, Rotunno M, et al. Environment And Genetics in study and cleared the article for publication but had no role in the analysis, Lung cancer Etiology (EAGLE) study: an integrative population-based decision to publish, or preparation of the article. case-control study of lung cancer. BMC Public Health. 2008;8:203–213. 23. Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Histological Notes Typing of Lung and Pleural Tumors. 3rd ed. Berlin, Germany: Springer; We thank the 13 hospitals that contributed to this study: A.O. Ospedale Niguarda Cà Granda, Milano; A.O. Spedali Civili, Brescia; Istituto Clinico 24. Smith JS, Lindsay L, Hoots B, et al. Human papillomavirus type Humanitas, Rozzano (Milano); Ospedale di Circolo e Fondazione Macchi, distribution in invasive cervical cancer and high-grade cervical lesions: a Varese; Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e meta-analysis update. Int J Cancer. 2007;121(3):621–632. Regina Elena, Milano; Istituto Scientifico Universitario Ospedale San Raffaele, 25. Gillison ML, Chaturvedi AK, Lowy DR. HPV prophylactic vaccines and Milano; A.O. Ospedale Luigi Sacco, Milano; A.O. San Paolo, Milano; A.O. the potential prevention of noncervical cancers in both men and women. Ospedale San Carlo Borromeo, Milano; IRCCS Policlinico San Matteo, Pavia; Cancer. 2008;113(10 suppl):3036–3046. A.O. San Gerardo, Monza; A.O. Ospedale Fatebenefratelli e Oftalmico, Milano; 26. Yuan CC, Miley W, Waters D. A quantification of human cells using an Ospedale San Giuseppe, Milano as listed on the EAGLE Web site (http://eagle. ERV-3 real time PCR assay. J Virol Methods. 2001;91(2):109–117. cancer.gov/, accessed January 14, 2011.). 27. Baay MF, Verhoeven V, Lambrechts HA, et al. Feasibility of collecting self-sampled vaginal swabs by mail: quantity and quality of genomic DNA. Eur J Clin Microbiol Infect Dis. 2009;28(11):1285–1289. 28. Gravitt PE, Peyton C, Wheeler C, et al. Reproducibility of HPV 16 and Affiliations of authors: Division of Cancer Epidemiology and Genetics, NCI/ HPV 18 viral load quantitation using TaqMan real-time PCR assays. NIH/DHHS, Bethesda, MD (JK, MR, AKC, AMG, AH, PRT, SW, MTL, NEC); J Virol Methods. 2003;112(1–2):23–33. Department of Internal Medicine, Ohio State University Comprehensive 29. Kleter B, van Doorn LJ, ter Schegget J, et al. Novel short-fragment PCR Cancer Center–James Cancer Hospital and Solove Research Institute, The assay for highly sensitive broad-spectrum detection of anogenital human Ohio State University, Columbus, OH (MLG, HS); DDL Diagnostic Laboratory, papillomaviruses. Am J Pathol. 1998;153(6):1731–1739. Voorburg, the Netherlands (L-JVD, WGVQ, LS); EPOCA Research Center, 30. Kleter B, van Doorn LJ, Schrauwen L, et al. Development and clinical Department of Occupational and Environmental Health, Università degli evaluation of a highly sensitive PCR-reverse hybridization line probe assay Studi di Milano, Milan, Italy (LT, PAB); Epidemiology Unit, Fondazione IRCCS for detection and identification of anogenital human papillomavirus. J Clin Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy (LT, Microbiol. 1999;37(8):2508–2517. PAB). jnci.oxfordjournals.org JNCI | Articles 507 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JNCI Journal of the National Cancer Institute Pubmed Central

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Published by Oxford University Press 2011.
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10.1093/jnci/djr003
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Abstract

DOI: 10.1093/jnci/djr003 Published by Oxford University Press 2011. Advance Access publication on February 3, 2011. This is an Open Access article distributed under the terms of the Creative Com mons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ARTICLE Jill Koshiol, Melissa Rotunno, Maura L. Gillison, Leen-Jan Van Doorn, Anil K. Chaturvedi, Letizia Tarantini, Hebin Song, Wim G. V. Quint, Linda Struijk, Alisa M. Goldstein, Allan Hildesheim, Philip R. Taylor, Sholom Wacholder, Pietro Alberto Bertazzi, Maria Teresa Landi, Neil E. Caporaso Manuscript received July 6, 2010; revised December 13, 2010; accepted December 30, 2010. Correspondence to: Jill Koshiol, PhD, Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Executive Blvd, Rm 7070, Bethesda, MD 20892-7248 (e-mail: koshiolj@mail.nih.gov). Background Lung 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. Methods We 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. Results Specimens 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. Conclusions When 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. J Natl Cancer Inst 2011;103:501–507 More than 1 million people die of lung cancer each year (1). integration into the cellular host DNA. Although geographic dif- Although smoking is the major etiologic factor, most smokers will ferences in the prevalence of HPV could potentially explain some not develop lung cancer (2), indicating that additional cofactors are variability in study results, interlaboratory variability and inade- needed for lung carcinogenesis. Lung cancer is also a major cause quate sample collection and handling leading to contamination may of cancer deaths even among never-smokers (3). also contribute to the variability in reported results (10). Therefore, Certain types of human papillomavirus (HPV) are r fi mly estab - the role of HPV in lung carcinogenesis remains unclear. lished as human carcinogens. These HPV types cause essentially all Part of the reason why HPV is hypothesized to be associated human cervical cancers (4,5) and are strongly implicated in the with lung carcinogenesis is because the respiratory tract is in close etiology of a substantial proportion of other anogenital cancers (6) proximity to the oropharynx, where HPV is known to cause a and oropharyngeal cancers (7,8). HPV has been hypothesized to substantial proportion of cancers (7,8). HPV has been detected in play a role in lung cancer pathogenesis, largely on the basis of more respiratory papillomas, which are sometimes found in the bronchi than 50 studies (9,10) in which HPV genomic DNA was detected and occasionally progress to malignancy, and also has been in 0%–100% (median = 16.7%) of lung tumor tissues. However, detected in respiratory tumors, as noted above (9,10). In addition, these studies are not den fi itive because of power constraints, re - HPV can transform bronchial cells in vitro (11). stricted study populations, use of nonspecic fi and/or insensitive Furthermore, cervical cancer survivors and immunosuppressed laboratory approaches, and use of HPV detection techniques that populations are at increased risk for lung cancer and established are limited to regions of the viral genome that may be lost with viral HPV-related malignancies (12–14). Although smoking is an jnci.oxfordjournals.org JNCI | Articles 501 The goal of our study was to complete a more definitive evalu - C O N T E X T A N D C A V E A T S ation of HPV in lung cancer. We tested for the presence of HPV DNA with highly sensitive polymerase chain reaction (PCR) and Prior knowledge took rigorous precautions against PCR contamination in tumor Human papillomavirus (HPV) is associated with cervical and oro- tissues from 450 lung cancer patients from the well-characterized pharyngeal cancers. A role for HPV in lung cancer has been pro- posed, although previous reports of HPV in lung tumor tissue have population–based case–control study, Environment and Genetics been inconclusive. in Lung Cancer Etiology (EAGLE). Study design Paraffin-embedded tissue specimens from 450 lung cancer patients Materials and Methods who were smokers or never-smokers enrolled in the Environment Study Population and Genetics in Lung Cancer Etiology study were evaluated for EAGLE is a large population-based case–control study with 2100 HPV DNA by real-time polymerase chain reaction to determine the HPV prevalence in tissues from lung cancer patients in a represen- lung cancer patients that was designed to investigate the genetic tative Western population. and environmental determinants of lung cancer and smoking per- sistence (22). As previously described (22), individuals with newly Contribution diagnosed lung cancer were recruited from April 22, 2002, to Two specimens were found to be HPV positive at low copy number February 28, 2005, from 13 hospitals in the Lombardy region of but were negative on additional genotype-specific testing and on Italy (see Notes). Each participant provided written informed con- testing for a broad spectrum of HPV types. The study found no evidence to support an association between HPV and lung cancer. sent. The study was approved by the institutional review board of each participating hospital and university in Italy and by the Implications National Cancer Institute, Bethesda, MD. Lung cancer was diag- HPV is not associated with lung carcinogenesis in lung cancer nosed by standard clinical criteria and confirmed by pathology patients from a representative Western population. reports from surgery, biopsy, or cytology samples (approximately Limitations 95% of patients) or through clinical history and imaging (approx- Multiple tissue specimens were used from each patient for analysis imately 5% of patients) (22). Tumor histology was classified by the and were not necessarily adjacent to or from the same paraffin 1999 World Health Organization Histological Typing of Lung block. Also, the possible contamination of specimens during col- and Pleural Tumors (23). The 13 Italian hospitals treat approxi- lection could not be determined. Because of geographic variances mately 80% of all patients with newly diagnosed lung cancer in the in the prevalence of HPV reported in lung tumors, further studies in area, which includes more than 1.3 million people aged 35–79 non-Western populations should be completed. years from five cities and surrounding towns and villages. EAGLE From the Editors had a participation rate of 86.6% among lung cancer patients. Thus, participants in the EAGLE study should be broadly repre- sentative of lung cancer patients in Western populations. We decided to test 450 adult male and female lung cancer established cofactor for cervical squamous cell carcinoma, it is not patients, including all 30 never-smokers and a random sample of associated with an increased risk of cervical adenocarcinoma (15). 420 smokers who had an adequate number of unstained tissue However, cervical adenocarcinoma survivors have approximately slides and at least one slide available that was stained with hema- twofold increased risk of lung cancer (16). In addition, HIV– toxylin and eosin. These stained slides were reviewed by H. Song AIDS patients, who have an increased risk of HPV and HPV- to establish the presence and estimate proportion of tumor tissue related malignancies (17), also have an increased risk of lung in the sample, but these slides were not necessarily adjacent to the cancer that has not been completely accounted for by increased tissue that was tested for HPV DNA. The samples were formalin smoking (13). These epidemiological data are consistent with a fixed for 399 patients and ethanol fixed for 51 patients. hypothesis that HPV contributes to respiratory carcinogenesis in a smoking-independent manner. HPV DNA Testing A difficulty with the hypothesis that HPV is a lung carcinogen All 450 patients were tested for HPV16 and HPV18 DNA because is the fact that HPV is not generally transmitted as an aerosol, these are the most common types found in cervical cancer (24) and making transmission to the lung difficult (18). However, the exis - account for the overwhelming majority of noncervical HPV– tence of rare recurrent respiratory papillomatosis demonstrates associated cancers (25). Type-specific PCR for HPV16 and that HPV can reach the respiratory tract. In addition, recurrent HPV18 was conducted in a laboratory at the Ohio State University, respiratory papillomatosis shows a second peak in young adults as described below. For a subset of 100 patients including all aged approximately 20 years (19) that is associated with oral sex never-smokers and a random selection of smokers, a second tissue and number of lifetime sex partners (20), consistent with sexual specimen was sent to DDL Diagnostic Laboratory (Voorburg, the transmission. The oral cavity, in which HPV infections are estab- Netherlands) for broad-spectrum HPV typing. lished largely through sexual transmission (21), may act as a reser- voir for transmission of HPV to the respiratory tract, possibly Type-Specific PCR for HPV16 and HPV18. All specimen receipt, through aspiration or mucosal transfer. The probability of such processing, and pre-amplification analysis procedures were transmission may be exceedingly low, however, considering the performed in ultraviolet light–irradiated laminar flow hoods in a rarity of recurrent respiratory papillomatosis. 502 Articles | JNCI Vol. 103, Issue 6 | March 16, 2011 laboratory with positive-pressure ventilation. All equipment and diploid genome equivalents of purified genomic DNA from tumor reagents were specific to the pre-amplification analysis laboratory samples that were evaluated for HPV16 and HPV18 DNA by real- with strict one-way transport of specimens from this laboratory to time PCR. All samples were tested once, and positive samples were a physically separate postamplification analysis laboratory. retested in duplicate. Although DNA was degraded because of Additional protocols to prevent specimen contamination included fixation, the mean yield was 54.5 ng/ mL. This subset analysis indi- sample processing in small batches, change of gloves after DNA cated that DNA integrity was sufficient to support amplification of extraction of each sample, exclusive use of barrier pipette tips, targets with amplicon size between approximately 58 and 135 bp. weekly cleaning of all laboratory surfaces and equipment with 70% The 58-bp ERV3 fragment was amplie fi d for the 431 remaining ethanol, and soaking of all racks and PCR plate holders in 10% specimens. DNA specimens with at least 100 evaluable cell equiva- bleach for a minimum of 10 minutes between each experiment. lents and a 260/280 ratio between 1.5 and 2.2 (27) were considered Additionally, one negative control per seven experimental samples to have adequate DNA quantity and quality for further analysis. was included in each column of each 96-well PCR plate. Type-specific TaqMan real-time PCR targeted to the DNA was extracted from 450 formalin- or ethanol-fixed paraffin- oncogenic E6 region of the genome for HPV16 (amplicon size = embedded tissue samples by means of paraffin removal with 201 bp) and the E7 region of the genome for HPV18 (amplicon octane, proteinase K digestion, phenol–chloroform extraction, and size = 137 bp) were performed in a 96-well plate format as previously ethanol precipitation. Briefly, after octane exposure, the sample described (28). To account for DNA degradation in formalin-fixed was washed with 100% ethanol, resuspended in 200 µL of diges- and paraffin-embedded tissues, all samples were also evaluated by tion buffer (50 mM Tris–HCl at pH 8.5, 1 mM EDTA, and pro- means of a TaqMan real-time PCR assay designed to amplify an teinase K at 20 µg/mL), and incubated for 48 hours at 55°C. If 81-bp amplicon in the E6 region of the genome for HPV16. tissue remained, 10 µL of proteinase K (400 µg/mL) was added, Primers and probes were manufactured at IDT Technologies and digestion was continued for an additional 24–48 hours. DNA (Coralville, IA) and sequences were: forward, 5′-GAGAAC was extracted in phenol–chloroform by use of Qiagen MaXtract TGCAATGTTTCAGGACC-3′; reverse, 5′-TGTATAGTTGT tubes (Qiagen, Germantown, MD) and ethanol precipitated in the TTGCAGCTCTGTGC-3′; and probe, 5′-56-FAMCAGGAGC presence of glycogen (0.02 mg/mL). Pellets were washed once in GACCCAGAAAGTTACCACAGTT-3BHQ1-3′. Standard curves 70% ethanol, dried at 37°C in a dry incubator, and resuspended in were generated in duplicate by use of a fivefold dilution series from 50 µL of diethylene pyrocarbonate-treated water. DNA quantity 2.5 × 10 copies of plasmid vector containing full-length viral and purity (calculated by use of the ratio of the absorbance at genome (pGEM HPV16) in a background of human placental 260 nm to that at 280 nm [260/280 ratio]) were measured with the DNA (5 ng/µL). Reactions that were performed in an Applied Nanodrop spectrophotometer (Thermo Fisher Scientific, Inc , Biosystems 7300 PCR system contained 2X TaqMan universal Wilmington, DE). PCR master mix (Applied Biosystems, Foster City, CA), 0.1 µmol An estimate of the number of cells analyzed in each PCR was of probe, 0.2 µmol of each primer, and 2 µL of purified tumor made by custom TaqMan real-time PCR assays targeting a single- DNA. Amplification conditions included a 12-minute incubation copy human gene on chromosome 7, human endogenous retrovi- at 95°C, followed by 50 cycles of 15 seconds at 95°C and rus 3 (ERV3) (26). In a pilot study of 19 specimens, DNA integrity 60 seconds at 60°C. The cycle threshold (CT) of unknown samples was evaluated through amplification of ERV3 sequences with was determined from an equation derived from a linear regression primers generating an amplicon of 58 , 135, or 354 base pairs (bp) through the log CT of the standard curve according to the manu- in size (Supplementary Table 1, available online). Primer and facturer’s recommendations. For positive samples, HPV viral load probe sequences for the 58-bp amplicon were as follows: forward was normalized to ERV3 copy number by real-time PCR, as a primer, 5′-GATAATTTCACACTAACCGCC-3′; reverse primer, measure of viral copy number per cell analyzed. Samples with any 5′-AGATGCTCTGACTTGATGGT-3′; and the probe sequence, detectable HPV16 or HPV18 DNA were tested in duplicate, 5′-56-FAM-CTCTTCCCTCGAACCTGC-3BHQ1-3′ (where with a mean viral load of greater than one viral copy per sample 56-FAM is 5′6-carboxy-fluorescein and 3BHQ1 is 3-Black Hole classified as real-time PCR positive. A mean viral load of greater Quencher-1). Primer and probe sequences for the 135-bp ampli- than or equal to one viral copy per cell was considered indicative con were as follows: forward primer, 5′-CATGGGAAGCAAG of a clonal viral–tumor relationship. GGAACTA-3′; reverse primer, 5′-CCCAGCGAGCAATACAG AATTT-3′; and probe, 5′-56-FAM-TCTTCCCTCGAACCTG Broad-Spectrum HPV Typing. Specimens from all 30 never- CACCATCAAGTCA-3BHQ1-3′. Primer and probe sequences smokers and 70 randomly sampled current and former smokers for the 354-bp amplicon were as follows: forward primer, were selected to test for broad-spectrum HPV typing at DDL 5′-CATGGGAAGCAAGGGAACTAATG-3′; reverse primer, Diagnostic Laboratory. HPV typing used PCR with short PCR 5′-GCCCACAGATCCAGTAGAGG-3; and probe were the same fragment (SPF ) primers, which targets the HPV L1 gene, and a as for the 135-bp amplicon above. Reaction conditions were as mixture of conservative probes that recognize at least 54 mucosal previously published (26). Briefly, purified genomic DNA (2 µL) HPV genotypes. We used the DNA enzyme immunoassay (DEIA) from lung cancer specimens was analyzed. A standard curve was to determine HPV DNA positivity by detecting amplimers, syn- generated in duplicate from a fivefold dilution series (from 150 000 thesized by biotinylated PCR primers, through hybridization to a to 1.92 cells) of a diploid human cell line, CCD-18LU (catalog mixture of HPV-specific probes, as described previously (29). number CCL-205, ATCC [American Type Culture Collection], Stringent PCR contamination precautions that were similar to the Manassas, VA). Results were reported as the number of human type-specific testing for HPV16 and HPV18 conducted at the jnci.oxfordjournals.org JNCI | Articles 503 Statistical Analysis Ohio State University were taken. All pre-amplification proce- Our goal in determining sample size was to be able to rule out a dures were performed in ultraviolet light–irradiated laminar flow true HPV prevalence greater than 1.0%. Using cii in Stata hoods that were separate from a postamplification analysis labora- (StataCorp LP, College Station, TX), we determined that zero tory. During DNA isolation, PCR amplification, and post-PCR HPV positives among 450 lung cancer patients would generate a analyses, specific negative (water blank) and positive (HPV DNA one-sided 97.5% confidence interval (CI) of 0% to 0.82% and that extracted from the HPV18-positive HeLa cell line) controls were one observed HPV-positive patient among 450 lung cancer included. We monitored potential contamination of samples with patients would provide a two-sided 95% confidence interval of negative controls and the sensitivity of the procedure with low- 0.0056% to 1.2%; zero positives among 400 lung cancer patients concentration positive controls. All reagents had been prepared in would provide a one-sided 97.5% confidence interval of 0% to a separate reagent laboratory and subjected to strict quality control 0.92%, and one observed HPV-positive patient would provide a to confirm efficacy and the absence of contaminants. two-sided 95% confidence interval of 0.0063% to 1.4%. The DNA amplification by PCR with SPF primers was followed chance of finding at least one positive when the true prevalence is by a reverse hybridization line-probe assay (LiPA , version 1; 1.0% or more was 98.9% with a sample size of 450 patients; thus, Labo Bio-Medical Products, Rijswijk, the Netherlands) for geno- finding no positives with a sensitive assay rules out even a low typing of 25 HPV types in SPF - and DEIA-positive samples. prevalence of infection with high confidence. These primers produce very short PCR fragments of approxi- In addition to the sample size calculations described above, we mately 65 bp, making them ideal for amplification in formalin- used the cii command in Stata (StataCorp LP ), version 9.0, to fixed specimens (29–31). Eight specimens did not appear to have estimate HPV prevalence and corresponding confidence tumors in the tissue specimen received for testing. DNA was intervals. 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 Results with a swab after confirming the presence of tumor cells in that Among the 450 lung cancer patients, 246 (54.7%) had adenocarci- part of the section. This part of the tissue was transferred to a noma and 137 (30.4%) had squamous cell carcinoma (Table 1). microtube (Sarstedt, Etten-Leur, the Netherlands) and digested Most patients had a history of tobacco smoking: 220 patients with proteinase K buffer (45 mM Tris–HCl at pH 8, 0.9 mM (48.9%) were current smokers, 198 patients (44.0%) were former EDTA, 0.45% Tween 20, and proteinase K at 1 mg/mL). smokers, and 30 patients (6.7%) were never-smokers. The median Proteinase K solution (100 µL) was added to each microtube and age at diagnosis of lung cancer was 67.6 years (range = 35.4–79.9 incubated for 16–24 hours at 56°C. Proteinase K solution was years), and the median body mass index was 25.3 kg/m (range = inactivated by incubating the tubes at 95°C for 10 minutes. 15.9–60.8 kg/m ). Among smokers, the median duration of smoking Resulting DNA preparations were stored at 220°C. During this was 45 years (range = 1–70 years), the median average smoking process, negative and positive DNA isolation controls were in- intensity was one pack per day (range = 0–3 packs per day), the cluded. In these samples, DNA quality and quantity were adequate median pack-years was 43.8 (range = 0.1–192 pack-years), and the as measured by real-time PCR for the human b-actin gene in the median age at initiation of smoking was 16 years of age (range = same DNA specimens as used for the SPF PCR. For one patient 6–45 years). Ninety-two patients (20.4%) were women. The ma- (a current smoker) where additional investigation was warranted jority of patients had less than a high school education (72.5% or due to weak HPV16 positivity in the sample tested at the Ohio 319 patients), drank alcohol (84.3% or 365 patients), were married State University, type-specific PCR for HPV16 was performed or cohabitating (82.2% or 356 patients), had abnormal respiratory with a 92-bp amplicon as previously described (32). Table 1. 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 HPV DNA status by PCR* testing, Smoking status, No. (%) No. (% HPV positive) Type-specific Broad-spectrum Lung cancer histology Never Former Current Unknown HPV16 and HPV18† HPV typing‡ Adenocarcinoma (n = 246) 25 (83.3) 103 (52.0) 117 (53.2) 1 (50.0) 221 (0.0) 63 (0.0) Squamous cell (n = 137) 2 (6.7) 68 (34.3) 66 (30.0) 1 (50.0) 123 (0.0) 27 (0.0) Large cell (n = 30) 1 (3.3) 10 (5.1) 19 (8.6) 0 (0.0) 24 (0.0) 0 (0.0) Small cell (n = 13) 0 (0.0) 5 (2.5) 8 (3.6) 0 (0.0) 11 (0.0) 0 (0.0) Other (n = 24)§ 2 (6.7) 12 (6.1) 10 (4.5) 0 (0.0) 20 (0.0) 2 (0.0) * 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). 504 Articles | JNCI Vol. 103, Issue 6 | March 16, 2011 function (51.7% or 186 patients), and had stage IIB disease or less Among the 399 specimens with adequate DNA, one female (71.3% or 321 patients) (Table 2). former smoker with adenocarcinoma was positive for HPV16 on The median number of cells evaluable for the HPV16 and initial testing. Given the very low viral load in this tumor (average HPV18 PCR as measured by ERV3 real-time PCR was 3680 cells = 0.4 per 1000 cell equivalents across three tests of the same (range = 1.9–399 750 cells). One specimen was negative for ERV3. sample), we tested another tumor tissue specimen from this The median 260/280 ratio was 1.63 (range = 1.23–1.94). Combining patient. We found no evidence of HPV16 or HPV18 DNA in this these data, 399 (88.7%) of 450 specimens had adequate DNA second specimen. The hematoxylin–eosin slide from this specimen quantity and quality for analysis (ie, 100 or more cell equivalents consisted of greater than 80% cancer cells, indicating there was and a 260/280 ratio between 1.5 and 2.2). On review of the 450 adequate tumor available to detect any HPV DNA if it were slides that were stained with hematoxylin and eosin, 412 specimens present. This patient was diagnosed with stage IB lung cancer at contained tumor cells. Of those tumor-positive specimens, 73.8% 72 years of age and had smoked for 48 years at an average intensity (n = 304 specimens) contained greater than 60% tumor. We in- of 0.75 packs per day (36 pack-years). cluded all specimens with adequate DNA (n = 399 specimens) in PCR analysis targeting an 81-bp amplicon in the HPV16 E6 the prevalence estimate. 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 Table 2. Descriptive characteristics of 450 lung cancer patients patient who was initially negative for HPV16 tested positive with from the population-based case–control Environment and this assay but at low copy number (131 per 1000 cell equivalents). Genetics in Lung cancer Etiology study* This second patient was a male with squamous cell carcinoma No. of patients diagnosed at 64 years of age who had smoked for 43 years at an Characteristic (% of total) average intensity of one pack per day (43 pack-years). The hema- Age, y toxylin–eosin slide from this patient also contained greater than <60 93 (20.7) 80% cancer cells. 60–69 181 (40.2) In the subset of 92 specimens tested with the broad-spectrum ≥70 176 (39.1) assay SPF -DEIA-LiPA (including those from the two patients 10 25 Sex Male 358 (79.6) who tested positive with low viral load, as described above), no Female 92 (20.4) specimens were positive for HPV16 DNA or for any HPV type. Area of Italy DDL Diagnostic Laboratory conducted type-specific testing for Brescia 72 (16.0) HPV16 as a follow-up test for the male patient identified as being Milano 268 (59.6) HPV positive by PCR analysis targeting an 81-bp amplicon in the Monza 54 (12.0) Pavia 22 (4.9) HPV16 E6 region. The specimen tested negative for HPV DNA Varese 34 (7.6) with this type-specific test. These results indicate that the preva - Education† lence of HPV DNA is 0.0% (Table 1) with an upper 97.5% confi - None 27 (6.1) dence interval of 0.92% for the 399 specimens with adequate Elementary school 161 (36.6) DNA, including 370 ever-smokers (upper 97.5% confidence Middle school 131 (29.8) High school (teaching training, job training, 105 (23.9) bound = 0.99%) and 27 never-smokers (upper 97.5% confidence college training) bound = 12.8%). University degree (bachelor, master, PhD) 16 (3.6) Alcohol consumption† No 68 (15.7) Discussion Yes 365 (84.3) Marital status† In this study, which is to our knowledge the largest study of HPV Married or cohabitating 356 (82.2) in lung tumor tissue from a Western country, we found no evi- Neither married nor cohabitating 77 (17.8) dence that HPV is associated with lung carcinogenesis. Extensive Spirometry-based respiratory function† laboratory efforts to avoid DNA contamination and state-of-the- Normal 174 (48.3) art, highly sensitive HPV DNA detection assays were performed. Mild COPD 51 (14.2) Moderate COPD 68 (18.9) The two tumors that tested positive had a very low viral load of less Severe and very severe COPD 17 (4.7) than one copy of HPV16 per cell, despite being composed of Potentially restrictive disease 50 (13.9) greater than 80% tumor cells. One of these patients tested nega- Stage tive in a separate tissue specimen, and the other was negative on IA 84 (18.7) additional type-specific HPV16 testing in a separate sample. None IB 126 (28.0) IIA 14 (3.1) of the subset of 92 patients was positive for any other HPV type by IIB 97 (21.6) the very sensitive SPF -DEIA-LiPA testing method. HPV- 10 25 IIIA 58 (12.9) related carcinogenesis at other anatomical sites indicates that HPV IIIB 43 (9.6) should be present in every tumor cell if it truly contributed to the IV 28 (6.2) development of that tumor (25). Thus, whereas low-level HPV * COPD = chronic obstructive pulmonary disease. positivity in one tissue sample and complete lack of HPV in a sep- † Numbers do not sum to total because of missing values. arate sample may be possible, such tumor heterogeneity is unlikely jnci.oxfordjournals.org JNCI | Articles 505 to reflect a truly causal association. On the basis of these results, found that nonsmoking female lung cancer patients were more the prevalence of HPV in lung tumor tissue from this population likely to have HPV-positive lung tumor tissue than male lung was essentially 0%. cancer patients, who were more likely to smoke (37). Environmental A limitation of this study was that the tissue specimens were not tobacco exposure is unlikely to account entirely for the increased necessarily adjacent or from the same block, although as described risk in never-smokers. Non–smoking-related factors must there- above, lack of uniform HPV results throughout the tumor would fore contribute to lung cancer among never-smokers. Given the suggest that HPV was not associated with tumorigenesis. Because high upper confidence limit of our prevalence estimate in never- tissue was collected for diverse purposes, it was not possible to smokers (12.8%), further study of HPV in lung tumor tissue from address contamination in specimen collection. However, all labo- never-smokers may be warranted. ratory assays were performed under stringent precautions to avoid Nevertheless, the prevalence of HPV can vary markedly within contamination. We also used viral load to assess whether detected the same country. For example, the PCR-based prevalence of HPV DNA was present at a meaningful level, as described above. HPV in lung tumor tissues from Japan ranges from 0.0% to 78.3% Although DNA degradation may occur in paraffin-embedded tis - (10). Although we found essentially no HPV in lung tumor tissues sues, DNA quality was confirmed by satisfactory 260/280 ratio and from Italy, others have reported 12.8%–21.1% HPV DNA posi- real-time PCR for the human b–actin gene and/or ERV-3 in 399 tivity in Italian lung cancer patients (10). Such disparities within of the 450 specimens. These results indicate that the contamina- the same geographic region emphasize the importance of taking tion precautions used during PCR analysis were largely sufficient precautions to avoid PCR contamination and prevent false-posi- to avoid contamination and that the DNA from the paraffin- tive results (38) when using sensitive HPV DNA detection assays embedded tissues was adequate for HPV DNA detection. to avoid false-negative results. Our study used such precautions in A particular strength of this study was that two independent two independent laboratories, verified DNA quality before HPV laboratories extracted DNA (at the Ohio State University using detection, and used multiple sensitive methods for HPV DNA phenol–chloroform extraction and at DDL Diagnostic Laboratories detection. Although a few previous Asian and European studies using crude extraction methods, as described in “Materials and have found 0% prevalence of HPV in lung tumor tissues, these Methods”) from different tissue specimens from the same patient studies included less than 100 patients. With nearly 400 patients and conducted separate PCR assays for HPV DNA. All 450 spec- evaluated, we had a sufficient sample size to detect any true preva - imens were tested for the E6 and E7 oncogenes of HPV16 and lence of HPV greater than 1%. Our study was twice as large as the HPV18, the two types most strongly associated with cancer out- next largest study in Europe (n = 218 specimens). side the cervix (25), which circumvented concerns about false- In conclusion, using multiple state-of-the-art methods to eval- negative results because of loss of the L1 gene through integration. uate the presence of HPV DNA in resected lung cancer tumors A substantial proportion (92 specimens from 450 lung cancer from a representative Western study population, we found no evi- patients) was tested for a broad range of HPV types with the dence that HPV is associated with the development of lung cancer. L1-based SPF -DEIA-LiPA system. The SPF -DEIA-LiPA 10 25 10 25 Although we detected essentially no HPV in specimens from the system is the gold standard for HPV DNA testing in paraffin- EAGLE study, evaluation in a larger population of never-smokers, embedded tissue because of its short PCR product (31) and in which the attributable risk of non–smoking-related risk factors ensured that specimens were tested for all carcinogenic and many is necessarily higher, may be informative. Differences in smoking noncarcinogenic HPV types. habits could potentially account for the higher prevalence of HPV Geographic differences in the prevalence of HPV in lung DNA detected in lung tumor tissue from Asian countries. For tumor tissue may be associated with variation in smoking habits, Western populations, however, this study found no data to support sexual behaviors, or other factors related to environmental expo- that HPV is associated with lung carcinogenesis. sures, culture, topography, or genetics (10). Asian studies typically References report higher PCR-based HPV prevalences of HPV in lung 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA tumors than European studies, with a summary meta-estimate of Cancer J Clin. 2005;55(2):74–108. 11.6% (95% CI = 9.5% to 14.2%) for HPV16 and 8.8% (95% 2. Thun MJ, Henley SJ, Calle EE. Tobacco use and cancer: an epidemiologic CI = 6.0% to 12.8%) for HPV18 in lung tumor tissues from Asia perspective for geneticists. Oncogene. 2002;21(48):7307–7325. compared with 3.5% (95% CI = 2.3% to 5.3%) for HPV16 and 3. Sun S, Schiller JH, Gazdar AF. Lung cancer in never smokers—a different 3.6% (95% CI = 2.3% to 5.7%) for HPV18 in lung tumor tissues disease. Nat Rev Cancer. 2007;7(10):778–790. 4. Bosch FX, de Sanjose S. The epidemiology of human papillomavirus in- from Europe (10). The largest PCR-based Asian studies found fection and cervical cancer. Dis Markers. 2007;23(4):213–227. HPV DNA in 42.0% (n = 92 specimens, 95% CI = 35.6% to 5. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is 48.6%) of 219 paraffin-embedded lung tumor tissues (33,34) and a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999; 44.1% (n = 138 specimens, 95% CI = 38.5% to 49.8%) of 313 189(1):12–19. fresh-frozen lung tumor tissues (10,35). Similar to our study, the 6. Munoz N, Castellsague X, de Gonzalez AB, Gissmann L. Chapter 1: HPV in the etiology of human cancer. Vaccine. 2006;24S3(23):S1–S10. largest previous European PCR-based study from France found a 7. Kreimer AR, Clifford GM, Boyle P, Franceschi S. Human papillomavirus very low prevalence of HPV DNA in 218 fresh-frozen lung tumor types in head and neck squamous cell carcinomas worldwide: a systematic tissues (1.8% prevalence, 95% CI = 0.7% to 4.8%, n = 4 positive review. Cancer Epidemiol Biomarkers Prev. 2005;14(2):467–475. samples) (10,36). Part of this geographic discrepancy may be 8. Marur S, D’Souza G, Westra WH, Forastiere AA. HPV-associated head because of differences in smoking habits. For example, Asian and neck cancer: a virus-related cancer epidemic. Lancet Oncol. women typically do not smoke. A study of lung cancer in Taiwan 2010;11(8):781–789. 506 Articles | JNCI Vol. 103, Issue 6 | March 16, 2011 9. Giuliani L, Favalli C, Syrjanen K, Ciotti M. Human papillomavirus infec- 31. Gravitt PE, Viscidi R. Chapter 5: measurement of exposure to human tions in lung cancer. Detection of E6 and E7 transcripts and review of the papillomaviruses. In: Rohan TE, Shah KV, eds. Cervical Cancer: From literature. Anticancer Res. 2007;27(4C):2697–2704. Etiology to Prevention (Cancer Prevention-Cancer Causes). Boston, MA: 10. Srinivasan M, Taioli E, Ragin CC. Human papillomavirus type 16 and 18 Kluwer Academic Publishers; 2004:119–141. in primary lung cancers—a meta-analysis. Carcinogenesis. 2009;30(10): 32. van Doorn LJ, Molijn A, Kleter B, Quint W, Colau B. Highly effective 1722–1728. detection of human papillomavirus 16 and 18 DNA by a testing algorithm 11. Willey JC, Broussoud A, Sleemi A, et al. Immortalization of normal combining broad-spectrum and type-specific PCR. J Clin Microbiol. human bronchial epithelial cells by human papillomaviruses 16 or 18. 2006;44(9):3292–3298. Cancer Res. 1991;51(19):5370–5377. 33. Miyagi J, Kinjo T, Tsuhako K, et al. Extremely high Langerhans cell 12. Caporaso NE, Dodd KW, Tucker MA, et al. Chapter 6: new malignancies infiltration contributes to the favourable prognosis of HPV-infected following cancer of the respiratory tract. In: Curtis RE, Freedman DM, squamous cell carcinoma and adenocarcinoma of the lung. Histopathology. Ron E, eds. New Malignancies Among Cancer Survivors: SEER Cancer 2001;38(4):355–367. Registries, 1973-2000. Bethesda, MD: National Cancer Institute; 34. Miyagi J, Tsuhako K, Kinjo T, Iwamasa T, Hirayasu T. Recent striking 2006:145–179. NIH Publication 05-5302. changes in histological differentiation and rate of human papillomavirus 13. Chaturvedi AK, Pfeiffer RM, Chang L, et al. Elevated risk of lung cancer infection in squamous cell carcinoma of the lung in Okinawa, a subtropical among people with AIDS. AIDS. 2007;21(2):207–213. island in southern Japan. J Clin Pathol. 2000;53(9):676–684. 14. Kalliala I, Dyba T, Nieminen P, Hakulinen T, Anttila A. Mortality in a 35. Wang Y, Wang A, Jiang R, et al. Human papillomavirus type 16 and 18 long-term follow-up after treatment of CIN. Int J Cancer. 2010;126(1): infection is associated with lung cancer patients from the central part of 224–231. China. Oncol Rep. 2008;20(2):333–339. 15. International Collaboration of Epidemiological Studies of Cervical 36. Coissard CJ, Besson G, Polette MC, et al. Prevalence of human papillo- Cancer. Comparison of risk factors for invasive squamous cell carcinoma maviruses in lung carcinomas: a study of 218 cases. Mod Pathol. 2005; and adenocarcinoma of the cervix: collaborative reanalysis of individual 18(12):1606–1609. data on 8,097 women with squamous cell carcinoma and 1,374 women 37. Cheng YW, Chiou HL, Sheu GT, et al. The association of human papil- with adenocarcinoma from 12 epidemiological studies. Int J Cancer. 2007; lomavirus 16/18 infection with lung cancer among nonsmoking Taiwanese 120(4):885–891. women. Cancer Res. 2001;61(7):2799–2803. 16. Chaturvedi AK, Kleinerman RA, Hildesheim A, et al. Second cancers after 38. Koshiol J, Kreimer AR. Lessons from Australia: HPV is not a major risk squamous cell carcinoma and adenocarcinoma of the cervix. J Clin Oncol. factor for esophageal squamous cell carcinoma. Cancer Epidemiol Biomarkers 2009;27(6):967–973. Prev. 2010;19(8):1889–1892. 17. Heard I. Prevention of cervical cancer in women with HIV. Curr Opin HIV AIDS. 2009;4(1):68–73. Funding 18. Gillison ML, Shah KV. Chapter 9: role of mucosal human papillomavirus This work was supported by an Intramural Research Award from the Division of in nongenital cancers. J Natl Cancer Inst Monogr. 2003;31):57–65. Cancer Epidemiology and Genetics, National Cancer Institute at the National 19. Shykhon M, Kuo M, Pearman K. Recurrent respiratory papillomatosis. Institutes of Health, and a National Cancer Institute Director’s Innovation Clin Otolaryngol Allied Sci. 2002;27(4):237–243. Award, National Cancer Institute at the National Institutes of Health to J.K.; 20. Kashima HK, Shah F, Lyles A, et al. A comparison of risk factors in General Funds from the Intramural Research Program of the National Cancer juvenile-onset and adult-onset recurrent respiratory papillomatosis. Institute at the National Institutes of Health; and the Cancer Prevention Laryngoscope. 1992;102(1):9–13. Fellowship Program, Office of Preventive Oncology, National Cancer Institute 21. Gillison ML. Current topics in the epidemiology of oral cavity and oro- at the National Institutes of Health, Bethesda, MD. The Division of Cancer pharyngeal cancers. Head Neck. 2007;29(8):779–792. Epidemiology and Genetics reviewed and approved the EAGLE case–control 22. Landi MT, Consonni D, Rotunno M, et al. Environment And Genetics in study and cleared the article for publication but had no role in the analysis, Lung cancer Etiology (EAGLE) study: an integrative population-based decision to publish, or preparation of the article. case-control study of lung cancer. BMC Public Health. 2008;8:203–213. 23. Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Histological Notes Typing of Lung and Pleural Tumors. 3rd ed. Berlin, Germany: Springer; We thank the 13 hospitals that contributed to this study: A.O. Ospedale Niguarda Cà Granda, Milano; A.O. Spedali Civili, Brescia; Istituto Clinico 24. Smith JS, Lindsay L, Hoots B, et al. Human papillomavirus type Humanitas, Rozzano (Milano); Ospedale di Circolo e Fondazione Macchi, distribution in invasive cervical cancer and high-grade cervical lesions: a Varese; Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e meta-analysis update. Int J Cancer. 2007;121(3):621–632. Regina Elena, Milano; Istituto Scientifico Universitario Ospedale San Raffaele, 25. Gillison ML, Chaturvedi AK, Lowy DR. HPV prophylactic vaccines and Milano; A.O. Ospedale Luigi Sacco, Milano; A.O. San Paolo, Milano; A.O. the potential prevention of noncervical cancers in both men and women. Ospedale San Carlo Borromeo, Milano; IRCCS Policlinico San Matteo, Pavia; Cancer. 2008;113(10 suppl):3036–3046. A.O. San Gerardo, Monza; A.O. Ospedale Fatebenefratelli e Oftalmico, Milano; 26. Yuan CC, Miley W, Waters D. A quantification of human cells using an Ospedale San Giuseppe, Milano as listed on the EAGLE Web site (http://eagle. ERV-3 real time PCR assay. J Virol Methods. 2001;91(2):109–117. cancer.gov/, accessed January 14, 2011.). 27. Baay MF, Verhoeven V, Lambrechts HA, et al. Feasibility of collecting self-sampled vaginal swabs by mail: quantity and quality of genomic DNA. Eur J Clin Microbiol Infect Dis. 2009;28(11):1285–1289. 28. Gravitt PE, Peyton C, Wheeler C, et al. Reproducibility of HPV 16 and Affiliations of authors: Division of Cancer Epidemiology and Genetics, NCI/ HPV 18 viral load quantitation using TaqMan real-time PCR assays. NIH/DHHS, Bethesda, MD (JK, MR, AKC, AMG, AH, PRT, SW, MTL, NEC); J Virol Methods. 2003;112(1–2):23–33. Department of Internal Medicine, Ohio State University Comprehensive 29. Kleter B, van Doorn LJ, ter Schegget J, et al. Novel short-fragment PCR Cancer Center–James Cancer Hospital and Solove Research Institute, The assay for highly sensitive broad-spectrum detection of anogenital human Ohio State University, Columbus, OH (MLG, HS); DDL Diagnostic Laboratory, papillomaviruses. Am J Pathol. 1998;153(6):1731–1739. Voorburg, the Netherlands (L-JVD, WGVQ, LS); EPOCA Research Center, 30. Kleter B, van Doorn LJ, Schrauwen L, et al. Development and clinical Department of Occupational and Environmental Health, Università degli evaluation of a highly sensitive PCR-reverse hybridization line probe assay Studi di Milano, Milan, Italy (LT, PAB); Epidemiology Unit, Fondazione IRCCS for detection and identification of anogenital human papillomavirus. J Clin Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy (LT, Microbiol. 1999;37(8):2508–2517. PAB). jnci.oxfordjournals.org JNCI | Articles 507

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