No role for human papillomavirus in esophageal squamous cell carcinoma in China

No role for human papillomavirus in esophageal squamous cellcarcinoma in China

Jill Koshiol1,*, Wen-Qiang Wei2,*, Aimee R. Kreimer1, Wen Chen2, Patti Gravitt4,5, Jian-Song Ren2, Christian C. Abnet1, Jian-Bing Wang2, Farin Kamangar1,6, Dong-Mei Lin3,Magnus von Knebel-Doeberitz7, Yu Zhang2, Raphael Viscidi8, Guo-Qing Wang2, Maura L.Gillison4,5,9, Mark J. Roth1, Zhi-Wei Dong2, Esther Kim4,5,9, Philip R. Taylor1, You-LinQiao2, and Sanford M. Dawsey11Division of Cancer Epidemiology and Genetics, Department of Health and Human Services,National Cancer Institute, National Institutes of Health, Bethesda, MD2Department of Cancer Epidemiology, Cancer Institute, Peking Union Medical College, ChineseAcademy of Medical Sciences, Beijing, China3Department of Pathology, Cancer Institute, Peking Union Medical College, Chinese Academy ofMedical Sciences, Beijing, China4Department of Epidemiology, Johns Hopkins University, Bloomberg School of Public Health, TheJohns Hopkins University, Baltimore, MD5Department of Moleculor Microbiology and Immunology, Bloomberg School of Public Health, TheJohns Hopkins University, Baltimore, MD6Department of Public Health Analysis, School of Community Health and Policy, Morgan StateUniversity, Baltimore, MD7Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Germany8Department of Pediatrics, School of Medicine, The Johns Hopkins University, Baltimore, MD9Department of Internal Medicine, Ohio State University Comprehensive Cancer Center—JamesCancer Hospital and Solove Research Institute, Ohio State University, Columbus, OH

AbstractCertain regions of China have high rates of esophageal squamous cell carcinoma (ESCC).Previous studies of human papillomavirus (HPV), a proposed causal factor, have produced highlyvariable results. We attempted to evaluate HPV and ESCC more definitively using extreme care toprevent DNA contamination. We collected tissue and serum in China from 272histopathologically-confirmed ESCC cases with rigorous attention to good molecular biologytechnique. We tested for HPV DNA in fresh-frozen tumor tissue using PCR with PGMY L1consensus primers and HPV16 and 18 type-specific E6 and E7 primers, and in formalin-fixedparaffin-embedded tumor tissue using SPF10 L1 primers. In HPV-positive cases, we evaluatedp16INK4a overexpression and HPV E6/E7 seropositivity as evidence of carcinogenic HPV activity.

© 2009 UICCCorrespondence to: Jill Koshiol, PhD, Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics,National Cancer Institute, 6120 Executive Blvd, Room 7070, Rockville, MD 20852-7248, USA, Tel: 301-402-9508, Fax:301-402-0817, [email protected]; or You-Lin Qiao, MD, PhD, Department of Cancer Epidemiology, Cancer Institute, ChineseAcademy of Medical Sciences, Beijing 100021, China, Tel: 86-10-8778-8489, Fax: 86-10-6771-3648, [email protected].*The first two authors contributed equally to this study.None of the other authors has a personal financial conflict of interest to report.

NIH Public AccessAuthor ManuscriptInt J Cancer. Author manuscript; available in PMC 2011 July 1.

Published in final edited form as:Int J Cancer. 2010 July 1; 127(1): 93–100. doi:10.1002/ijc.25023.

NIH

-PA Author Manuscript

NIH


NIH


β-globin, and thus DNA, was adequate in 98.2% of the frozen tumor tissues (267/272). Of these,99.6% (95% confidence interval (CI) = 97.9–100.0%) were negative for HPV DNA by PGMY,and 100% (95% CI = 98.6–100%) were negative by HPV16/18 E6/E7 PCR. In the correspondingformalin-fixed paraffin-embedded tumor specimens, 99.3% (95% CI = 97.3–99.9%) were HPVnegative by SPF10. By PGMY, 1 case tested weakly positive for HPV89, a noncancer causingHPV type. By SPF10, 2 cases tested weakly positive: 1 for HPV16 and 1 for HPV31. No HPVDNA-positive case had evidence of HPV oncogene activity as measured by p16INK4a

overexpression or E6/E7 seropositivity. This study provides the most definitive evidence to datethat HPV is not involved in ESCC carcinogenesis in China. HPV DNA contamination cannot beruled out as an explanation for high HPV prevalence in ESCC tissue studies with less stringenttissue procurement and processing protocols.

Keywordshuman papillomavirus; esophageal squamous cell carcinoma

Esophageal cancer rates in Linxian, Henan Province, China, are some of the highest in theworld.1 All of the esophageal cancer cases are esophageal squamous cell carcinomas(ESCC). No dominant risk factor for ESCC has been identified in this region. The major riskfactors for ESCC in Western countries, smoking and alcohol consumption,2 are not stronglyassociated with ESCC in Linxian.3 The magnitude of other risk factors for ESCC in Linxianalso tends to be modest.3–7 Therefore, additional major risk factors may be causing themajority of the cases in this area.

Human papillomavirus (HPV) has been suggested as a risk factor for ESCC. Although over70 studies have tested for HPV in ESCC tissue,8 the role of HPV in the development ofESCC is still hotly debated.9 The arguments in support of a causal role for HPV in ESCCinclude: (i) the close proximity and histological similarity of esophageal and oral squamousepithelium,9 where HPV is known to cause tonsillar and oropharyngeal cancers10,11; (ii) theexistence of potentially HPV-associated benign esophageal squamous papillomas8,9; (iii)evidence for clonality of esophageal tumor cells9; (iv) the association between HPV andesophageal cancer in bovine animals8,9; (v) HPV detection in esophageal cancer tissues andprecursor lesions8,9; and (vi) in vitro evidence of HPV-induced transformation of esophagealcells.8 Arguments against the involvement of HPV in esophageal carcinogenesis include: (i)variation in HPV prevalence in case-series of ESCC from 0 to 100%9; (ii) inconsistentassociations between HPV exposure (as measured by serology) and ESCC, with estimatesfrom large studies tending toward the null12; and (iii) lack of documented associationsbetween ESCC and sexual behavior, immunosuppression or previous HPV-associatedmalignancy.9

Estimates of the prevalence of HPV in esophageal tumors range widely, even within thesame country.8 A few possible reasons for this variation between studies include small studysizes, different HPV detection assays, interlaboratory variability, and suboptimal samplecollection and handling leading to contamination.12–14 Contamination of specimens incollection, processing and testing can lead to false-positive results.14,15 This potential forcontamination is of particular concern for studies of HPV because HPV is a common humaninfection that can involve a variety of epithelial tissues and because DNA is resistant tocleaning with detergent. 16–18 While true population differences are possible, estimates ofthe prevalence of HPV DNA in ESCC tumor specimens reported from 2 adjacent high-riskcounties (Linxian and Anyang) in Henan Province, China, range from 0% (0/35) to 94%(29/31).19–24 In fact, the extremes of this range both come from Linxian, which suggests thatthis variation is not due solely to differences in study populations.

Koshiol et al.

Int J Cancer. Author manuscript; available in PMC 2011 July 1.

NIH


NIH


NIH


We have previously conducted 2 studies of HPV and ESCC in Linxian. One was aprospective study that evaluated the association of seropositivity for antibodies against HPVTypes 16, 18, and 73 L1 and L2 viral capsid proteins with risk of ESCC.12 This study foundno association. However, L1/L2 serology has limited sensitivity25 and cannot distinguishesophageal exposure to HPV from genital exposure. Furthermore, although HPV16 and 18account for 70% of cervical cancers and the vast majority of extra-cervical HPV-associatedcancers,26 the World Health Organization has classified 13 HPV types as carcinogenic orprobably carcinogenic. 27 Thus, a more definitive study may need to evaluate a broaderspectrum of HPV types. To that end, we used Hybrid Capture 2, which tests for all 13carcinogenic HPV types, to determine the prevalence of HPV in esophageal cytologyspecimens.28 This study found that the prevalence of HPV did not vary by disease severity.However, a previous study of oral cancer patients found poor correlation of HPV detectionin matched cytologic and tumor specimens,29 suggesting that a tissue-based study mayprovide a more definitive evaluation of HPV and ESCC.

As 1 step toward a more definitive evaluation of HPV in ESCC, we collected surgicalresection specimens and serum from ESCC cases in Linxian with rigorous attention toprevent DNA contamination. We then tested for HPV DNA using the most sensitiveavailable detection methods and evaluated HPV DNA-positive cases for evidence of anactive HPV infection that could have contributed to the development of cancer in that case.

Material and MethodsStudy population

We recruited 272 consecutive incident histopathologically-confirmed ESCC cases who were≥18 years of age and undergoing surgical resection at Yaocun Commune Hospital inLinxian, China, from mid-October 2006 through March 2007. Yaocun Commune Hospital isa regional referral center that performs over 1,000 resections for esophageal or gastriccancer each year. All subjects gave written informed consent. The protocol was approved bythe Institutional Review Boards of the US National Cancer Institute (NCI) and the CancerInstitute of the Chinese Academy of Medical Sciences (CICAMS). Demographic data,current or ever smoking, current or ever drinking alcohol, and family history of cancer wereabstracted from medical records.

Specimen collection and processingSpecimens were collected and processed using a standardized protocol designed to minimizethe possibility of tissue contamination by environmental HPV. Immediately after removalfrom the vasculature, the unopened esophagus was brought in a sterile pan directly from theoperating room to the tissue processing room, where all working surfaces were washed with20% bleach and 70% ethanol before each case. The esophagectomy specimen was receivedon a sterile disposable pad and then transferred to a fresh sterile disposable pad forprocessing the tumor tissue. Using sterile forceps, scissors and scalpels, the esophagus wasopened, and adjacent samples of nonnecrotic tumor tissue were cut and placed in prelabeledcryovials containing no preservative (n = 2) and 10% neutral buffered formalin (n = 2). Thevials containing no preservative were immediately submersed in liquid nitrogen. Theesophagus was then transferred to a fresh sterile disposable pad for processing the nontumortissue. Using new sterile gloves and new sterile supplies, adjacent samples ofmacroscopically normal nontumor tissue at least 2 cm from the nearest visible tumor werecut and processed as described above. Between each case, new sterile sleeve covers wereused to cover the arms of the technician. In over 90% of the cases, both tumor and non-tumor tissues were snap frozen in liquid nitrogen within 30 min of removal of the esophagus

Koshiol et al.


NIH


NIH


NIH


from the vasculature. At the end of each day, all snap frozen cryovials were transferred to a−70°C freezer for storage.

The fixed tissues were processed in new disposable cassettes and embedded in new paraffinusing sterilized forceps and embedding molds. A total of 10 paraffin sections were cut fromeach formalin-fixed ESCC tissue block using a new disposable microtome blade in a strictlyclean environment. The 1st and 10th 4-µM sections were cut for hematoxylin and eosin(H&E) staining. The 8th and 9th 4-µM sections were attached to silanated slides forimmunohistochemical assays. Two groups of three 8-µM paraffin sections (the 2nd to 4thand 5th to 7th sections) were placed in 2 clean conical tubes for HPV DNA measurement.Finally, an HPV negative quality control block was cut between every 10 ESCC blocks toevaluate potential cross-contamination.

CICAMS and NCI pathologists (DML, MJR, SMD) confirmed the presence of nonnecroticESCC in the H&E stained slides of each formalin-fixed tumor block; the same formalin-fixed tumor block was used for the SPF10 assays. Adjacent samples of frozen tissue wereused for the PGMY and HPV16 and 18 type-specific E6/E7 PCR assays. To further ensurethat the frozen samples tested with PCR contained tumor tissue, we cut flankingcryosections in 64 (22%) of the cases. Nonnecrotic ESCC was present in all of these tissues.

HPV DNA testingDNA extraction/purification—Frozen tissues were homogenized in digestion buffer witha final concentration of 400 µg ml−1 of proteinase K and 0.1% Laureth-12, using adisposable pestle in a sterile 1.5-ml microcentrifuge tube and digested at 55°C 24–48 hr untilthe tissue was fully dissolved. DNA was purified using phenol : chloroform : isoamylorganic extraction in Qiagen high density MaXtract microcentrifuge tubes. The aqueouslayer was removed into a new, sterile microcentrifuge tube using a disposable transferpipette, and DNA was precipitated with ammonium acetate and ethanol overnight at −20°C.DNA was pelleted by centrifugation at 4°C, washed with 70% ethanol, and resuspended in100 µl of loTE.

As previously described,30–32 paraffin-embedded formalin-fixed tissue sections were soakedin digestion buffer (250 µl of 1 mg ml−1 proteinase K in 45mM Tris-HCl, pH 8.0, 0.9 mMEDTA, and 0.45% Tween 20) on a 70°C dry block for ~23 hr and then heated to 95°C for 10min to inactivate the proteinase K. Ten microliters of the digested DNA was diluted 10-foldin DEPC-treated water.

HPV DNA detection and genotyping—A 50-µl aliquot of the purified DNA from thefrozen tissue was tested for HPV DNA at Johns Hopkins University using the Roche HPVLinear Array Assay according to the manufacturer’s instructions. 33 The Roche Linear Arraytest is based on consensus amplification using PGMY primers and has high sensitivity forthe detection of a wide range of HPV types (HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42,45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 82v(IS039), 83, 84, and 89 (CP6108)). Compared to other systems, the Linear Array has anincreased ability to differentiate between types and to detect multiple HPV types.34 The β-globin gene was coamplified. Samples positive for β-globin were considered of sufficientquality for analysis.35 Four positive controls, 10 and 100 HPV16-plasmids and 10 and 100HPV18-plasmids diluted in a background of 50 ng of human placental DNA, were placeddirectly into PCR. Because of concerns that HPV integration might disrupt or delete the L1region, leading to false-negative results with L1-based primers, 36,37 all specimens were alsotested for the presence of the E6 and E7 oncogenes of HPV16 and HPV18, the 2 majorcarcinogenic types in cervical and head and neck cancer,38,39 using type-specific TaqManassays targeting the E6/E7 open reading frame (ORF).40,41

Koshiol et al.


NIH


NIH


NIH


A 10-µl aliquot of the DNA dilution from the formalin-fixed paraffin-embedded tissuespecimens was tested for HPV DNA at CICAMS using PCR with SPF10 primers (DDLDiagnostic Laboratories, Netherlands), followed by the reverse line probe assay (LiPA) forgenotyping of 25 HPV Types 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53,54, 56, 58, 59, 66, 68, 70, 74.30 This system also targets the L1 region of the HPV genomeand is ideal for use in formalin-fixed tissue because of its short amplimer (65 bp).34 DNAextraction controls included HPV16-positive tumor tissue from a SiHa xenograft mouse,HPV18-positive tumor tissue from a HeLa xenograft mouse, and an HPV-negative paraffinsection processed in the same manner as the ESCC tumor tissue. PCR controls includedHPV18-positive (HeLa) cells at a concentration 10–100 times the limit of detection for LiPA(<10 HPV DNA copies per reaction) and a negative control (DEPC).

At both laboratories, meticulous processes were applied to avoid cross-contamination in allspecimen processing and HPV testing procedures. New filter tips, powder-free gloves, andsterile conical tubes were used for each case. The microtome, hood, and biosafety cabinetwere also periodically cleaned with 20% bleach and 70% ethanol.

Evaluation of HPV oncogene activityCases positive for HPV DNA were evaluated for evidence of HPV-induced carcinogenesisby immunohistochemical analysis of p16INK4a overexpression, a well-established marker forHPV oncogene expression.42–44 4-µm tissue sections were stained with a monoclonalantibody E6H4 provided by mtm-laboratories AG (Heidelberg, Germany) according tostandard procedures recommended by the supplier. Sections of an HPV-negative colorectalcancer and a HPV 16-positive cervical cancer biopsy were used as negative and positivecontrols, respectively. In addition, serum from HPV DNA-positive cases was tested for anti-E6 and anti-E7 antibodies (available for HPV types 16 and 18), which indicates the presenceof an HPV-associated tumor,45,46 using enzyme-linked immunosorbent assays withglutathione S-transferase fusion proteins provided by Dr. Michael Pawlita (DKFZ, GermanCancer Research Center, Germany), as previously described.47,48 Positive controls includedknown seropositive samples from patients with HPV-associated oral cancers previouslytested by Dr. Raphael Viscidi at the Johns Hopkins University.

Statistical analysisThe prevalence and 2-sided 95% confidence intervals (CIs) of HPV in the ESCC tissue werecalculated in STATA 9.2 (StataCorp, College Station, TX). Maps of the distribution ofESCC cases were made using Epi Info 3.5.1 (CDC, USA).

ResultsOf the total 272 cases, 10 (3.7%) came from Linxian, 65 (23.9%) came from other countiesor cities in the high-risk Taihang mountain region of which Linxian is a part, and 197(72.4%) came from outside of this area (Figs. 1a and 1b). The median age was 60 years(range: 38–79). More than two-thirds of the cases were male, and nearly all were married(Table 1). One hundred and sixty (58.8%) of the patients were ever smokers. All but 2 ofthose were males. Similarly, 110 (40.4%) of the patients were ever drinkers, all of whomwere male. Overall, 102 (37.5%) of the patients, 83 of whom were female, never smokednor drank. Sixty-seven patients (24.6%) had a family history of cancer, including 55 (20.2%)with relatives who had esophageal cancer.

The frozen tissue produced strong β-globin signals for 95.6% of cases (N = 260), weak β-globin signals for 2.6% (N = 7), and no β-globin signal for 1.8% (N = 5). Thus, β-globin was

Koshiol et al.


NIH


NIH


NIH


adequate in 98.2% of cases (267/272), which demonstrates the adequacy of the DNA inthese tissues.

Among the 267 cases with adequate β-globin, 266 (99.6%, 95% CI: 97.9–100.0%) werenegative for HPV DNA by PCR using the PGMY consensus primers and Linear Array. Onemale case tested weakly positive for HPV89 (CP6108), a non-cancer causing HPV type. Nocases were positive by HPV16 or HPV18 E6 or E7-based PCR (100.0% negative, 95% CI:98.6–100.0%).

Using PCR with SPF10 primers followed by LiPA genotyping, 265/267 cases (99.3%, 95%CI: 97.3–99.9%) were negative for HPV DNA. Only 2 cases tested weakly positive: 1female for HPV16 and 1 male for HPV31. All of the HPV negative quality control blockstested negative.

None of the tissues were HPV DNA-positive in both laboratories. Of the 3 HPV DNA-positive cases that were positive in 1 laboratory, none exhibited p16INK4a proteinoverexpression. In addition, the HPV16-positive case was seronegative for HPV16 E6 andE7 antibodies.

DiscussionIn this large study, we found little evidence of HPV involvement in the etiology of ESCC.All but 3 cases were HPV negative by PCR using 2 very sensitive consensus L1 detectionprimer systems and HPV16-and HPV18-specific E6 and E7 primers. Each of the 3 positivecases was weakly positive in 1 L1 PCR assay and negative in the other L1 PCR assay, andnone of them produced any evidence of HPV oncogene activity as indicated by HPVoncogene expression (p16INK4a protein overexpression) or E6/E7 seropositivity. Theseresults support our 2 previous serology-and cytology-based studies in Linxian, which alsosuggested that HPV is not a significant cause of ESCC in this population.12,28

Every case in this analysis contained histopathologically confirmed, nonnecrotic ESCC. Toaddress concerns over the potential loss of L1 through viral integration, we complementedthe PGMY L1 and SPF10 L1 PCR assays, which were performed at 2 independentinstitutions and are highly sensitive for a broad spectrum of HPV types,34 with additionalPCR targeted to the E6 and E7 regions of HPV16 and HPV18, the 2 major carcinogenictypes in cervical and head and neck cancers. Thus, our study is unlikely to have producedfalse negatives due to lack of tumor tissue or primer sensitivity.

One feature of this protocol that may be different from the HPV studies previouslyperformed in the high-risk region around Linxian is the extreme care we took to preventDNA contamination throughout specimen collection, processing, and testing. This cautionwas prompted by a suspicion that the variability in previous results from this area might berelated to environmental contamination during these successive steps.14–18 Using thiscareful approach, only 3 (1.1%) of 267 cases showed any evidence of HPV, and despite ourrigorous protocols, environmental contamination cannot be ruled out as an explanation ofthese 3 low-copy positive results. While HPV89 is not detected in cancer in humans,49 it isrelatively prevalent in cervical samples from the general population.50 Thus, otherspecimens processed during the same time frame as this study could have led to low levelcontamination. Given that this HPV type is non-carcinogenic and that the banddemonstrating its presence in the PGMY assay of 1 case was very weak, it seems likely thatthe HPV89 positivity was due to contamination. HPV16 and HPV31 are well-knowncarcinogenic HPV types, but the evidence for their presence in the SPF10 testing was alsoweak, and the involved cases showed no evidence of HPV in the PGMY or HPV16/18 E6/E7 tissue assays. Finally, none of these 3 cases showed evidence of HPV oncogene activity,

Koshiol et al.


NIH


NIH


NIH


as evaluated by p16INK4a protein overexpression in all 3 cases and HPV E6/E7seropositivity in the HPV16-positive case. Taken together, these results suggest that thehighly sensitive PGMY and SPF10 primers may have detected minute amounts of HPVDNA in these cases that were present in the sample due to contamination from laboratorycontrols or other environmental sources, rather than from actual established HPV infections.

Patients in our study came from 13 of the 31 Provinces, Municipalities, and Regions ofChina, including both the high-risk Taihang mountain region (27.6%) and other areas(72.4%). In addition, cases came from counties with low (<2/100,000) and high(≥32/100,000) cervical cancer mortality rates.51 Given that the negative results of this studywere not restricted to a single county or a single high-risk population, it seems unlikely thatHPV is a significant causal factor for ESCC throughout China. Thus, true geographicetiologic heterogeneity is an unlikely explanation for discrepancies in HPV prevalence inESCC tissues found in previous studies. Strict review of study methodologies isrecommended to more fully understand discrepant HPV/ESCC findings.

This study is one of the largest tumor tissue studies of HPV and esophageal cancer to date.Paying careful attention to good molecular biology procedures and using the most sensitiveHPV DNA detection and confirmation techniques available, we found that the prevalence ofHPV DNA in the ESCC cases approached 0%. This study provides the most definitiveevidence to date that HPV is not involved in esophageal carcinogenesis.

AcknowledgmentsThe authors thank Dr. Hormuzd Katki for providing biostatistical expertise during the design phase of this study.This work was supported by an Intramural Research Award to Dr. Koshiol from the Division of CancerEpidemiology and Genetics, National Cancer Institute, National Institutes of Health; the Cancer PreventionFellowship Program, National Cancer Institute, National Institutes of Health; a research contract [N01-RC-47702]from the National Cancer Institute to the Cancer Institute at the Chinese Academy of Medical Sciences; GeneralFunds from the Intramural Research Program of NCI; and a research grant [JK2006B05] from the Cancer Institute,Peking Union Medical College and Chinese Academy of Medical Sciences. Dr. Gravitt has been a paid consultantand has received research funding from Roche Molecular Systems, who manufacture the HPV Linear Array testused in this study. Dr. von Knebel Doeberitz is a member of the board of mtm-laboratories AG, the company thatprovided reagents for the analysis of p16INK4a overexpression in the tumor samples.

Grant sponsor: National Cancer Institute to the Cancer Institute at the Chinese Academy of Medical Sciences;Grant number: N01-RC-47702; Grant sponsor: Cancer Institute, Peking Union Medical College and ChineseAcademy of Medical Sciences; Grant number: JK2006B05; Grant sponsors: Division of Cancer Epidemiologyand Genetics, National Cancer Institute, National Institutes of Health, Cancer Prevention Fellowship Program,National Cancer Institute, National Institutes of Health, Intramural Research Program of NCI, Roche MolecularSystems

References1. Ke L. Mortality and incidence trends from esophagus cancer in selected geographic areas of China

circa 1970–90. Int J Cancer. 2002; 102:271–274. [PubMed: 12397650]2. Freedman ND, Abnet CC, Leitzmann MF, Mouw T, Subar AF, Hollenbeck AR, Schatzkin A. A

prospective study of tobacco, alcohol, and the risk of esophageal and gastric cancer subtypes. Am JEpidemiol. 2007; 165:1424–1433. [PubMed: 17420181]

3. Tran GD, Sun XD, Abnet CC, Fan JH, Dawsey SM, Dong ZW, Mark SD, Qiao YL, Taylor PR.Prospective study of risk factors for esophageal and gastric cancers in the Linxian generalpopulation trial cohort in China. Int J Cancer. 2005; 113:456–463. [PubMed: 15455378]

4. Mark SD, Qiao YL, Dawsey SM, Wu YP, Katki H, Gunter EW, Fraumeni JF Jr, Blot WJ, DongZW, Taylor PR. Prospective study of serum selenium levels and incident esophageal and gastriccancers. J Natl Cancer Inst. 2000; 92:1753–1763. [PubMed: 11058618]

Koshiol et al.


NIH


NIH


NIH


5. Taylor PR, Qiao YL, Abnet CC, Dawsey SM, Yang CS, Gunter EW, Wang W, Blot WJ, Dong ZW,Mark SD. Prospective study of serum vitamin E levels and esophageal and gastric cancers. J NatlCancer Inst. 2003; 95:1414–1416. [PubMed: 13130117]

6. Abnet CC, Lai B, Qiao YL, Vogt S, Luo XM, Taylor PR, Dong ZW, Mark SD, Dawsey SM. Zincconcentration in esophageal biopsy specimens measured by X-ray fluorescence and esophagealcancer risk. J Natl Cancer Inst. 2005; 97:301–306. [PubMed: 15713965]

7. Abnet CC, Qiao YL, Dawsey SM, Dong ZW, Taylor PR, Mark SD. Tooth loss is associated withincreased risk of total death and death from upper gastrointestinal cancer, heart disease, and strokein a Chinese population-based cohort. Int J Epidemiol. 2005; 34:467–474. [PubMed: 15659476]

8. Syrjanen KJ. HPV infections and oesophageal cancer. J Clin Pathol. 2002; 55:721–728. [PubMed:12354793]

9. Gillison ML, Shah KV. Chapter 9: role of mucosal human papillomavirus in nongenital cancers. JNatl Cancer Inst Monogr. 2003; 31:57–65. [PubMed: 12807947]

10. Dahlstrand H, Nasman A, Romanitan M, Lindquist D, Ramqvist T, Dalianis T. Humanpapillomavirus accounts both for increased incidence and better prognosis in tonsillar cancer.Anticancer Res. 2008; 28:1133–1138. [PubMed: 18505048]

11. Kreimer AR, Alberg AJ, Daniel R, Gravitt PE, Viscidi R, Garrett ES, Shah KV, Gillison ML. Oralhuman papillomavirus infection in adults is associated with sexual behavior and HIV serostatus. JInfect Dis. 2004; 189:686–698. [PubMed: 14767823]

12. Kamangar F, Qiao YL, Schiller JT, Dawsey SM, Fears T, Sun XD, Abnet CC, Zhao P, Taylor PR,Mark SD. Human papillomavirus serology and the risk of esophageal and gastric cancers: resultsfrom a cohort in a high-risk region in China. Int J Cancer. 2006; 119:579–584. [PubMed:16496409]

13. Poljak M, Cerar A, Seme K. Human papillomavirus infection in esophageal carcinomas: a study of121 lesions using multiple broad-spectrum polymerase chain reactions and literature review. HumPathol. 1998; 29:266–271. [PubMed: 9496830]

14. Hubbard RA. Human papillomavirus testing methods. Arch Pathol Lab Med. 2003; 127:940–945.[PubMed: 12873165]

15. Federschneider JM, Yuan L, Brodsky J, Breslin G, Betensky RA, Crum CP. The borderline orweakly positive Hybrid Capture II HPV test: a statistical and comparative (PCR) analysis. Am JObstet Gynecol. 2004; 191:757–761. [PubMed: 15467536]

16. Ferenczy A, Bergeron C, Richart RM. Human papillomavirus DNA in fomites on objects used forthe management of patients with genital human papillomavirus infections. Obstet Gynecol. 1989;74:950–954. [PubMed: 2555753]

17. Sonnex C, Strauss S, Gray JJ. Detection of human papillomavirus DNA on the fingers of patientswith genital warts. Sex Transm Infect. 1999; 75:317–319. [PubMed: 10616355]

18. Strauss S, Stephen H, Sonnex C, Gray J. Contamination of environmental surfaces by genitalhuman papillomaviruses (HPV): a follow up study. Sex Transm Infect. 2003; 79:426–427.[PubMed: 14573848]

19. Lu S, Luo F, Li H. Detection of human papilloma virus in esophageal squamous cell carcinomaand adjacent tissue specimens in Linxian. Zhonghua Zhong Liu Za Zhi. 1995; 17:321–324.[PubMed: 8697965]

20. de Villiers EM, Lavergne D, Chang F, Syrjanen K, Tosi P, Cintorino M, Santopietro R, Syrjanen S.An interlaboratory study to determine the presence of human papillomavirus DNA in esophagealcarcinoma from China. Int J Cancer. 1999; 81:225–228. [PubMed: 10188723]

21. Chang F, Syrjanen S, Shen Q, Cintorino M, Santopietro R, Tosi P, Syrjanen K. Humanpapillomavirus involvement in esophageal carcinogenesis in the high-incidence area of China. Astudy of 700 cases by screening and type-specific in situ hybridization. Scand J Gastroenterol.2000; 35:123–130. [PubMed: 10720108]

22. Si HX, Tsao SW, Poon CS, Wang LD, Wong YC, Cheung AL. Viral load of HPV in esophagealsquamous cell carcinoma. Int J Cancer. 2003; 103:496–500. [PubMed: 12478665]

23. Cao B, Tian X, Li Y, Jiang P, Ning T, Xing H, Zhao Y, Zhang C, Shi X, Chen D, Shen Y, Ke Y.LMP7/TAP2 gene polymorphisms and HPV infection in esophageal carcinoma patients from ahigh incidence area in China. Carcinogenesis. 2005; 26:1280–1284. [PubMed: 15774487]

Koshiol et al.


NIH


NIH


NIH


24. Li S, Li Y, Wang LD, Wu XZ, Zhou L, Zhao XY, Liu HT, Zeng Y. Detection of humanpapillomavirus in tissues of esophageal carcinomas by polymerase chain reaction. Zhonghua ShiYan He Lin Chuang Bing Du Xue Za Zhi. 2008; 22:251–253. [PubMed: 19105334]

25. Iftner T, Villa LL. Chapter 12: human papillomavirus technologies. J Natl Cancer Inst Monogr.2003; 31:80–88. [PubMed: 12807950]

26. Gillison ML, Chaturvedi AK, Lowy DR. HPV prophylactic vaccines and the potential preventionof noncervical cancers in both men and women. Cancer. 2008; 113:3036–3046. [PubMed:18980286]

27. Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Benbrahim-Tallaa L, Guha N,Freeman C, Galichet L, Cogliano V. A review of human carcinogens—Part B: biological agents.Lancet Oncol. 2009; 10:321–322. [PubMed: 19350698]

28. Gao GF, Roth MJ, Wei WQ, Abnet CC, Chen F, Lu N, Zhao FH, Li XQ, Wang GQ, Taylor PR,Pan QJ, Chen W, et al. No association between HPV infection and the neoplastic progression ofesophageal squamous cell carcinoma: result from a cross-sectional study in a high-risk region ofChina. Int J Cancer. 2006; 119:1354–1359. [PubMed: 16615110]

29. Herrero R, Castellsague X, Pawlita M, Lissowska J, Kee F, Balaram P, Rajkumar T, Sridhar H,Rose B, Pintos J, Fernandez L, Idris A, et al. Human papillomavirus and oral cancer: theinternational agency for research on cancer multicenter study. J Natl Cancer Inst. 2003; 95:1772–1783. [PubMed: 14652239]

30. Kleter B, van Doorn LJ, Schrauwen L, Molijn A, Sastrowijoto S, ter Schegget J, Lindeman J, terHarmsel B, Burger M, Quint W. Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital humanpapillomavirus. J Clin Microbiol. 1999; 37:2508–2517. [PubMed: 10405393]

31. Kleter B, van Doorn LJ, ter Schegget J, Schrauwen L, van Krimpen K, Burger M, ter Harmsel B,Quint W. Novel short-fragment PCR assay for highly sensitive broad-spectrum detection ofanogenital human papillomaviruses. Am J Pathol. 1998; 153:1731–1739. [PubMed: 9846964]

32. Chen W, Zhang X, Molijn A, Jenkins D, Shi JF, Quint W, Schmidt JE, Wang P, Liu YL, Li LK,Shi H, Liu JH, et al. Human papillomavirus type-distribution in cervical cancer in China: theimportance of HPV 16 and 18. Cancer Causes Control. 2009; 20:1705–1713. [PubMed: 19705288]

33. Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutlée F, Hildesheim A, Schiffman MH, ScottDR, Apple RJ. Improved amplification of genital human papillomaviruses. J Clin Microbiol. 2000;38:357–361. [PubMed: 10618116]

34. Gravitt, PE.; Viscidi, R. Chapter 5: measurement of exposure to human papillomaviruses. In:Rohan, TE.; Shah, KV., editors. Cervical cancer: from etiology to prevention (cancer prevention-cancer causes). Boston: Kluwer Academic Publishers; 2004. p. 119-141.

35. Gravitt PE, Peyton CL, Apple RJ, Wheeler CM. Genotyping of 27 human papillomavirus types byusing L1 consensus PCR products by a single-hybridization, reverse line blot detection method. JClin Microbiol. 1998; 36:3020–3027. [PubMed: 9738060]

36. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J,Meijer CJ, Munoz N. Human papillomavirus is a necessary cause of invasive cervical cancerworldwide. J Pathol. 1999; 189:12–19. [PubMed: 10451482]

37. Li T, Lu ZM, Chen KN, Guo M, Xing HP, Mei Q, Yang HH, Lechner JF, Ke Y. Humanpapillomavirus type 16 is an important infectious factor in the high incidence of esophageal cancerin Anyang area of China. Carcinogenesis. 2001; 22:929–934. [PubMed: 11375901]

38. Kreimer AR, Clifford GM, Boyle P, Franceschi S. Human papillomavirus types in head and necksquamous cell carcinomas worldwide: a systematic review. Cancer Epidemiol Biomarkers Prev.2005; 14:467–475. [PubMed: 15734974]

39. Smith JS, Lindsay L, Hoots B, Keys J, Franceschi S, Winer R, Clifford GM. Humanpapillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: ameta-analysis update. Int J Cancer. 2007; 121:621–632. [PubMed: 17405118]

40. Sotlar K, Stubner A, Diemer D, Menton S, Menton M, Dietz K, Wallwiener D, Kandolf R,Bultmann B. Detection of high-risk human papillomavirus E6 and E7 oncogene transcripts incervical scrapes by nested RT-polymerase chain reaction. J Med Virol. 2004; 74:107–116.[PubMed: 15258976]

Koshiol et al.


NIH


NIH


NIH


41. Sotlar K, Diemer D, Dethleffs A, Hack Y, Stubner A, Vollmer N, Menton S, Menton M, Dietz K,Wallwiener D, Kandolf R, Bultmann B. Detection and typing of human papillomavirus by E6nested multiplex PCR. J Clin Microbiol. 2004; 42:3176–3184. [PubMed: 15243079]

42. Klaes R, Friedrich T, Spitkovsky D, Ridder R, Rudy W, Petry U, Dallenbach-Hellweg G, SchmidtD, von Knebel Doeberitz M. Overexpression of p16(INK4A) as a specific marker for dysplasticand neoplastic epithelial cells of the cervix uteri. Int J Cancer. 2001; 92:276–284. [PubMed:11291057]

43. Sano T, Oyama T, Kashiwabara K, Fukuda T, Nakajima T. Expression status of p16 protein isassociated with human papillomavirus oncogenic potential in cervical and genital lesions. Am JPathol. 1998; 153:1741–1748. [PubMed: 9846965]

44. von Knebel Doeberitz M. New markers for cervical dysplasia to visualise the genomic chaoscreated by aberrant oncogenic papillomavirus infections. Eur J Cancer. 2002; 38:2229–2242.[PubMed: 12441259]

45. Sun Y, Eluf-Neto J, Bosch FX, Munoz N, Booth M, Walboomers JM, Shah KV, Viscidi RP.Human papillomavirus-related serological markers of invasive cervical carcinoma in Brazil.Cancer Epidemiol Biomarkers Prev. 1994; 3:341–347. [PubMed: 8061584]

46. Meschede W, Zumbach K, Braspenning J, Scheffner M, Benitez-Bribiesca L, Luande J, GissmannL, Pawlita M. Antibodies against early proteins of human papillomaviruses as diagnostic markersfor invasive cervical cancer. J Clin Microbiol. 1998; 36:475–480. [PubMed: 9466762]

47. D’souza G, Kreimer AR, Viscidi R, Pawlita M, Fakhry C, Koch WM, Westra WH, Gillison ML.Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med. 2007;356:1944–1956. [PubMed: 17494927]

48. Sehr P, Zumbach K, Pawlita M. A generic capture ELISA for recombinant proteins fused toglutathione S-transferase: validation for HPV serology. J Immunol Methods. 2001; 253:153–162.[PubMed: 11384677]

49. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ.Epidemiologic classification of human papillomavirus types associated with cervical cancer. NEngl J Med. 2003; 348:518–527. [PubMed: 12571259]

50. Castle PE, Porras C, Quint WG, Rodriguez AC, Schiffman M, Gravitt PE, Gonzalez P, Katki HA,Silva S, Freer E, Van Doorn LJ, Jimenez S, et al. Comparison of two PCR-based humanpapillomavirus genotyping methods. J Clin Microbiol. 2008; 46:3437–3445. [PubMed: 18716224]

51. Li, JY.; Liu, BQ.; Li, GY.; Chen, ZJ.; Sun, XD.; Rong, SD. Atlas of cancer mortality in thePeople’s Republic of China. Shanghai: China Map Press; 1979.

Koshiol et al.


NIH


NIH


NIH


Figure 1.Distribution of 272 pathologically confirmed esophageal squamous cell carcinoma (ESCC)cases presenting for surgery at Yaocun Commune Hospital in Linxian, China, from October2006 through March 2007. (a) The distribution of cases in all of China on a map showing allprovincial and county boundaries. (b) The distribution of cases in the Taihang mountainregion (indicated by hatch marks), where ESCC rates are especially high, and nearbyprovinces.

Koshiol et al.


NIH


NIH


NIH


NIH


NIH


NIH


Koshiol et al.

Table 1

Distribution of case characteristics among 272 esophageal squamous cell carcinoma cases presenting forsurgery at Yaocun Commune Hospital in Linxian, China, from October 2006 through March 2007

Characteristic N %

Age

<55 66 24.3

55–<60 64 23.5

60–<65 61 22.4

≥65 81 29.8

Gender

Male 187 68.8

Female 85 31.3

Marital status

Married 266 97.8

Widowed 6 2.2

Currently smoking

No 250 91.9

Yes 22 8.1

Ever smoked

No 112 41.2

Yes 160 58.8

Currently drinking alcohol

No 245 90.1

Yes 27 9.9

Ever drank alcohol

No 162 59.6

Yes 110 40.4

Family history of cancer

No 205 75.4

Yes 67 24.6


No role for human papillomavirus in esophageal squamous cell carcinoma in China

Documents

Transcript of No role for human papillomavirus in esophageal squamous cell carcinoma in China