ATN holds an NHMRC Training Fellowship (630724 - Australian Based Public Health Fellowship) and has previously received research funding for other projects from a manufacturer of HPV vaccine. HS currently holds a National Health and Medical Research Council (NHMRC) Training Fellowship (1012631) - Australian Based Public Health Fellowship). SG has received advisory board fees and grants support from CSL and GlaxoSmithKline, and lecture fees from Merck, GSK and Sanofi Pasteur; in addition, she has also received funding through her institution to conduct HPV vaccine studies for MSD and GSK. She is a member of the Merck Global Advisory Board as well as the Merck Scientific Advisory Committee for HPV. CRM has been on advisory boards for Merck, GSK and Pfizer for vaccines other than HPV. She has received funding for investigator-driven research on HPV vaccine from Merck. She has received funding for investigator driven research for other vaccines from GSK, CSL Biotherapies and Pfizer. The other authors have no competing interests to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: CRM BR ATN SL. Performed the experiments: BR SL. Analyzed the data: BR SL. Contributed reagents/materials/analysis tools: SL BR IR ATN SNT. Wrote the paper: SL CRM BR ATN ST SMG HS. Critical review and comments: BR IR ATN SNT SMG ES HS PJC AM CRM. Design of consrt diagram: AM. Editing of manuscript: AM SL.
The aetiological role of human papillomavirus (HPV) in oesophageal squamous cell carcinoma (OSCC) has been widely researched for more than three decades, with conflicting findings. In the absence of a large, adequately powered single case-control study, a meta-analysis of all available case-control studies is the most rigorous way of identifying any potential association between HPV and OSCC. We present the first global meta-analysis of case-control studies investigating the role of HPV in OSCC.
Case-control studies investigating OSCC tissue for presence of HPV DNA were identified. 21 case-control studies analyzing a total of 1223 cases and 1415 controls, met our inclusion criteria. HPV detection rates were tabulated for each study and all studies were assessed for quality. The random effects method was used to pool the odds ratios (OR).
From all OSCC specimens included in this meta-analysis, 35% (426/1223) were positive for HPV DNA. The pooled OR for an HPV-OSCC association was 3.04 (95% CI 2.20 to 4.20). Meta-regression analysis did not find a significant association between OR and any of the quality domains. Influence analysis was non-significant for the effect of individual studies on the pooled estimate. Studies conducted in countries with low to medium OSCC incidence showed a stronger relationship (OR 4.65, 95% CI 2.47 to 8.76) than regions of high OSCC incidence (OR 2.65, 95% CI 1.80 to 3.91).
Uncertainty around the aetiological role of HPV in OSCC is due largely to the small number and scale of appropriately designed studies. Our meta-analysis of these studies suggests that HPV increases the risk of OSCC three-fold. This study provides the strongest evidence to date of an HPV-OSCC association. The importance of these findings is that prophylactic vaccination could be of public health benefit in prevention of OSCC in countries with high OSCC incidence.
Oesophageal cancer is the eighth most common malignancy worldwide, with an annual incidence of over 500,000
The multifactorial aetiology of OSCC is thought to contribute to its highly variable incidence rates across the globe with up to a 500 fold difference between high risk areas such as the Transkei region of South Africa, the Caspian Littoral of Iran and Northern China and low incidence regions such as Western Africa
In 1982, Syrjänen observed characteristic HPV related morphological changes, usually found in condylomas, in both benign and cancerous oesophageal tissue
While the role of certain oncogenic HPV types in some oropharyngeal and anogenital cancers has been acknowledged by the International Agency on Research on Cancer (IARC), there has been no consensus about a potential aetiologic relationship between HPV and OSCC
The development of prophylactic HPV vaccines, Gardasil® (Merck Sharpe Dohme) and Cervarix® (GlaxoSmithKline), in recent years has been of significant benefit in the fight against cervical cancer and other anogenital cancers. The association of HPV with non-smoking-related head and neck cancers, especially tonsillar, is also now firmly established and the impact of vaccination on the incidence of these cancers is awaited. Clinical trials of HPV vaccines against non-cervical cancers are lacking. However prophylactic efficacy of the quadrivalent HPV vaccine has proven its efficacy on HPV-related vulvar and vaginal lesions with efficacy from approximately 35%–94% in randomised controlled trials
Case-control is the most suitable methodology for the investigation of an HPV-OSCC association. Yet the majority of studies carried out in this area to date have been small, poorly designed case series unsuitable to answer any questions of aetiology, because measures of association cannot be calculated without a control group for comparison. However, a number of small-scale case-control studies have been conducted. We performed a meta-analysis of case-control studies on this topic.
A literature search of the MEDLINE, PUBMED and EMBASE databases was performed to identify published studies in peer-reviewed journals, which investigated for a potential association between HPV and OSCC. Key search terms were “human papillomavirus”, “papillomavirus infections”, “(o)esophageal neoplasms”, “carcinoma, squamous cell”. Only English language papers were included. Articles were sourced from the earliest dates available in each database until February 2012. In addition, reference lists from all case-control studies were reviewed and hand searches of key journals publishing in this area (Annals of Oncology, Lancet Oncology, Anticancer Research, Gastroenterology, International Journal of Cancer, BMC Cancer, Diseases of the Esophagus, Cancer Epidemiology Biomarkers & Prevention and Journal of Clinical Pathology) were performed to retrieve any articles, which were not electronically indexed. No additional data was identified in searches for unpublished papers and abstracts on this topic.
All published studies identified were reviewed by one author (SSL) and included if they examined OSCC and normal oesophageal tissue for the presence of HPV DNA. Animal models, reports on morphology, in vitro and serological studies were excluded. A total of 1223 OSCC and 1415 oesophageal control specimens were tested for the presence of HPV in the 21 case-control studies included in this meta-analysis.
Cases were defined as patients with a histological diagnosis of OSCC and controls were described as healthy subjects with no pre-existing or concurrent chronic medical conditions. There is a potential for cross-contamination and spread of HPV from tumour tissue to adjacent non-malignant oesophageal tissue, creating false positive results in detection of HPV DNA in non-tumour tissue. As a result, 19 articles that classified control tissue as macroscopically normal oesophageal tissue adjacent to the OSCC tumour did not qualify for this meta-analysis.
Summative information from the 21 case-control studies was recorded (
REFERENCE | COUNTRY | HPV DETECTON METHOD | HPV TYPES DETECTED | POSITIVE NO OF CASES (%) | POSITIVE NO OF CONTROLS (%) | OR (95% CONFIDENCE INTERVAL)1,2 | P VALUE | TOTAL QUALITY SCORE3 |
Williamson |
S. Africa | PCR | Various | 6/14 (43) | 6/41 (15) | 4.38 (1.11–17.18) | 0.0272 | 46 |
Cooper |
S. Africa | ISH | 6,11,18,31,33 | 25/48 (52) | 0/2 (0) | Incalculable | 0.1489 | 52 |
Khurshid |
Japan | PCR | CP,16,18 | 17/27 (63) | 3/12 (25) | 5.1 (1.11–23.37) | 0.0286 | 60 |
Agarwal |
India | ISH, IHC | 16,18 | 19/30 (63) | 2/10 (20) | 6.91 (1.24–38.52) | 0.0175 | 33 |
Farhadi |
Iran | PCR | 16,18 | 8/38 (21) | 5/38 (13) | 1.76 (0.52–5.97) | 0.3608 | 44 |
Antonsson |
Australia | PCR | 16, 35 | 8/222 (4) | 0/55 (0) | Incalculable | 0.1531 | 61 |
Fidalgo |
Portugal | PCR | 16,18 | 9/16 (56) | 0/10 (0) | Incalculable | 0.0034 | 39 |
Lenhart |
USA | ISH, IHC | 6,11,16,18,31,33 | 4/12 (33) | 0/12 (0) | Incalculable | 0.0285 | 54 |
Koh |
Korea | PCR | 16 | 0/102 (0) | 0/40 (0) | Incalculable | Incalculable | 35 |
Lambot |
Belgium | PCR | CP | 1/21 (2) | 0/5 (0) | Incalculable | 0.6188 | 22 |
Benamouzig |
France | ISH | 6,11,16,18,31,33 | 4/12 (33) | 1/24 (4) | 11.5 (1.11–118.71) | 0.0171 | 35 |
Ashworth., 1993 |
UK | ISH | 6,11,16,18,31,33 | 0/4 (0) | 0/10 (0) | Incalculable | Incalculable | 28 |
Weston |
Brazil | HCII | HR & LR | 1/40 (2.5) | 1/10 (10) | 0.23 (0.01–4.05) | 0.2790 | 44 |
Souto Damin |
Brazil | PCR | 16,18 | 26/165 (16) | 0/26 (0) | Incalculable | 0.0294 | 47 |
Lyronis |
Greece | PCR | 16,18,other | 17/30 (56) | 6/27 (22) | 4.58 (1.44–14.59) | 0.0081 | 44 |
Li |
China | PCR, ISH | 16 | 2/2 (100) | 66/112 (59) | Incalculable | 0.2406 | 53 |
Guimaraes |
China | PCR | CP | 2/32 (6) | 4/57 (7) | 0.88 (0.15–5.11) | 0.8898 | 65 |
Cao |
China | PCR | 16,18 | 207/265 (78) | 203/357 (57) | 2.71 (1.89–3.88) | <0.0001 | 46 |
Gao |
China | ISH | nil | 0/4 (0) | 61/475 (13) | Incalculable | Incalculable | 67 |
Liu |
China | PCR | 16 | 35/69 (51) | 2/32 (6) | 15.44 (3.42–69.70) | <0.0001 | 46 |
Zhang |
China | PCR | 16,18,58 | 35/70 (50) | 20/60 (33) | 2.00 (0.98–4.08) | 0.0552 | 55 |
OR – odds ratio; CP – consensus primers; HCII – Hybrid Capture 2; IHC – immunohistochemistry; ISH – in situ hybridization; LR – Low–risk HPV types; HR- High risk HPV types; PCR – Polymerase chain reaction. aSome of the data included in the table have been described in one of our previous publications
The study did not involve collection of raw data from individual researchers, as the data required to conduct the meta-analysis were present in the published papers.
Existing tools for quality assessment of meta-analysis were not used in this study as they do not adequately address specific quality aspects considered important in the investigation of an HPV-OSCC association. Quality assessment was performed using a standardised scoring instrument developed by the authors (BR, SL, AN and ST), based on a previously developed quality assessment tool used in a published meta-analysis
Each study was assessed according to our scoring instrument and assigned a score from a maximum of 100 points. The assessment form was comprised of four main sections of quality assessment including selection of study population (40 points), measurement of exposure (HPV) and outcome (OSCC) factors (40 points), adjustment for confounders (15 points) and analysis of results (5 points). Evaluation of selection and measurement issues were given the highest weighting as these are of relatively greater significance in observational studies. The analysis category was allocated the lowest weighting on the basis that a study which is awarded high scores in the three remaining domains of quality assessment could theoretically have data re-analysed. Each of the four main sections were further sub-divided into individually scored items, which were weighted according to relevance for quality assessment and summed to provide the overall section score. For instance, within the measurement category approximately 65% of marks were ascribed to accuracy of exposure (HPV) assessment while the remaining 35% were allocated to measurement of outcome (OSCC) because the specific HPV identification techniques which are utilised are of paramount importance in any study aiming to report the presence of the virus in oesophageal tissue.
The quality evaluation forms were accompanied by a detailed introduction to the various components of assessment and written instructions on how to complete the scoring instrument. Each study was scored in a blinded fashion by two independent assessors (SL and IR). Upon completion of the scoring process, inconsistencies were discussed by the scorers and a single best answer agreed upon. In the instance that an agreement could not be reached, a third scorer (BR) was consulted to resolve the discrepancy. An assessment of inter-observer correlation was conducted.
Average quality scores and inter-observer agreement on scoring for all studies was assessed by calculating intra-class correlation coefficient, using the SPSS software
As only two studies
Meta-regression analysis of the association between the OR and the total and four quality scoring domains was carried out to investigate the potential effect of study quality on effect estimates. A cumulative meta-analysis was also conducted to investigate the cumulative evidence at the time that each study was published and to show the trend of results over time. The impact of each individual study on the pooled OR was investigated by performing an influence analysis, which omitted one study at a time in the calculation of the summary outcome. Existing publication bias was assessed with Begg’s and Egger’s tests and by examining for irregularities in funnel plots demonstrating the relationship between the individual log ORs and their standard errors
Based on GLOBOCAN guidelines
From literature searches, a total of 130 published studies evaluating a possible HPV-OSCC link were identified. Of these, 86 studies were case series, 4 were case reports and 19 studies examined were ineligible as they classified as controls, specimens such as para-oesophageal tissue, oesophageal tissue from patients with oesophagitis and known head and neck malignancies. A consort diagram outlining the selection of studies for inclusion is shown in
The averaged quality scores for all case-control studies are summarised by section in
Average (%) of the maximum category quality score (range) | ||
Categories of quality scoring (maximum points value) | Case-control studies (n = 21) | Inter-cluster correlation (ICCs) for inter-observer agreement (95% CI) |
Selection (40 points) | 29 (0–67) | 0.93 (0.82 to 0.97) |
Measurement (40 points) | 82 (56–100) | 0.53 (−0.11 to 0.81) |
Adjustment for confounding (15 points) | 11 (0–73) | 0.91 (0.77 to 0.96) |
Analysis (5 points) | 10 (0–100) | 0.88 (0.70 to 0.95) |
Total (100 points) | 46 (22–67) | 0.87 (0.62 to 0.95) |
The total inter-class correlation (ICC) of 0.87 (95% CI 0.62 to 0.95), showing very good inter-observer agreement, indicates reliability of the developed scoring instrument (
Individual and pooled OR estimates derived from a random effect model analysis have been illustrated in a Forest plot (
The ORs for the case-control studies analysed, ranged from 0.23 to 26.6. The pooled estimate was 3.04 (95% CI 2.20 to 4.20), indicating a significant association between HPV and OSCC. Cochrane’s Q test for heterogeneity was not significant across all studies (Q = 21.54, p-value 0.366) and the I2 value was 7.15%. This can be interpreted as 7.15% of the variation across the studies being attributed to heterogeneity rather than chance. An I2 value of 25% is generally considered to be low, suggesting that it is less likely that any variability in this meta-analysis is due to chance
There were 11 studies
The meta-regression analysis investigated the association between study-specific ORs and the quality scoring domains i.e. Selection, Measurement, Confounding, Analysis and overall study quality, to test whether any of the quality assessment sections were associated to the ORs from the individual studies (
Item in quality score | Regression co-efficient (95% CI) | P-value |
Selection | −0.0165737 (−0.05 to 0.02) | 0.325 |
Measurement | 0.0433383 (−0.03 to 0.12) | 0.274 |
Confounding | −0.028375 (−0.11 to 0.06) | 0.521 |
Analysis | −0.0407042 (−0.15 to 0.06) | 0.449 |
Total score | −0.0261726 (−0.07 to 0.01) | 0.212 |
A cumulative random-effects meta-analysis of the 21 studies revealed the trend of results over time. All studies demonstrated a positive HPV-OSCC association. Earlier studies indicated a stronger association of HPV with OSCC, with the earliest study in 1991
The meta-analysis result of the pooled OR was not significantly affected by omission of any of the 21 individual studies analysed (
There was no evidence of publication bias as demonstrated by the non-significant p-values for both Begg’s (0.39) and Egger’s test (0.48), and near-symmetric funnel plot (
Nine studies analysed OSCC specimens from high incidence OSCC regions
This meta-analysis demonstrates that HPV increases the risk of OSCC by three-fold and provides the strongest evidence to date of a potential role for HPV in the aetiology of OSCC. While causation cannot be clearly established, it is a major step toward resolving a longstanding uncertainty. With most cancers, the question of aetiology can only be addressed by an observational epidemiologic study, since it is not ethical or feasible to use any other study design to determine aetiology. To date, the association of smoking and lung cancer is a statistical association found in cohort and case control studies, such as the pivotal case-control and cohort studies of Doll and Hill, which to this date remain the strongest evidence linking smoking to lung cancer
Despite there being 130 studies on the topic, the question of whether HPV plays a role in the aetiology of OSCC has been poorly studied. Suboptimal study design, heterogeneous and small-scale studies, inconsistent laboratory methods for HPV detection and variations in specimen retrieval and storage have all contributed to the lack of clarity and inability to resolve this question to date. We found that the majority of the 130 studies identified are case series, which are unable to answer questions of aetiology because a measure of association cannot be calculated without a control group
In conducting this meta-analysis, we discovered that there are very few case-control studies done on the subject, and of the 21 studies most are small scale, with the largest recruiting only 265 subjects. Case-control study design is particularly time-efficient and economical when investigating diseases with long latency periods such as OSCC, since the case subjects have already been diagnosed with the condition at the start of the investigation
Interestingly, of the 21 case-control studies, all presented data from which a measure of association such as an OR could be calculated, but only 2/21 papers presented an OR calculated by the authors. This again highlights the lack of multi-disciplinary, methodological expertise which has gone into evaluating this research question. Our study is therefore a significant contribution to advancing knowledge by utilising the available case control studies to show a convincing and strong statistical association of HPV with OSCC. HPV was detected in a significantly higher proportion of OSCC samples than control tissue, and if this result is representative of the general population, it may be possible that HPV acts as a carcinogen or important co-factor in the pathogenesis of a significant proportion of OSCC cases across the globe.
We believe this is a major step forward in the understanding of this subject, supporting an aetiological role of HPV in OSCC, and that there is some ethical imperative to resolving the question, given the availability of a preventive HPV vaccine, which could have a major impact in preventing OSCC in high incidence countries such as South Africa, Iran and China
The significant pooled OR (3.042, 95% CI 2.2–4.2) which we obtained from the random effects analysis, supported by an I2 value of 7.1%, is strongly indicative of an aetiological role for HPV in OSCC. This result is further validated by our analysis of all subcategories. Our influence calculations demonstrate that no single study on its own, affected the summary effect significantly more or less than any other study included in the meta-analysis, which further supports the robustness of the study. In addition, there was no evidence of publication bias and our meta-regression analysis revealed that study quality did not influence the ORs. The required manipulation of data for studies with incalculable ORs, also did not affect the effect measure, as separate analysis of both studies with calculable and incalculable ORs yielded independently significant pooled ORs suggestive of an association between HPV and OSCC. All these results in combination, support and strengthen our finding of an HPV-OSCC link.
General trends have shown that subjects from areas with a relatively higher incidence of OSCC demonstrate higher rates of HPV DNA detection in OSCC tissue, in comparison to those from regions of low OSCC incidence
Our results suggest that while all studies showed a positive correlation between HPV and OSCC, the association was stronger in earlier studies compared to later studies. There is no evidence that this may be due to HPV detection methodology i.e. amplification versus non-amplification of DNA, as studies carried out in the early 1990s also used DNA amplification techniques
A limitation of this study is that most of the available case control studies, in addition to not calculating a measure of association, did not measure potential confounders or effect modifiers or adjust for their effects. Age, gender, smoking, alcohol consumption, family history of oesophageal cancer, pre-existing immunosuppression prior to cancer diagnosis, a history of thoracic irradiation, socio-economic status, diets high in red and processed meat, consumption of hot food and beverages, pickled foods and diets low in fresh fruit and vegetables are all confounding for effect-modifying actors which may have an impact on the analysis of a HPV-OSCC link. The study did not involve collection of individual raw data, however, all provided aggregate data categorised into exposed and ill; exposed and not ill; non-exposed and ill; and non-exposed and not ill; therefore allowing the meta-analysis to be done. Only five of the studies presented (or purported to collect) data on co-factors, effect modifiers or confounders such as smoking or alcohol, so adjustment for these factors was not possible in the meta-analysis, even if raw data were used. We are not aware of any known confounders which could result in such a strong association of HPV with OSCC. Any future single case control study to address this issue would need to be substantially larger than any of the currently published studies in order to add to the knowledge in light of this meta-analysis. We would recommend that such a study should collect data on potential confounders and effect modifiers and that these be adjusted for when examining the effect of HPV. A final limitation is the varying ratio of cases to controls in the studies, which suggest ad-hoc or convenience-based design rather than carefully planned study designs. However, we included all 21 studies meeting the inclusion criteria, regardless of the ratio of cases to controls.
Based on the findings outlined in GLOBOCAN 2008 and the cancer registry data from Cancer Incidence in Five Continents, it has been estimated that HPV is responsible for approximately 5.1% of the global cancer burden and contributes 20%–50% of non-anogenital cancers
Ours is the first meta-analysis of case-control studies on this topic. A recent meta-analysis of HPV and OSCC did not restrict analysis to any particular study design
This study is the most definitive contribution to date about a question which has defied answers for 30 years: the association of HPV with OSCC. We found, with a significant, robust and strong statistical measure of association, that HPV is associated with a 3-fold increase in the risk of OSCC. There is an imperative for this study to be considered by IARC because of the availability of a prophylactic vaccine against HPV, the late presentation and poor prognosis of OSCC and a large burden of OSCC mortality in many countries. It further adds to the support of HPV vaccination as a cancer-preventing vaccine for children of both genders, to broaden the preventive targets of the vaccine.
(DOCX)