Oesophageal cancer (OC) is currently the sixth most common cause of cancer-related death in the world, claiming the lives of 406,000 people in 2008 [
The multifactorial aetiology of OSCC may explain its highly variable geographical distribution with up to a 500-fold difference in incidence between high-risk areas such as the Transkei region of South Africa, the Caspian Littoral of Iran, and Northern China and low-risk regions such as Western Africa [
The International Agency on Research on Cancer (IARC) has acknowledged HPV involvement in head and neck, particularly oropharyngeal, tonsillar, and oral SCC, but has not yet made a conclusive statement about a causal relationship between HPV and OSCC [
The inconsistent observations between individual studies may be attributed to many factors including differences in study design, laboratory techniques, sample types, testing methodology, and the dynamic aetiology of OSCC [
We conducted a case-control study to investigate whether HPV is associated with OSCC in Australia. Case specimens were obtained retrospectively from tumor banks at five hospitals nationwide. These included three teaching hospitals in New South Wales, one in Queensland, and a cancer hospital in Victoria. Control subjects were recruited prospectively in 2010, at the endoscopy clinics of two of the hospitals in NSW.
Case subjects were defined as patients who had a histopathologically confirmed diagnosis of OSCC. Five tumour banks across the country were approached and any OSCC specimens available were requested and obtained. Case specimens included 23 genomes amplified, 26 fresh frozen, and 50 formalin-fixed and paraffin-embedded samples. Formalin-fixed and paraffin-embedded samples were reviewed by a pathologist to ensure that appropriate sections were obtained for testing. Case specimens used in this study dated from 1989 to 2009.
Controls were defined as healthy subjects with no previous history of gastric or oesophageal malignancy and no underlying chronic illness. In addition, those with any history of antecedent or concurrent precursor lesions for adenocarcinoma, such as Barrett’s oesophagus, were excluded from recruitment. Patients who were not suitable for oesophageal biopsy, such as those with a predisposition to bleeding, were also excluded. Subjects were randomly sampled from patients undergoing routine endoscopy for investigation of presumed nonmalignant conditions such as gastrooesophageal reflux disease. Areas of oesophagus biopsied were macroscopically normal in appearance. Three biopsies were obtained from the upper, middle and lower thirds of the oesophagus, for each control.
Of a total of 126 controls who were approached, two were excluded due to a previous history of Barrett’s oesophagus, three were excluded due to concurrent warfarin therapy which contraindicated biopsy during procedure, and twenty-two people declined invitation to take part in the study.
A range of commercial and in-house adapted HPV detection methods were utilized to test all case and control specimens. All testing was carried out at the HPV Labnet, the World Health Organization Western Pacific regional HPV reference laboratory at the Royal Women’s Hospital, Victoria, Australia.
For paraffin-embedded archival samples, two commercially available assays utilizing PCR and targeting 65 and 150 bp region of L1 were used. The smaller amplicons are particularly suited for archival tissue [
Fresh biopsy samples were tested using well-established PGMY09/11 primers either in a PCR-ELISA format as described previously [
Nucleic acid extracted from FFPE was tested using the PapType high-risk HPV detection and genotyping kit (Genera Biosystems, Melbourne, Australia) and with the INNO-LiPA HPV genotyping extra (INNO-LiPA) (Innogenetics, Ghent, Belgium) as both methods amplify a relatively small gene region and are preferred for samples that may be degraded or contain low levels of DNA [
PapType detects 14 high-risk and 2 low-risk HPV genotypes using fluorescently labelled primers to simultaneously amplify a 150 base-pair (bp) region of the HPV L1 gene and includes a 288 bp region of the human-specific gene, myosin light chain (MLC-1) as an internal control. PCR product is hybridized to HPV type-specific probes bound to labelled silica microspheres. These detection beads are distinguishable by their size and fluorescent intensity using flow cytometry (BD FACSArray Bioanalyzer, Becton Dickinson, Franklin Lakes, New Jersey, USA) and the data interpreted using QPlots (Genera Biosystems).
InnoLipa is a multiplex PCR-based assay (SPF10-modified primers), followed by reverse line blot hybridization, detects and genotypes 28 different HPV genotypes, including 18 high-risk HPVs (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82), 6 low-risk HPVs (6, 11, 40, 43, 44, 54, and 70), and 3 other non-classified HPVs (69, 71, and 74), targeting a 65 bp fragment of the HPV L1 ORF. DNA and WGA DNA samples were tested using PapType as described above, because DNA volumes were limited.
Nucleic acid from biopsy samples and whole genome amplified DNA were amplified using HPV L1 consensus primer set PGMY09-PGMY11 [
Any biopsy samples that tested positive for HPV by either PapType or the PGMY09-PGMY11 PCR-ELISA were subsequently tested using the LINEAR ARRAY HPV Genotyping Test (Roche) with the modification of using an automated blot processor, BeeBlot (Bee Robotics Ltd., Gwynedd, United Kingdom), for the hybridization and washing steps, as previously validated by the Royale Women’s Hospital laboratory [
In addition to being tested by Linear Array using 50 µL of DNA as per manufacturer’s instructions, these samples were also tested using 10 µL DNA (plus 40 µL water) to minimize the potential effect on PCR competition by an overabundance of human genomic DNA in the sample. Both high
Based on available literature, we estimated the presence of HPV DNA to be at least 15% in cases and less than 3% in controls. To detect this difference with 95% confidence and 80% power, 55 subjects per arm are required. We therefore aimed to recruit 100 cases and 100 controls. Due to the small number of annual OSCC cases across Australia, all cancer samples included in this study were archival specimens from tumor banks.
Data collection for cases included retrieval of information primarily from hospital medical records and, where possible, direct patient contact via telephone interviews. One investigator (SL) reviewed medical records and collated the various sources of data for OSCC cases. Detailed data collection for case subjects was not possible for several reasons. Many of the OSCC patients, whose tissues were used in our study as cases, were deceased at the time this study was conducted and therefore information could only be obtained from hospital medical records and patient files from the consulting rooms. In addition, some hospitals had destroyed medical records of patients whose admissions had occurred more than 10 years ago. Several patients had been treated at multiple medical facilities across the state and the country and internationally, at various stages of their illness, thereby making it difficult to obtain a complete record. Finally, some OSCC patients either were not contactable or declined invitations to participate in a phone interview. In contrast, all relevant information was collected prospectively for each of the 100 control subjects during their visit to endoscopy clinics. Control data were collected prospectively prior to oesophageal biopsy by an investigator-administered survey by two investigators, SL and AM.
As the data collection for cases was retrospective (and many cases were deceased) and that for controls was prospective, there was a substantial difference in the quality and quantity of information obtained between cases and controls. As a result of the inconsistency and incompleteness of data in cases compared to control subjects in the study, data analysis for other risk factors (such as smoking, alcohol, and diet) for OSCC was not possible. Therefore, this is mainly a descriptive study concentrating primarily on prevalence of HPV DNA in OSCC compared to normal oesophageal tissue.
OSCC is a rare cancer in Australia, and collection of control specimens entails an invasive oesophageal procedure on healthy subjects. As such, large studies are unfeasible. In order to improve the statistical power, a pooled analysis was conducted combining the results of the only other Australian case-control studies [
Ethics approval for this study was obtained from the lead ethics committee at Northern Sydney Central Coast Health in Sydney, Australia. In addition, ethics approval was obtained from each of the five sites of specimen collection. All participants in the study were provided with printed information regarding the study and written consent was obtained prior to recruitment. Reading material provided to recruits pertaining to the study and all patient data collection forms were approved by the Lead Ethics Committee.
Table
Characteristics of cases and controls.
Variable | Cases (%)* | Controls (%) |
|
---|---|---|---|
Mean age | 67.5 | 52.3 | N/A |
Male | 50/99 (51) | 52/100 (52) | 0.83 |
Deceased | 39/54 (72) | 0/100 (0) | <0.001 |
HPV | 1/99 (1) | 0/100 (0) | 0.31 |
History of other ADCs | 10/58 (17) | 0/100 (0) | <0.001 |
Smoking | 55/84 (65) | 45/100 (45) | 0.005 |
Alcohol | 55/74 (74) | 81/100 (81) | 0.29 |
N/A: not applicable; ADCs: Aerodigestive cancers.
*Although a total of 99 case specimens were included in our study, poor quality of obtainable data meant that not all information was recorded for every case. Therefore, variation of denominators in this column indicates the total number of cases for which information on the corresponding variable could be determined.
Two-thirds (65%) of case subjects smoked during their lifetime compared to 45% of controls (
HPV DNA was detected in only one of the case samples and none from the control samples tested, a rate of 1010 per 100,000 (95% CI: 30–5500). The HPV genotype detected in the case subject was the oncogenic type HPV16. The patient with the HPV positive OSCC sample was a Caucasian female, aged 62 years at the time of diagnosis of her OSCC. She had an invasive, moderately differentiated SCC of the esophagus and died within six weeks of diagnosis. She had a 45 pack-year history of smoking and was a smoker at the time of her diagnosis. She also had a history of heavy alcohol use.
The pooled analysis with the only other Australian case-control study [
Most developed countries are known as low-risk for OSCC due to stable or declining OSCC incidence rates over the last few years. In Australia, the major risk factors for OSCC appear to be smoking and alcohol. Our results suggest that, in this multifactorial cancer, oncogenic HPV may well be a risk factor, but the study was underpowered. The confidence limits around the rate of HPV detection in OSCC in our study ranged from 30 to 5500 per 100,000 cancers, and the rate is consistent with that found in the previous Australian case-control study [
From 1986 to present, there have been only three studies conducted in Australia, to determine whether HPV is a causative factor in OSCC. A variety of methods have been used for HPV detection in OSCC specimens, producing mixed results. The first study carried out in 1986 employed filter in situ hybridization (FISH) methodology and detected HPV in 5/10 (50%) of OSCC specimens and no HPV in macroscopically normal oesophageal tissue biopsied adjacent to the OSCC tumor [
The pooled analysis of this study with ours and the odds ratio of 9.35 are suggestive of HPV as a risk factor for OSCC but did not reach statistical significance. Clearly, OSCC is a multifactorial cancer, and other risk factors such as alcohol and smoking predominate in countries like Australia. However, the consistency of findings between these two studies and the pooled analysis point, as well as our meta-analysis [
Our study adds to the small body of work which currently exists on this topic in the Australian population. In terms of prevalence of HPV in OSCC, there have been some low-risk settings such as USA, Germany, Portuga, and Italy, where high HPV detection rates have been reported in OSCC samples [
A limitation of this study is the small sample size, and the fact that only one sample was positive for HPV. Studies of OSCC and HPV are difficult and invasive, making large sample size unfeasible. Collection of control specimens entails an oesophageal biopsy, which is an invasive procedure conducted in a healthy subject, and makes it difficult to increase the number of controls to improve statistical power. In fact, our study had almost double the number of controls of the other Australian study [
In summary, our study adds to the very few studies done in Australia, a low-risk country for OSCC. The findings, whilst not conclusive, are consistent with the findings of our meta-analysis and indicate that there may be a role of HPV in OSCC [
Suzanne M. Garland has received advisory board fees and grant support from CSL and GlaxoSmithKline and lecture fees from Merck, GSK, and Sanofi Pasteur; in addition, she has 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. C. Raina MacIntyre has received funds from GSK and Merck for investigator-driven research on several vaccines. Holly Seale currently holds a National Health and Medical Research Council (NHMRC) Training Fellowship (1012631 - Australian Based Public Health Fellowship).
Surabhi S. Liyanage has contributed to data collection and data analysis and is primary author. Eva Segelov contributed to critical review of the paper and organising specimen collection at St. Vincent's clinic. Aisha Malik contributed to data collection. Suzanne M. Garland contributed to critical review of the paper. Sepehr N. Tabrizi contributed to the analysis of specimens and critical review of the paper. Eleanor Cummins contributed to the analysis of specimens and critical review of the paper. Holly Seale contributed to the critical review of the paper. Bayzidur Rahman contributed to the critical review of the paper. Aye Moa contributed to the critical review and editing of the paper. Andrew P. Barbour contributed to the provision of specimens and critical review of the paper. Philip J. Crowe contributed to the critical review of the paper. C. Raina MacIntyre contributed to the conception and design of study, critical review, and editing of the paper.
The authors would like to thank Prof. Ross Smith and A/Prof Garett Smith (Royal North Shore Hospital) and Dr. Wayne Phillips and Dr. Kate Brettingham-Moore (Peter MacCallum Cancer Centre) for their help in obtaining OSCC specimens from tumour banks as well as assistance in retrieval of patient data and records. The authors would like to thank the following gastroenterologists at St. Vincent's Clinic for their contribution to the study: Dr. David J Byrnes, Dr. Robert Feller, Dr. Alissa Walsh, Dr. Simon Benstock, Dr. Alan Meagher, Dr. Steven Mistilis, Dr. Christopher Vickers, Dr. Paul Edwards, Dr. Antony Wettstein, Dr. Carolyn Bariol, and Dr. David Frommer. The authors would also like to extend their sincere thanks to Drs. Peter Earls and Zerlene Lim at St. Vincent’s Hospital in Sydney as well as Prof. Elizabeth Salisbury at Prince of Wales Hospital, Sydney, for their time and efforts in preparation of OSCC specimens for testing and also to Dr. Tania Tabone at the Royal Women’s Hospital in Melbourne for her contribution to specimen testing for this study.