The authors have declared that no competing interests exist.
Conceived and designed the experiments: HL TD JL HD. Performed the experiments: HL JD AN EA CR TR TD HD. Analyzed the data: HL YL JL HD. Contributed reagents/materials/analysis tools: TD JL HD. Wrote the paper: HL JL HD.
The prevalence and role of human papillomavirus (HPV) in the aetiology of oesophageal squamous cell carcinoma is uncertain. Based on the presence of HPV in the oral cavity and its causal association with squamous cell carcinoma of the oropharynx, we hypothesised that HPV is more strongly associated with proximal than distal oesophageal squamous cell carcinoma.
A population-based study comparing HPV infection in relation to tumour site in patients diagnosed with oesophageal squamous cell carcinomas in the Stockholm County in 1999–2006. Multiplex polymerase chain reaction genotyping (PCR) with Luminex was conducted on pre-treatment endoscopic biopsies to identify type specify HPV. Carcinogenic activity of HPV was assessed by p16INK4a expression. Multivariable logistic regression was used to calculate odds ratios and 95% confidence intervals.
Among 204 patients, 20 (10%) had tumours harbouring HPV DNA, almost all (90%) of HPV high-risk type, mainly HPV16. Tumours containing HPV were not overrepresented in the upper compared to the middle or lower third of the oesophagus (odds ratio 0.6, 95% confidence interval 0.2–1.9). P16INK4a expression was similarly common (24% and 16%) in the HPV-positive and HPV-negative groups.
This study found a limited presence of HPV in oesophageal squamous cell carcinoma of uncertain oncogenic relevance and did not demonstrate that HPV was more strongly associated with proximal than distal tumours.
The introduction of preventive vaccines against human papillomavirus (HPV) has increased the incentive to clarify possible causal links between HPV and non-cervical carcinomas, for example oesophageal squamous cell carcinoma cancer. Oesophageal cancer is the 8th most common type of cancer and the 6th most common cause of death from cancer, globally,
This population-based study included patients diagnosed with oesophageal squamous cell carcinoma in the Stockholm County in Sweden during the period 1999 to 2006. The patients were identified through the Swedish Cancer Register, a nationwide register initiated in 1958 with a completeness of 96% for all cancers
Detection of HPV in the tumour biopsies was conducted as follows. Formalin-fixated paraffin embedded biopsies were sectioned into 4×15 µm slices on glass, and macro-dissected to make sure that the section contained at least 70% tumour cells. To minimise contamination, blanks were added after every 5th sample and treated in the same way as regular samples during slicing and micro-dissection. DNA extraction was performed using the High Pure RNA Paraffin Kit (Roche's) without DNAse. PCR was performed by adding 10 µl of DNA sample and 40 µl of reaction mixture, containing broad-spectrum GP5+/6+ primers (BGP5+/BGP6+), to the Qiagen Multiplex PCR Master Mix (Qiagen, Hilden, Germany)
To assess biological activity of HPV in the tumours, p16INK4a, a surrogate marker for HPV activity,
Fisher's exact test was used to identify predictors for HPV-positive tumours. A p-value below 0.05 was considered statistically significant. Unconditional logistic regression was used to calculate relative risk between HPV infection and risk of various tumour sites in the oesophagus, expressed as odds ratios (OR) with 95% confidence intervals (CI). Using multivariable modelling, ORs were adjusted for age (grouped into <60, 60–70, or >70 years), sex, and tumour differentiation (high, middle, or low). Any missing data was grouped into a separate category in these analyses.
The study was approved by the Regional Ethics Review Board in Stockholm.
Among 348 new cases of oesophageal squamous cell carcinoma in the Stockholm County between 1999 and 2006, 67 (19%) were excluded because of; tumour misclassification (n = 3, 1%), diagnosis based on autopsy results (n = 11, 3%), or failure to identify any endoscopic biopsy (n = 53, 15%). Among 281 (81%) eligible patients, 77 (27%) were classified as non-participants; 51 (18%) because of lack of tumour material and 26 (9%) because of tumour material being unavailable. Finally, 204 patients (73%) remained for the present study. Characteristics of the participants, non-participants and excluded patients are presented in
Variable | Participants | Non-participants/excluded* |
Number (%) | Number (%) | |
|
204 (59) | 144 (41) |
|
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Men | 127 (62) | 94 (65) |
Women | 77 (38) | 50 (35) |
|
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<60 | 37 (18) | 23 (16) |
60–70 | 76 (37) | 52 (36) |
>70 | 91 (45) | 69 (48) |
|
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Upper third | 53 (26) | 23 (16) |
Middle third | 64 (31) | 45 (31) |
Lower third | 83 (41) | 45 (31) |
Unspecified | 2 (1) | 5 (4) |
Missing | 2 (1) | 26 (18) |
Nonparticipants include low DNA level (n = 51, 18%) and unable to collect the endoscopic biopsy (n = 26, 9%). Excluded participants include tumour misclassification (n = 3, 1%), tumour detected at autopsy (n = 11, 3%) and unavailable endoscopic material (n = 53, 15%).
#Tumour location was similar in the participants and non-participant/excluded groups (p = 0.113, Fisheŕs exact test) except for more missing in the non-participant/excluded group p<0.001, Fisheŕs exact test).
Characteristics of patients with HPV-positive and HPV-negative tumours are presented in
Variable | HPV negative | HPV positive | p-value |
Number (%) | Number (%) | ||
|
184 (90) | 20 (10) | |
|
|||
Men | 113 (61) | 14 (70) | 0.628 |
Women | 71 (39) | 6 (30) | |
|
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<60 | 32 (17) | 5 (25) | 0.426 |
60–70 | 71 (39) | 5 (25) | |
>70 | 81 (44) | 10 (50) | |
|
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Upper third | 49 (27) | 4 (20) | 0.151 |
Middle third | 53 (29) | 11 (55) | |
Lower third | 78 (42) | 5 (25) | |
|
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High | 10 (5) | 2 (9) | 0.426 |
Middle | 100 (54) | 13 (62) | |
Low | 72 (39) | 6 (29) | |
|
113 (87) | 17 (13) | |
|
|||
Negative | 95 (84) | 13 (76) | 0.488 |
Positive | 18 (16) | 4 (24) |
p16 analysis was conducted on 130 (64%) out of 204 patients.
Percentages not adding to 100% are due to missing data.
Patients with tumours containing HPV DNA did not have any increased risk of having a tumour located in the upper third of the oesophagus compared to a more distal site (adjusted OR 0.6, 95% CI 0.2–1.9,
Variable | Upper | Middle/lower | Crude model | Adjusted model |
Number (%) | Number (%) | OR 95% CI* | OR 95% CI# | |
|
53 (27) | 147 (74) | ||
|
||||
Negative | 49 (92) | 131 (89) | Reference | Reference |
Positive | 4 (8) | 16 (11) | 0.7 (0.2–2.1) | 0.6 (0.2–1.9) |
Variable | Upper/middle | Lower | Crude model | Adjusted model |
Number (%) | Number (%) | OR 95% CI* | OR 95% CI# | |
|
117 (59) | 83 (42) | ||
|
||||
Negative | 102 (87) | 78 (94) | Reference | Reference |
Positive | 15 (13) | 5 (6) | 2.2 (0.8–6.4) | 2.1 (0.7–6.3) |
No adjustments made.
# Adjustments made for sex, age and tumour differentiation.
This study indicates a limited presence of human papillomavirus (HPV) DNA in oesophageal squamous cell carcinoma in a Swedish population, and does not provide support for the hypothesis of an increased prevalence of squamous cell carcinomas infected with HPV in the proximal compared to the distal oesophagus. Furthermore, the p16INK4a data do not support a carcinogenetic role in the oesophageal squamous cell cancers harbouring high-risk HPV.
Strengths of the present study include its population-based design, the high number of patients included compared to other studies in the same area, the valid assessment of the exposure and outcome, and the ability to adjust for potential confounding factors. The use of oesophageal squamous cell carcinoma patients as both case and control subjects ensured similar risk factor profiles, such as for example smoking and alcohol use, and thus reduced confounding. The use of endoscopic biopsies in all participants circumvented biased detection of HPV infection between tumour sites. The biopsies were taken prior to any preoperative treatment, which ensured that the studied tumour material was not affected by e.g. chemotherapy or radiotherapy. Finally, the detection of HPV DNA using Luminex on PCR product is an established method with a high sensitivity.
Limitations include a low statistical power, which was largely due to insufficient DNA material in some biopsies and a low prevalence of HPV infection in the tumours. Furthermore, due to the limited size of the tumour biopsies, we could not assay for HPV E6 and E7 mRNA as marker for active HPV in tumour cells, instead we analysed for p16 INK4a, which has previously been used as a surrogate marker for HPV in cervical cancer
This study did not reveal any higher occurrence of HPV infection in the tumour location of the upper third of the oesophagus as hypothesised. Instead, most tumours positive for HPV DNA were located in the middle third of the oesophagus. This might merely be an effect of chance, or speculatively this might be a true finding. The middle part of the oesophagus, the transition zone, holds no significant contraction amplitude when measuring peristaltic contractions,
To our knowledge, this is the first study comparing the association between HPV infection and the development of squamous cell carcinoma in different locations in the oesophagus as the primary endpoint. Previous studies show diversity in frequency of tumours containing HPV DNA. A review article from 2002 looking at frequency of HPV DNA in oesophageal tumours showed great diversity of between 0 and 70% of tumours containing HPV DNA.
Although 90% of the HPV positive tumours harboured high-risk HPV types (and 5% putative high-risk types), we found no correlation between the subset of tumours containing HPV DNA and p16 INK4a expression. The low portion (24%) of overexpression of p16INK4a in the HPV positive cancer biopsies indicate that HPV may not be involved in tumour progression in oesophageal cancer to a large extent. These results are in line with a recent case-control study of 222 patients with oesophageal squamous cell carcinoma.
In conclusion, this population-based study from Sweden found limited presence of HPV with uncertain oncogenic relevance in oesophageal squamous cell carcinoma and could not demonstrate that HPV was more likely associated to proximal than distal squamous cell carcinomas of the oesophagus.
The presence of human papillomavirus (HPV) in oesophageal squamous cell carcinoma was not correlated to the tumour location within the oesophagus