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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 871-875

Detection of human papilloma virus in patients with squamous cell carcinoma of the esophagus planned for definitive chemo-radiotherapy, and a study of their clinical characteristics


1 Department of Radiotherapy, Kasturba Medical College, Mangalore, Karnataka, India
2 Department of Radiotherapy, Kasturba Medical College, Manipal, Karnataka, India

Date of Web Publication25-Jul-2016

Correspondence Address:
Krishna Sharan
Department of Radiotherapy, Shirdi Saibaba Cancer Hospital, Manipal, Udupi - 576 104, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.168971

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 > Abstract 


Context: To identify the incidence of human papilloma virus (HPV) infection in squamous esophageal cancer.
Aims: To identify high-risk (HR) HPV positivity rates in patients with squamous carcinoma esophagus and to compare their characteristics with HPV negative counterparts.
Settings and Design: A prospective study, in which tumor biopsies of 18 consecutive patients with squamous carcinoma of the esophagus treated with definitive chemo-radiotherapy (CT-RT) were evaluated for the presence of HPV.
Subjects and Methods: Tumor biopsies of 18 consecutive patients with squamous carcinoma esophagus treated with definitive CT-RT were assessed for presence of HR HPV DNA by hybrid DNA capture technique (Digene-HC2). The clinical characteristics and treatment outcomes of the two groups were then compared.
Statistical Analysis Used: Pearson's Chi-squared test, Kaplan–Mier survival curve/log rank test.
Results: Nine patients (50%) tested positive for HR HPV. The clinical features including age, gender, grade, location, and tumor extent were similar between the two groups. All the three patients with residual disease at the end of treatment tested positive for HPV (P = 0.058). At a mean follow-up of 52 weeks, the estimated median recurrence free survival was 37 weeks (95% confidence interval (CI): 13.6–60.4) among HPV positive patients compared to 53 weeks (95% CI: 29.6–76.4 weeks) for the HPV negative (P = 0.93).
Conclusions: There appears to be a high incidence of HPV among patients with squamous oesophageal cancer in coastal Karnataka. Further studies are required to evaluate its causative role and prognostic implications.

Keywords: Chemo-radiotherapy, human papilloma virus, squamous esophageal cancer


How to cite this article:
Prakash Saxena P U, Fernandes DJ, Vidyasagar MS, Singh A, Sharan K. Detection of human papilloma virus in patients with squamous cell carcinoma of the esophagus planned for definitive chemo-radiotherapy, and a study of their clinical characteristics. J Can Res Ther 2016;12:871-5

How to cite this URL:
Prakash Saxena P U, Fernandes DJ, Vidyasagar MS, Singh A, Sharan K. Detection of human papilloma virus in patients with squamous cell carcinoma of the esophagus planned for definitive chemo-radiotherapy, and a study of their clinical characteristics. J Can Res Ther [serial online] 2016 [cited 2019 Dec 11];12:871-5. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/871/168971




 > Introduction Top


Human papilloma virus (HPV) has been known to be one of the causative agents in development of cervical cancers, vulvovaginal cancers, and anal carcinomas. High-risk (HR) strains such as type 16 and 18 are responsible for up to 80% of these malignancies. Of late, the role ofHPV as one of the causative agents in oropharyngeal malignancies (squamous cell carcinomas) has been established.[1],[2],[3] Though the method of transmission is yet to be defined, most studies implicate oral sex (from mouth-to-genital or mouth-to-anus contact).[4] That the esophagus is anatomically a continuous structure with the upper aerodigestive tract and the fact that squamous cell carcinoma is a common histopathological sub-type leads to the hypothesis that HPV may be associated with esophageal cancers as well. In this study, we tried to analyze the presence of HR HPV DNA (type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) in esophageal squamous carcinomas and the outcome of such patients treated with definitive chemo-radiotherapy (CT-RT).


 > Subjects and Methods Top


Twenty-one consecutive cases of biopsy proven squamous cell carcinomas of the esophagus reporting between January and June 2013 to the center were included in this study. All the patients were planned for definitive CT-RT. Metastatic disease at presentation, poor general condition prohibiting definitive CT-RT and patients reporting for adjuvant radiotherapy were excluded from the study.

Treatment

All patients were treated on Elekta Precise Linear Accelerator by three-dimensional-conformal radiotherapy technique. Patients were treated 5 days a week to a total dose of 50.4 Gy delivered over 5.5 weeks at the rate of 180 cGy per fraction. The chemotherapy sensitizer protocol included weekly cisplatin 40 mg/m 2. Barium swallow and/or esophagogastroscopy was performed for response assessment 1 month after patients had completed treatment. Surgery was only performed as a salvage, in patients with local residual or recurrent disease.

Analysis of biopsy specimen for human papilloma virus

Since we didn't intend to perform any additional intervention for the sake of study, formalin fixed paraffin embedded tissue biopsy samples of the patients obtained at the time of initial diagnosis were used for identification of presence of HPV DNA. The protocol used for DNA extraction from formalin fixed paraffin embedded biopsy sample is as follows: 5–6 sections of 20 microns width of paraffin embedded tissue were put in a properly labeled tube and deparaffinized using 3 mL xylene. The tubes were then incubated at 55°C for 15 min and then centrifuged for 5 min at 3000 rpm. Unused xylene was removed and the tissue sections were rehydrated by incubating the tissue sections in 3 mL of 100% ethanol at 55°C for 5 min at 3000 rpm. The remaining ethanol was removed by careful aspiration. Following this, 3 mL of 70% ethanol was added to the rehydrated sections and the sections were again incubated at 55°C for 5 min at 3000 rpm. Three mL of autoclaved MQ water was then added to the rehydrated solution and again the specimens were incubated as above. The MQ water was then aspirated and discarded. One hundred and eighty microliters of ATL (tissue lysis buffer) and proteinase K (final concentration 150 µg/mL) was added to the sections and the tubes were incubated on a dry bath at 55°C for 12–16 h. The tubes were intermittently shaken to ensure even digestion. Following this if particulate matter was still visible then another 20 µL of proteinase K was added and the specimen incubated for further 2–4 h at 55°C following which 200 µL of lysis buffer was added and incubated at 70°C for 10 min. The digested solution was then taken out from the dry bath and equal volume of 100% ethanol was added to the lysate. Seven hundred microliters of the mixture was then centrifuged in QIAamp spin column at 10,000 rpm for 1 min. The flow through was discarded and the step was repeated with the leftover volume. A fresh collection tube was placed below the column and 500 µL of buffer AW1 was added and again centrifuged at 10,000 rpm for 1 min. After discarding the flow through, 500 µL of buffer AW2 was added and centrifuged at 13,000 rpm for 5 min. A dry spin at 13,000 rpm was given for 1 min and the resultant flow through was discarded. A fresh collection tube was used for collecting DNA. Twenty-five to 45 µL of buffer AE was added to the center of the silica membrane of filter column incubated for 5 min at room temperature. The tubes were then centrifuged at 10,000 rpm for one min at RT and the flow through was retained. The DNA was adsorbed by running in 0.8% agarose gel. Properly labeled tubes were then stored at −70o C deep freezer until used. Hybrid DNA Capture technique (Digene-HC2)[5] was used for identifying HPV DNA in the sample. The HR HPV types included in the test were: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. A cut-off ratio of 0–0.99 was considered negative for HR HPV; a value of 1.0 or above was considered as positive for HR HPV based upon the semi-quantitative viral load, represented as a numeric positive cut-off value. A cut-off ratio of 1.0 corresponds to a viral DNA load of around 5,000 copies/assay or 1 pg/mL at a threshold of finding a clinical disease.

Statistical analysis

The statistical analysis was done using IBM-SPSS statistical software (Version 20). The association between patient characteristics and HPV status was analyzed using Pearson's Chi-squared test. A P ≤ 0.05 was taken as statistically significant. Log-rank test was used to identify any differences in relapse free survival between the two groups.


 > Results Top


Twenty-one patients presenting with squamous cell carcinoma of the esophagus were tested for HPV DNA in their tumour biopsies. Of these, two patients didn't receive radiotherapy and one was found to be metastatic at presentation. Consequently, 18 patients were enrolled into the study. Nine of these (50%) tested positive for HPV DNA. All the patients had presented with complaints of dysphagia for solids. The median duration of symptoms was similar for HPV positive and HPV negative cohorts, at 9 weeks. Clinical parameters of the patients and their relationship with HPV positivity is compiled in [Table 1].
Table 1: Comparison of clinical features among HPV positive and negative patient groups

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All the patients completed the prescribed treatment, with good compliance. Except for two patients, all received at least four cycles of concurrent weekly chemosensitizers. Four patients had radiotherapy dose escalation at discretion of the treating physician for varying reasons including bulky tumor at presentation, cervical location or young age with good tolerance to treatment. Dose escalation wasachieved either by intraluminal high dose rate brachytherapy (8 Gy/2#; three patients) or external radiotherapy (additional 9 Gy/5#/1 week; one patient, with cervical esophageal primary).

At 1 month following completion of treatment, only three patients had residual disease at the primary site. All the three patients with residual disease tested positive for HPV, whereas no HPV negative patient had a residual primary on treatment completion. This higher incidence of residual disease among HPV positive patients approached statistical significance (P = 0.058).

With a mean follow-up duration of 52 weeks, 11 patients (61%) were found to either have a residual or recurrent disease. The estimated median relapse free survival for the study population was 45 weeks (95% confidence interval [CI]: 11.7–78.2 weeks). The relapse free survival plot of the cohort is shown in [Figure 1]. Of the patients with recurrence, 10 (56%) had isolated local recurrence or residual disease, three (17%) presented with distant recurrence, and one patient had both local and distant failure. Of the four patients who had radiotherapy dose escalation, two patients were HPV positive, and both had no evidence of recurrence at 73 and 105 weeks of follow-up. Of the two HPV negative patients, one had a local recurrence at 37 weeks of follow-up, while the other patient remained disease free at 61 weeks of follow-up.
Figure 1: Recurrence free survival of the entire cohort (median survival = 45 weeks; confidence interval: 11.7–78.2)

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Though the estimated median recurrence free survival was 37 weeks (95% CI: 13.6–60.4 weeks) among HPV positive patients compared to 53 weeks (95% CI: 29.6–76.4 weeks) for the HPV negative, the result was not statistically significant (P = 0.93). The Kaplan–Meir relapse free survival curves of the two patient groups is shown in [Figure 2].
Figure 2: Relapse free survival among human papilloma virus positive (green) and human papilloma virus negative (blue) patients. (P = 0.93)

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 > Discussion Top


Since the establishing of its association with cervical cancer, and more recently, oropharyngeal cancer, HPV is emerging as a risk factor for esophageal squamous carcinoma. A meta-analysis of 15 case control studies assessing the etiological role of HPV concluded that 14 of the 15 studies showed a significant increase in risk of esophageal cancer among HPV positive population, with a pooled odds ratio of 3.69.[6] This pilot study attempted to identify the incidence of HPV positivity in squamous cell carcinoma of the esophagus, and its correlation to patient characteristics and treatment outcomes. Half of the patients taken into the study tested positive for HPV. The prevalence of HPV in squamous carcinomas of the esophagus has been reported by a meta-analysis to vary geographically, from around 10% in the USA to nearly 42% in China.[7] The report suggested that esophageal squamous cell carcinomas potentially had different etiological factors in low and high incidence geographic regions, with HPV playing an important role only in the latter.[7]

Various clinical factors, including gender, history of smoking or alcohol consumption, location, extent of disease, or histological grade of primary seemed to have no correlation with the prevalence of HPV in our study. While the small sample size in this study limits its capacity to evaluate the differences in clinical variables between HPV positive and negative patients, there was little to differentiate between the two groups, except that HPV positive patients tended to be older and more likely to have grade 2 or higher primaries. Other studies have also shown no consistently different epidemiological characteristics of HPV positive esophageal squamous carcinomas.[8]

It has been hypothesized that HPV could be more common in upper esophagus, because of the presence of the virus in the oral cavity and its association with oropharyngeal carcinoma. A report from the Northern China suggested that HPV positivity was higher in upper esophagus.[9] However another study from Sweden, specifically evaluating HPV prevalence by anatomical site of oesophageal cancer suggested no relationship with location of primary.[10]

With respect to outcome of treatment, there was no significant difference in relapse free survival among the two groups. The HPV positive patients appeared more likely to have residual disease, and a shorter relapse free survival. There are conflicting reports on the prognostic value of HPV in esophageal cancers. Furihata et.al. evaluated the presence of HPV and aberrant expression of p53 in squamous esophageal cancers and found that patients testing negative for HPV and having normal p53 expression had a significantly superior survival.[11] In a similar study by Dreilich et.al., neither HPV positivity nor HPV viral load seemed to have any effect on outcomes of squamous oesophageal carcinomas treated with definitive, neoadjuvant, or palliative CT-RT.[12] On the other hand, in a study by Cao et.al., tumor HPV status was found to be an independent prognostic factor; patients with HPV-positive tumors had a 63% lower risk of death than the HPV-negative.[9] The reason to this variability in prognostic significance of HPV between different research groups needs additional evaluation. In addition, since there has been no consistently different biological course of HPV positive disease, there is limited research to suggest whether it has a predictive role in these patients. Further studies are needed to clarify if HPV positive esophageal carcinoma behaves differently, or might benefit from altered treatment strategy, as has been observed in oropharyngeal carcinomas.

Though there are several limitations of this study, the most important one is the small sample size, which prevented from effectively analysing for biological differences between HPV positive and negative patients. Furthermore, the method we used for detecting HPV DNA signal was a nonpolymerase chain reaction (PCR) based technique (HC2 is a nonradioactive, chemiluminescence method) and is based upon linear signal amplification of the target DNA (HPV genome). Its clinical sensitivity to detect CIN 2 and 3 and above (carcinomas) is greater than 96%. However if PCR based techniques are used the proportion of HPV positive cases detected may even be higher. In addition, paraffin fixed tissue was used in the study as against fresh biopsy specimen recommended by Digene. Nevertheless, we believe that with adequate care to prevent the shearing of DNA by avoiding any vigorous shaking, accuracy can be retained.


 > Conclusion Top


Nearly half of the esophageal cancer patients taken into this study had evidence of HPV in their tumors. However, the presenting features and “treatment response” of the HPV positive patients did not seem to be different as compared to their HPV negative counterparts. This high a degree of HPV positivity in our sampled population, approaching the highest reported prevalence rates, warrants further evaluation into the causative role of HPV in the South-Western belt of India, and the potential benefit of vaccination in reducing the burden of squamous cell carcinomas of the esophagus.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

1.
Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000;92:709-20.  Back to cited text no. 1
    
2.
Psyrri A, Gouveris P, Vermorken JB. Human papillomavirus-related head and neck tumors: Clinical and research implication. CurrOpinOncol 2009;21:201-5.  Back to cited text no. 2
    
3.
D'Souza G, Kreimer AR, Viscidi R, Pawlita M, Fakhry C, Koch WM, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med 2007;356:1944-56.  Back to cited text no. 3
    
4.
Heck JE, Berthiller J, Vaccarella S, Winn DM, Smith EM, Shan'gina O, et al. Sexual behaviours and the risk of head and neck cancers: A pooled analysis in the International Head and Neck Cancer Epidemiology (INHANCE) consortium. Int J Epidemiol 2010;39:166-81.  Back to cited text no. 4
    
5.
Sambrook J, Russell DW. Preparation and analysis of eukaryotic genomic DNA. In: Argentine J, editor. Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001.  Back to cited text no. 5
    
6.
Liyanage SS, Rahman B, Gao Z, Zheng Y, Ridda I, Moa A, et al. Evidence for the aetiology of human papillomavirus in oesophageal squamous cell carcinoma in the Chinese population: A meta-analysis. BMJ Open 2013;3:e003604.  Back to cited text no. 6
    
7.
Syrjänen K. Geographic origin is a significant determinant of human papillomavirus prevalence in oesophageal squamous cell carcinoma: Systematic review and meta-analysis. Scand J Infect Dis 2013;45:1-18.  Back to cited text no. 7
    
8.
Wang X, Tian X, Liu F, Zhao Y, Sun M, Chen D, et al. Detection of HPV DNA in esophageal cancer specimens from different regions and ethnic groups: A descriptive study. BMC Cancer 2010;10:19.  Back to cited text no. 8
    
9.
Cao F, Han H, Zhang F, Wang B, Ma W, Wang Y, et al. HPV infection in esophageal squamous cell carcinoma and its relationship to the prognosis of patients in northern China. ScientificWorldJournal 2014;2014:804738.  Back to cited text no. 9
    
10.
Löfdahl HE, Du J, Näsman A, Andersson E, Rubio CA, Lu Y, et al. Prevalence of human papillomavirus (HPV) in oesophageal squamous cell carcinoma in relation to anatomical site of the tumour. PLoS One 2012;7:e46538.  Back to cited text no. 10
    
11.
Furihata M, Ohtsuki Y, Ogoshi S, Takahashi A, Tamiya T, Ogata T. Prognostic significance of human papillomavirus genomes (type-16, -18) and aberrant expression of p53 protein in human esophageal cancer. Int J Cancer 1993;54:226-30.  Back to cited text no. 11
    
12.
Dreilich M, Bergqvist M, Moberg M, Brattström D, Gustavsson I, Bergström S, et al. High-risk human papilloma virus (HPV) and survival in patients with esophageal carcinoma: A pilot study. BMC Cancer 2006;6:94.  Back to cited text no. 12
    


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