|Ahead of print publication
Human papillomavirus infection and p53 mutation in esophageal squamous cell carcinoma and its impact on treatment outcome
Tanmay Singh1, Nadia Shirazi2, Saurabh Bansal1, Meenu Gupta1, Sunil Saini3, Mushtaq Ahmad1
1 Department of Radiation Oncology, Cancer Research Institute, Swami Rama Himalayan University, Dehradun, Uttarakhand, India
2 Department of Pathology, Cancer Research Institute, Swami Rama Himalayan University, Dehradun, Uttarakhand, India
3 Department of Surgical Oncology, Cancer Research Institute, Swami Rama Himalayan University, Dehradun, Uttarakhand, India
Department of Radiation Oncology, Cancer Research Institute, Swami Rama Himalayan University, Jolly Grant, Doiwala, Dehradun - 248 140, Uttarakhand
Source of Support: None, Conflict of Interest: None
Introduction: Human papillomavirus (HPV) is emerging as a risk factor for esophageal squamous carcinoma. The prognostic value of the HPV status has been investigated. However, the results are much controversial.
Aim: This study aims to document the association of HPV infection and mutation of p53 gene in esophageal squamous cell carcinoma (ESCC) and its impact on treatment outcome.
Subjects and Methods: The study was conducted over a period of 12 months. A total of 30 cases of ESCC who were primarily to be treated with radiotherapy/chemoradiotherapy were included in the study. All the tissue samples for biopsy were subjected to immunohistochemistry to study p53 and p16 expression, which is a surrogate marker for HPV. The patients were treated by radiotherapy alone or concurrent chemoradiotherapy depending on performance status and stage of disease. The impact of p16 and p53 on overall survival (OS) and disease-free survival (DFS) was determined.
Results: The median OS of HPV-positive patients was 22 months (95% confidence interval [CI] 12–31) as compared to 13 months (95% CI 7–18) for HPV-negative patients (P
= 0.298). The median DFS for HPV-positive patients was 16 months (95% CI 7–24) as compared to 5 months (95% CI 4–6) for HPV-negative patients (P
= 0.048). The median OS of p53-positive patients was 13 months (95% CI 6.7–19) as compared to 22 months (95% CI 12.7–31.2) for p53-negative patients (P
= 0.080). The median DFS for p53-positive patients was 5 months (95% CI 3.7–6.2) as compared to 22 months (95% CI 15.7–29.4) for p53-negative patients (P
Conclusion: Clinical findings of our result can be used to sum up that both HPV infection and p53 mutation status are reliable biomarkers and can help clinicians to predict treatment outcome and prognosticate patients better.
Keywords: Esophageal cancer, human papillomavirus, P53
|How to cite this URL:|
Singh T, Shirazi N, Bansal S, Gupta M, Saini S, Ahmad M. Human papillomavirus infection and p53 mutation in esophageal squamous cell carcinoma and its impact on treatment outcome. J Can Res Ther [Epub ahead of print] [cited 2019 Aug 23]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=263860
| > Introduction|| |
The management of patients with esophageal squamous cell carcinoma (ESCC) requires a multidisciplinary approach, including surgery, chemotherapy, and radiotherapy. In the last 30 years, the potential of human papillomavirus (HPV) or oncogenes and tumor suppressor genes like p53, as cofactors in the tumorigenic process of esophageal carcinomas has been widely studied and is the focus of ongoing research. Many retrospective studies have shown that HPV infection increases the radiosensitivity of head-and-neck squamous cell carcinoma (HNSCC), cervical cancer and anal cancer. However, conflicting results have been observed on the treatment response in HPV-infected ESCC. Kumar et al. and Wang et al. reported that HPV-positive ESCC patients had a significantly favorable survival rate and better treatment response compared to patients with HPV negative tumors, especially those who received chemoradiation therapy. However, Dreilich et al. reported that HPV16 infection status did not have a significant effect on the survival and treatment response (radiotherapy or chemotherapy). Considering the HPV-E6-P53 signal pathway, the function of P53 and its interaction with HPV infection may play an important role in the process of changing radiosensitivity. P53 is the most commonly mutated tumor suppressor gene, with a mutation frequency ranging from 75% to 85% in HNSCC, which is higher than that observed in ESCC (0%–70%).,, There is a paucity of data on the effect of radiotherapy on the treatment outcome of patients testing positive for HPV and p53 mutation in cases of ESCC.
Thus, the aim of the present study is to document the association of HPV infection and mutation of p53 gene in ESCC and its impact on treatment outcome.
| > Subjects and Methods|| |
The study was conducted over a period of 12 months from January 2015 to December 2015 at the Cancer Research Institute, Swami Rama Himalayan University, Dehradun India. Thirty patients with primary biopsy-proven diagnosis of ESCC after obtaining written informed consent were included in the study. All the patients were planned for definitive chemoradiotherapy. This study was approved by the ethics committee of our institute.
The inclusion criteria were age 18–70 years with newly histologically diagnosed having esophageal squamous cell cancer. Patients <18 years of age or pregnant or with any past histroy of malignancy or with previous oncological treatment or psychiatric illness or having tissue diagnosis other than squamous cell carcinoma or with distant metastasis or with morbid conditions or Eastern Cooperative Oncology Group score >3 or active untreated infection or blood urea and serum creatinine higher than twice the normal value or liver function tests more than twice of the normal value were all excluded from the study.
Patients treatment was defined by tumor board. Patients were tested for HPV positivity and p53 mutation. The pretreatment workup consisted of complete history and physical examination, upper gastrointestinal endoscopy, routine blood counts, liver function tests and serum creatinine, imaging with CECT thorax and abdomen, and histopathology and immunohistochemistry (IHC). The patients were treated by radical concurrent chemoradiotherapy. Chemoradiotherapy consisted of 50–50.4 Gy of radiation at 180 cGy/day, 5 days/week for 5 weeks with concurrent chemotherapy (cisplatin at 75 mg/m2 and 5FU at 750 mg/m2 on days 1–4 and 29–32). During radiotherapy, the blood counts and serum creatinine levels were checked every week. Finally, evaluation of response was done using the new response evaluation criteria in solid tumors revised RECIST guidelines.
Evaluation and interpretation of p53 and p16
IHC for p53 was carried out using p53 (clone-DO7) antibody (Biogenex). IHC staining was performed according to standard operating protocols that were standardized in the department of pathology. The protocol included deparaffinization, antigen retrieval, blocking of endogenous enzyme activity, rehydration and incubation with primary antibody, and further treatment with high-sensitivity polydetector HRP/DAB system (Bio-SB). Immunohistochemical expression for p53 was graded weak, moderate, or strong according to nuclear and cytoplasmic staining intensity using clinically established criteria. p53 was scored positive when >50% of tumor cells presented with a strong nuclear stain. Tumors that did not meet this threshold of detection were classified as p53 negative.
To assess the p53 staining positivity, the number of p53-positive nuclei out of 200 were counted and expressed in percentage and scoring was done as follows:
0 → Negative 1 → <10% 2 → 10%–50% 3 → >50% p53 expression was considered negative for score 0, 1, 2, and positive for score 3.
IHC for p16, a surrogate marker for HPV 16, was carried out with p16 rabbit/mouse monoclonal ready to use antibody(CDINK4a) (Biogenex) using epitope retrieval technique. Squamous cell carcinomas known to be positive for p16 were used as positive control. For negative controls, the antibody was omitted during the immunohistochemical study. Immunohistochemical expression for p16 was graded weak, moderate, or strong according to nuclear and cytoplasmic staining intensity using clinically established criteria. p16 was scored positive when >50% of tumor cells presented with a strong nuclear stain. Tumors that did not meet this threshold of detection were classified as p16 negative. All scores were based on examining the whole section in each biopsy under a multiheaded microscope by three observers, who were blinded to clinical patient information. Immunostaining of the sections was reviewed and a strong nuclear as well as cytoplasmic staining was considered as positive reaction.
0 – No staining, 1 – Weak staining, 2 – Intermediate, 3 – Strong staining.
Scoring was classified as follows:
0–2 was low p16INK4a expression and 3 was high p16INK4a expression. p16 score of 3 was considered as positive. Score of 0–2 was considered as negative.
The time from initial diagnosis to the last return visit or death was tracked, namely, the overall survival (OS). Related data were recorded and Kaplan–Meier survival curve was drawn.
A database was constituted using available software solutions SPSS Version 22 (IBM Corp, Armonk, NY, USA.) and electronic spreadsheets (MS Excel) to store and manage the collected data. Parametric and nonparametric test were used to determine the level of significance for categorical variable. Survival analysis was used to check the outcome of radiotherapy using log-rank test. Values of P < 0.05 were considered statistically significant.
| > Results|| |
We enrolled 30 patients for this study. The mean age was 55.3 years (range 33-70 years, median 60 years) and the male: female ratio was 2:1. The baseline characteristics and the response assessment after treatment of the 30 patients is described in Table 1.
Analysis of HPV and p53
p16 was used as a surrogate IHC marker for HPV infection. The p16 scoring was done in all patients. One patient had 0 score, 5 patients had 1+ score, ten patients had 2+ score, and 3+ score was seen in 14 patients. p16 positivity was seen in 14 patients (46.67%).
On analysis of patient population, 14 (46.67%) patients showed HPV positivity. About 55% males were positive for HPV as compared to 30% females who were positive for HPV. OS was 57.1% for HPV-positive patients as compared to 43.75% for HPV-negative patients. The median OS and disease-free survival (DFS) were superior in the patient population that tested positive for HPV infection [Figure 1] and [Figure 2].
|Figure 1: Median overall survival in human papillomavirus-positive and human papillomavirus-negative patients|
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|Figure 2: Median disease-free survival in human papillomavirus-positive and human papillomavirus-negative patients|
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On p53 analysis, two patients had 0 score, six patients had 1+ score, two patients had 2+ score, and 3+ score was seen in twenty patients. P53 positivity was seen in 20 patients (66.67%). Nearly 60% males were positive for p53 as compared to 80% females who tested positive for p53 mutation. Among the p53 positive population, the OS was 45% as compared to 60% in p53 negative population. The median OS and DFS were inferior in p53 mutation-positive patient population [Figure 3] and [Figure 4].
|Figure 3: Median overall survival in p53-positive and p53-negative patients|
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|Figure 4: Median disease-free survival in p53-positive and p53-negative patients|
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Further analysis of the sample population on the basis of both HPV infection and p53 mutation was done and the results are tabulated in [Table 2]. When comparing the median OS of the sample population, higher median OS was associated with HPV-positive and p53-negative population (P = 0.194) [Figure 5]. A strong correlation was seen with higher median DFS associated with HPV-positive and p53-negative population (P = 0.047) [Figure 6].
|Table 2: The analysis of patient population on the basis of both human papillomavirus and p53 status|
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|Figure 6: Median disease-free survival for various p16 and p53 combinations|
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| > Discussion|| |
In this study, all the patients were treated with curative intent. 90% of the patients received concurrent chemotherapy. The median dose of radiation received by the patients was 50.4 Gy which is the recommended dose for treating patients with radical chemoradiotherapy.
The median follow-up of our study was 12 months. At the time of last visit, 50% of the patients were alive. On further analysis, 60% of them were alive without disease and 40% were alive with the disease. The median OS of the patients in our study was 16 months. There are very few studies done depicting the incidence of HPV in esophageal cancer its relationship with the treatment outcome. In this study, HPV was found to be positive in 46.7% of the patients, which was comparable to the incidence of HPV in the Indian population (44%) as reported in a study by Katiyar et al. HPV was found to be positive in 55% of the male population and 30% of the female population which was comparable to the study done by Cao et al.
At the time of the last visit, among the patients who were alive, 53.3% were HPV positive. The median OS of HPV-positive patients was estimated to be 22 months as compared to 13 months of HPV-negative patients. Thus, the patients who were HPV positive showed better median OS benefit of 9 months. The results of the present study were comparable to the results reported by Cao et al., who reported an OS benefit for HPV-positive esophageal cancer patients (64.1% vs. 45.5%). Wang et al. also reported that among the patients treated with chemoradiotherapy, patients who were HPV positive had a better OS as compared to patients who were HPV negative (55% vs. 21%).
DFS was compared for the patients according to their HPV status; we found that patients who were HPV positive had a better median DFS (16 months) as compared to patients who were HPV negative (5 months). The value for this analysis was slightly significant (0.048). Cao et al. reported that patients who were HPV positive had a better response to chemoradiotherapy and had a significantly improved 5-year OS of 64.1% versus 45.5% for HPV negative tumors.
In this study, we also tested another marker, that is, p53, its incidence, and its influence, if any, on treatment outcome. The incidence of p53 in the study population was found to be 66.7% which was comparable to the reported incidence in the study done by Hu et al., who reported the incidence between 70% and 80%. p53 positivity was more in females (80%) as compared to males (60%) which was in accordance with a study done by Mir et al., which reported that the incidence of p53 positivity was twice in females as compared to males (58% vs. 25%).
At the time of the last follow-up, 45% of the patients who had tested positive for p53 mutation were alive and 55% had expired. We found that the median OS of the patients who were positive for p53 mutation was inferior to the patients who were p53 negative (13 months vs. 22 months). On comparing the DFS, our results showed that the patients who were p53 positive had a shorter DFS (5 vs. 22 months) and this was found to be statistically significant (P = 0.014). This was comparable to the results reported by Makino et al., who postulated that the patients who were positive for p53 mutation had a poorer response to chemoradiotherapy and a poorer OS (40.5% vs. 78.6%).
We also analyzed the patients as per the combination of their HPV and p53 status and found that the median OS was maximum in the group of patients who were HPV positive and p53 negative (22 months) followed by group of patients who were HPV negative and p53 positive (13 months) and finally by group of patients who were HPV and p53 positive (9 months). However, this did not reach statistical significance (P = 0.194).
On comparing the DFS between the various groups, we found that the patients who were HPV positive and p53 negative had the maximum DFS of 22 months followed by 6 months for HPV positive and p53 positive and 5 months for HPV-negative and p53-positive patients. This comparison reached statistical significance (P = 0.047).
In this study, the median OS calculated for the complete patient population was 16 months and median DFS was 12 months. This was in correlation with the general poor outcomes for patients of cancer esophagus, as reported in the available literature. Additional variables of potential prognostic importance, such as weight loss, anemia, performance status, dietary habits, geographical region, pathological type, and sexual behavior were lacking in our study. Sample size limits the number of variables that could be included in our models. Factors not included in our models may be important and affect survival.
| > Conclusion|| |
Our results showed that the OS time of the HPV and p53 expression group was longer than that of HPV- and p53-negative group, but the difference showed no statistical significance by log-rank detection. There was significantly better DFS with both HPV and p53 expression group as compared to HPV- and p53-negative group. Clinical outcome of the present study can be used to sum up that both HPV infection and p53 mutation status are useful and reliable biomarkers and can help clinicians to predict treatment outcome and prognosticate patients better. However, further studies with larger sample size and longer follow-up are needed to arrive at a definitive conclusion. Furthermore, the role of viruses when investigated in combination with other factors can provide a direction for future clinical research.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Shukla S, Bharti AC, Mahata S, Hussain S, Kumar R, Hedau S, et al.
Infection of human papillomaviruses in cancers of different human organ sites. Indian J Med Res 2009;130:222-33.
] [Full text]
Hong A, Zhang X, Jones D, Veillard AS, Zhang M, Martin A, et al.
Relationships between p53 mutation, HPV status and outcome in oropharyngeal squamous cell carcinoma. Radiother Oncol 2016;118:342-9.
Harima Y, Sawada S, Nagata K, Sougawa M, Ohnishi T. Human papillomavirus (HPV) DNA associated with prognosis of cervical cancer after radiotherapy. Int J Radiat Oncol Biol Phys 2002;52:1345-51.
Koerber SA, Schoneweg C, Slynko A, Krug D, Haefner MF, Herfarth K, et al.
Influence of human papillomavirus and p16(INK4a) on treatment outcome of patients with anal cancer. Radiother Oncol 2014;113:331-6.
Kumar R, Ghosh SK, Verma AK, Talukdar A, Deka MK, Wagh M, et al.
P16 expression as a surrogate marker for HPV infection in esophageal squamous cell carcinoma can predict response to neo-adjuvant chemotherapy. Asian Pac J Cancer Prev 2015;16:7161-5.
Wang WL, Wang YC, Lee CT, Chang CY, Lo JL, Kuo YH, et al.
The impact of human papillomavirus infection on the survival and treatment response of patients with esophageal cancers. J Dig Dis 2015;16:256-63.
Dreilich M, Bergqvist M, Moberg M, Brattström D, Gustavsson I, Bergström S, et al.
High-risk human papillomavirus (HPV) and survival in patients with esophageal carcinoma: A pilot study. BMC Cancer 2006;6:94.
Pickering CR, Zhang J, Yoo SY, Bengtsson L, Moorthy S, Neskey DM, et al.
Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers. Cancer Discov 2013;3:770-81.
Cancer Genome Atlas Network. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature 2015;517:576-82.
Lyronis ID, Baritaki S, Bizakis I, Tsardi M, Spandidos DA. Evaluation of the prevalence of human papillomavirus and Epstein-Barr virus in esophageal squamous cell carcinomas. Int J Biol Markers 2005;20:5-10.
White RE, Mungatana C, Mutuma G, Robert ME, Daniel RW, Topazian MD, et al.
Absence of human papillomavirus in esophageal carcinomas from Southwestern Kenya. Dis Esophagus 2005;18:28-30.
Zhang D, Zhang W, Liu W, Mao Y, Fu Z, Liu J, et al.
Human papillomavirus infection increases the chemoradiation response of esophageal squamous cell carcinoma based on P53 mutation. Radiother Oncol 2017;124:155-60.
Schwartz' LH, Litiere S, Vries ED, Ford R, Gwyther S, Mandrekar S, et al
. RECIST 1.1 – Update and Clarification: From the RECIST Committee. Eur J Cancer. 2016 Jul; 62: 132-7.
Dey B, Raphael V, Khonglah Y, Lynrah KG. Immunohistochemical analysis of P53 and PRB in esophageal squamous cell carcinoma. J Clin Diagn Res 2014;8:FC01-3.
Cao F, Zhang W, Zhang F, Han H, Xu J, Cheng Y, et al.
Prognostic significance of high-risk human papillomavirus and p16(INK4A) in patients with esophageal squamous cell carcinoma. Int J Clin Exp Med 2014;7:3430-8.
Halperin EC, Wazer DE, Perez CA. Principles and Practice of Radiation Oncology. 6th
ed. Philadelphia: Wolters and Kluwers; 2013. p. 6448-51.
Katiyar S, Hedau S, Jain N, Kar P, Khuroo MS, Mohanta J, et al.
P53 gene mutation and human papillomavirus (HPV) infection in esophageal carcinoma from three different endemic geographic regions of India. Cancer Lett 2005;218:69-79.
Hu N, Huang J, Emmert-Buck MR, Tang ZZ, Roth MJ, Wang C, et al.
Frequent inactivation of the TP53 gene in esophageal squamous cell carcinoma from a high-risk population in China. Clin Cancer Res 2001;7:883-91.
Mir MM, Dar NA, Gochhait S, Zargar SA, Ahangar AG, Bamezai RN, et al.
P53 mutation profile of squamous cell carcinomas of the esophagus in Kashmir (India): A high-incidence area. Int J Cancer 2005;116:62-8.
Makino T, Yamasaki M, Miyata H, Yoshioka S, Takiguchi S, Fujiwara Y, et al.
P53 mutation status predicts pathological response to chemoradiotherapy in locally advanced esophageal cancer. Ann Surg Oncol 2010;17:804-11.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]