HPV-16 infection modifies overall survival of Puerto Rican HNSCC patients
- Bianca Rivera-Peña†1Email authorView ORCID ID profile,
- Francisco J. Ruíz-Fullana†2,
- Germán L. Vélez-Reyes1,
- Rosa J. Rodriguez-Benitez3,
- María J. Marcos-Martínez4, 5,
- Juan Trinidad-Pinedo2 and
- Adriana Báez2, 6
Received: 14 May 2016
Accepted: 29 July 2016
Published: 24 August 2016
Abstract
Background
HPV-16 modifies the overall survival (OS) of patients with oropharyngeal cancer (OPSCC). HPV-16 has been established as risk factor for OPSCC, but HPV-16 infection may also reside in the larynx and oral cavity. We evaluated HPV-16 status on OS of Head and Neck Squamous Cell Carcinoma (HNSCC) patients.
Methods
HPV-16 infection was confirmed by amplification of E6 and E7 viral oncogenes through PCR assay and E6 IHC in 185 HNSCC samples. Associations between HPV-16 status and clinicopathological parameters were performed using Fisher’s exact test and x2. Survival analysis was completed using Kaplan-Meier estimator and multivariate Cox regression analysis.
Results
OS of HPV-16 positive patients was longer compared to HPV-16 negative patients (P = 0.002). HPV-16 positive tumors of the larynx (LSCC) and pharynx (PSCC) showed improved OS compared to HPV-16 negative tumors. Also, HPV-16 positive patients exposed to radiotherapy presented a better survival.
Conclusions
HPV-16 status has a positive prognostic value in HNSCC. Addition of HPV-16 status to the TNM staging can provide better assessment in prognosis and guide treatment for HNSCC patients.
Keywords
Background
HNSCC is the seventh most common type of cancer diagnosed, and it is ranked as the eighth cause of cancer death worldwide [1, 2]. This cancer includes tumors from the oral cavity (OSCC) (ICD-10-C14.8), pharynx (PSCC) (ICD-10-C14.0), larynx (LSCC) (ICD-10-C32.9), and the paranasal sinuses (ICD-10-C31.9) [3]. HNSCC is predominantly diagnosed in patients over 60 years old; however, a growing number of HNSCC patients are being diagnosed at younger ages [4]. Historically, HNSCC has been more frequently diagnosed in men, with a male–female ratio of about 4:1. However, this ratio is rapidly changing because more women are exposing themselves to tobacco and alcohol [5]. The overall-5-year survival (OS) for HNSCC patients is 65.9 %, for all HNSCC sites and stages [6], with a median survival of 2.5 years after treatment.
In the United States, Puerto Rican Hispanics, African-Americans, and economically disadvantaged Whites are at greater risk of developing HNSCC. The incidence of HNSCC in Puerto Ricans is 2.5 higher than Hispanics in the US [7]. The incidence of OSCC or PSCC is approximately 72 % higher in Puerto Ricans than in US Hispanics. Similarly, the incidence rate of LSCC is 51 % higher than among Hispanics living in the Unites States [7].
The etiology of HNSCC involves a variety of toxic, environmental, and viral agents [5]. Studies have established that smoking and alcohol consumption are the major risk factors for the development of HNSCC [8–10]. Currently, human papillomavirus (HPV) infection has also been recognized as a risk factor for HNSCC, particularly for OPSCC [11–13]. There are more than 180 types of HPVs described, of which 30 types are considered high risk, including HPV-16 and HPV-18 [14, 15]. The malignant transformation of HPV integration is mediated by HPV oncoproteins E6 and E7 [14]. HPV-16 E6 protein has been associated to the abnormal degradation of the p53 protein, leading to a disruption in G1/S cell cycle control [16]. Also, HPV-16 E7 oncoprotein binds to the phosphorylated form of pRb protein, which inactivates pRb and a disruption in the G1/S transition occurs [17]. Both events cause an abnormal promotion of cell proliferation due to disruptions in the cell cycle control mechanisms. HPV-16 DNA has been detected in almost 35 % of HNSCC patients, and evidence has accumulated showing that HPV is etiologic for OPSCC [18, 19]. It has been proposed that HPV-16 positive HNSCC patients have a distinct cancer progression and prognosis than HPV-16 negative HNSCC patients [20]. HPV-16 positive patients tend to be diagnosed at a younger age when compared to HPV-16 negative patients [20, 21]. Additionally, the presence of HPV-16 in HNSCC patients has been correlated to the presence of local metastases, positive lymph nodes, and a more advanced tumor stage at the time of diagnosis [22]. Clinically, HPV-16 positive HNSCC patients have a better prognosis than HPV-16 negative patients [11, 13, 23, 24].
The complex anatomical structure of the head and neck area makes it very challenging for clinicians to determine the primary site of HNSCC [25]. Detection of HNSCC involves clinical and histological examinations of suspicious tissue, but, at times, unnoticed malignant lesions remain undetected. HNSCC tumors arising from each anatomical site have a unique progression, epidemiology, and therapeutic approach. HNSCC prognostication is based on the TNM Classification of Malignant Tumors (TNM) according to the sub-site [26]. The TNM system is useful to describe the extent of the disease, estimate the likely prognosis, and plan treatment. Treatment strategies rely on TNM, possible side effects, and the patient’s preferences and overall health. Since HNSCC is often discovered in advance stages (III and IV), the most urgent problem is the need to identify an effective diagnostic marker for early detection, and prediction of outcome. Therefore, the purpose of this study was to evaluate whether addition of HPV-16 status to the TNM staging system will help predict better the OS of HNSCC Puerto Rican patients.
Methods
Study design
Study cohort clinicopathological characteristics
Characteristics | N = 185 |
|---|---|
Age (y) | |
Mean ± SD | 62.72 ± 12.13 |
Range | 24-98 |
Sex, n (%) | |
Male | 164 (88.6) |
Female | 21 (11.4) |
Primary Tumor Site, n (%) | |
Larynx | 83 (44.9) |
Oral Cavity | 68 (36.7) |
Oropharynx | 17 (9.2) |
Hypopharynx | 17 (9.2) |
HPV-16 Status, n (%)* | |
HPV-16 + | 97 (52.4) |
HPV-16 - | 88 (47.6) |
Tumor Stage, n (%) | |
I, II | 47 (25.4) |
III, IV | 138 (74.6) |
Tumor Grade, n (%) | |
Well | 43 (23.2) |
Moderate | 107 (57.8) |
Poor | 16 (8.7) |
SCC | 19 (10.3) |
Nodal Involvement | |
Yes | 64 (34.6) |
No | 121 (65.4) |
Heavy Smoking, n (%) | 163 (88.1) |
Heavy Drinking, n (%) | 154 (83.2) |
DNA extraction
Genomic DNA from all tumor samples was isolated using the DNA Isolation kit for Cells and Tissues (Roche, Indianapolis, IN) according to the manufacturer instructions. DNA concentration was measured with NanoDrop 8000 UV–vis Spectrophotometer (Thermo Scientific, Waltham, MA).
Detection of HPV16 DNA
HPV-16 status had been pre-screened by Gp5+/6+ consensus PCR followed by HPV-16 E6/E7 type-specific PCR, and results were confirmed for this study with a TaqMan-based qPCR targeted at HPV-16 E6 and E7 viral oncogenes. The HPV-16 E6 specific primer set included a forward primer 5′-gcacagagctgcaaacaactataca-3′, a reverse primer 5′-tcccgaaaagcaaagtcatatacc-3′, and a probe oligo 5′-tgtactgcaagcaacagttactgcgacgt-3′. The HPV-16 E7 specific primer set included a forward primer 5′-gatgaaatagatggtccagc-3′, a reverse primer 5′-gctttgtacgcaaccgaagc-3′, and a probe oligo 5′-cggacagagcccattacaatattgtaacc-3′. Quality and amount of input DNA samples were tested in each qPCR assay with β-actin gene primers with a forward primer 5′-gcccatctacgaggggta-3′, a reverse primer 5′-ccttaatgtcacgcacga-3′, and a probe oligo 5′-accaccacggccgagcgg-3′. Reaction mixtures with SiHa DNA (1–2 copies of HPV-16) and K562 DNA (HPV-16 negative) were used as positive and negative control, respectively. qPCR reactions were carried out in a 96-well optical tray with a final volume of 25 μL. Each reaction consisted of 600 nM of each primer, 200 nM of each probe (Taqman, Applied Biosystems, Grand Island, NY), 1X of TaqMan Universal PCR Master Mix (Applied Biosystems), which contains the Taq Polymerase, dNTPs, and ROX reference dye, and 75 ng of genomic DNA. DNA was amplified in a 7500 Real Time PCR System (Applied Biosystems, Grand Island, NY). Thermal cycling conditions were: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and an annealing temperature of 60 °C for 1 min. All HNSCC samples classified as HPV-16 positive had amplification of E6 and E7 viral oncogenes through qPCR assay.
Statistical analysis
Data from independent groups was compared using Fisher exact test or x2, as appropriate. Odds ratio (OR) calculations for clinicopathological parameters were performed using binary logistic. Overall survival (OS) was measured in months from the date of diagnosis until death, if occurred. Survival analyses were performed using Kaplan-Meier curves. Log-rank Mantel-Cox and Gehan-Breslow Wilcoxon tests were used to determine the significance between two survival curves. Established prognostic factors having an impact on HNSCC survival were analyzed in a multivariate Cox regression analysis. Statistical significance was established to be p ≤ 0.05. Statistical analyses were performed using IBM SPSS Version 22 (IBM Corp; Armonk, NY).
Results
HPV-16 status in a cohort of HNSCC Puerto Rican patients
Patient characteristics are shown in Table 1. The mean age was 62.72 years (range 24–98 SD: 12.13). Of the 185 HNSCC patients included in the study, 88.6 % were male and 11.4 % were female. Three-quarters (74.6 %) of the patients presented tumors with advance staging (III and IV) and 25.4 % were in early stages (I and II) of the disease. The HNSCC sub-site distribution was 44.9 % LSCC, 36.7 % OSCC and 18.4 % PSCC. The PSCC sub-site includes cases from the oropharynx and hypopharynx. Patients with oropharyngeal and hypopharyngeal cancers were combined under PSCC for the statistical analysis in view of the relatively small number of cases of each sub-site in our study cohort. However, the distribution of oropharyngeal and hypopharyngeal cancers is shown in Table 1. Smoking and drinking habits of our study cohort were defined according to the substance usage reported by each consented patient. Heavy smoking patients reported smoking a pack or more of cigarettes per day and heavy drinking patients reported 15 drinks or more per week. The majority of our HNSCC patients were heavy smokers (88.1 %) and heavy drinkers (83.2 %).
Adjusted OR’s and 95 % CIs of HNSCC patients according to HPV-16 status and clinicopathological parameters
Variable | HPV-16 + (N = 97) | HPV-16 - (N = 88) | OR value, 95 % CI | P value | ||
|---|---|---|---|---|---|---|
No. | Percent | No. | Percent | |||
Age (y) | ||||||
≤ 60 | 45 | 46.4 | 38 | 43.2 | ||
> 60 | 52 | 53.6 | 50 | 56.8 | 1.14 [0.637 – 2.035] | 0.767 |
Sex | ||||||
Male | 89 | 91.8 | 75 | 85.2 | ||
Female | 8 | 8.2 | 13 | 14.8 | 1.93 [0.759 – 4.902] | 0.173 |
Tumor site | ||||||
Larynx | 41 | 42.3 | 42 | 47.7 | 0.866 [0.304 – 2.470] | 0.788 |
Oral Cavity | 36 | 37.1 | 32 | 36.4 | 0.998 [0.338 – 2.942] | 0.996 |
Pharynx | 20 | 20.6 | 14 | 15.9 | 1.675 [0.420 – 6.681] | 0.465 |
Stage of Disease | ||||||
I,II | 30 | 30.9 | 17 | 19.3 | ||
III,IV | 67 | 69.1 | 71 | 80.7 | 1.87 [0.945 – 3.700] | 0.091 |
Tumor grade | ||||||
SCC | 11 | 11.3 | 8 | 9.1 | ||
Well | 22 | 22.7 | 21 | 23.9 | 0.875 [0.226 – 3.385] | 0.847 |
Moderate | 55 | 56.7 | 52 | 59.1 | 0.729 [0.269 – 1.973] | 0.534 |
Poor | 9 | 9.3 | 7 | 7.9 | 0.720 [0.240 – 2.161] | 0.558 |
Nodal involvement | ||||||
Yes | 30 | 16.2 | 34 | 18.4 | ||
No | 67 | 36.2 | 54 | 29.2 | 0.711 [0.387-1.306] | 0.272 |
Tobacco status | ||||||
Yes | 88 | 90.7 | 75 | 85.2 | ||
No | 9 | 9.3 | 13 | 14.8 | 1.70 [0.686 – 4.186] | 0.265 |
Alcohol status | ||||||
Yes | 82 | 84.5 | 72 | 81.8 | ||
No | 15 | 15.5 | 16 | 18.2 | 1.22 [0.561 – 2.630] | 0.695 |
Survival analysis of HNSCC patients according to HPV-16 status
Overall survival of HNSCC patients according to HPV-16 status. Kaplan-Meier survival curve analysis of HNSCC patients according to HPV-16 status. Analysis includes all 185 HNSCC patients for all anatomical sub-sites and stages
Multivariate Cox analysis
Covariate | Coefficient | Standard error | P value | HR value, 95 % CI |
|---|---|---|---|---|
Positive nodal status | 0.308 | 0.209 | 0.141 | 1.361 [0.903 – 2.052] |
Tumor stage | 0.034 | 0.099 | 0.733 | 1.034 [0.852 – 1.257] |
Tumor grade | ||||
Well | 0.089 | 0.442 | 0.841 | 1.093 [0.460 – 2.598] |
Moderate | 0.348 | 0.322 | 0.279 | 1.417 [0.754 – 2.662] |
Poor | 0.496 | 0.344 | 0.149 | 1.642 [0.837 – 3.222] |
Tumor site | ||||
Larynx | - 0.170 | 0.337 | 0.614 | 0.844 [0.436 – 1.634] |
Oral Cavity | - 0.176 | 0.350 | 0.616 | 0.839 [0.423 – 1.665] |
Pharynx | 0.237 | 0.423 | 0.575 | 1.267 [0.554 – 2.901] |
Heavy smoking | 0.202 | 0.329 | 0.539 | 1.224 [0.642 – 2.333] |
Heavy drinking | 0.217 | 0.249 | 0.384 | 1.242 [0.762 – 2.024] |
HPV-16 | 0.555 | 0.182 | 0.002 | 1.742 [1.219 – 2.488] |
Sex | - 0.247 | 0.324 | 0.447 | 0.781 [0.414 – 1.475] |
Age | 0.026 | 0.007 | 0.000 | 1.026 [1.011 – 1.041] |
Overall survival of HNSCC patients according to HPV-16 status in each HNSCC anatomical sub-sites. Kaplan-Meier survival curve analysis of HPV-16 positive patients versus HPV-16 negative patients according to HNSCC anatomical sub-site. a Larynx (LSCC); b Pharynx (PSCC); c Oral Cavity (OSCC)
Overall survival of HNSCC patients according to HPV-16 status and tumor staging. Kaplan-Meier survival curve analysis of HNSCC HPV-16 positive patients versus HPV-16 negative patients distributed by tumor staging. a Early Stages (I and II); b Late Stages (III and IV)
Overall survival of HNSCC patients exposed to radiation according to HPV-16 status and HNSCC sub-sites. Kaplan-Meier survival curve analysis of HNSCC patients exposed to radiotherapy (RTX) adjuvant to surgery. Patients were distributed by HPV-16 status and anatomical sub-site. a All RTX patients, which includes all HNSCC sub-sites and stages; b Larynx RTX patients; c Pharynx RTX patients; d Oral Cavity RTX patients
Overall survival of HNSCC patients with heavy smoking history according to HPV-16 status. Kaplan-Meier survival curve analysis of HPV-16 positive versus HPV-16 negative HNSCC patients with a history of heavy smoking
Overall survival of HNSCC patients with heavy alcohol consumption history according to HPV-16 status. Kaplan-Meier survival curve analysis of HPV-16 positive versus HPV-16 negative HNSCC patients with a history of heavy alcohol consumption
Discussion
Our study shows that HPV-16 positivity modifies the OS of HNSCC patients in two anatomical sub-sites, PSCC and LSCC, but not in OSCC (Fig. 2). Previous studies have shown that HPV-16 infection in LSCC and PSCC increased the survival of HNSCC patients [11, 31]. In contrast, a recent study shows that HPV-16 infection in OSCC does not cause an increase in survival, supporting our findings [32].
The key mechanism for which HPV-16 infection gives a better prognosis to HNSCC patients is still unknown. However, it has been proposed that HPV-16 positive cells have an increased sensitivity to cancer therapies, a slower cellular growth rate, an enhanced immune response towards the virus, or a combination of these factors [33]. Additionally, it has been shown that HPV-16 positive cells have fewer DNA copy number alterations, less genome-wide hypomethylation, less TP53 mutations, and lower expression of EGFR [34]. Because of those differences, two HNSCC carcinogenesis models have being proposed. The first model suggests that progression of HNSCC, not infected by HPV-16, may be due to the amplification or loss of large parts of chromosome arms 3p, 9p, 11p and 17p [35–37]. In contrast, the second model of HNSCC carcinogenesis suggests that tumors infected with HPV-16 have a lower level of chromosomal loss at these regions, which may be the cause for a better survival in these patients [37].
It has been proposed that HPV-16 positivity in HNSCC produces distinct tumor sub-site differences when exposure risks are combined, suggesting that different molecular pathways are involved [38]. When we evaluated HPV-16 presence in each HNSCC anatomic sub-site, we demonstrated that HPV-16 status has a unique impact in the patient’s survival. HPV-16 positive LSCC and PSCC patients have an improved survival, in contrast to OSCC patients which did not show an improved survival. This difference may arise due to smoking and drinking habits of our study population. Our study cohort is composed of heavy smokers (88.1 %) and heavy drinkers (83.2 %). It has been proposed that HNSCC carcinogenesis in HPV-16 positive tumors, with a history of heavy smoking, may arise upon HPV-16 infection in pre-neoplastic tissue already having a number of genetic alterations, for instance, p53 mutations or an increase in EGFR expression [39]. If such alterations are acquired prior to HPV-16 infection, it may impart some of the molecular characteristics of HPV-16 negative tumors, thus resulting in poor outcome and prognosis [40]. Also, it has been suggested that tobacco, alcohol and HPV-16 are independent risk factors for HNSCC, producing distinct tumor groups with different prognosis and guide of treatment [41].
HPV-16 status is an important factor for establishing the prognosis and treatment of HNSCC. Our results showed that HPV-16 positive patients had a better response to radiotherapy when compared to HPV-16 negative patients. This increase in radiosensitivity could be mediated through wild–type p53-mediated apoptosis in HPV-16 positive cells and a lower chromosomal loss [42].
The primary limitations of this study are the small number of early stages (I and II) HNSCC samples, and a small group of PSCC’s in our study cohort. These limitations may explain why we did not observe a statistical difference in early stage tumors by HPV-16 status. Our HNSCC patient population is composed mostly of late stage tumors (III, IV), predominantly from LSCC and OSCC. This issue occurs because our head and neck cancer service is attached to a supraterciary level hospital care center which is responsible for the management of complex cancer cases for the whole island of Puerto Rico.
Conclusions
In this study we showed that, HPV-16 presence, in HNSCC tumors, causes an increase in OS and increases radiosensitivity of tumor cells. Interestingly, we have shown that HPV-16 is present, not only in OPSCC as previously described, but it was also detected in LSCC and OSCC. Although the TNM classification has been effective for prognostication of HNSCC, HPV-16 detection may serve as a potential biomarker, in combination with the TNM, to better establish the prognosis of LSCC and PSCC and guide treatment. Future work should be directed to understand how HPV-16 affects HNSCC carcinogenesis, and how its infection modifies the disease progression increasing the OS in these patients.
Abbreviations
HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; IHC, immunohistochemistry; LSCC, larynx squamous cell carcinoma; OPSCC, oropharynx squamous cell carcinoma; OS, overall survival; OSCC, oral cavity squamous cell carcinoma; PSCC, pharynx squamous cell carcinoma; qPCR, quantitative real-time polymerase chain reaction; RTX, radiotherapy; TNM, TNM classification of malignant tumors
Declarations
Acknowledgements
The research described was supported by the University of Puerto Rico School of Medicine Otolaryngology Section, NIH/National Cancer Institute grants P20CA91402, U54CA96297; NIH/National Institute of General Medical Sciences grant S06GM8224. This research also used core facilities supported by NIH/NCRR G12RR03051 and NIH/NIMHDD 8G12MD007600. Additionally, we want to thank Antonio Arrieta, MSII, for the assistance on the statistical part of this study.
Availability of data and materials
The authors are cognizant and agree to abide to NIH data sharing policies. Raw data from this study will be made accessible only to those who agree to defined conditions of use, and to individuals who meet professional criteria as appropriate.
Authors’ contributions
BR, FR, JT & AB performed the study design. FR, JT & AB consented HNSCC patients to the study and collected all HNSCC tumor samples. MM carried out the histopathological analysis of the samples. BR & GV carried out the DNA extraction and HPV screening using qPCR on all samples. RR, BR & FR performed the statistical analysis. All authors drafted, revised and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests with the presented study.
Ethics approval and consent to participate
All study procedures have the approval of the University of Puerto Rico-Medical Sciences Campus IRB (MSC-IRB Protocol 2770103). Informed consent was obtained from all study participants.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
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