The aim of this study is to improve knowledge regarding the behaviour of HPV associated malignancies located in a posterior region, marking the transition between two territories – maxillofacial and ENT. Determinations of HPV status and genotyping, P16 and P53 expression were performed in 50 patients with malignancies located at the junction between the oropharynx and retromolar trigone, and were then correlated with the treatment response, prognosis and survival of the selected cases. Results showed better treatment outcome and improved prognosis in HPV positive patients compared to HPV negative, and a strong a link between HPV presence and P16 expression. Multimodal treatment including surgery, radiotherapy and chemotherapy provided the best results, although surgery was only an option in a small number of cases, due to the advanced presenting stage and extension in the surrounding tissues, considering the complex anatomy of the area. In the context of the increasing incidence of HPV positive head and neck cancer, HPV testing together with molecular profiling for P16 and P53 tumour markers could help diagnose malignancies in the initial stages, and also provide important clues towards a targeted, more efficient treatment.
Keywords
HPV, cancer, radiotherapy, prognosis, oropharynx
Introduction
Cancers located at the junction between the retromolar trigone and the oropharynx have unique characteristics regarding local spread with significant implications on the possibility of surgical treatment. Due to the posterior location and absence of obvious symptoms in the early stages, patients tend to present with locally advanced tumours. Surgical access, oncological safety and proper closure are difficult to ensure in extended tumours. For this reason, the oncological treatment becomes of outmost importance and should provide predictable positive results and increased survival.
In the recent years there has been increasing interest regarding the molecular profiling of head and neck cancers in the attempt of reaching a more targeted, individualised oncologic treatment that would lead to favourable outcomes and increased overall survival of patients [1]
HPV related cancers are increasing in frequency and tend to involve younger patients. Increased knowledge of the mechanisms involved could not only improve diagnosis, treatment protocols, and prognosis, but it could also improve prophylaxis of the disease by the use of existing vaccines. Molecular profiling of HPV positive head and neck cancer patients can provide explanations for the particular course of the disease and the proven better prognosis of those patients. Certain tumour markers like P53 and P16 are being increasingly studied for correlations between the behaviour of HPV positive head and neck cancer, treatment response and survival. The outcome of these studies could help the development of treatment protocols best suited for individual cases.
Materials and Methods
This study was performed on the basis of obtained informed consent from each participant. It was approved by the Ethics Committee of St. Spiridon Hospital, Iasi, Romania, and that of The University of Medicine and Pharmacy Gr. T Popa, Iasi, Romania.
Study population
The study population consisted of 46 men and 4 women, aged between 39 and 78 years, admitted in the Maxillofacial (OMF) and Otolaryngology (ENT) Departments of the St. Spiridon Hospital, Iasi, Romania, in between 2013-2015. The cases were selected by the help of a standard questionnaire. A written informed consent was obtained from all participants.
The inclusion criteria were the presence of histologically confirmedoropharyngeal cancer or cancer located at the junction between the retromolar trigone and the oropharynx. Patients not able to provide informed consentdue to medical comorbidities, and other types of oral and pharyngeal cancers were excluded from the study.
The diagnosis and staging was established by a multidisciplinary team, including ENT, OMF and Oncology, on the basis of the clinical features, performed biopsy, presence of HPV and tumour markers, and the highlighting of the local spread by the help of CT.
Each case was discussed in the oncologic board and the treatment protocol was advised in accordance with the NCCN Clinical Practice Guidelines in Oncology, Head and Neck Cancers.
The treatment of each case was noted, as well as the evolution regarding the presence or absence of local recurrence and the overall survival.
In all patients we performed the detection of HPV and of the tumour markers P16 and P53.
HPV determination
The samples were collected by the help of the IVD HPV screening kit from AID-Diagnostika, Germany, by collecting scrapings of epithelial cells from the oral cavity using special tubes. For genotyping we used the IVD kit “Opegen” Spain, allowing the genotyping of 19 HPV strains of medium and high risk.
We isolated the genomic DNA from the cytology product, which was then amplified by the multiplex PCR technique, according to the HPV4A ACE SCREENING kit from Seeplex, or subsequently hybridized on strips from the HIGH PAPILLOMA STRIP (Operon) kit, and the obtained product was migrated in agarose gel (2%). The HPV genotyping was performed by the qualitative SSO (reverse blot) method and by the Multiplex PCR qualitative method.
P53 and P16 detection
The existing biopsy tissues from the included patients were histologically processed for determining the presence of P53 and P16. From the paraffin blocks immunohistochemical reactions were carried out using as primary antibodies: anti-p53 monoclonal antibody (clone DO-7, Novocastra, Leica Biosystems Newcastle, UK, dilution 1:800, 30 minutes at 25°C) and anti-p16 monoclonal antibody (clone G175-405, catalogue no. 550834, BD Pharmingen, 1:25 dilution, 60 minutes at 25°C).The immunohistochemical technique included the following steps: deparafination, hydrating, exposing the antigenic sites, neutralizing the endogenous peroxidase, incubation with the primary antibody, visualization with Novolink Polymer Detection System, developing with DAB and counterstaining with Mayer haematoxylin (Fig. 1, 2).
Figure 1 – HPV positive oral squamous cell carcinoma: (A) p53 positive immunostaining x 200; (B) p16 positive immunostaining x 200
Figure 2 – HPV negative oral squamous cell carcinoma: (A) p53 positive immunostaining x 100; (B) p16 positive immunostaining x 100
Figure 3 – HPV positive oral squamous cell carcinoma: (A) p53 negative immunostaining x 100; (B) p16 positive immunostaining x 100
Results
From the 50 patients tested so far (46 males and 4 females), 16 presented HPV infection, out of which 2 females. The genotypes found among the detected patients were: HPV 16 (2 cases), HPV 18 (1 case), HPV 31(1 case), HPV 33 (2 cases), HPV 51 (4 cases), HPV 66 (6 cases) (Table 1, Chart 1). Most HPV positive patients in our study were under the age of 60 at the time of diagnosis with the youngest patient aged 39.
From the total number, 32 patients were P53 positive (28 males and 4 females) and 18 had a negative P53 status. P16 status was positive in 43 cases (39 men and 4 females) and negative in 7. All HPV positive patients had P16 positive status, while only 11 were P53 positive.
Table 1 – Distribution of HPV positive cases according to P53 and P16 status;
Figure 4 – Distribution of HPV positive cases according to P53 and P16 status;
Most tumours were moderately differentiated (35), with only 9 well differentiated and 6 poorly differentiated.
9 of the included patients underwent initial surgical treatment, of which 7 HPV positive patients. All 50 patients had radiotherapy, while only 16 underwent chemotherapy.
A good prognosis was noticed in 28 cases, of which 15 HPV positive patients. From the 50 patients, 16 cases relapsed (1 HPV positive) and 6 patients died (HPV negative) during the study interval (Table 2).
Table 2 – Prognosis according to HPV status
Best results were noticed in patients undergoing surgical treatment followed by radiotherapy or radio-chemotherapy. Patients who underwent surgical treatment had better survival rates than patients who did not. The cases treated by chemotherapy alone had lower survival rates than the ones benefitting from associated chemotherapy and radiotherapy. The same analysis showed that patients with radiotherapy had higher survival than the ones without. Most cases with good prognosis had moderately differentiated tumours and underwent either radiotherapy or chemo-radiotherapy. Recurrences and deaths were noted in patients that underwent radiotherapy alone.
Table 3 – Prognosis and evolution of the selected patients
Results following treatment were overall superior for the HPV positive group, including postoperative outcomes. Most HPV positive patients had good prognosis following treatment and increased survival, as opposed to HPV negative cases in which recurrences and deaths had the highest frequency.
P53 positive patients had better overall prognosis (Table 4), similarly to P16 positive cases (Table 4). Most P53 cases that had good prognosis underwent either surgery associated with radiotherapy, or surgery followed by radiotherapy and chemotherapy.
Table 4 – Prognosis following treatment according to P53 status
Table 5 – Prognosis following treatment according to P16 status
Discussion
HPV positive tumour cells overexpress the P16 protein in a diffuse manner [2, 3]. This overexpression is directly related to the molecular process involved in the carcinogenesis induced by the major HPV oncogenes (E6 and E7). The P16 protein is both a key element of the negative feedback mechanism of mitosis, which is mainly aimed at promoting the inhibition by Rb of the transition to the cell cycle, and a regulator of cell growth factor [4]. Inactivation of P16 by genetic or epigenetic changes contributes to cancer cell growth in HPV-negative oropharyngeal cancers [5].
As a result of the involvement of HPV in oropharyngeal carcinogenesis, HPV epidemiology and specificity of tumour localization, the HPV genome expression and the expression of protein P16 are highly correlated. For this reason, the immunohistochemical staining of p16 may be a surrogate marker for the presence of the HPV genome [6]. This was also true regarding our study since we found all HPV positive cases were also P16 positive, while not all HPV positive cases were also P53 positive.
Given the high rate of false negative results of PCR and the low sensitivity of in situ hybridization techniques, it is important to perform HPV testing in oropharyngeal cancers by using at least two different techniques. Therefore, it is recommended to use both the viral genome detection techniques, and techniques that show theoverexpression of the P16 protein [6]. Considering the increasing number of HPV positive head and neck cancers, also called a recent “epidemicâ€, HPV testing should be performed routinely and screening programs should be developed for high-risk patients.
The inactivation, degradation or mutation of the P53 gene can result in the disturbance of its functions, resulting in cellular proliferation, accumulation of defective DNA and prolonged survival of affected cells. Still, the loss of P53 function is not enough for the development of cancer. Other cytogenetic alterations are necessary to carry out the malignant transformation [7, 8].P53 mutations with high molecular expression are involved in malignancies found in chronic smokers [3, 6].
Molecular profiling can be a useful tool in determining elements relating to treatment response and prognosis with implications in treatment decision. Consistent with the outcomes of our study, HPV positive cancer patients with a greater expression of P16 and lower P53 expression respond better to treatment and have animproved prognosis.
The most common subtype of HPV detected according to the literature is HPV 16, consisting about 90% of all HPV- positive squamous cell carcinoma of the head and neck [7, 9]. This is not consistent with our studies. Out of the 16 HPV positive patients, only two were HPV 16 subtype. This could plead for a geographical distribution of HPV subtypes, which could help improve prophylaxis by the help of geographically specific vaccines. Further studies with larger number of cases are needed for rendering a more relevant statistical analysis regarding all factors.
In oropharyngeal squamous cell tumours, determination of baseline HPV status, demonstrated by immunohistochemical expression of p16INK4a, and detection of HPV DNA by PCR can be used as prognostic indicators. Smoking and chronic alcohol consumption, are well-known, universally accepted risk factors for squamous cell carcinomas of the head and neck, that frequently associate to the HPV status[10]. Further studies are needed to determine the role of additional risk factors in the appearance of HPV induced tumours.
Traditionally, most cases of oropharyngeal cancer were associated with smoking and alcohol abuse. This leads to the loss of P16 and P53 gene mutation. The decrease in tobacco consumption coupled with an increase in HPV-positive patients changed the frequency pattern of oropharyngeal cancers, now appearing more often in non-smokers.
Additionally, patients with HPV positive cancers tend to be younger than those with HPV negative tumours, raising the presumption of dysfunctional sexual habits [11]. Most HPV positive patients in our study were under the age of 60 at the time of diagnosis with the youngest patient aged 39.
HPV positive cancers are associated with a very good survival, despite an advanced tumour stage. This is particularly important since the posterior location of oropharyngeal-retromolar trigone malignancies can lead to presentation in more advanced stages- the main reason for the small number of patients included in our study that underwent initial surgical resection. Best outcomes were obtained by association of surgery, radiotherapy and chemotherapy.Most HPV positive patients had good prognosis following treatment, including postoperative and increased survival, as opposed to HPV negative cases in which recurrences and deaths had the highest frequency.
The risk of nodal metastasis increases with the tumour stage. Patients with P16 positive oropharyngeal tumours have been stated less likely to have persisting lymph node metastases following chemo-radiotherapy and it was hypothesized that a cervical neck dissection can therefore be avoided [4, 5].During the time of the study there was only one recurrence in an HPV positive, P16 positive patient. This underlines the importance of molecular profiling in head and neck cancer for providing the best treatment strategy.The inclusion of high-risk HPV positive patients into screening programs could help the diagnosis of the disease in an incipient stage with increased chances of achieving prolonged survival.
Although the number of patients included in the study was small, there was a clear prevalence of improved outcomes of HPV positive subjects and good prognosis related to the expression of P16 protein. This is consistent with data obtained from other studies [12]. The current system of staging for oropharyngeal cancer should be amended to better reflect the prognosis regarding HPV status leading also to a more defined guide to the targeted treatment of these cases [13].
Conclusions
In our study we proved the involvement of HPV in the genesis of cancers located at the junction between the retromolar trigone and the oropharynx, a particular area due to its location at the border of two territories, OMF and ENT. Additionally, we found implications of HPV status, P16 and P53 expression regarding prognosis and related to the treatment applied in the selected cases. The correlations found were mostly according to the results of other studies. Subsequent research is needed for translating the information regarding molecular profiling and prognosis into relevant treatment protocols.
Overall it can be stated that the involvement of HPV in oropharyngeal malignancies showed favourable response to treatment and a good overall prognosis by the expression of P16, which can be considered a marker for identifying HPV positive malignancies in the region of the head and neck, a common finding with other studies in this regard[8, 14].
References
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Conflict of interests
The authors have stated explicitly that there are no conflicts of interest connected to this article.
Acknowledgments
The authors thank and express their gratitude to the participants for their contributions to the present study.
Tables
Table 1 – Distribution of HPV positive cases according to P53 and P16 status
Cases HPV (+) Cases
HPV(+) Cases
P 53 (+) Cases
P 16 (+)
HPV (+) 16 2 1 2
HPV (+) 18 1 1 1
HPV (+) 31 1 – 1
HPV (+) 33 2 2 2
HPV (+) 51 4 3 4
HPV (+) 66 6 4 6
TOTAL 16 11 16
Table 2 – Prognosis according to HPV status
HPV Total
Negative 16 18 31 33 51 66
Prognosis Good 13 2 0 1 2 4 6 28
Recurrence 15 0 1 0 0 0 0 16
Death 6 0 0 0 0 0 0 6
Total 34 2 1 1 2 4 6 50
Table 3 – Prognosis and evolution of the selected patients
Surgery+RT+CHT
Good 4
Recurrence –
Death –
RT+CHT
Good 9
Recurrence 3
Death –
Surgery +RT
Good 5
Recurrence –
Death –
RT alone
Good 12
Recurrence 9
Death 8
Table 4- Prognosis following treatment according to P53 status
P53 status
Prognosis Good Recurrence Death
Positive 23 Surg+RT+CHT 3 9 Surg+RT+CHT 0 3 Surg+RT+CHT 0
RT+CHT 6 RT+CHT 3 RT+CHT 0
Surg+RT 5 Surg+RT 0 Surg+RT 0
RT alone 9 RT alone 6 RT alone 3
Negative 6 Surg+RT+CHT 0 5 Surg+RT+CHT 1 5 Surg+RT+CHT 0
RT+CHT 3 RT+CHT 0 RT+CHT 0
Surg+RT 1 Surg+RT 0 Surg+RT 0
RT alone 2 RT alone 4 RT alone 5
Table 5 – Prognosis following treatment according to P16 status
P16 status
Prognosis Good Recurrence Death
Positive 27 Surg+RT+CHT 3 10 Surg+RT+CHT 0 6 Surg+RT+CHT 0
RT+CHT 7 RT+CHT 3 RT+CHT 0
Surg+RT 6 Surg+RT 0 Surg+RT 0
RT alone 11 RT alone 7 RT alone 6
Negative 2 Surg+RT+CHT 0 3 Surg+RT+CHT 1 2 Surg+RT+CHT 0
RT+CHT 1 RT+CHT 0 RT+CHT 0
Surg+RT 0 Surg+RT 0 Surg+RT 0
RT alone 1 RT alone 2 RT alone 2
Figure captions
Figure 1 – HPV positive oral squamous cell carcinoma: (A) p53 positive immunostaining x 200; (B) p16 positive immunostaining x 200
Figure 2 – HPV negative oral squamous cell carcinoma: (A) p53 positive immunostaining x 100; (B) p16 positive immunostaining x 100
Figure 3 – HPV positive oral squamous cell carcinoma: (A) p53 negative immunostaining x 100; (B) p16 positive immunostaining x 100
Figure 4 – Distribution of HPV positive cases according to P53 and P16 status
Corresponding author:
Mihail Dan Cobzeanu, Professor, MD, PhD, Department of Otorhinolaryngology, “Grigore T. Popa†University of Medicine and Pharmacy, Iassy, Romania 16 University Street, 700115 Iassy, Romania; phone: 0040-722628390; e-mail: cobzeanu_dan@yahoo.com
Received:
Accepted: