Abstract
Background
Juvenile nasopharyngeal angiofibroma (JNA) has witnessed a four-fold increase in the incidence at our facility in the current decade as compared to the 1980s. With high global incidence of human pappilloma virus (HPV) related oropharyngeal cancer in India, we hypothesize its implication in JNA as it has not yet been reported.
Methods
Clinico–Surgical variables of 6 patients of JNA were included for correlation and their tissue samples were subjected to western blotting (WB), polymerase chain reaction and immunoflorescence to demonstrate a definite association with HPV. In addition 6 control samples (adenoids) underwent WB analysis.
Observations
A universal presence of HPV with JNA is novel ‘discovery’ and has suggested a possibility of a definite association. Only a single case suggested weak infection. None of the controls suggested infection, thus ruling out the presence of HPV in nasopharynx of normal population.
Interpretation
With the dawn of this definite association, no specific conclusions can yet be drawn but a whole plethora of questions have emerged with our novel ‘discovery’.
Juvenile nasopharyngeal angiofibroma (JNA) is the most common tumour of nasopharynx amongst adolescents and is well known for its extreme vascularity (manifesting as profuse epistaxis and intraoperative haemorrhage) and controversial etiology. It accounts overall for 0.05% of all head and neck tumours but is seen more commonly in India. Recently we have noticed a four-fold rise in the incidence of JNA at our facility in the current decade as compared to the 1980s. This suggests some evolving host–agent–environmental interaction or probably a novel etiologic agent operating in this part of the world. HPV is known for its tumorigenic effect and viruses as such are associated with a variety of nasopharyngeal tumours. For example Human herpes virus-type 8 (HHV-8) with Kaposi sarcoma , Epstein–Barr virus as an etiologic agent in nasopharyngeal cancer, Burkitt lymphoma and Hodgkin lymphoma. With rising incidence of HPV-related head and neck squamous cell cancer (HNSCC), it is likely to affect the postnasal space. Hence the possibility of its implication in JNA cannot be ignored. There is a scarcity of literature implicating association of any virus with JNA as only a single such report has revealed that JNA does not appear to be associated with either HHV-8 or EBV. To the best of our knowledge NO study has yet reported an association with HPV. Hence in the current scenario we hypothesized its association and found a definite evidence of the presence of HPV in JNA tissue.
1
Material and methods
In accordance with our hypotheses we subjected 3 formalin preserved JNA specimen for laboratory assessment of HPV in the tissue. By curiosity we tried a western blotting in the first specimen (sample 3) and surprisingly found positive result. Subsequently we subjected samples 1 & 2 to immunoflorescence and again found the evidences of HPV inside the tumor tissue in both the samples. After the confirmation of its presence in all the formalin fixed specimens and with an aim to overcome the possibility of a false positive conclusion based upon a single technique use in each one of them, we subjected another set of 3 fresh tissue specimens of JNA for a comprehensive estimation of HPV. The laboratory methods undertaken for the presence of the HPV infection in the tissue sample included PCR, western blotting & immunofluorescence. To rule out the possibility of a natural presence of HPV in the nasopharynx of our population we subjected an additional 5 specimens of adenoids to western blotting. These were obtained during routine adenoidectomy at our facility and the protocol of selection of patients was as per the surgical guidelines. An attempt was further made to correlate the clinical and surgical behaviour of these cases with HPV involvement.
Clinical workup: was carried out at the department of Otorhinolaryngology, King George Medical University Lucknow in routine patients of JNA. Accordingly the history, demographics, symptoms, signs, imaging, radiological staging, other investigations, surgical details including intraoperative bleeding and tumour volume were noted. The treatment of choice was open surgery where intra-and post-operative transfusion of packed RBC was undertaken as per the requirement. Following surgery a 0.5 cm tissue sample was transported in RNA litter under cold chain (frozen cryogels in an insulated box) to CDRI for molecular assessment.
Molecular assessment: was undertaken at the CDRI Lucknow. After screening the formalin fixed 3 samples for presence of HPV a comprehensive analysis of the other 3 fresh specimen was undertaken through western blotting, PCR & immunofluorescence.
DNA extraction and PCR: 10 mg of the tissue was chopped & 200 μl of buffer A (40 mM Tris, ph 8.0; 5 mM Nacl; 1 mM EDTA, ph 7.5), 10 μl of proteinase K and 30 μl of 20% SDS were added to it and incubated at 37 °C overnight. 200 μl of buffer B (4 mM Tris, ph 8.0; 1.5 mM Nacl; 1.2 mM EDTA, ph 7.5), equal volume of chloroform: isoamyl alcohol was added after 24 h incubation and kept for 5 min. The samples were then centrifuged at 12,000 rpm for 10 min at 10 o C & upper aqueous layer was collected. The upper aqueous layer was collected separately and equal volume of phenol: chloroform: isoamyl alcohol. (P:C:I) in the ratio of 25:24:1 was added and then centrifuged at 12,000 rpm for 10 min at 10 o C. Upper aqueous phase was collected again and double volume of chilled ethanol was added. The samples were then kept overnight at 4 o C for precipitation. In the morning these samples were centrifuged at 12,000 rpm for 10 min. Pellet obtained was washed with 70% ethanol, air dried and dissolved in Nuclease Free Water (NFW) & quantified by Nanodrop (Thermoscientific).
Primers designed for the most common HPV types-HPV6, 11, 16, 18, 31 and 33 were used in the polymerase chain reaction-based assays for the detection of the HPV DNA in the patient samples. All the samples were checked for DNA integrity using β globin primers. PCR amplification was carried out in a 12.5 μl reaction volume containing of genomic DNA (500 ng/μl), 0.5 μl of each primers (10 p.mol), 6.25 μl PCR Master Mix (2 ×) (Thermo Scientific) and the total volume was adjusted to 12.5 μl using Nuclease free water. The amplified gene fragments were observed of 273 bp for HPV and 268 bp for β globin. All the amplified products were visualized on 2% agarose gels.
1.1
Western Blotting
Nasopharyngeal angiofibroma tissue samples were weighed around 10 mg and homogenized in 100 μl of RIPA lysis buffer containing protease inhibitor cocktail (Pierce). The lysate was then centrifuged and supernatant containing extracted protein was collected. The extracted protein was quantified using the Bradford method and was denatured with Laemmli buffer by addition in 1:1 ratio. 50 μg protein was loaded into each well and electrophoresed on 12.5% SDS-PAGE gel. The SDS-PAGE separated proteins were transferred onto nitrocellulose membrane (GE Healthcare) by electro-blotting. The blotted membrane was stained with ponceau stain (Sigma). It was then washed with 1 × PBS and kept for Blocking in 2% BSA for 2 h. The blot was then incubated with HPV primary antibody (ab70, Abcam) for overnight at 4 °C. Now the membrane was washed with PBST and subjected to anti-rabbit secondary antibody conjugated with HRP for 4 h at room temperature. Thereafter membrane was washed and protein bands were detected by using ECL reagent and developed with the aid of chemi-documentation system (Image Quant LAS4000, GE Life Science, PA).
1.2
Immunofluorescence
The tissue samples of nasopharyngeal angiofibroma patients were fixed in 4% formalin tissue and dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin. Sections (7 μm) were cut, mounted onto slides, deparaffinized, rehydrated and blocked with blocking solution containing 10% fetal bovine serum (FBS) in PBS. Sections were then incubated with HPV primary antibody (ab70, Abcam) for overnight at 4 °C followed by washing with PBST. Sections were then subjected to anti-mouse secondary antibody conjugated with Cy3 for 4 h at room temperature in dark and washed with PBST. The nucleus was stained with DAPI followed by washing & mounted with the cover slip along with Prolong Gold Antifade Reagent (Invitrogen) and sealed with nail polish. Sections were observed under epifluorescence microscope using a Nikon microscope.
2
Observations
The presence of HPV in JNA tissue and that too in all the cases was absolutely unexpected. The clinical and molecular observations are summarised in the Table 1 . The first 3 cases showed HPV in formalin fixed tissue that were difficult to work up than the later 3 fresh tissue frozen specimen transported in RNA litter. To further make the comparison in both the groups similar we included one recurrent case in each.
| Case (corresponding to sample) number | 1 | 2 | 3 | 4 | 5 | 6 |
|---|---|---|---|---|---|---|
| Age | 12 y | 15 y | 16 y | 24 y | 12 y | 18 y |
| Sex | Male | Male | Male | Male | Male | Male |
| Duration of nasal obstruction | 3 m | 2 m | 2 m | 4 m | 1 y | 3 m |
| Duration of headache | 3 m | 1 m | 1 m | 10 days | 3 m | 15 d |
| Degree of epistaxis | Mild | Mild | mild | Mild | Mild | Mild |
| Stage (Mishra et al. ) | IIIA | IIIB | IIIA | I | IIIA | IIIA |
| Tumour volume | 94 ml | 42 cm 3 | 40 cm 3 | 5 ml | 28 ml | 22 ml |
| Intraoperative blood loss | 1193 ml | 1765 ml | 311 | 5 ml | 1160 ml | 950 ml |
| Recurrent | No | Yes | No | Yes | No | No |
| Evidences of HPV on Immunohistochemistry | Yes | Yes | ND | Yes | Yes | Not evident |
| Evidences of HPV on polymerase chain reaction (PCR) | ND | ND | ND | Yes | Yes | Yes (faded signal) |
| Evidences of HPV on western blot | ND | ND | Yes | Yes | Yes | Yes |
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