To study HPV and EBV in squamous cell carcinoma of upper aerodigestive tract by immunohistochemistry markers

Primary objective

To determine the presence or absence of HPV and EBV in SCC of UADT by IHC.

Secondary objective

To determine the association of HPV with EBV in SCC of UADT by IHC.

Study design

Cross-sectional study.

Inclusion criteria

All patients with biopsy confirmed UADT squamous cell carcinoma who underwent radical primary surgery (Figure 1, Figure 2, Figure 3) including neck dissection.

Exclusion criteria

All small biopsies with inadequate material without TNM staging of SCC of UADT specimens were excluded from the study.

Study duration

October 2018- November, 2021.

Sampling technique

Consecutive sampling.

Sampling size

30.

Statistical method

Chi square test, Independent sample t test, Fisher exact t test.

Method of collection of data

A sample of 30 cases were taken with their clinical details and where adequate biopsy available for paraffin embedded tissue. All the clinical details were either collected from Laboratory information services and medical record section. The required sections were stained with Hematoxylin and Eosin. Pathological grading of tumor was done by Broder’s classification. Tumor, nodal, metastasis was used for pathological staging.

Procedure for p16 and LMP1 immunohistochemistry (IHC) staining

IHC was performed on 4 µm thick sections on poly-l-lysine coated slides, deparaffinised with xylene and rehydrated in graded ethanol concentrations to distilled water.

Antigen retrieval was done with EDTA/ Tris buffer at pH 8.5 – 9 in a pressure cooker for 20 minutes. The sections are incubated with primary antibody p16 (DAKO, p16-INK4(MX007)- MAD-000690QD-R-3) and LMP1 (Epstein-Barr Virus /LMP1 (DAKO, CS1-4)- MAD- 001619QD-R-3) for 30 minutes followed by primary antibody amplifier for 15 minutes, then secondary antibody for another 30 minutes. This is followed by Diaminobenzidine as chromogen. Counter staining is done with Harris Hematoxylin, followed by clearing, mounting and labelling.

Figure 1

Left mandibulectomy

Figure 2

Laryngectomy

Figure 3

Left segmental mandibulectomy

Squamous cell carcinoma of cervix was taken as a positive control for p16 IHC. p16 IHC was considered positive if more than 70% of the tumor cells showing nuclear and cytoplasmic staining (Figure 4, Figure 5).7, 8

Classic Reed Sternberg cell in Hodgkin lymphoma was taken as a positive control for LMP1 marker. LMP was considers positive based on Allred scoring (Figure 9, Figure 10).9, 10

Figure 4

p16 IHC control- Squamous cell carcinoma of cervix(10x), showing >90% cells positivity

Figure 5

Diffuse p16 positivity in >80% of cells, strong cytoplasmic and nuclear staining

Figure 6

Diffuse p16 positivity in >80% of cells, strong cytoplasmic and nuclear staining

Figure 7

p16 positive, strong nuclear positivity, moderate cytoplasmic positivity (400x magnification [40x objective])

Figure 8

p16 negative, no nuclear positivity and no cytoplasmic positivity (100x magnification [10x objective])

Patient demographics

The age of the patients ranges from 40 years to 88 years with a mean age of around 60. Majority of the patients (16/30) were in the age group between 50-70 years accounting for more than 50% of the total cases (Table 1). Out of the 30 patients 21 (70%) were male and 9 (30%) were female. Male to female ratio was 2.3: 1 with higher prevalence seen in males (Table 2). There were patients (63.33%) with history of tobacco in form of either cigarette, beedi and chewable form (Table 3). Of the 19 cases with history of tobacco 13 were male and 6 were female. Among the non-tobacco users, 7 are male and 4 are female. Tumor is located in oral cavity in 19 (63%) cases, larynx 8 (26%) cases and hypopharynx 3 (10%) cases (Table 4). Majority of cases 25 (83%) were moderately differentiated SCC, followed by well differentiated 3 (10%) and poorly differentiated 2 (6.6%). Lymphovascular invasion was identified in 18 (60%) cases and perineural invasion was identified in 8 (27%) cases. Majority of tumors presented at stage 3 (15/30 cases, 50%), followed by stage 2 (9/30 cases, 30%).

P16, LMP1 IHC correlation with clinicopathological variables

HPV p16 IHC positivity was seen in 5/30 (16.7%) cases (Figure 6, Figure 7, Figure 8) and none showed positivity for EBV with LMP1 marker. p16 positivity was seen in elderly population (55-88 years) of which three were males and two were females. Three had history of tobacco and two didn’t. Majority of p16 positive tumors were in oral cavity (3/5 cases, 60%), followed by 1 (20%) in larynx and 1 (20%) in hypopharynx. All the cases which showed p16 positivity were well differentiated SCC (5/5 cases, 100%). Lymphovascular invasion (LVI) was identified in 4/5 (80%) and perineural invasion (PNI) was seen in 2/5 (40%) cases in p16 IHC positive SCC. LMP1 marker for EBV was negative in all the 30 cases studied (Figure 11).

Figure 9

LMP1 control (100x magnification [10x objective])

Figure 10

LMP1 control (400x magnification [40x objective])

Figure 11

LMP1 negative (10x)- in squamous cell carcinoma (100x magnification [10x objective])

Site of distribution of squamous cell carcinoma of UADT

Majority of tumors are found in oral cavity (63%) in the present study which is similar to other studies like Basu R et al, El-Mofty S et al and S Nair et al. Other studies (Table 5) like those of Dahlstrom et al, Cadoni et al and Riener M et al are discordant with our studies, showing tumors predominantly in oropharynx.

HPV presence by p16 IHC in Squamous cell carcinoma in UADT

In the present study 5 cases (5/30 cases, 16.66%) showed strong and diffuse (>70%) nuclear and cytoplasmic p16 positivity. There are few (3/30 cases, 10%) cases where there is a diffuse staining of nucleus and cytoplasm ranging from 20% - 30% with moderate to strong positivity, these unequivocal cases are considered negative. The remainder of the cases were considered negative for p16 IHC where less than 20% percent of tumor cells are staining regardless of intensity.

Majority of the cases which are positive for p16 IHC are present in oral cavity (3/5 cases, 60%), one in pharynx (1/5 cases, 20%) and one in larynx (1/5 cases, 20%) which is consistent with study of Verma et al. Other studies (Table 5) like Riener M et al showed p16 IHC positivity predominantly in Pharynx (Oropharynx) followed by oral cavity.

HPV prevalence in OPSCC was highest in North America (65.8%) but higher in men than women and lowest in Asia (28.9%) where HPV prevalence in women was highest (61.5%) and higher in women than men. OPSCC has been found to have a disproportional rise in younger individuals under 45 years of age without significant cigarette or alcohol exposure. In this subpopulation females are more affected than males. Patients with HPV positive SCC were three times more often to report having had oral sex as those with HPV negative SCC. This subpopulation has a distinct clinical profile, with an earlier diagnosis, a higher risk of modest regional spread at the time of diagnosis, and a better prognosis and response to therapy.25 Unlike other studies where HPV positivity was present in younger population below 50 years, in the current study we identified HPV positivity in much older population (above 55 years, 55, 61, 67, 70 and 88 years respectively). HPV-positive SCC were more likely to have an AJCC tumor stage of T3 & T4 than HPV-negative tumors (T2). (26) In the current study we identified patients with HPV positive SCC at higher, T3 stage.

According to few studies, patients with HPV-positive oropharyngeal cancer as detected by PCR, in situ hybridization, or P16 immunohistochemistry on tumor samples, have a considerably better overall and disease-free survival than those with HPV-negative oropharyngeal cancer.25 When compared to HPV-negative cancer, HPV-positive oropharyngeal carcinoma is more sensitive to radiation and anticancer medicines, and has a better prognosis.26

LMP1 IHC expression in SCC of UADT

There are several studies which showed the expression of LMP1 IHC for EBV in nasopharyngeal carcinoma, ranging from 0% in few studies to about 78%.10, 27

The presence of EBV was linked to neoplastic states of the oral cavity by Cruz I et al, but the information obtained does not suggest a direct involvement for EBV in the malignant transformation of normal epithelial cells. It rather suggests that the appearance of EBV in OSCC samples is due to immune suppression leading to shedding of the virus in the oral cavity.28

D Goldenberg et al, in their study observed that EBV was present in a small fraction of cases of head and neck tumors and they were detected at low levels, suggesting that the virus may not be contributing to tumorigenesis of head and neck SCC.29

Here in our present study, the cases we got were from oral cavity, larynx and hypopharynx. All of these cases showed LMP1 IHC negative. The lack of positivity is due to lack of cases especially from the nasopharyngeal and oropharyngeal areas. Other reason might be clearing of the infection by the host immune system or failure of the IHC to identify the latent infection, where the viral DNA is integrated in host DNA silently, in such cases serum immunoglobulin, polymerase chain reaction (PCR) and Fluorescent in Situ Hybridization (FISH) could be useful in identification.