Perineural Invasion in Oral Squamous cell Carcinoma: Incidence, prognostic Impact and Molecular Insight.

BACKGROUND
The objective of this study was to characterize the incidence and prognostic correlation of perineural invasion in oral squamous cell carcinoma and determine whether nerve growth factor and its receptor tyrosine Kinase A expression could be used as biological markers for perineural invasion.


METHODS
A retrospective review of pathology reports of 430 patients with oral squamous cell carcinoma who were treated from 1992 to 2014 in Tayside, Scotland was carried out. The expression of nerve growth factor and tyrosine Kinase A was assessed with immunohistochemistry in 132 tissue sections of oral squamous cell carcinoma.


RESULTS
Perineural invasion was identified in 17.4% of oral squamous cell carcinomas. High expression of nerve growth factor and tyrosine Kinase A was seen in 84% and 92% of oral squamous cell carcinoma respectively. Tumours with perineural invasion expressed nerve growth factor and tyrosine Kinase A with a greater frequency than tumours without perineural invasion. Perineural invasion and high expression of nerve growth factor were significantly associated with pain. Perineural invasion was significantly associated with stage IV tumours and poor disease-specific survival.


CONCLUSIONS
A higher level of expression of nerve growth factor and tyrosine Kinase A may predict perineural invasion and therefore may be considered as biological markers for perineural invasion in oral squamous cell carcinoma. Perineural invasion and nerve growth factor overexpression may contribute to the pain generation in oral cancer patients. Perineural invasion and nerve growth factor expression can predict the aggressiveness and prognosis of oral squamous cell carcinoma patients.

perineural spread and neurotropism, is used to describe the process of cancer cells "invasion in, around and through the nerves". 5 PNI in cancer is also defined as "tumour in close proximity to nerve and involving at least 33% of its circumference or tumour cells within any of the 3 layers of the nerve sheath". 4 There is considerable variability in the reported incidence rates of PNI in OSCC worldwide.
At sites such as the tongue and/or floor of the mouth, PNI was detected in up to 70% of OSCC, whereas cancer of the lower lip has a lower rate of PNI at 5.2%. 6 PNI exhibited by some OSCC has been known as an independent predictor of poor prognosis and an indicator of aggressive behaviour. [7][8][9] The presence of PNI is significantly correlated with advanced T and N tumour staging, extranodal extension, poor tumour differentiation, lymphovascular invasion and increased depth of invasion, and therefore, PNI seems to be critical to prognosis in OSCC. 10,11 PNI is known to be implicated in pain generation in patients with adenoid cystic carcinoma, pancreatic cancer and OSCC. [12][13][14] A number of neurotrophic factors such as nerve growth factor (NGF) and its high affinity tyrosine kinase A (TrkA) receptor are likely to be involved in the possible molecular mechanism of PNI. 15 Few reports have revealed the association between the NGF and/or TrkA with PNI in OSCC. [16][17][18][19][20] Given the paucity of information on the role of these factors in PNI and their possible role in the development of pain in OSCC, this study was undertaken to characterise the incidence and prognostic correlation of PNI in OSCC and to determine whether NGF and its receptor TrkA expressions could be used as potential markers for PNI in ex-vivo OSCC.

| Patients and tissue collections
A retrospective review was undertaken of the histopathology reports of 430 patients with OSCC from 1992 to 2014 in Tayside, Scotland, who were reviewed or a minimum of 5 years or until death.

| Immunohistochemistry
The FFPE tissue blocks were sectioned at 5 µm, deparaffinised in xy-  were ductal epithelial cells in salivary gland tissues. 16 The NGF and TrkA antibodies were blocked using the respective blocking peptide (#365944P and #7268P, respectively, Santa Cruz biotechnology) by adding twice the volume of peptide as the volume of antibody used.
These tissues were used as negative control.

| Immunohistochemical score
Assessment of the staining was carried out according to the scoring system reported previously in the literature with some modification. 16 Stained sections were scanned using a light microscope at low power. Tumour cells exhibiting a brown cytoplasmic, nuclear and/or surface membrane staining were counted as positive.
Analyses of intra-class correlation by the main observer twice and inter-class correlation by three independent observers were carried out to test the scoring results consistency gave a Cronbach's alpha of more than 0.8. NGF and TrkA staining scoring was performed as follows: the percentage of tissue staining was designated as 1 when 0%-25% of tumour cells were stained, 2 when 25%-50% of tumour cells were stained and 3 when >50% of tumour cells were stained.
The intensity of tissue staining was categorised as 1 for weak staining, 2 for moderate staining and 3 for strong staining. The final scoring was determined according to the product of staining intensity and the percentage of tissue staining ranging from 0-6 categorised as low expression (IHC = 0-2) and high expression (IHC = 3-6).

| Statistical analysis
Data were analysed using SPSS package (IBM statistics version 22).
Data related to categorical variables were described in terms of numbers with percentage and as mean with standard deviation (±) were calculated using the Kaplan-Meier method, the difference in survival rate was assessed by log-rank test and a P-value of <.05 was considered statistically significant.

| Relationship between NGF and TrkA expression with clinicopathological parameters in OSCC samples
PNI-positive OSCC expressed NGF and TrkA with a greater frequency than PNI-negative OSCC (P < .5). There were significant differences between NGF/TrkA expression levels, DOI > 4 mm and lymphovascular invasion (P < .05). Furthermore, patients who presented with pain had a significantly higher NGF expression in their tumour compared with patients with low NGF expression who presented with pain (P < .5). Correlation between NGF and TrkA expression levels and clinicopathological parameters including the presence and absence of PNI of 132 OSCC samples is shown in Table 3.

| Survival analysis
The presence of PNI was associated with poor DSS in OSCC (P < .05).
The trend for OS and DFS was similar but not significant. The over-

| D ISCUSS I ON
PNI is observed when cancer cells invade and grow along nerve tracts away from the primary tumour and is recognised as one of the most important prognostic factors in OSCC. 9 Our study found that the incidence of PNI was 17.4% in OSCC, and this is consistent with the trend of overall frequency of PNI that ranges from 2.5% to 71% in OSCC. 6 The vast majority of PNI-positive OSCC were tumours arising in the tongue and floor of the mouth. These results are consistent with other studies where heterogeneous anatomical sites were employed. 6 Some studies have shown that infiltration of the perineural space of the nerves by tumour cells correlates with tumour size, depth and pattern of invasion, the degree of tumour differentiation, presence of nodal metastasis and presence of extranodal extension in OSCC. 10,11,25 Our study demonstrates a significant correlation between the presence of PNI and these histological features in OSCC.
The process of PNI requires activation of some neurotrophic growth factors such as NGF and its TrkA receptor that may play a role in the mechanism of PNI. 26  OSCC. [16][17][18][19][20] In the present study, we demonstrated NGF and TrkA expression in OSCC; interestingly, the expression levels were significantly higher in PNI-positive compared with PNI-negative OSCC.
Moreover, there was a positive relationship between the expression of NGF and TrkA in OSCC in agreement with another study on OSCC. 16

Besides the cancer cells, immunoreactivity of NGF and
TrkA was remarkably present in nerve tissues in OSCC in this study.
Therefore, this may suggest that there is a close interaction between the cancer cells and neural tissue, where NGF is expressed by cancer cells causing tumour cells and nerves to grow together and enhance their physical contact, and therefore induce PNI. This hypothesis is supported by a previous study showed the influence of NGF/TrkA interactions between cancer cells and neural tissues in OSCC that may facilitate the development of PNI. 16 The clinical features of PNI have been studied in different types of cancer, and it is well known that PNI is often clinically silent. 27 However, in this study, PNI is notably accompanied by pain in OSCC patients with PNI. Another study also reported that pain was a symptom in the majority of patients with PNI-positive OSCC at the time of presentation. 28 NGF and its receptor TrkA are believed to be major mediators of pain in pancreatic and prostate cancer. 13,29 To our knowledge, this is the first report to show that overexpression of NGF might influence pain generation in patients with OSCC.
A previous study has shown an association of NGF overexpression with some pathological parameters in OSCC; however, TrkA immunoreactivity was not associated with any pathological factors in the same study. 19 Interestingly, our findings demonstrated a signif-