Oral cancer is a major global health burden with higher rates of occurrence reported in the recent decades especially among Asian countries. Significantly higher proportion of the oral cancers are related to areca nut and tobacco chewing. ^{1 – 3} Despite the advances in medical technology and therapeutics, the survival and disease outcome of oral cancer still remains within 5% to 50%. ⁴

Oral cancer has a complex multifactorial pathological basis with host immune responses having a role play in influencing the biological behavior of the tumor. Recent evidence has emphasized on the significance of systemic inflammatory response and the effect of cytokines in determining the disease progression and outcome. ^{5 , 6}

Interleukin-6 (IL-6) is considered as a multifactorial cytokine that controls various steps of inflammatory cascade. IL-6 is capable of influencing the biological activities of the cell by acting on the autocrine and paracrine pathway, thereby influencing the transduction process on the cell receptors and is also a crucial factor contributing to the differentiation and growth of various types of cells, including endothelial cells, B-cells, T-cells, neuronal cells, and osteoclasts. In terms of their activity with regard to tumors, IL-6 can mediate various oncogenic processes such as growth, apoptosis, migration, invasion, proliferation, progression, angiogenesis and differentiation of the tumour cells by the activation of inflammasomes. ⁷

Apart from the cytokines, various inflammatory cells also play a vital role in the regulation of oncogenesis. Neutrophils are the most common cell type associated with acute phase of inflammation, whereas lymphocytes are associated with chronic inflammation. The neutrophil–lymphocyte ratio (NLR) is regarded as a marker related to the presence and prognosis of several types of cancer by demonstrating the equilibrium between the activation of the inflammatory pathway and the anti-tumour immune function. ^{8 , 9}

Platelets also have a direct role in promoting, invasion and spread of the tumor cells. The complex interaction of platelets, interleukins and myeloid metalloproteins are also related to the progression of tumors. The multifaceted role of platelets in the progression of cancer is well recognized and there are several evidences reporting platelet–lymphocyte ratio (PLR) as a prognostic marker of various cancers including head and neck cancers. ^{10 , 11}

The diagnostic and prognostic markers associated with cancers are studied extensively in the recent decades. Though, there is evidence in the literature about the prognostic value of NLR and PLR, studies examining their significance before and after surgery are limited. Hence, the current study was undertaken for comparative evaluation of serum IL-6, NLR and PLR in pre- and post-surgery oral squamous cell carcinoma.

Comparative evaluation of serum IL-6 with NLR and PLR in pre- and post-surgical OSCC patients.

1.

To estimate pre- and post-surgical serum levels of IL-6, PLR, NLR in OSCC patients

The demographic features and characteristics of the subjects included in the study were enumerated in Table 1. The study population comprised of 17.5% of females and 82.5% of males. The mean duration of patients suffering with OSCC as reported by the patients was 5.08±6.46 months, Majority of the study subjects had the habit of consumption of multiple areca nut with or without tobacco products and most patients presented with the ulcercero-proliferative type of OSCC. The clinical, imaging and histopathological based grading distribution was given in Table 2. Overall, there were no subjects in stage I, II and IVB. 52.5% of the study population had stage III OSCC and 47.5% had stage IVA OSCC based on the AJCCTNM staging of Oral Cancer.

On comparison of the IL-6, NLR and PLR based on the staging of OSCC ( Table 3). The mean preoperative NLR in stage III was 2.58±0.80 and stage IVA was 3.38±0.67, with a statistically significant difference (P=0.002). The mean post operative NLR in stage III was 2.73±0.94 and stage IVA was 2.60±0.85. However, there was no statistical significance observed (P=0.635). The mean preoperative PLR in stage III was 134.27±37.60 and stage IVA was 181.15±32.57 with a statistically significant difference (P<0.001). The mean post-operative PLR in stage III was 113.8108±39.65309 and stage IVA was 135.06±40.49, but there was no statistical significance observed (P=0.102).

There was no statistical significance noted among various histological grades of OSCC with IL-6, NLR and PLR both pre- and post-operatively.

On comparison between the pre- and post-operative IL-6, NLR and PLR ( Table 4). The mean preoperative IL-6 was 30.66±455.15 and the post operative IL-6 was 60.86±76.92. The difference was statistically significant (p=0.047). Statistically significant difference was also noted between the mean preoperative PLR (156.54±42.15) and the post-operative PLR (123.90±40.97) (P=0.001). But there was no significant difference noted between the mean preoperative NLR and the post operative NLR (P=0.134).

Inflammation is considered as a host response that aims at restoring normalcy of the injured or altered tissue. But chronic and persistent inflammation can result in neoplastic progression of the affected cells, because of the effect of the inflammatory cells. ¹⁴ The state of inflammation has an influential role in the tumor microenvironment contributing to the progression and spread of the tumor. Various pro-inflammatory markers such as interleukins, TNFα, interferon γ and inflammatory cell mediators such as neutrophils, lymphocytes, monocytes and mast cells are known to be involved in the tumor associated inflammatory status. ^{15 , 16}

The present study explore the role of IL-6, NLR and PLR in OSCC in both pre- and post-operative status. In the present study, we observed the mean age of patients with OSCC to be 48.47±10.7 years which represents the 5th decade. This is in accordance with several previous studies by Aruna et al. ¹⁷ and Shenoi et al. ¹⁸ who had reported similar findings. A male:female ratio of 4.7:1 was observed in this study, which is correlated to previous study. ¹⁹ It was observed that the mean duration of the disease was reported as 5.08±6.46 months which represents the state of delayed diagnosis of OSCC from the onset of the symptoms. Similarly, Naseer et al. ²⁰ in their analysis had reported a delay in the diagnosis of OSCC about 4 to 6 months due to the low socioeconomic status of patients and lack of patients availing early medical assistance. Majority of the patients were diagnosed with stage III (52.5%) and IVA (47.5%) of OSCC which supports the fact that most cases of OSCC in India are diagnosed only at the later stages. ^{21 , 22}

IL-6 levels in OSCC subjects were evaluated both pre- and post-operatively. There was a lack of significant difference noted both pre and post operatively between the overall staging of OSCC and histopathological grades of OSCC. However, Dineshkumar et al. ²³ had reported a significant downregulation of serum IL-6 from well-differentiated to poorly differentiated OSCC. In the present study lower levels of pre and post operative IL-6 were observed in poorly differentiated OSCC when compared with that of well differentiated type, that might be related to the presence of increase in the central necrosis in tumor mass. However, our results were not statistically significant. Chang et al. ²⁴ and Panneer Selvam et al. ²⁵ in their study, reported IL-6 concentrations to increase with advancing overall stage as in the present study subjects were only belonging to stage III and stage IV that is advanced stage as compared to stage I and stage II. Increase in serum IL-6 corresponding to the increase in the tumor size and depth have been reported in the previous studies. ^{26 , 27} Increase in IL-6 with advancing stage is also attributed to the lymph node metastasis and invasion of OSCC by the activation of NLRP3 and inflammasomes. ²⁸ Upregulation of IL-6 is known to have an inducing effect on the vascular endothelial growth factor (VEGF)-C resulting in the lymphatic spread of the tumor. ⁷ Therefore, agents inhibiting the IL-6-induced signaling pathways are considered in the targeted therapy against OSCC to gain control of spread of the tumor.

In the present study, the mean preoperative IL-6 levels in OSCC subjects was 30.66±455.15 whereas the post operative levels was 60.86±76.92, which was much higher than in healthy individuals. ^{29 , 30} However, there is a lack of studies comparing the pre-and post-operative levels of IL-6 in OSCC and the post-operative levels are considered to be directly related to the age and magnitude of post operative complications and stress among the patients, as IL-6 is known to vary largely depending on the post-surgical host inflammatory status. ³¹

NLR is the measure of the differences between the absolute neutrophil counts to the absolute lymphocyte count. It is well recognized as a marker denoting the status of systemic inflammation. It is a well-known prognostic indicator of HNC. ³² A study by Mahalakshmi et al. ³³ reported the mean range of NLR as 1.7–2.3 in healthy individuals of a similar population. Another study by Singh et al. ³⁴ reported a mean value of 1.82 in normal healthy individuals. The NLR values among the study subjects with OSCC were higher than that of normal values reported in healthy individuals was seen in previous studies. Evaluation of NLR values in OSCC showed a significant difference in preoperative NLR based on the staging of the tumor, with higher NLR in advanced stage (Stage IVA). But there were no significant statistical findings in the post-operative NLR based on the staging and histopathological type of OSCC. Similar findings have been reported by Tazeen et al. ³⁵ and Bobdey et al. ³⁶ who reported high preoperative NLR to be associated significantly with advancing stage of the tumour. Hasegava et al. ³⁷ reported high NLR to correlate with worsening of the differentiation of OSCC which is contrasting to our findings. The inconsistency and difference in the observations can be attributed to the age, associated co-morbid conditions and other inflammatory factors in the study population, as NLR tends to be highly influenced by other systemic and local inflammatory conditions. The increase in the NLR in advancing stages is directly related to the neutrophilia with relative lymphopenia, that can be related to the neutrophil recruiting action of the tumor cells and tumor induced destruction of the lymphocytes as the tumor progresses. The increase in the NLR is brought about by the presence of neutrophilia and relative lymphopenia that manifests owing to the various immuno-inflammatory changes that take place in the tumor microenvironment, that can be hypothesized as the reason behind increased NLR in advanced TNM stage of OSCC.

Platelets are major components of blood mainly responsible for haemostasis and blood coagulation. Platelets along with lymphocytes serve as markers representing the tumor-related inflammatory status in various cancers including oral cancer. ³⁸ The present study evaluated the PLR values in OSCC both pre- and post-operatively. A notable difference was noted in the pre and post operative NLR between Stage III and Stage IVA of OSCC. However, no notable difference in the post operative PLR based on the staging was observed. Also no noticeable difference in pre and post operative PLR based on the histopathological type of OSCC was observed. Tazeen et al. ³⁵ reported a positive correlation of pre-treatment PLR with increasing stage of the tumor, which can be matched with our finding. A significant association between PLR and histopathological degree of differentiation of OSCC was also reported by Tazeen et al. ³⁵ which is in contrast to the findings of present study. Malik et al. ³⁹ had reported positive correlation of pretreatment PLR increase in staging of the tumor, similar to our finding. In the present study, there was a significant difference noted in comparison between the pre and postoperative PLR, that could be related to the improvement in terms of reduction in the tumor associated host inflammatory status. However, there are no much evidence in the literature comparing the preoperative PLR with post operative PLR. The present study did not find significant difference in the NLR pre- and post-operatively, but significant difference was observed in PLR, suggesting that PLR could be superior in terms of representing the tumor related inflammatory status, similar to the findings of Acharya et al. ⁴⁰ and Tazeen et al. ³⁵

Overall, from the observations of present study, it is evident that pre-operative or pre-treatment IL-6 levels, NLR and PLR can be used as markers to assess the stage of the tumor as higher values of NLR and PLR are associated with high stages of tumor. Evaluation of post-operative IL-6 and PLR can aid in understanding of the post-operative and post-treatment inflammatory status in individuals affected with OSCC.

However, this study was bound for limitations such as a single Institute study and presence of underlying areca nut habit with or without tobacco associated metabolic diseases that may serve as a confounding factor. Further, prospective multi-centric studies are required for evaluating the pre- and post-operative IL-6, NLR and PLR in terms of overall survival and disease free survival of OSCC to aid in future understanding of the clinical utility of these pro-inflammatory cytokines and inflammatory mediators.

Evaluation of preoperative IL-6, NLR and PLR can facilitate a better understanding the role of pro-inflammatory cytokines in tumor progression. NLR and PLR are useful markers representing the stage of the tumor as higher values of NLR and PLR are associated with high stages of tumor. Pre- and post-operative IL-6 and PLR can also be useful in post-operative and post-treatment inflammatory status in individuals affected with OSCC. The understanding of immune mediated inflammatory response in OSCC can serve as marker for selecting appropriate post-operative chemotherapeutic measures to exert a positive influence in the disease outcome and quality of life.