Depth of Invasion as Tumor Indicator in Cervical Lymph Node Metastasis in OSCC GENDER INVOLVEMENT IN OSCC

This work was carried out in collaboration among all authors. Author FA designed the study, wrote the protocol and wrote the first draft of the manuscript. Author SF managed the analysis of the study and result write up. Author AZ had performed data entry, statistical analysis. Authors SB, SS and HB managed the literature searches. All authors read and approved the final manuscript. ABSTRACT Squamous cell carcinoma of oral cavity is the 6 th common cancer of the world and a major source of (89.3%) was the most predominnant grade observed in this study. on the basis of DOI the patients were divided into 2 groups. One Group with Depth of invasion upto 4 mm and second group with Depth of invasion >4 mm. depth of invasion above 4 mm was found in 82.1% and without nodal metastasis(40.7%). The mean tumor size was was found to be 4.36± 2.433 mm and tumor thickness was 1.95±1.48 mm. and the most common anatomical site was found to be buccal mucosa (73.6%). T Stage and Nodal status,Tumour thicknes showed significant association with depth of invasion with p values 0.001, 0.023 and 0.002 respectively. Conclusion: The tumor depth of invasion is found to be an important variable that can be related with metastasis in Oral cancer as our study showed its significant association with lymph node meatastasis.


INTRODUCTION
Oral squamous cell carcinoma develops from epithelial lining of the oral cavity.It is the 6 th commont cancer of the world and a major source of malignancy around the world [1][2][3]. Burden of disease in oral cancer is highly increased in the less developed countries of asia around 40% as compared to the west where over all prevalnace is 2 to 5% [4].
Early oral cancer detection and diagnosis leads to a better prognosis, increases the chances of patient survival and plays a significant role in successful clinical treatment outcome. Delayed detection may lead to treatment failure and death [5]. The current available treatment options for the oral cancers are not satisfactory, and the survival rate from past 5 years has not improved significantly over the past two-decades [5][6][7][8].
Lymph node metastasis is the most common source of oral cancer metastasis.Surgical resection of tumour with neck dissection is the most important treatment of choice for the patient survival in oral cancer.Elective neck dissection gives information of pathological neck nodes involvement, hence remove undetectable cancerous cells in the lymphatic vessels and also helps to add additional therapy.The 5-year survival rate for patients of oral squamous cell carcinomas with lymph node metastasis is upto 50% [9][10][11].
According to AJCC 8th edition Depth of invasion is measured as "distance from horizon of basement membrane of adjacent squamous mucosa to deepest point of tumor in perpendicular direction through a plumbline" [12]. Now-a-days ,the best prognostic indicators for the occult lymph node metastasis are Depth of Invasion and lymph node biopsies. Sentinel lymph node biopsies are highly accurate for identification of the lymph nodes metastasis. The sentinel lymph node biopsy is a delicate technique which requires accuracy. Whereas, Depth Of Invasion is used as a marker for elective neck dissection [10].
The purpose of this study was to assess the association of DOI and the risk of occult lymph nodes metastasis in the early oral cancer diagnosis.

MATERIALS AND METHODS
A Cross sectional study was conducted at Ziauddin hospital, North nazimabad after Ethical Review Committee approval (Reference Code: 0330618FAOM) The diagnostic histological slides of 140 patients with OSCC managed between January 2021 and January 2022 from the Ziauddin Hospital North Nazimabad histopathology department were collected from the hospitals' archives.
A total of 140 paraffin embedded foramlin fixed OSSC tissue slides were included. Patients with any other cancer of oral cavity were excluded from study. 2 investigators (F.A. and S.F.) evaluated H&E slides by light microscopy which were further reviewed by a senior pathologist (S.B). A pilot study of 20 cases was conducted to standardize the evaluation criteria for all 3 investigators. At the time of the evaluation all 3 investigators were blinded to the clinicopathologic data.
All Patients file records were recorded.which includes age of the patient,gender, tumor site,tumor staging(TNM),the DOI, Lymph node involvement, tumour differentiation grades, perineural invasion and lymphovascular invasion.

Measurement of Depth of Invasion:
The hematoxylin and eosin slides were used to measured depth of invasion which was measured in accordance with AJCC 8 th edition as "a plumb-line from as the distance from the inner most invasion area to the basement membrane adjacent to normal mucosa ". A cutoff value of 4mm was used for depth of invasion [10,11].

RESULTS AND DISCUSSION
The present study included 140 OSCC patient with mean age of 46.87 ± 12.25 years. 80.7% of patients were males. the most common anatomical site was found to be buccal mucosa (73.6%). The mean tumor size was 4.36± 2.433 mm and tumor thickness was 1.95±1.48 mm. the most common grade of OSCC was found to be moderately differentiated grade present in 89.3% of patients. 50% of patients had T4 stage followed by 25% of patients with T2 Stage. Lymph node metastasis was present in 59.3% of cases. on the basis of DOI the patients were divided into 2 groups. One group with depth of invasion upto 4 mm and second group with depth of invasion >4 mm. depth of invasion up to 4mm was present in 17.9% of patients whereas above 4 mm was found in 82.1%.
The association of depth of invasion with clinicopathological parameters was assessed. There is a significant association of depth of invasion with Tumor stage (P value 0.001), lymph node involvement (P value 0.023) and thickness of tumor ( P value 0.002) respectively. Literature suggested that depth of invasion of tumor is used as a tumor predictor and prognostic indicator in decision making of End node dissection in the early stages of oral squamous cell carcinoma. The optimal cut off point of depth of invasion was taken 4mm. The histomorphometric parameter of the tumor are best recognized after the surgical resection of the tumour.therefore treatment planned of such cancer patients depend upon the surgical resected tumour specimens results [10,15].
Numerous studies reported tumor depth of invasion and tumor thickness are not the same. ''Depth of invasion'' means the extent of cancerous lesion growth into the tissue beneath an epithelial surface [11,16].
Literature suggested that the Depth of invasion of tumor as a occult lymph node metastasis predictor, In early oral cancer diagnosis END is used as a tumour predictor. For indurated tumours, The DOI is considered a better prognostic factor than Tumour Thickness. The 8 th edition of AJCC guideline clearly stated the Depth of invasion definition as distance from horizon of basement membrane of adjacent squamous mucosa to deepest point of tumor in perpendicular direction through a plumbline Our study showed with depth of invasion>4mm raised significantly the risk of nodal involvement. some studies do not confirm cut off value of 4mm. Desparity exist in literature number of studies showed large variance. Some studied reported cut off value of <5mDOI Some showed cut off value of > 5mm. However, Some showed 7.25mm cut off. However, Some reported 4mm cut off. Some did not specify any cut off value for DOI, Some showed DOI value cutt off varied from different subsites for tongue 2mm, floor of mouth 3mm.The reason for lower cut off value for tongue as compared to other subsite was the higher risk of lymph node metastasis due to its rich vascular and lymphatic supply and floor of the mouth [10,[17][18][19][20][21].
Literature revealed there was a significant correlation between the tumor thickness and cancer metastasis at an early stage. Studies showed a strong association co-exist between the tumor depth of invasion and nodal status involvement, Which were concurrent with our study. Their study showed a depth of invasion of 4 mm to be a valuable cut-off for the occurrence of lymph node metastasis. They have recommended that oral cancers with 4 mm DOI are at higher risk of metastasis and should be selected for elective nodal dissection [22]. but no significant association were reported with pathological tumor size and tumor grade in tumor metastasis. Despite our study showed significant assoiciation of tumor size and tumor grade.The reason for that was tumor thickness became more significantly co-related with larger tumor size (T2-T4) which were concurrent with other studies [10,11].
In our study, We had OSCC patients with tumor size measuring > 4 cm and upto 4mm.The strength of this study was measuring the depth of invasion for all OSCC cases by AJCC guide line. The depth of invasion was measured by AJCC guideline on H & E slides. By specialistoral pathologist. Limitation of this study was retrospective single centered study. A multicentic prospective study with standard protocols should be carried out in future in order to correlate depth of invasion with the radiological aspects and histopathological determinants of tumor [18].

CONCLUSION
The DOI is an important tumor predictive factor for occult neck metastasis in Oral Cancer. Our study showed significant association of DOI with lymph node metastasis. OSCC patients with a Depth of invasion >4 mm had increased the probability of lymph node metastasis than those having depth of invasion upto 4 mm.

CONSENT
Informed written consent was obtained from patients guardian.

ETHICAL APPROVAL
The study was accepted by the Ziauddin University Ethics Review Comimitee. Ethical approval was obtained (Reference Code: 0330618FAOM) It was carried out in line with the Helsinki Declaration, and all participants gave their informed written permission.