India shares a major burden of oral and oropharyngeal cancers of the world. ¹ Even with the major advances in the surgical techniques and nonsurgical modalities such as various forms of radiotherapy, chemotherapy and targeted therapy, overall outcome in terms of 5 year survival remains at 56% in oral and oropharyngeal cancers.² The prognosis is still worse in Indian patients  with high incidences of p53 mutations.³ Delay in detection of these cases is one of the prime reason for such outcomes. Cancers at other sites such as cervix and breast do have better outcomes due to routine screening of early stage lesions which are detected early of any developing malignant changes in the lesion. Similar results of improved survivals are anticipated in a scenario of detectable cyto-morphological changes that can be easily detected and prognosticated^4,5. This will help in detecting the malignancies at an early stage, will reduce the overall disease burden and will improve the overall treatment outcome of oral and oropharyngeal cancers.

Nuclear and cytological markers of developing neoplastic transformations includes morphological features including increased nuclear cytoplasmic ratios, increased nuclear diameter and decreased cellular diameters; featured of proliferative activity such as increased number of regions with affinity for silver stains (AgNOR) and percentage of cells with >3 AgNOR avid regions; and meta nuclear alterations including increased number of micronuclei, cells with karyorrhexis and bi-nucleated cells. Micronucleus is a marker of chromosomal damage, genome instability and carcinogenesis, integrating acquired mutations and genetic susceptibility towards mutations.^6,7 Possible role of identification of such cytological nuclear features has been proven in buccal cells in literature to identify DNA damage⁸ and  in cases of buccal cancer⁴. However, there is still lack of literature about its diagnostic and prognostic significance in premalignant lesions where these can act as indicators of DNA damage and carcinogenesis for early detection of developing malignancies. The prognostic significance of cyto-morphologic nuclear and micro-nuclear changes in oral premalignant lesions is also needed to be established.

Hence we plan to conduct this study in order to see for various cytomorphologic and nuclear changes in oral and oropharyngeal premalignant and malignant lesions, to establish its diagnostic significance for early diagnosis and management in premalignant lesions in developing malignancies and prognostic significance in early malignant lesions of oral cavity and oropharynx.

Methods:

The various method for studying these nuclear and micro nuclear changes includes molecular biologic techniques such as flow cytometry, FISH, routine stains, DNA specific stains, Fluorescent stains and immune stains. Nonspecific routine stains include Giemsa, H & E, Crystal violet, Azan and Papanicolaou etc. DNA specific stains include diamine phenylindole (DAPI), Hoechst 33528, Ethidium Iodide/Bromide, Acridine Orange and Propidium Iodide etc. Automation is possible in diamine phenylindole (DAPI), Hoechst 33528, Ethidium Iodide/Bromide and Propidium Iodide. We intend to include Acridine orange, Fuelgen, Papanicolaou, AgNOR and CK-8 antibodies in cell blocks or standard histopathologic examinations of the identified patients for our analysis.

Post recruitment and consent patients will be taken for definitive diagnosis and management as per the routine departmental protocols and send for scrap cytology. The smears hence collected will be Collection of exfoliated cells. Subjects will be asked to rinse their mouth gently with tap water. To obtain the smear of exfoliated cells from the oral cavity (buccal mucosa in control group), a slightly moistened wooden spatula will be used. For pre malignant lesions representative site selected will be areas of leucoplakia, erythroplakia & oral sub mucous fibrosis in oral cavity and oropharynx. In OSCC patients, oral mucosal cells will be scraped from the margins of the lesion for obtaining the smear. The cells will be immediately smeared on pre cleaned microscopic slides. Just prior to drying, the smears will be fixed with commercially available spray fixative for 15 min. The slides will be labelled and preserved in dust-free boxes until evaluation. Biopsy & histopathological examination will be carried out in relevant cases based upon cytopathological findings.

Biopsy procedure and histopathological grading

Incisional biopsies will be taken from the representative sites with all aseptic precautions. The tissue specimen will be labelled, fixed in 10% neutral buffered formalin for 24 h, and paraffin embedded. The wax blocks will be cut to obtain two tissue sections of 4-5 μm thickness for each block and the sections will be stained by haematoxylin and eosin. Immunohistochemistry (CK8) will be performed on relevant cases.

Cytological preparation and evaluation

The smears will be stained by Papanicolaou technique using a commercially available staining kit RAPIDPAP, Feulgen stain, AgNOR stain & acridine orange. From each slide, 500 cells will be examined under the light microscope using low magnification (×400) for screening and high magnification (×1000) for counting of MN.

Scoring criteria

The criteria which was developed by Tolbert et al. was used for counting the MN. Screening of each slide was made in a zigzag manner from one end, toward the other end of the slide.

Tolbert et al. criteria parameters for identifying MN are as follows:

1.      Rounded smooth perimeter suggestive of a membrane.

2.      Less than a third the diameter of associated nucleus, but large enough to discern shape and colour.

3.      Staining intensity similar to nucleus.

4.      Same focal plane as nucleus.

5.      Texture similar to nucleus.

6.      The absence of overlap with or bridge to the nucleus.

Only those structures fulfilling the above-mentioned criteria were recorded as MN.

            For Silver avid regions we intend to calculate number of Ag avid regions and its presence of absence.

            The nuclear features and dimensions will be recorded using the standard software available for calculating nuclear dimensions.

Inclusion and exclusion criteria (Post-cytology)

Micronucleated cells will be counted out of 500 intact epithelial cells, and they were expressed as percentages. Nuclear blebbing (MN-like structure connected with the main nucleus with a bridge) are not considered. Clumps of cells with obscured nuclear or cytoplasmic boundaries and overlapping of cells are avoided and separated or cells lying singly are preferred for counting of MN. Dead or degenerated cells, apoptotic cells, and cytoplasmic fragments are excluded from evaluation.

Data analysis plan

As per the requirement relevant statistical data analysis will be performed using

Independent Samples Test (Student t test)/ chi square for comparison, Pearson’s and spearman’s coefficients for correlation, multiple logistic regressions and ANNOVA of one way, Friedman and kruswal wallis type as desired by the type of the data and situation for multiple groups will be used.

At the end a ROC curve will be plot for the final scores to determine a threshold above which a definite biopsy and referral can be planned for such lesions.