Cytomorphological features of presence and number of AgNOR avid regions and micronuclear score compared to CK-8 immunoreactive controls in premalignant and early malignant lesions of oral cavity and oropharynx

Indiashares a major burden of oral and oropharyngeal cancers of the world. 1 Even with the major advances in thesurgical techniques and nonsurgical modalities such as various forms of radiotherapy,chemotherapy and targeted therapy, overall outcome in terms of 5 year survivalremains at 56% in oral and oropharyngeal cancers.2 The prognosis is still worse inIndian patients  with high incidences ofp53 mutations.3 Delay in detection of these casesis one of the prime reason for such outcomes. Cancers at other sites such ascervix and breast do have better outcomes due to routine screening of earlystage lesions which are detected early of any developing malignant changes inthe lesion. Similar results of improved survivals are anticipated in a scenarioof detectable cyto-morphological changes that can be easily detected andprognosticated4,5. This will help in detecting themalignancies at an early stage, will reduce the overall disease burden and willimprove the overall treatment outcome of oral and oropharyngeal cancers.

Nuclearand cytological markers of developing neoplastic transformations includesmorphological features including increased nuclear cytoplasmic ratios,increased nuclear diameter and decreased cellular diameters; featured ofproliferative activity such as increased number of regions with affinity forsilver stains (AgNOR) and percentage of cells with >3 AgNOR avid regions;and meta nuclear alterations including increased number of micronuclei, cellswith karyorrhexis and bi-nucleated cells. Micronucleus is a marker ofchromosomal damage, genome instability and carcinogenesis, integrating acquiredmutations and genetic susceptibility towards mutations.6,7 Possible role of identification ofsuch cytological nuclear features has been proven in buccal cells in literatureto identify DNA damage8 and  in cases of buccal cancer4. However, there is still lack ofliterature about its diagnostic and prognostic significance in premalignantlesions where these can act as indicators of DNA damage and carcinogenesis forearly detection of developing malignancies. The prognostic significance ofcyto-morphologic nuclear and micro-nuclear changes in oral premalignant lesionsis also needed to be established.

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

Methods:

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

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

Biopsy procedureand histopathological gradingIncisional biopsies will be takenfrom the representative sites with all aseptic precautions. The tissue specimenwill be labelled, fixed in 10% neutral buffered formalin for 24 h, and paraffinembedded. The wax blocks will be cut to obtain two tissue sections of 4-5 μmthickness for each block and the sections will be stained by haematoxylin andeosin. Immunohistochemistry (CK8) will be performed on relevant cases.

Cytological preparation and evaluationThe smears will be stained byPapanicolaou technique using a commercially available staining kit RAPIDPAP,Feulgen stain, AgNOR stain & acridine orange. From each slide, 500 cellswill be examined under the light microscope using low magnification (×400) forscreening and high magnification (×1000) for counting of MN.

 Scoring criteriaThe criteria which was developed byTolbert et al. was used for counting the MN. Screening of each slidewas made in a zigzag manner from one end, toward the other end of the slide.

Tolbert et al. criteria parameters for identifyingMN are as follows:

1.      Roundedsmooth perimeter suggestive of a membrane.

2.      Lessthan a third the diameter of associated nucleus, but large enough to discernshape and colour.

3.      Stainingintensity similar to nucleus.

4.      Samefocal plane as nucleus.

5.      Texturesimilar to nucleus.

6.      Theabsence of overlap with or bridge to the nucleus.

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

ForSilver avid regions we intend to calculate number of Ag avid regions and itspresence of absence.

Thenuclear features and dimensions will be recorded using the standard softwareavailable for calculating nuclear dimensions.

Inclusion and exclusion criteria (Post-cytology)Micronucleated cells will be counted out of 500 intactepithelial cells, and they were expressed as percentages. Nuclear blebbing(MN-like structure connected with the main nucleus with a bridge) are notconsidered. Clumps of cells with obscured nuclear or cytoplasmic boundaries andoverlapping of cells are avoided and separated or cells lying singly arepreferred for counting of MN. Dead or degenerated cells, apoptotic cells, andcytoplasmic fragments are excluded from evaluation.

Mile stone

Targets

|0-6 months

1.      Sample collection.

2.      Procurement of stains, antibodies and detection kit.

  |7-18 months

1.      Standardization of the staining & IHC protocol

2.      Prospective case analysis by staining & IHC

|19-24 months

Compilation of data and submission along with knowledge outcome in terms of a paper.

  Data analysis plan

As per the requirement relevantstatistical data analysis will be performed using

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

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