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ORIGINAL ARTICLE

Flow cytometry in oral cytology: Improved brush biopsy-based delineation of oral malignant and potentially malignant lesions

Pavithra Srinivasan1† ,  P. Sunny P. Sunny1,2† ,  Vaishnav Vasudevan1 ,  Bonney Bonney1 ,  Aditi Hariharan1 ,  Mendonca Mendonca1 ,  Uma Mohan1 ,  Subhashini Raghavan3 ,  Shubha Gurudath3 ,  Keerthi Gurushanth3 ,  Ashwini Hallikeri4 ,  Vivek Shetty2 ,  Vidya Bhushan2 ,  Yogesh Dokhe2 ,  Naveen B. Shivanand2 ,  Satyajit Topajiche4 ,  Pavithra Chandrashekhar4 ,  Vijay Pillai2 ,  Praveen Birur1,3 ,  Christian Brand5 ,  Thomas Reiner6 ,  Amritha Suresh1,2* ,  Moni A. Kuriakose1,2*
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1 Integrated Head and Neck Oncology Program laboratory, Mazumdar Shaw Medical Foundation, Narayana Health, Bangalore, Karnataka, India
2 Department of Head and Neck Oncology, Mazumdar Shaw Medical Center, Narayana Health, Bangalore, Karnataka, India
3 Department of Oral Medicine and Radiology, Karnataka Lingayat Education Society’s Institute of Dental Sciences, Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka, India
4 Department of Oral Pathology, Karnataka Lingayat Education Society’s Institute of Dental Sciences, Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka, India
5 Summit Biomedical Imaging LLC, New York, United States of America
6 Memorial Sloan Kettering Cancer Center, New York, United States of America
†These authors contributed equally to this work.
JCTR, 025180023 https://doi.org/10.36922/JCTR025180023
© Invalid date by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution -Noncommercial 4.0 International License (CC-by the license) ( https://creativecommons.org/licenses/by-nc/4.0/ )
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Abstract

Background: Brush biopsy is a minimally invasive method for early detection of oral squamous cell carcinoma (OSCC). Enhanced accuracy for clinical utility depends on analysis of the whole cell population and automated cohort classifications. Aim: This study aims to delineate OSCC, high-grade dysplasia (HGD), and low-risk lesions (LRLs) by profiling single-cell level alterations using multiplexed flow cytometry. Methods: Brush-biopsy samples were analyzed from patients with LRL, HGD, and OSCC. Flow cytometry analysis was standardized to ascertain cell distribution, heterogeneity, and epithelial cell content. Markers were used for epithelial cell (Pan-Cytokeratin [Pan-CK]/ propidium iodide [PI]) and atypical cell (Sambucus–Nigra–Agglutinin-1 [SNA-1]/ polyadenosine diphosphate-ribose polymerase inhibitor [PARPi-FL]) delineation. In addition, scatter properties and molecular-equivalence fluorescence (MEF) values of markers were analyzed for cohort classification. Results: Brush-biopsy samples from OSCC/HGD patients showed heterogeneity in the percentage of Pan-CK+ve/PI+ve cells. Significant variation in MEF values of SNA-1/PARPi-FL/PI delineated the OSCC cohort (area under the curve > 0.85). Furthermore, the markers in combination with scatter properties delineated OSCC (multivariate logistic regression; sensitivity: 90%, specificity: 82%). The analysis of the forward-scatter height-to-area ratio delineated HGD from low-risk lesions by capturing the morphology-based cellular differences. Conclusions: These results suggest that a flow cytometry-based analysis of brushbiopsy samples may serve as an adjunct tool for risk stratification of oral lesions. Relevance for patients: This study provides evidence towards the application of flow cytometry as an objective, quantitative adjunct to conventional cytology, and improves early detection and risk stratification of oral lesions using a minimally invasive sampling method, thereby supporting timely clinical decision-making and patient management.

Graphical abstract
Keywords
Flow cytometry
Oral cancer
Oral potentially malignant disorders
High-grade dysplasia
FlowCal
Funding
This study was supported by the Biotechnology Industry Research Assistance Council, India (https://birac.nic.s/; GCE-India/R4/2018/006) under the Grand Challenges Explorations program.
Conflict of interest
The authors declare no conflict of interest.
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