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

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 brush-biopsy 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.

Keywords

Flow cytometry / Oral cancer / Oral potentially malignant disorders / High-grade dysplasia / FlowCal

Acknowledgments

We acknowledge the Mazumdar Shaw Medical Foundation for its support in facility usage. We acknowledge Ms. Mythri C and Ms. Shivani for formatting and uploading the documents.

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.

Author contributions

Conceptualization: Thomas Reiner, Amritha Suresh, Moni A Kuriakose Data curation: Pavithra Srinivasan, Sumsum P. Sunny, Aditi Hariharan, Vaishnav Vasudevan, Bonney L. James, Satyajit Topajiche, Uma Mohan, Pavithra Chandrashekhar, Vijay Pillai, Praveen Birur, Amritha Suresh Formal analysis: Pavithra Srinivasan, Sumsum P. Sunny, Aditi Hariharan, Uma Mohan, Vaishnav Vasudevan, Bonney L. James, Amritha Suresh, Pramila Mendonca, Subhashini Raghavan, Shubha Gurudath, Keerthi Gurushanth, Vivek Shetty, Vidya Bhushan R, Yogesh Dokhe, Naveen B. Shivanand, Ashwini Hallikeri Investigation: Sumsum P. Sunny, Pramila Mendonca, Subhashini Raghavan, Shubha Gurudath, Keerthi Gurushanth, Vivek Shetty, Vidya Bhushan, Yogesh Dokhe, Naveen B. Shivanand, Ashwini Hallikeri Methodology: Christian Brand, Thomas Reiner, Amritha Suresh, Moni A Kuriakose, Pavithra Srinivasan, Sumsum P. Sunny, Aditi Hariharan, Vaishnav Vasudevan, Bonney L. James, Satyajit Topajiche, Uma Mohan, Pavithra Chandrashekhar, Vijay Pillai, Praveen Birur Writing–original draft: Pavithra Srinivasan, Sumsum P. Sunny, Vaishnav Vasudevan, Bonney L. James Writing–review & editing: Amritha Suresh, Moni A Kuriakose, Christian Brand, Vijay Pillai, Praveen Birur, Thomas Reiner

Ethics approval and consent to participate

The study received ethical approval from the institutional ethics committees of Mazumdar Shaw Medical Center (NHH/MEC-A14/EA/2019) and Karnataka Lingayat Education Society’s Institute of Dental Sciences (KLE/JAN-2022/04), Bengaluru, and written informed consent was obtained from all participants prior to enrolment.

Consent for publication

Written informed consent was obtained from all participants for the publication of anonymized data.

Availability of data

The datasets generated during the current study are available from the corresponding author on reasonable request. Processed data are provided in the figures and tables.

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