Evaluation of Static DNA Ploidy Analysis Using Conventional Brush Biopsy-Based Cytology Samples as an Adjuvant Diagnostic Tool for the Detection of a Malignant Transformation in Potentially Oral Malignant Diseases: A Prospective Study

Abstract

Background: The accuracy of DNA image cytometry as an investigation method for potentially malignant disorders of the oral cavity is currently still a subject of controversy, due to inconsistently applied definitions of DNA aneuploidy, small cohorts and different application techniques of the method. The aim of this study was to examine the accuracy of the method as a supplementary diagnostic tool in addition to the cytological examination using internationally consented definitions for DNA aneuploidy.

Methods: A total of 602 samples from 467 patients with various oral lesions were included in this prospective study. Brush biopsies from each patient were first cytologically examined and categorized by a pathologist, second evaluated using DNA image cytometry, and finally compared to either histological biopsy result or clinical outcome.

Results: Using the standard definition of DNA aneuploidy, we achieved a sensitivity of 93.5%, a positive predictive value for the detection of malignant cells of 98.0%, and an area under the curve of 0.96 of DNA ploidy analysis for the detection of severe oral epithelial dysplasia, carcinoma in situ or oral squamous cell carcinoma. Importantly, using logistic regression and a two-step model, we were able to describe the increased association between DNA-ICM and the detection of malignant cells (OR = 201.6) as a secondary predictor in addition to cytology (OR = 11.90).

Conclusion: In summary, this study has shown that DNA ploidy analysis based on conventional specimens of oral brush biopsies is a highly sensitive, non-invasive, patient-friendly method that should be considered as an additional diagnostic tool for detecting malignant changes in the oral cavity.

Keywords: DNA image cytometry; aneuploidy; brush biopsy; mass screening tool; oral cancer; oral potentially malignant disorders.

Figure 2

Sample with a doubtful cytological diagnosis and euploid–polyploid DNA histogram. Two euploid DNA stemlines at 2c and 4c and single cells with DNA contents up to 8.3c are shown. DNA content in c-units (x-axis), number of cells (y-axis), reference cells (green), analysis cells (red), biggest stemline (yellow). In this patient case, a 66-year-old female patient showed an ulceration on the right edge of the tongue, which was histologically diagnosed as granulation tissue and healed after 6 weeks without irritation.

Figure 3

Sample with a suspicious cytological diagnosis and presence of DNA aneuploidy as a sign of malignant transformation. An atypical stemline at 3.52c is shown with an associated doubling peak and two cells with a DNA content >9c. DNA content in c-units (x-axis), number of cells (y-axis), reference cells (green), analysis cells (red), biggest stemline (yellow). In the follow-up of this sample, a second carcinoma of the floor of the mouth was diagnosed in an 86-year-old male patient.

Figure 4

Sample with a positive cytological diagnosis and DNA aneuploidy as a sign of malignant transformation. Atypical stemline distribution at 3.24c and 6.5c. In addition, several cells with a DNA content >9c are shown. DNA content in c-units (x-axis), number of cells (y-axis), reference cells (green), analysis cells (red), biggest stemline (yellow). This sample is a carcinoma of the soft palate in a 68-year-old male patient.

Figure 1

Presentation of the results of the oral brush biopsy, the subsequent DNA ploidy analysis and their follow-up outcome.

Figure 5

ROC (receiver operating characteristics) curves for cytological examination (left) of the cells and DNA image cytometry (right).

Figure 6

Model 1: calculation of the logistic regression with the cytological examination as the sole predictor (OR = 171.11). Model 2: addition of the second predictor. DNA-ICM: increased association between DNA-ICM and detection of malignant cells (OR = 201.6) in addition to decreased association for cytological examination (OR = 11.90).