A New Multi-Color FISH Assay for Brush Biopsy-Based Detection of Chromosomal Aneuploidy in Oral (Pre)Cancer in Patients with Fanconi Anemia

SIMPLE SUMMARY: Patients with Fanconi Anemia (FA) are at high risk of developing oral squamous cell carcinoma with poor prognosis. Oral brush biopsy–based cytology represents an attractive non-invasive advantage in the characterization of (pre) malignant oral lesions, but a sufficient number of abnormal cells is mandatory to make the diagnosis. The aim of this 2-phases retrospective study is to introduce a new multi-color FISH assay including a 9p21 FISH assay to determine the oral lesions that require treatment, with just a few cells. We demonstrate a good accuracy of the multicolor FISH assay if applied on oral brush biopsy-based specimens from FA patients, using different algorithms to determine chromosomal aneuploidy. Nevertheless, some false positive results were observed and the detection of a genetically altered field in the oral cavity as a possible reason for what may hamper the application of multi-color FISH is discussed. ABSTRACT: Background: Fanconi anemia (FA) is a rare inherited DNA instability disorder with a remarkably elevated risk of oral squamous cell carcinoma. These cancers can be detected with oral brush biopsy-based cytology even at early stages. This study aims to determine the diagnostic accuracy of a new multi-color fluorescent in situ hybridization (FISH) assay consisting of probes for CCND1, TERC, MYC and centromere of chromosome 6, as well as a 9p21 FISH assay consisting of probes for CDKN2A and centromere of chromosome 9 for the detection of oral (pre) malignant lesions in FA. Methods: (I) Cutoffs for the dichotomization of positive or negative multi-color FISH results are determined and (II) retrospectively validated by using archived oral brush biopsy specimens from individuals with Fanconi anemia. In addition, the specimens for cutoff determination were re-hybridized with the 9p21 FISH assay. Results: A cutoff of six or more chromosomal aneuploid cells for a positive FISH result was determined in the cutoff study on 160 biopsy specimens. The validating of this cutoff on 152 specimens showed at best a sensitivity of 87% and a specificity of 82.9%. Conclusion: Multi-color FISH is a sufficient tool to detect chromosomal aneuploidy in oral (pre) malignant lesions of individuals with Fanconi anemia. However, some false positive results may hamper the application as an adjuvant method to oral brush biopsy-based cytology in an oral cancer surveillance program.