DNA aneuploidy and tissue architecture in oral potentially malignant disorders with epithelial dysplasia assessed by a 10 locus FISH panel

Abstract

Subjectivity in oral dysplasia grading has prompted evaluation of molecular-based tests to predict malignant transformation. Aneuploidy detected by DNA image-based cytometry (ICM) is currently the best predictor but fails to detect certain high risk lesions. A novel multiplex fluorescence in situ hybridization (FISH) panel was used to explore possible explanations by detecting aneuploidy at the single cell level. FISH was compared to reference standard DNA ICM in 19 oral lesions with epithelial dysplasia and used to characterize the cellular architecture. Copy number variation at 3q28, 7p11.2, 8q24.3, 11q13.3 and 20q13.12 and matched chromosome specific loci were assessed by dual-color FISH to assess numerical and spatial patterns of copy number increase and gene amplification. FISH revealed wide variation in copy number at different loci. Only low level copy number gain was present and often in only a small proportion of cells, although usually with all or all but one locus (9/12). Four cases showed gene amplification, one at two loci. Some probes revealed an internal presumed clonal structure within lesions not apparent in routine histological examination. Both methods produced similar diagnostic results with concordance in detection of aneuploidy by both methods in 17 out of 19 samples (89%). We have shown that oral dysplastic lesions may contain very few aneuploid cells at a cellular level, high copy number gain is rare and changes appear to arise from large chromosomal fragment duplications. Single stem lines are relatively homogeneous for loci with copy number gain but there is a subclonal structure revealed by gene amplification in some lesions.

The genetic basis of oral leukoplakia and its key role in understanding oral carcinogenesis

“…In line with that, in silico analyses of transcriptome data suggest that in some cases the premalignant lesions and the carcinomas are not clonally related to each other 47 . Furthermore, the results of a recent study using a FISH panel in oral epithelial dysplasia reinforce that dysplasia progression to OSCC can occur through neutral clonal evolution (ie randomly), instead of through non‐random selection 22 . However, more studies are necessary to exclude the possibility of non‐random selection of genetic alterations in OL progression.…”

“…Interestingly, aneuploid cells formed a minority cell population in all cases and were shown to be interspersed with normal diploid cells in the oral dysplastic epithelium 22 . Notably, the authors also observed agreement in aneuploidy results by the FISH panel and image‐based cytometry in 17 of 19 samples 22 …”

“…Multiple copy number gain involved only CCND1 and EGFR, with patterns suggestive of a clonal architecture. While CCND1 amplification co‐localized with severe dysplasia, EGFR amplification did not correlate with dysplasia 22 …”

“…A multiplex FISH panel has been recently used to detect aneuploidy at a single cell level in epithelial dysplasia, providing information on the spatial architecture of dysplasia not observed by other methods 22 . Interestingly, aneuploid cells formed a minority cell population in all cases and were shown to be interspersed with normal diploid cells in the oral dysplastic epithelium 22 .…”

“…Dual‑colour FISH was used to assess the numerical and spatial patterns of CNA, revealing wide variation in copy number at different loci in oral epithelial dysplasia 22 . Multiple copy number gain involved only CCND1 and EGFR, with patterns suggestive of a clonal architecture.…”