Correlation Between Dysplasia and Ploidy Status in Oral Leukoplakia

Oral leukoplakia and other potentially malignant disorders (PMD) may progress to oral squamous cell carcinoma (OSCC). The gold standard for assessing the potential for malignant transformation remains histologic examination with the aim of grading the dysplastic changes. However, not all lesions with dysplasia will progress to OSCC. DNA ploidy has been suggested as a method to predict the clinical behaviour of PMD. This study reports on the use of high-resolution flow cytometry to determine the ploidy status of formalin-fixed, paraffin-embedded material from PMD compared to their dysplasia grade on histology. Aneuploidy was found in 13 % of mild, 31 % of moderate, and 54 % of severe dysplasia cases. This difference was statistically significant (p = 0.011). The differences in ploidy status were more significant when grouping the dysplasia into low-risk and high-risk categories (p = 0.008). These findings indicate that the ploidy status of PMD as determined by high-resolution flow cytometry may be of value in predicting biological behaviour in PMD such as leukoplakia

A novel approach to simultaneously scan genes at fragile sites

<p>Abstract</p> <p>Background</p> <p>Fragile sites are regions of the genome sensitive to replication stress and to exposure to environmental carcinogens. The two most commonly expressed fragile sites FRA3B and FRA16D host the histidine triad (<it>FHIT</it>) and WW domain containing oxidoreductase (<it>WWOX</it>) genes respectively. There is growing evidence that both genes contribute to cancer development and they are frequently altered by allelic and homozygous deletions in a variety of tumors. Their status is linked to prognosis in several malignancies and they are thought to be involved in early tumorigenesis.</p> <p>The loci for <it>FHIT </it>and <it>WWOX </it>both span over a megabase but the genes encode for small transcripts. Thus the screening of intragenic deletion can be difficult and has relied on loss of heterozygosity LOH assays, or genomic arrays.</p> <p>Methods</p> <p>Multiplex ligation dependent probe amplification MLPA, allows for the detection of deletions/duplications and relative quantification of up to 40 specific probes in a single assay. A <it>FHIT/WWOX </it>MLPA assay was designed, applied and validated in five esophageal squamous cell carcinoma ESCC, cell lines established in South Africa where this cancer is of high prevalence. Sixteen probes covered all <it>FHIT </it>exons and 7 probes covered <it>WWOX</it>.</p> <p>Results</p> <p>Both homozygous and hemizygous deletions were detected in <it>FHIT</it>, in four of the cell lines with a preferential deletion of exons 5 and 4. Chromosome 3 short arm was present in normal copy number indicating that deletions were site specific. In contrast <it>WWOX </it>was not altered in any cell lines. RT-PCR expression pattern paralleled the pattern of deletions. Ten primary ESCC tumor specimens were subsequently screened with this assay. <it>FHIT </it>exon deletions were found in four of them.</p> <p>Conclusion</p> <p>This method offers an alternative to loss of heterozygosity studies. Simultaneous scanning of <it>FHIT </it>and <it>WWOX </it>exons in the context of early tumorigenesis and tumor progression, may help clarify the mechanistic events related to cancer development which are not revealed by imuno histochemistry assays. The presence of site specific deletions of <it>FHIT </it>in these cell lines and primary tumors support its possible role in South African ESCC and justifies a wider screening.</p