Source: Laboratory Investigation (2005) 85, 1481–1488. published online 29 August 2005
Authors: Jantine F Bremmer et al.

Early diagnosis of oral squamous cell carcinoma (OSCC) may have a major impact on survival and quality of life. Recent studies have shown that the majority of OSCC is preceded by precursor lesions characterized by genetic alterations. The aim of this study was to develop and evaluate a noninvasive screening test for oral preneoplastic lesions, based on genetic alterations as marker.

Various methods to obtain a high yield of cells by brushing a small area of the oral mucosa were compared. A novel genetic assay, multiplex ligation-dependent probe amplification (MLPA), was applied that enables the measurement of gains and losses at 40 different chromosomal locations in one PCR reaction using 150 ng DNA. MLPA was performed on DNA of normal and dysplastic oral mucosa as well as of OSCC with the intention to select a specific probe set for accurate detection of precursor lesions in the oral cavity.

The assay was correlated to loss of heterozygosity analysis using microsatellite markers, and evaluated on noncancer subjects and patients with oral leukoplakia. A noninvasive sampling method was developed with DNA yields ranging from 150 to 600 ng. Using 120 probes, we could detect large differences with MLPA in the number of alterations between normal vs dysplastic and dysplastic vs tumor tissue with P-values <0.001.

A significant correlation was found between the number of alterations as detected by MLPA and the analysis for allelic loss. The available data enabled the selection of a set of 42 MLPA probes, which had the power to optimally discriminate between normal and dysplastic tissue. Our data show that MLPA is a sensitive, reliable, high-throughput and easy-to-perform technique, enabling the detection of genetic alterations on small noninvasive samples and can be considered a promising method for population-based screening of preneoplastic lesions in the oral cavity.

Authors:
Jantine F Bremmer1, Boudewijn J M Braakhuis2, Henrique J Ruijter-Schippers1,2, Arjen Brink1,2, Helena M B Duarte3, Dirk J Kuik4, Elisabeth Bloemena1, C René Leemans2, Isaäc van der Waal1 and Ruud H Brakenhoff2

Authors’ affiliations:
1Department of Oromaxillofacial Surgery and Oral Pathology, VU University Medical Center, Amsterdam, The Netherlands
2Department of Otolaryngology/Head-Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands
3MRC-Holland, Amsterdam, The Netherlands
4Department of Clinical Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands