A noninvasive genetic screening test to detect oral preneoplastic lesions.
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.
Bremmer
Jantine F
JF
Department of Oromaxillofacial Surgery and Oral Pathology, VU University Medical Center, Amsterdam, The Netherlands.
Braakhuis
Boudewijn J M
BJ
Ruijter-Schippers
Henrique J
HJ
Brink
Arjen
A
Duarte
Helena M B
HM
Kuik
Dirk J
DJ
Bloemena
Elisabeth
E
Leemans
C René
CR
van der Waal
Isaäc
I
Brakenhoff
Ruud H
RH
D002294:Carcinoma, Squamous Cell / Q000175:diagnosis* / Q000235:genetics; D002869:Chromosome Aberrations; D004247:DNA / Q000032:analysis; D042241:Early Diagnosis; D005819:Genetic Markers; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007971:Leukoplakia / Q000235:genetics / Q000473:pathology*; D019656:Loss of Heterozygosity; D042282:Microdissection; D015336:Molecular Probe Techniques; D009061:Mouth Mucosa / Q000473:pathology; D009062:Mouth Neoplasms / Q000175:diagnosis* / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D011230:Precancerous Conditions / Q000175:diagnosis*; D015203:Reproducibility of Results
D005819:Genetic Markers; D004247:DNA