Sensitivity and limitations of high throughput fluorescent microsatellite analysis for the detection of allelic imbalance: Application in lung tumors

We have used two hexaplex fluorescent microsatellite assays and analysis on an automatic sequencer to determine allelic imbalance in lung tumors. The markers used are located close to tumor-suppressor genes, DNA repair genes and regions frequently lost in lung cancer. We present a reference interval and quantify the reproducibility of the assays as assessed by multiple rep eat reactions for normal DNAs. The cut-off value was calculated to 0.77 (23 % reduction of one allele intensity) which, to the best of our knowledge, i s currently the lowest reported cut-off. Using these parameters we analysed 85 lung carcinomas, Eighty-three samples (97.6%) showed allelic imbalance in at least one locus. It is of note that by using a selection of only 6 ma rkers, imbalance was detected in 81 (95.2%) of the samples. Loci 9p21 and 9 p23 exhibited the greatest imbalance (77% and 75% respectively). The fracti onal allele loss (FAL) for the 3p markers examined was greater in squamous cell carcinomas than adenocarcinomas (t-test, p = 0.0001) while no such dif ference was observed for 9p. The degree of imbalance of different markers w ithin the same sample was divergent, indicating heterogeneity of genomic st atus (losses, amplifications, aneuploidy) in these tumors. In conclusion, w e have established a robust experimental platform with high throughput, sen sitivity and specificity for the detection of allelic imbalance in lung tum ors. Such assays may be useful for the detection of allelic imbalance in cl inical samples to trace genetically abnormal cells and thus assist in the i dentification of individuals at a high risk for developing lung cancer.