Detection of genetic alterations in esophageal squamous cell carcinomas and adjacent normal epithelia by comparative DNA fingerprinting using inter-simple sequence repeat PCR

In this study, we screened 19 esophageal squamous cell carcinomas (ESCCs) f or the detection of genetic alterations using inter-simple sequence repeat PCR, a DNA fingerprinting approach, Three simple repetitive unanchored prim ers representing tri- and tetranucleotide repeats [(GTG)(5), (GACA)(4), and (GATA)(4)] were used, and evidence of gains and losses of chromosomal sequ ences were detected in all tumors (19 of 19 cases) for at least one of the primers. In 13 of these cases, apparently normal marginal epithelia adjacen t to the tumors were also collected and examined. Eight of the 13 (62%) pat ients showed matching somatic mutations in the marginal epithelia adjacent to the tumors. Five of these 8 (63%) marginal epithelial samples were histo logically normal, two were dysplastic, and one had extremely rare tumor cel ls. In 3 of these 13 (23%) cases, the profile bands were also seen to quant itatively increase in intensity, progressing from normal epithelia to margi nal epithelia to tumors. Ten profile bands shelving gains and one profile b and showing loss in tumors compared with the corresponding normal epithelia were cloned, and their origins were determined by sequencing, The DNA sequ ence of one of the profile bands showing gain in the tumor could be matched to an expressed sequence tag sequence that has been mapped to the 7q22 reg ion, a genomic amplification novel to ESCC, The sequence of the other profi le band showing gain in the tumor could be matched to a nonexonic sequence of chromosome 20, whereas the sequences of the remaining profile bands coul d not be matched with any known sequences after comparison with the genomic sequence data in the European Molecular Biology Laboratory and GenBank dat abases. The bonafide nature of the gains or losses of 11 profile bands in t he original cases was confirmed by direct genomic PCR amplification. The fr equencies of these specific gene alterations in tumors were then analyzed i n a total of 60 ESCCs, which included 41 additional cases of ESCC, Signific antly, 26 of 60 (43%) tumors showed the DNA amplification for the expressed sequence tag sequence of chromosome 7, whereas the frequency of other indi vidual gene alterations ranged from 7% to 15%. It is concluded that the int er-simple sequence repeat PCR strategy is adequate for the detection of som atic mutations in tumors, most of which are quantitative alterations in ano nymous genomic sequences. This approach is also suitable for detection of s omatic mutations preceding the onset of morphologically detectable neoplasi a in ESCC,