The requirement for well spread out chromosomes for the cytogenetic analysis of primary gastrointestinal tumors led us to develop new techniques. These techniques involved two main procedures: (1) preliminary incubation with culture medium in the presence of collagenase, Dispase, and colcemid, for 3 h, and (2) treatment with an extremely hypotonic solution (0.044M KCl) for 30 min. The techniques were applied to 11 gastrointestinal malignancies (including 1 early gastric cancer and 1 metastatic liver lesion of colon cancer) and significant increases (P < 0.01) in the number of metaphases of analyzable karyotypes were obtained, compared with a previous method in which the standard hypotonic molarity of KCL (0.075 M) was employed. The mean value for metaphase numbers of the analyzable karyotypes was 37.0 +/- 3.7% in the 5 gastric cancers and 44.7 +/- 4.8% in the 5 colon cancers and 1 metastatic lesion. These values were three times and more than twice, respectively, the values obtained by the previous method. A fluorescence in situ hybridization (FISH) study was carried out on one cologenic tumor, the alpha-satellite centromere-specific probe 17 being used. Deletion of the long arm of chromosome 17 was demonstrated. The method proposed here could yield a sufficient number of metaphases without the use of tissue culture that might cause alteration of karyotype. It can be employed with small biopsy specimens and in studies utilizing the FISH technique.

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Cytogenetic studies of biopsy specimens endoscopically obtained from gastric cancers were performed in our laboratory. The necessity for well-spread chromosomes for analysis has resulted in the development of a new technique, in which culture medium containing acetylcholine (Ach) is used; with this new technique, the number of metaphases of analyzable karyotypes was significantly increased (P < 0.01 compared with a previous method in which Ach was not used). The mean ratio of metaphase numbers of analyzable karyotypes in four cases in which Ach was used was 38.1 +/- 8.1%, a value more than four times the number in the seven cases in which Ach was not used. It is possible that Ach may decrease the viscosity of the cytoplasm in gastric cancer cells by disrupting microfilaments.