Tissue subtraction DNA in situ hybridization (TSDISH): in situ detection of DNA abnormalities in formalin-fixed paraffin-embedded tissue sections by subtraction hybridization with whole genomic DNA.
We have developed a novel in situ histochemical method of screening for genetic alterations of human malignancies by subtraction hybridization of genomic DNA without employing specific probes to give a colorimetric reaction. We identified a t(13;14) chromosome abnormality in the chromosome spread of a patient with multiple myeloma. In situ hybridization of a whole cell preparation of MCF-7 cell demonstrated reaction products as intranuclear dots-in all MCF-7 cells. We subsequently examined the cells of different foci of esophageal squamous cell carcinoma(10) and esophageal biopsy specimens (9) by this method. Hybridization with genomic DNA from the patients demonstrated no reaction products in the stromal cells of the esophagus. However, hybridization with reference DNA from a healthy individual demonstrated intranuclear reaction products in the stromal cells, possibly due to individual genomic differences. There were more intranuclear reaction products in the carcinoma cells than in the stromal cells when hybridized with reference DNA. When hybridized with the reference DNA above, the cells of the non-pathologic epithelium of 8 of 10 malignant esophagi demonstrated significantly more reaction product than the stromal cells of the some specimens. This was not detected in the cells of normal epithelium obtained from non-cancerous esophagi suggesting the accumulation of genetic alterations of the non-malignant epithelium of the cancerous esophagus. This method is thought to detect DNA alterations, including those which have not been previously identified, using genomic DNA for hybridization, and the results can be correlated with the morphological findings. Application of this in situ method, together with other molecular genetic techniques may contribute to the analysis of various genetic alterations of human malignancies using archival material.
Itakura
Y
Y
Department of Pathology, Tohoku University School of Medicine, Sendai, Japan.
Sasano
H
H
Mori
S
S
Nagura
H
H
Shimizu
S
S
D002869:Chromosome Aberrations; D004273:DNA, Neoplasm / Q000032:analysis*; D004938:Esophageal Neoplasms / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm