Putative precursor cancer cells in human colorectal cancer tissue.

Multistage carcinogenesis is an important concept in cancer biology. Each new stage is triggered by the acquisition of an additional genetic aberration, leading to clonal expansion of the cancer cell. The resulting tumor mass consists of cancer cells with all genetic aberrations, but may include precursor cells at some point of carcinogenesis. We analyzed six colorectal cancer tissues with APC, K-ras, and p53 mutations. From each sample, 40-50 areas (100x100x40microm) consisting only of cancer cells were microdissected, and genomic DNA was purified. Ratios of mutated and normal alleles were quantitated by the SNaPshot assay, a primer extension assay. In five tumor tissues, we identified cancer cell subpopulations corresponding to putative precursors, i.e., cells with mutations in one or two of the three genes. All samples were likely to be of monoclonal origin, and temporal sequences of the mutations could be deduced from the mutation patterns of putative precursors. The orders of mutation events were variable. However, the two carcinoma tissues accompanying adenoma regions started with the APC mutation, not contradicting the previous studies. The analysis also revealed considerable heterogeneity in allele ratios of one or two of the chromosomes. The current findings are promising to uncover the process of carcinogenesis directly from the tumor tissue of the patient.