T he standard model invoked to explain tumor progression, the increase of biological malignancy with time, is clonal evolution, first proposed by Nowell in 1976 (1). Successive acquisition of mutations generates diverse clones, and it is postulated that the emergence of a dominant clone underlies the biological and clinical properties of a tumor at a point in its natural history (2). The study of structural genetic alterations found in human tumors provides further support for the clonal evolution model (3, 4), and limited observations suggest that the tempo of tumor progression may follow a punctuated pattern with long periods of stasis and periods of rapid genetic change in the tumor cells (5). In contrast to the concept of the emergence of a dominant clone, it is becoming increasingly apparent not only that microheterogeneity of tumor cells can be demonstrated at the phenotypic level, but that genetic heterogeneity is present in many advanced tumor types. Recent reports demonstrating genetic diversity within loci that play a key mechanistic role in tumor formation [e.g., K-ras, p53, transforming growth factor-␤RII (TGF␤RII), and BAX] present a paradox (6-8). How can the prolonged coexistence of genetically diverse populations and the concomitant lack of dominance be explained? After studying microdiversity in tumors exhibiting a microsatellite instability (MSI), we present a possible answer to this question. In silico modeling of the geography of clonal diversity suggests that the same fundamental principles that explain lack of dominance in complex ecosystems underlie the maintenance of clonal diversity in human tumors. Materials and MethodsHuman Tissues and Microdissection. Annotated, anonimized tissues were obtained after approval by the human investigations committee at the Ciutat Sanitària i Universitària de Bellvitge, Barcelona. The six tumors were part of a group of colorectal neoplasms previously studied for MSI (9). All were adenocarcinomas with variable amounts of mucin production. Four tumors were staged as Duke's B (T-2, T-3, T-5, and T-6), and two tumors belonged to the Duke's C with visceral metastases (T-1 and T-4). For microdissection, archival paraffin tissue blocks were cut at a thickness of 6 m and stained with methyl green. Microdissection was carried out by laser capture (PixCell; Arcturus Engineering, Mountain View, CA), by using a ϫ40 lens and paying attention to the exclusion of stromal cells. Intraepithelial lymphocytes in tumor glands were not identified. The micrographs in Fig. 1a show how a tumoral glandular structure is acquired: from the field shown at Left, a single structure is lifted from the tissue (Center). The result (Right) verifies the purity of the microdissected cell population. Each pool of cells analyzed represents the population of tumor cells enclosed in a contiguous surface of 100 m 2 .Genotyping. DNA extraction with proteinase K, as manufacturers recommend, was followed by PCR amplification by using 40 cycles of three loci: bat26 (a 26 A tract within an intron of ...

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To assess the presence of MSI in colorectal cancer, we have developed a simple, sensitive, and specific approach based on the apparent good fit of the data to a two-population model. Its application to a prospective series of patients with colorectal carcinomas demonstrates that the presence of MSI characterizes a subset of less aggressive tumors.