Heterogeneous tumor clones as an explanation of discordance between plasma DNA and tumor DNA alterations

Genetic and epigenetic alterations have been proved to be present in plasma DNA of patients with malignancies, ranging between 20% and 80%, depending on tumor type and detection methods (Chen et al., 1996; Anker et al., 1997; Esteller et al., 1999; Silva et al., 1999b). A previous study, published in Genes Chromosomes and Cancer (Silva et al., 1999b), found TP53 gene mutations in tumor and plasma DNA in a small series of patients with breast cancer and small-cell lung cancer. No microsatellite markers were used in this study. Although almost all circulating DNA alterations match tumor DNA changes (as in the former report), the majority of studies show a small number of alterations present only in plasma, mainly when microsatellite polymorphic markers are used to characterize tumor and plasma DNA (Goessl et al., 1998; Silva et al., 1999a). This finding also has been noted in leukemia patients, in whom NRAS mutations detected in plasma DNA were sometimes absent in the DNA of peripheral blood cells or even in bone marrow samples (Vasioukhin et al., 1994). Some authors suggest that these nonmatched alterations are pitfalls, arguing that the DNA is not pure enough and that small quantities of template can result in polymerase chain reaction artifacts (Chen et al., 1999; Mayall et al., 1999; Coulet et al., 2000). On the other hand, other investigators propose the existence of heterogeneous tumor clones as an alternative explanation (Chen et al., 1996; Goessl et al., 1998), although this possibility has not been proved. To clarify this issue, we analyzed six microsatellite markers—D10S541, D10S2491, D10S583, D17S855, D17S1323, and D17S1322—and KRAS2 mutations in tumor samples from 42 colon cancer patients (in 19 cases, the samples were taken without contiguity from the same tumor, and in 23 patients they were chosen from synchronous multifocal tumors). Microsatellite analysis was done in nondenaturing 8% polyacrylamide followed by a silver-staining method. KRAS2 changes were identified by single-strand conformational polymorphism (SSCP). Samples that showed a differential band at SSCP were sequenced by the deoxynucleotide triphosphate (dNTP) method with the Sequenase Kit (United States Biochemical Corp., Cleveland, OH) to identify the mutations. Second, we tested for the presence of the same DNA tumor alterations in the plasma of patients who had different alteration patterns among their tumor samples. Fourteen (33%) patients had heterogeneous patterns among their samples in at least one of the markers, suggesting the presence of different cell clones in the tumor of each patient. Eight were multifocal neoplasias, and six were tumors with multiple samples. The plasma study was done on seven blood samples available from these patients. In five samples, three corresponding to patients with multifocal tumors and two to patients with different tumor fragments, we found that the genetic alterations detected in plasma DNA were present only in some of the tumor samples analyzed (Table 1). Usually, molecular studies check a small portion of the tumor, but it is possible that the supposedly homogeneous neoplastic tissue could be a mixture of different proliferating clones. In conclusion, although polymerase chain reaction artifact can be linked to plasma DNA changes that are not present in tumor tissues, and plasma analysis must be done carefully to select adequate markers and avoid false-positive results, our results show that the existence of different neoplastic clones could explain some nonmatched alterations between plasma and tumor DNA, on the basis of the heterogeneous genetic appearance of tumor cells.