[CANCER RESEARCH 62, 1961–1965, April 1, 2002] Advances in Brief Low Mutation Incidence in Polymorphic Noncoding Short Mononucleotide Repeats in Gastrointestinal Cancer of the Microsatellite Mutator Phenotype Pathway 1 Koichi Suzuki, Tomoko Dai, Ikuko Suzuki, Yuichi Dai, Kentaro Yamashita, and Manuel Perucho 2 The Burnham Institute, La Jolla Cancer Center, La Jolla, California 92037 Abstract Frameshifts in short mononucleotide tracts (SMT) in genes, such as TGFRII and BAX, are common in gastrointestinal tumors of the micro- satellite mutator phenotype (MMP). The significance of less common mutations has been recently challenged because frequencies as high as 50% were reported in some noncoding SMTs in MMP colon cancer cell lines (L. Zhang, et al., Cancer Res., 61: 3801–3805, 2001). We did not confirm these findings after examining >50 MMP gastrointestinal cancers for mutations in eight SMT loci with the highest reported frequencies. In three of these loci, no clonal mutations were detected, and they were infrequent (2.9 – 6.7%) in the other five. Length polymorphisms are fre- quent (25.7– 43.9%) in one-half of these SMTs, suggesting an explanation for the discrepancy. Because of the peculiar features of MMP tumors, low prevalence of mutations in cancer genes may not be a disqualifying criterion for their functionality. Introduction A widespread genomic instability known as MSI 3 is germane to the pathogenesis of some colon cancers (1–3). MSI is the landmark of sporadic and hereditary gastrointestinal cancer of the MMP pathway (4). These hundreds of thousands of microsatellite contraction-expan- sion mutations originate by the genetic or epigenetic inactivation of the DNA MMR machinery (5, 6). Once the mutator phenotype un- folds in precursor normal stem cells, cancer of the MMP pathway arises after mutations in genes involved in cell growth or survival (“cancer genes”) drive tumor development and progression (1, 4, 7). Implicit in this concept is the assumption that these tumors display distinctive features in genotype and phenotype because they harbor a defined spectrum of mutated cancer genes that are specific targets for MMP (4). However, the determination of these target cancer genes has been difficult (8, 9). The exacerbated mutator phenotype of these tumors generates many neutral and irrelevant mutations, complicating the task of distinguishing cause from consequence. One of the criteria commonly used relies on the presence of a significant prevalence of frameshift mutations in SMT present in some cancer genes (10, 11). This criterion is sustained by the observation that these mutations are rare in similar noncoding SMTs and in other genes without obvious links to oncogenesis (10 –12). This interpretation has been challenged in a recent issue of Cancer Research by Zhang et al. (13). The authors analyzed the incidence of mutations in several nonfunctional short mononucleotide repeat se- quences in DNA MMR-deficient colon cancer cell lines and mouse xenografts. The mutability of these noncoding SMTs exhibited high variability. Whereas some of the loci analyzed exhibited a low mu- tation frequency (from 0 –5%), several of the mononucleotide loci analyzed exhibited mutation incidences that were surprisingly high (25–54.2%). These findings have important implications for understanding the mechanisms underlying cancer pathogenesis of the MMP pathway. If some noncoding SMTs are mutated in up to 50% of the tumors, the functionality of less prevalent frameshifts in similar SMTs within some cancer genes would be seriously questioned. Zhang et al. (13) thus caution that “a significant prevalence of mutation in a given gene in MSI cancers is not a reliable indicator that such genes are targets rather than passengers, even when the mutated tract is small.” In our experience, noncoding repeats that are eight nucleotides long display very few mutations in MMP tumors (11, 12). Consequently, we determine in our panel of primary tumors the mutation incidence of the SMTs Zhang et al. reported to undergo the highest incidence of mutations in the tumor cell lines and mouse xenografts that they analyzed (13). We have not confirmed these findings, and instead, we found a high frequency of length polymorphisms in many of these SMTs. Materials and Methods Tumor Samples. Tumor samples were obtained as frozen specimens from the Southern Division of the Cooperative Human Tissue Network (University of Alabama, Birmingham, AL), the National Cancer Center Research Institute (Tokyo, Japan), and Sapporo Medical University (Sapporo, Japan). The origin of the colorectal tumor samples has been described previously (11, 12). Genomic DNA was extracted with phenol-chloroform and diluted to a con- centration of 20 ng/l before PCR amplification. PCR Amplification. The sequence of the PCR primers for seven of the chromosome 22 mononucleotide loci reported by Zhang et al. (13) to exhibit the highest mutation incidence were as follows: SMT4,5'-CCTAGGTTGTGGGTG- TATG-3' and 5'-CCTACTCCAGTGTGGTCG-3'; SMT6,5'-GAGAGCATA- AGTCACTCAAC-3' and 5'-CACTAGAAATTGCTGAGCCAG-3'; SMT14,5'- CCAAGGACCACGCATCTAC-3' and 5'-TTCCCTTGGCGCCTCACTG-3'; SMT15, 5'-GAGAGAGATAGTGGAAGG-3' and 5'-CAGGGATGGCTA- CATAATTTG-3'; SMT16,5'-CAGTGGAAATTGTTCGCC-3' and 5'-CAC- CAGTGACTTACATCAC-3'; SMT28, 5'-TGCACAGGTTCCACCCTCC-3' and 5'-CCCCATTCTGTCCTGGCC-3'; and SMT29,5'-GAGATGTACAGCT- CAACTC-3' and 5'-TTACTATTGATGTGGCTGGG-3'. PCR was carried out with Vent DNA polymerase (New England Biolabs, Beverly, MA) and AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) for 35 cycles in the presence of 0.1 Ci of [ 32 P]dCTP. A four-stage touch-down protocol was performed following the conditions described by Zhang et al. (13). PCR products were electrophoresed in denaturing 6% polyacrylamide gels (National Diagnostic, Atlanta, GA). The gels were dried on filter paper and subjected to autoradiography. Sequencing Analysis. Sequencing was performed as described previously (11). The PCR products were eluted from the gels and amplified. DNA was also reamplified and purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA) and sequenced with the ABI PRISMTM dye terminator cycle sequencing kit (Perkin-Elmer, Foster City, CA). Determination of Mutation Clonality. To estimate the allelic status of the SMT mutations, we assessed the extent of contaminating normal tissue in the Received 12/10/01; accepted 2/5/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants CA 63585 and CA38579 (to M. P.). K. S. was supported in part by a fellowship of the Japan-North America Medical Exchange Foundation. 2 To whom requests for reprints should be addressed, at The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 646-3112; Fax: (858) 646- 3190; E-mail: mperucho@ljcrf.edu. 3 The abbreviations used are: MSI, microsatellite instability; MMP, microsatellite mutator phenotype; MMR, mismatch repair; SMT, short mononucleotide tracts. 1961 Research. on January 22, 2022. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from