Virchows Archiv (1995) 426:223-227 © Springer-Verlag 1995 Jtirgen Schlegel • Tina Bocker • Hubert Zirngibl Ferdinand Hofst~idter • Josef Rtischoff Detection of microsatellite instability in human colorectal carcinomas using a non-radioactive PCR-based screening technique Received: 6 September 1994 / Accepted: 21 December 1994 Abstract The aim of the present study was to establish a rapid, non-radioactive screening method for the detection of microsatellite instability (MIN). MIN is the primary characteristic of the mutator phenotype in tumours consti- tuting hereditary non-polyposis colon cancers (HNPCC). We investigated 30 patients suffering from colorectal can- cer using a non-radioactive PCR-based technique. MIN was present in 7 of 30 (23%) of the cases. There was a statistically significant correlation between MIN and lo- calization of the tumour. Five of 7 (72%) tumours with MIN but only 4 of 23 (17%) turnouts without MIN were localized in the proximal colon (P<0.01). There was a tendency to higher MIN frequency in turnouts of patients with familial clustering of cancers. However, this was sta- tistically not significant (P>0.05). In addition, no correla- tion between MIN and tumour grade and stage was found. For the investigations in the present study we used a non-radioactive PCR-based method followed by denatu- rating polyacrylamide gel electrophoresis and silver stain- ing. This method is highly sensitive and reproducible. Thus, PCR-based analysis using a non-radioactive stain- ing technique represents a comprehensive tool for MIN screening in diagnostic pathology. Key words Colon cancer. Hereditary non-polyposis colon cancer • Microsatellite • Polymerase chain reaction Introduction Genomic instability due to spontaneous errors in DNA replication has been implicated in neoplastic transforma- tion and as a mechanism to explain chromosomal altera- tions in cancer cells [4]. It has been shown that gene am- J. Schlegel (~) • T. Bocker - E Hofstadter • J. Rtischoff Universit~itsklinik, Institut ftir Pathologie, D-93042 Regensburg, Germany H. Zirngabel Chirurgische Universit~itsklinik, Universit~it Regensburg, Germany plifications and deletions of putative turnout suppressor genes accumulate during carcinogenesis of many human malignant tumours [5]. However, the mechanisms leading to genomic instability are poorly understood. Widespread alterations in simple repeated sequences have been found in several tumour types [7, 11, 13, 16]. Such alterations are most easily observed as changes in the lengths of mi- crosatellite sequences between tumour DNA and DNA from non-neoplastic tissue of the same individual. This type of genomic sequence consists of one- to six-nucleo- tide motifs, which are tandemly repeated numerous times. The dinucleotide repeat (CA)n constitutes one of the most abundant classes of repetitive DNA sequences in the hu- man genome, with an estimated occurrence of 105 loci [21]. Recently, it has been shown that microsatellite insta- bility (MIN), that is expansion or reduction of the number of dinucleotide repeats, is the major characteristic of the mutator phenotype in tumours constituting hereditary non-polyposis colon cancers (HNPCC) [1, 8, 17]. In these tumours MIN is due to aberrant DNA repair enzymes. Mutations have been reported in the human routS and mutL homologous genes hMSH2 and hMLH in patients suffering from HNPCC [6, 12]. In some HNPCC families there is also a linkage to the chromosomal locations of the two genes on chromosome 2 and 3, respectively [14]. Thus, detection of MIN in colorectal turnouts would be of interest in diagnostic pathology and may allow a risk as- sessment for disease. However, in all investigations re- garding MIN radioactive detection systems were used, with clear limitations for routine diagnosis since they re- quire special laboratory equipment and are expensive and time consuming. Here we report a rapid, non-radioactive screening method which allows the detection of MIN in diagnostic pathology. Materials and methods Biopsy specimens of surgically removed colorectal carcinomas from 30 consecutive patients operated at the Department of Sur- gery, University of Regensburg (20 male, 10 female) were snap-