Materials. Paraffin-embedded tissue samples from 107 randomly selected patients with colorectal cancer, who were diagnosed and operated during the past 5 years at the Surgery Clinic, Faculty of Medicine, Skopje, Republic of Macedonia, were included in the study. Histopathological diagnosis was done at the Institute of Oncology and Radiotherapy, Faculty of Medicine, Skopje. Clinical and histopathological features of colorectal cancers included in the study, such as age, gender, localization, size, histopathological grade and clinical stage of the tumors, were obtained from the patients' records.

Methods. Six 5 mm thick sections from both paraffin-embedded tumor tissue samples and normal tissues of each patient were used for DNA extraction. The sections were examined with a microscope, and homogeneous malignant and normal tissues of each patient were scraped off with a scalpel into separate tubes. Following deparaffinization with xylol, DNA was extracted using the Proteinase K digestion, phenol/chloroform extraction and ethanol precipitation method [18].

In the introductory phase of the study, the MSI status of the tumors was evaluated by nonradioactive PCR-PAGE (polyacrylamide gel electrophoresis) analysis of seven microsatellite markers with procedures routinely in use in our laboratory [19]. Initially, DNA from the tumor and normal tissues of each patient was amplified separately for five markers (BAT26, D17S250, D18S58, D18S61, DCC). Samples that showed instability in only one marker were further analyzed for two loci (D2S123, D5S346). For each analysis, 300 ng of purified DNA were amplified in a final volume of 25 mL in a mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 200 mM each of the four dNTPs, 25 pM of each primer and 1.5 U of Taq poly­merase. The PCR amplification consisted of 30 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute. Two mL of each reaction were heat-denatured in 2 vol. of loading dye (95% formamide, 1 mM EDTA, 0.05% bromephenol blue and 0.05% xylen cyanol), quickly chilled on ice and load­ed onto 6% denaturing polyacrylamide gels (40 x 20 cm x 0.4 mm) containing 6 M urea and 32% formamide. The electrophoresis was run at 70 W constant power for 2 hours and the fragments were transferred to positively charged nylon membranes (Hybond+; Amersham Pharmacia Biotech Ltd., Amersham, Buckinghamshire, UK). The membranes were hybridized with non-radioactively labeled internal oligonucleotide probes and detected by chemiluminiscence, as described in detail elsewhere [19].

The results obtained with the nonradioactive PCR-PAGE method were compared with the results obtained with fluorescent multiplex PCR/capillary electrophoresis assay. In this assay, the five loci were amplified in two multiplex PC reactions: BAT26, D18S58 and D18S61 (reaction 1); DCC and D17S250 (reaction 2). One primer of each primer pair was labeled at the 5’end with either 6’-FAM (BAT26, D18S58 and DCC) or HEX (D18S61 and D17S250) fluorescent dyes (MWG-Biotech AG, Ebersberg, Germany). The PCR conditions were similar to those described above. Fluorescently labeled products were detected using the ABI PRISM 310 genetic analyzer and GeneScan Software (Applied BioSystems, Foster City, CA, USA).

Tumors were classified as MSI-H if two or more of the five markers showed instability, and MSS if none of the five markers used was unstable. Tumors with instability in only one locus were further analyzed with two markers (D5S346 and D2S123) and were either included in the MSI-H group, when instability occurred in another locus, or they were termed as MSI-L if MSI was still present in one locus.