The files of 164 patients treated for sporadic UUT-TCC over 12 years were reviewed; all tumour blocks retrieved were screened for MSI. Paired DNA from tumours and normal tissues were amplified by PCR using five microsatellite markers from the Bethesda panel : BAT25 (4q12), BAT26 (2p16), D2S123 (2p16), d5s346 (5q21–22) and D17S250 (17q) (for primer sequences, sense/antisense, see http://www.gdb.org). In accordance with National Cancer Institute consensus , any pair of samples of normal DNA and tumour DNA that had instability at two or more of these five loci was scored as having high-frequency MSI, whereas a sample pair with no instability at these five loci was scored as having MSI. Any sample pair having instability at one of the five loci was tested again at that locus to exclude artefact. If MSI was confirmed additional loci were tested to determine whether the phenotype of the sample was low-frequency (1–4 loci) or high-frequency MSI (five or more loci). For additional loci, we used markers that we had already tested in UUT-TCC [10,14]: MFD15 (1q23), APC (5q22), BAT40 (1p13.1), d18s58 (18q22), D18S69 (18q21), d10s197 (10p12), MYC1L (1p34), UT5320 (8q24), ACTBP2 (6q13), CFS1R (5q33-q35), D20S82 (20p12), d11s488 (11q24) and D9S242 (9q33). PCR amplification was carried out with ≈ 10 ng of DNA in a 20-µL final volume of reaction mixture (0.25 mmol/L dNTP in 1 mol/L Tris, 0.9 mol/L boric acid, 0.01 mol/L EDTA, 20 pmol of each primer (MWG Biotech, Ebersberg, Germany), 0.75 µL of DMSO, and 1 U Taq Polymerase (Qbiogen, Illkirch, France). Cycling parameters were described previously ; 1 µL of PCR product was added to 1 µL blue Dextran and 3 µL formamide. After a 2-min denaturation step at 94°C, the mixture was immediately immersed in an ice bath. The amplified fragments were separated by denaturing gel electrophoresis in Tris-borate-edetic acid buffer/4% polyacrylamide (acryl-to-bisacryl 29 : 1), 6 mol/L urea (gel) using an PRISM 377 Genetic Analyser (Applied Biosystems, Palo Alto, California); GeneScan 3.1 Fragment Analysis software (Applied Biosystems) was used to analyse the data.
Twenty-seven patients (16%) had high MSI levels; the following data were collated: age, personal or family history of a HNPCC-associated tumour, history of other cancers, tumour stage (TNM 1997) and grade. None of these 27 patients met the Amsterdam clinical criteria for HNPCC . These 27 patients had their DNA sequenced to detect hMSH2 mutation, which were found in three (11%). Consequently, clinical criteria were defined to suspect a predisposition for hereditary UUT-TCC, i.e. a personal or familial history of HNPCC-associated cancer and/or aged < 60 years . Only those patients who met these criteria and had no hMSH2 mutation were included in the present study.
For genetic testing, blood samples were obtained from all participating subjects and kept frozen at − 30 °C until DNA extraction. DNA was isolated from peripheral blood lymphocytes using a purification kit (QIAamp blood kit, Qiagen, Courtaboeuf, France). All coding exons of the hMSH6 and hMLH1 were sequenced by PCR amplification with intronic flanking primers, as described elsewhere . Briefly, primers were designed using the human genome sequence (GenBank NM_000179). Sequencing reactions were conducted with the ABI Prism Big Dye Terminator Cycle sequencing kit and analysed on an ABI310 sequence analyser (both Applied Biosystems).