This study was approved by the ethical committee of the Medical Faculty of the Ruhr-University Bochum. All patients underwent interdisciplinary counselling by a geneticist, clinician and psychologist. Patients included in this study fulfilled the Amsterdam or Bethesda criteria [1
]. Substantial pedigree information moreover including the revised Bethesda criteria [6
] is given in table . After given informed consent blood samples were drawn for genetic testing. Genomic DNA was extracted using standard protocol [7
Pedigree information and clinical diagnosis of the investigated patients
Formalin fixed and paraffin embedded tumor tissues were obtained from different primary pathologists and sent upon request to the local reference pathology of the Familial Colorectal Cancer Center of the Ruhr University Bochum. Tumors were reexamined for histomorphological HNPCC features, immunohistochemical MMR repair protein expression and microsatellite instability.
Tumor and surrounding normal tissue were microdissected by a skilled pathologist. Tumor cell cellularity was at least 70%. DNA was isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Microsatellite markers BAT-25, BAT-26, D5S346, D17S250, D2S123 and BAT-40 were amplified. The markers included the NIH reference panel according to the international guidelines for the evaluation of MSI in colorectal cancer (Boland et al., 1998). Primer sequences are available on request. Tumors were classified as having high grade microsatellite instability (MSI-H) if at least 30% of the markers showed instabilities. Microsatellite-PCR reactions were performed in 96-well microtiter plates, in 20 mmol/L Tris-HCl, pH 8.4, 5 mmol/L KCl, 1.5 mmol/L MgCl2, 100 ng of each primer, 200 mmol/L dNTPs, 60 mmol/L TMAC (Sigma, Taufkirchen, Germany), 1.5% formamide, 2 ml DNA template (tumor or normal tissue), and 1.5 units Taq DNA polymerase (Gibco BRL, Karlsruhe, Germany), in a final volume of 15 ml. Reactions were performed in a Hybaid Touchdown temperature cycler (MWG-Biotech, Ebersberg, Germany), for 40 cycles of 94°C for 15 seconds, different annealing temperatures for each marker for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. BAT-25, BAT-26, BAT-40 and D2S123 were amplified with an annealing temperature of 60°C, whereas D5S346 was performed at 58°C and D17S250 at 53°C. PCR products were separated on 6% polyacrylamide, 8 mol/L urea gels and DNA fragments were visualized by silver staining.
For immunohistochemistry 3 μm thick sections of formalin fixed paraffin embedded tumor tissue were mounted on poly-L-lysine capillary slides and dried overnight at 37°C. Paraffine sections were dewaxed with xylene, rehydrated in a graded series of alcohol and finally washed in Tris-HCl (pH 7.6) for 10 min. The following steps were performed at room temperature in an automated staining system (TechMate 500, Dako, Glostup, Denmark). To avoid unspecific staining, sections were blocked with buffer 1 (Dako) for 5 min prior to incubation with the primary antibody at the appropriate dilution in blocking buffer (Zytomed, USA) for 30 min at room temperature. As primary antibodies mouse monoclonal antibodies for MLH1 (BD Biosciences, 1:20), MSH2 (BD Biosciences, 1:20) and MSH6 (BD Biosciences, 1:50) were applied. After several rinses in buffer (Buffer Kit, Dako) the immunoreaction was demonstrated using the APAAP Kit (Dako) according to the specifications of the manufacturer. Fast Red (Dako) was used as alkaline-phosphatase substrate. Finally sections were rinsed in distilled water and counterstained with Mayer's haematoxylin (Dako). Staining was considered only informative when there was normal nuclear staining in adjacent non-neoplastic cells, which served as internal control. Less than <1% of stained tumor cells was considered as loss of expression of a MMR protein, whereas 10–100% of stained tumor cells was considered as normal staining.
Mutation analysis of MLH1, MSH2 and MSH6 genes
Exons and flanking intronic regions were sequenced by using ABI standard protocols. Primers are available on request. For all systems forward and reverse strands were sequenced. Sequencing was performed corresponding to the immunohistochemical result. If MSH2 staining was reduced or negative and MSH6 staining negative as well, the MSH2
gene was sequenced at first. Pathogenic relevant mutations were sequenced twice and confirmed in a second blood sample of the patient. Mutations were named according to the nomenclature of den Dunnen and Antonarakis [8