BMC Cancer 2002, 2 http://www.biomedcentral.com/1471-2407/2/25 Table 2: Antibodies and conditions used for immunohistochemistry Antibody p53 -catenin MLH1 MSH2 Clone DO-7 14 G168-5 FE-11 Company DAKO A/S, Glostrup, Denmark Transduction laboratories Lexington, KY, USA PharMingen, San Diego CA, USA Oncogene Research Products, Bos- ton, MA, USA Dilution 1:300 1:5000 1:100 1:100 Buffer Citrate Citrate EDTA EDTA 3' (forward) and 5'-TGAGCTCGAGTCATTGCATAC-3' (re- verse), followed by size separation in acrylamide gels. KRAS mutations KRAS was analyzed for point mutations in codons 12, 13 and 61 by sequencing. PCR amplification was performed in two fragments using the primer pairs K1 (covering co- dons 12–13) 5'-GGCCTGCTGAAAATGACTGA-3' (for- ward) and 5'-GTCCTGCACCAGTAATATGC-3' (reverse) and K2 (covering codon 61) 5'-TTCCTACAGGAAGCAAG- TAG-3' (forward) and 5'-CACAAAGAAAGCCCTCCCCA- 3' (reverse). Immunohistochemistry for TP53, -catenin, MLH1 and MSH2 Fresh 5- M sections from paraffin embedded tumor blocks were transferred to glass slides (DAKO ChemMate capillary gap microscope slides, 75 M, DAKO A/S, Den- mark). Following deparaffinisation in xylene and rehydra- tion through graded ethanol, the sections were microwave treated at 750 W in citrate or EDTA buffer (Table 2) for 15 min to achieve antigen retrieval. Staining was performed in an automated immunostainer (TechMate™ 500 Plus, DAKO A/S, Denmark) using the biotin-streptavidin-per- oxidase procedure with diaminobenzidine as the chro- mogen. Endogenous peroxidase activity was blocked by incubation in H2O2. All antibodies used were commer- cially available mouse monoclonal IgG's (Table 2). After counterstaining with hematoxylin the slides were dehy- drated in ascending concentrations of alcohol and mount- ed. The immunohistochemical staining (Fig. 1) was independently evaluated by two of the investigators (E.F. and M.N.). The staining for MLH1 and MSH2 was nuclear and was classified as present or absent in the tumor tissue, and retained expression was in all tumors identified in surrounding normal crypts and in the tumor stroma. Nu- clear p53 staining was evaluated in 5 high power fields (HPFs) (x40) and was classified as positive if >5% of the tumor cell nuclei were stained. Immunohistochemical staining for -catenin normally results in a membraneous and/or a weak cytoplasmic staining, whereas a mutation leading to -catenin stabilization is reflected as an in- creased cytoplasmic or nuclear staining. We classified the predominant -catenin staining pattern in 5 HPFs accord- ing to cellular localization as membraneous, cytoplasmic or nuclear. DNA flow cytometry 50- M sections from paraffin embedded tumor tissue were used for flow cytometric DNA analysis. The nuclear suspension was stained with propidium iodide for the DNA quantification in an Ortho-Cytoron Absolute flow cytometer (Ortho Diagnostics, Raritan, NJ, USA). Up to 20,000 nuclei were analyzed per sample, which created a histogram profile with 256 channel resolution. Chicken and trout red blood cells were used as internal controls. A cell population was defined as a G0/G1 peak with a corre- sponding G2 peak. Tumors with a single G0/G1 peak were classified as DNA diploid (DNA-index of 1.0). If more than one cell population was present the sample was clas- sified as aneudiploid. The degree of DNA aneuploidy was expressed as the DNA index (DI) which represents the po- sition of the aneuploid peak in relation to the diploid ref- erence cells. ModFitLT 3.0 software from Verity Software House Inc. (Topsham, ME, USA) was used for the analysis of the DNA histograms. Results The results of the molecular genetic characterization of the tumors are presented in Table 3. In summary, aneuploidy was found in 21/30 tumors (7 of which were near-diploid with a DI of 1.1–1.2) whereas 9 tumors (including the 3 MSI tumors) were diploid. No tumor displayed any muta- tion involving exon 3 of CTNNB1, but immunohisto- chemical staining for -catenin revealed a membraneous staining in 5 tumors, a cytoplasmic staining in 19 tumors and a nuclear staining for -catenin in 6 tumors. p53 was immunohistochemically expressed in 18/30 tumors; strong wide-spread nuclear staining was found in 16 tu- mors and a weaker staining in >5% of the tumor cells in 2 tumors. KRAS mutations were identified in 6 patients (20%); all mutations were missense mutations, 5 muta- Page 4 of 8 (page number not for citation purposes)