BMC Cancer 2002, 2 http://www.biomedcentral.com/1471-2407/2/25 which is characterized by development of colorectal can- cer at a mean age of 43 years and an increased risk also for cancer of the endometrium, ovaries, urothelium and the small intestine [2]. HNPCC has in population-based stud- ies been demonstrated to account for about 2% of color- ectal cancer, but has been estimated to cause about 1/3 of the colorectal cancer cases occurring at young age [3,4]. HNPCC is caused by germline mutations in one of several mismatch-repair (MMR) genes, most commonly in the genes MLH1 and MSH2. Studies of colorectal cancers from young patients have revealed MSI frequencies be- tween 17 and 58% (compared to about 15% in sporadic colorectal cancer), with the higher frequencies found in patients below age 35 at diagnosis [5–10]. Colorectal cancer cells evade growth control through a se- ries of genetic changes, which have been recognized to af- fect distinctive pathways. The chromosomal instability (CIN) pathway is involved in about 70–80% of the tu- mors and is characterized by aneuploidy, multiple chro- mosomal rearrangements, and an accumulation of somatic mutations in oncogenes such as KRAS and -cat- enin and tumor suppressor genes such as TP53 and the ad- enomatous polyposis coli (APC) gene [11]. The microsatellite instability (MSI) pathway characterizes about 15% of the colorectal cancers and is associated with diploid or near-diploid tumors and a defective MMR ma- chinery, which is reflected as MSI in the tumor tissue. MSI tumors are more likely to be right-sided with mucinous histology, poor differentiation and tumor-infiltrating lymphocytes [12]. MSI is only infrequently caused by germline HNPCC-causing MMR gene mutations, but is more commonly due to somatic hypermethylation of the MLH1 promoter [13]. Differences in aetiology, clinical behaviour and histopa- thology between colon cancer and rectal cancer suggest different preferred tumorigenic pathways in these tumor types. The few molecular genetic studies that have sepa- rately analyzed these clinical entities demonstrate a lower frequency of MSI and KRAS mutations and a higher fre- quency of aneuploid tumors, p53 mutation/overexpres- sion and -catenin overexpression in rectal cancer. These findings are likely to reflect that CIN and the TP53 path- ways contribute more than MSI to rectal cancer develop- ment [3,14–18]. However, the importance of MSI versus CIN in rectal cancer development in young patients has to our knowledge not been characterized. We have therefore studied 30 rectal cancers from patients below 50 years of age, an age limit chosen in accordance with the Amster- dam criteria for the classification of HNPCC [2] with re- spect to flow cytometric DNA content, MSI, KRAS mutations and immunohistochemical expression of the MMR proteins MLH1 and MSH2 and the tumor suppres- sor proteins p53 and -catenin. The aim was to clarify pos- sible differences between colon cancer and rectal cancer in young patients as regards the contribution of the MSI and the CIN pathways to tumor formation and to estimate the proportion of cases attributable to defective MMR. Methods Patients Thirty patients diagnosed with histologically verified ade- nocarcinomas of the rectum before age 50 were recruited for the study (Table 1). Tumor stage was Dukes' A in 6 pa- tients, B in 6, C in 12 and D in 6 patients. Tumor location was low (<5 cm from the anal verge) in 5 patients, middle rectum (5–10 cm) in 13, and high (10–15 cm) in 12 pa- tients. Tumor histopathology was reviewed by a gastroin- testinal pathologist (B.H.) and differentiation was classified as moderate in 23 tumors and mucinous /signet ring cell in 5 tumors, as well differentiated in 1 tumor and as poor in 1 tumor. The patients had a mean age of 42 (range 25–49) years and the material included 9 women and 21 men. Family histories of cancer were available from 28 of the 30 patients; 20 had no history of familial colorectal and/or endometrial cancer, 6 had one or more relatives with colorectal and/or endometrial cancer at any age and 2 patients fulfilled the Amsterdam II criteria for the classification of HNPCC, whereas data on family his- tory was not available from 2 patients (Table 1) [2]. Adju- vant treatment with 5-fluorouracil (5-FU) and leucovorin (in one case combined with oxaliplatin) was given to 16 patients. Median follow-up for the survivors was 46 (range 19–85) months. Six patients had metastasis at di- agnosis and 9 patients died from tumor median 21 (range 3–40) months after diagnosis (Table 1). Paraffin embed- ded tumor specimens were retrieved, and were sectioned and stained with Hematoxylin & Eosin to verify that rep- resentative tumor tissue was present in the sample. Three 10 M paraffin sections were used for DNA extraction (as described below) and 3 M sections were used for immu- nohistochemical staining. Permission for the study was granted by the Lund university ethics committee. DNA-extraction, PCR amplification and DNA sequencing DNA was extracted from three 10- M sections of paraffin embedded tumor tissue using proteinase-K incubation for 2–12 hours at 65 C. After incubation, the samples were boiled for 10 min to inactivate the enzyme, quickly centri- fuged and 1 L was used for the PCR amplifications. PCR was performed using AmpliTaq Gould (Perkin Elmer – Roche, Branchburg, NJ, USA) and the reagents supplied according to the PCR programme: initial denaturation (94 C, 7 min) followed by 35 cycles of denaturation (94 C, 15 sec), annealing (45–50 C, 15 sec) and exten- sion (72 C, 15 sec) whereafter a final elongation step at 72 C for 10 min was added followed by cooling at 4 C. All primer sequences and exact PCR conditions are availa- ble from the authors upon request. DNA sequencing was Page 2 of 8 (page number not for citation purposes)