Breast cancer samples
A total of 155 breast cancer DNA samples were available for the study. The samples were derived from patients unselected for a possible family history of cancer and diagnosed with mammary gland adenocarcinoma between 1988 and 1994 at the Oulu University Hospital. The individuals had a mean age at diagnosis of 55 years (range 29–89 years) [9]. Tumor samples were evaluated by a pathologist prior to DNA extraction and contained at minimum 30% of tumor cells. A total of 34 samples (21.9%) had a tumor content < 50% (approximately 40% of tumor cells). On average the tumor cell content was 50–70%, which should enable detection of somatic mutations by sequencing. Patients gave their informed consent, and approval to perform the study was obtained from the Ethical Board of the Northern Ostrobothnia Health Care District (11/2000, 88/2000) and the Finnish Ministry of Social Affairs and Health (Dnr 46/07/98).
Prostate cancer samples
Genome Phi-amplified (GenomiPhi Amplification kit, Amersham, GE Healthcare, UK) unselected prostate tumour samples (n = 71) from the Tampere region were analysed. Of these 71 samples, 30 were prostatectomy specimens from previously untreated patients. 14 hormone-refractory specimens were obtained from transurethral resection. These patients had been treated by androgen withdrawal, on average, for 38 months (range: 15–68 months). In addition, 6 benign prostate hyperplasia specimens, 16 prostate cancer xenografts (LuCaP 23.1, 23.8, 35, 35V, 49, 58, 69, 70, 73, 77, 78, 81, 92.1, 96, 105, 115, kindly provided by Dr. Robert Vessella, University of Washington, Seattle, WA, USA), and 5 prostate cancer cell lines (DU145, LAPC4, LNCaP, PC-3 and 22Rv1) were available. DU145, LNCaP, PC-3 and 22Rv1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and LAPC4 was kindly provided by Dr. Charles Sawyers (MSKCC, New York, NY, USA). Prior to DNA extraction the samples were evaluated by a pathologist to confirm sufficient content of malignant tissue. All tumor samples contained >60% of malignant cells. The use of clinical tumor material has been approved by the Ethical Committee of Tampere University Hospital. All patients participating in this study gave informed consent.
PCR and sequencing
MYH11 (NM_002474 and NM_022844) exons 9, 12, 24, 27, 28, 29, 38 and 40 (isoform SM2) were analyzed using PCR and genomic sequencing. The first coding exon was termed exon 1. MYH11 encodes almost 2000 amino acids (42 exons), and targeted mutation analysis was performed focusing only on the regions that were important in colorectal tumor formation. The mutations which were previously functionally examined were located in exons 12 (R500L), 24 (K1044N) and 40 (SM2, c.5798del/insC) [8].
Sequencing primers have been described previously [8]. PCRs were performed in 25 μl volume containing 20 ng of genomic DNA, 1× PCR buffer (Applied Biosystems, AB, Foster City, California), 200 μmol/l each dNTP (Finnzymes, Espoo, Finland), 0.25 μmol/l both primers, and 1.25 units AmpliTaq Gold DNA polymerase. Samples were purified using ExoSAP-IT PCR purification kit (USB Corporation, Cleveland, Ohio), and sequenced using AB 3730 BD3.1 sequencing chemistry and 5.1 sequencing analysis software (Applied Biosystems, Branchburg, NJ). Genomic (un-amplified) DNA was available from the prostate samples for further analyses.