Supplemental Data for Demichelis, Fall, et al.
Description of Watchful Waiting Cohort. The study setting has been described in detail previously(Andren et al., 2006; Johansson et al., 1992; Johansson et al., 1989; Johansson et al., 2004; Johansson et al., 1997). In brief, the cohort comprises all men with early prostate cancer (T1a-b, Nx, M0) diagnosed by transurethral resection of the prostate (TURP) or transvesical adenoma enucleation for symptomatic benign prostatic hyperplasia (i.e., lower urinary track symptoms) at the Örebro University Hospital, Sweden, which serves a defined catchment area of about 190,000 inhabitants. Between 1977 and 1991 a total of 1,230 patients were diagnosed with prostate cancer. Of these, 252 were diagnosed through TURP or transvesical adenoma enucleation. At the time, there were no private urologists practicing in the area, and no screening for prostate cancer took place during the study period. Baseline evaluation at diagnosis included physical examination, chest radiography, bone scan and skeletal radiography (if needed). Nodal staging was not carried out. Because this evaluation provided no evidence for distant metastases, patients were followed expectantly (without curative treatment) and received clinical exams, laboratory tests and bone scans every 6 months during the first 2 years after diagnosis and subsequently at 12-month intervals. Symptomatic disease progression was treated with androgen deprivation therapy or palliative radiotherapy.
The patients were followed until death from prostate cancer, or censored at time of other death or until end of observation period in October 2005. The medical records of all deceased patients were reviewed. An autopsy was performed if the cause of death was not obvious on clinical grounds alone. Prostate cancer was recorded as the underlying cause of death, a contributory cause of death or unrelated to death. If treatment of the prostate cancer was related to death (e.g. cardiovascular complications following estrogens administration) prostate cancer was recorded as a contributory cause. A validation study regarding cause of death compared to the Swedish Death Register showed greater than 90% concordance, with no systematic under- or over-reporting of any cause of death(Johansson et al., 1989). Follow-up of the cohort with respect to mortality was 100% and no patients were lost to follow-up through October 2005. The endpoint of the present study was lethal prostate cancer, defined as development of distant metastases or prostate cancer as the underlying cause of death (median follow-up time 9.1 years, maximum 28 years).
Description of the ERG break apart FISH assay previously described in Perner et al. (Perner et al., 2006). For analyzing the ERG rearrangement on chromosome 21q22.2, a break apart probe assay was applied, consisting of the biotin-14-dCTP labeled BAC clone RP11-24A11 (eventually detected with cy3-avidin to produce a red signal) and the digoxigenin-dUTP labeled BAC clone RP11-137J13 (eventually detected with FITC-antidigoxygenin to produce a green signal), spanning the neighboring centromeric and telomeric region of the ERG locus, respectively. All BAC clones were obtained from the BACPAC Resource Center, Children’s Hospital Oakland Research Institute (CHORI), Oakland, CA. Prior to tissue analysis, the integrity and purity of all probes were verified by hybridization to normal peripheral lymphocyte metaphase spreads. Tissue hybridization, washing, and fluorescence detection were performed as described previously(Garraway et al., 2005; Rubin et al., 2004).
A break apart assay is in general significantly easier to interpret as compared to a fusion assay and will detect rearrangement in all cases. Thus, a break apart assays is more specific and much faster to evaluate(Bridge et al., 2006). The samples were analyzed under a 100x oil immersion objective on an Olympus BX-51 fluorescence microscope equipped with appropriate filters, a CCD (charge-coupled device) camera and the CytoVision FISH imaging and capturing software (Applied Imaging, San Jose, CA). Evaluation of the tests was independently performed by two pathologists (SP and J-M M) both with experience in analyzing interphase FISH experiments. For each case, we attempted to score a minimum 100 nuclei per case.
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