Implications of the human genome for understanding human biology and medicine.

Clinical researchers, practicing physicians, patients, and the general public now live in a world in which the 2.9 billion nucleotide codes of the human genome are available as a resource for scientific discovery. Some of the findings from the sequencing of the human genome were expected, confirming knowledge presaged by many decades of research in both human and comparative genetics. Other findings are unexpected in their scientific and philosophical implications. In either case, the availability of the human genome is likely to have significant implications, first for clinical research and then for the practice of medicine. This article provides our reflections on what the new genomic knowledge might mean for the future of medicine and how the new knowledge relates to what we knew in the era before the availability of the genome sequence. In addition, practicing physicians in many communities are traditionally also ambassadors of science, called on to translate arcane data or the complex ramifications of biology into a language understood by the public at large. This article also may be useful for physicians who serve in this capacity in their communities. We address the following issues: the number of protein-coding genes in the human genome and certain classes of noncoding repeat elements in the genome; features of genome evolution, including large-scale duplications; an overview of the predicted protein set to highlight prominent differences between the human genome and other sequenced eukaryotic genomes; and DNA variation in the human genome. In addition, we show how this information lays the foundations for ongoing and future endeavors that will revolutionize biomedical research and our understanding of human health.

Sequence analysis of the human genome: implications for the understanding of nervous system function and disease.

The recent publication of the sequence of the human genome will accelerate the discovery of new genetic susceptibility factors for human disease, leading to the development of novel diagnostics and therapeutics. The exhaustive analysis of the human genome sequence will be the focus of the biomedical research community for many years to come. In particular, comparative analysis of the available eukaryotic genome sequences is an important approach to further our understanding of gene structure, function, and evolution. Our initial analysis of the human genome sequence has revealed many interesting features that are relevant to nervous system function, evolution, and disease. We analyzed the prominent features of predicted human proteins involved in neuronal function and prepared a comparative analysis of 146 human genes that have alleles (or mutations) conferring susceptibility for 168 neurologic diseases.

Mutation screening of the TNFRSF11A gene encoding receptor activator of NF kappa B (RANK) in familial and sporadic Paget's disease of bone and osteosarcoma.

Paget's disease of bone (PDB) is a common disorder characterized by focal areas of increased and disorganized osteoclastic bone resorption, leading to bone pain, deformity, pathological fracture, and an increased risk of osteosarcoma. Genetic factors play an important role in the pathogenesis of Paget's disease. In some families, the disease has been found to be linked to a susceptibility locus on chromosome 18q21-22, which also contains the gene responsible for familial expansile osteolysis (FEO)--a rare bone dysplasia with many similarities to Paget's disease. Insertion mutations of the TNFRSF11A gene encoding Receptor Activator of NF kappa B (RANK) have recently been found to be responsible for FEO and rare cases of early onset familial Paget's disease. Loss of heterozygosity (LOH) affecting the PDB/FEO critical region has also been described in osteosarcomas suggesting that TNFRSF11A might also be involved in the development of osteosarcoma. In order to investigate the possible role of TNFRSF11A in the pathogenesis of Paget's disease and osteosarcoma, we conducted mutation screening of the TNFRSF11A gene in patients with familial and sporadic Paget's disease as well as DNA extracted from Pagetic bone lesions, an osteosarcoma arising in Pagetic bone and six osteosarcoma cell lines. No specific abnormalities of the TNFRSF11A gene were identified in a Pagetic osteosarcoma, the osteosarcoma cell lines, DNA extracted from Pagetic bone lesions, or DNA extracted from peripheral blood in patients with familial or sporadic Paget's disease including several individuals with early onset Paget's disease. These data indicate that TNFRSF11A mutations contribute neither to the vast majority of cases of sporadic or familial PDB, nor to the development of osteosarcoma.

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Microbial disease in humans: A genomic perspective.

The approach of whole-genome shotgun sequencing coupled with the availability of computational algorithms to facilitate the assembly, gene prediction, and functional annotation of entire genomes has sparked a revolution in our understanding of the biology of free-living organisms. More than 40 bacterial genomes have been sequenced to date, of which several are important human pathogens. The capacity to sequence and assemble entire genomes of bacteria, pathogenic protozoans, and fungi in a rapid and cost-effective way has energized every aspect of microbial science. Comparative genome analysis allows us to dissect the evolutionary forces at work and provides insights into adaptations of microbes to their unique ecological niches. Factors that shape host-pathogen interactions and their outcomes include genetic polymorphisms in the microbial pathogen and host, both of which can impact on microbial virulence or host immune responses to infection. The availability of the genome sequence of entire organisms, together with the use of high-throughput sequence-based genomic technologies to define microbial and host physiological states, provides the unparalleled opportunity to better define clinical outcomes in the field of infectious diseases. There is one overarching lesson: completion of the genomic sequence of any species answers many questions, while at the same time it invites totally new questions.

Whole genomes: the foundation of new biology and medicine.

Our genomic DNA sequence provides a unique glimpse of the provenance and evolution of our species, the migration of peoples, and the causation of disease. Understanding the genome may help resolve previously unanswerable questions, including perhaps which human characteristics are innate or acquired. Such an understanding will make it possible to study how genomic DNA sequence varies among populations and among individuals, including the role of such variation in the pathogenesis of important illnesses and responses to pharmaceuticals. The study of the genome and the associated proteomics of free-living organisms will eventually make it possible to localize and annotate every human gene, as well as the regulatory elements that control the timing, organ-site specificity, extent of gene expression, protein levels, and post-translational modifications. For any given physiological process, we will have a new paradigm for addressing its evolution, development, function, and mechanism.

Sequencing the entire genomes of free-living organisms: the foundation of pharmacology in the new millennium.

The power and effectiveness of clinical pharmacology are about to be transformed with a speed that earlier in this decade could not have been foreseen even by the most astute visionaries. In the very near future, we will have at our disposal the reference DNA sequence for the entire human genome, estimated to contain approximately 3.5 billion bp. At the same time, the science of whole genome sequencing is fostering the computational science of bioinformatics needed to develop practical applications for pharmacology and toxicology. Indeed, it is likely that pharmacology, toxicology, bioinformatics, and genomics will merge into a new branch of medical science for studying and developing pharmaceuticals from molecule to bedside.

Unusual case of non-exophytic invasive penile squamous cell cancer arising from a chronic sinus tract.

We present an unusual case of an extremely well-differentiated but deeply invasive squamous cell carcinoma of the penis without an obvious external lesion, arising from a chronic draining sinus tract. This case highlights how a confounding clinical history, physical examination and initial biopsies may lead to a significant delay in diagnosis. This delay may have resulted in tumour growth and the need for a more extensive partial penectomy than would have occurred had the diagnosis been made more promptly. Finally, this case demonstrates the key diagnostic utility of deep core biopsies of the penis in situations where a cutaneous lesion does not exist.

In vitro sperm-binding assay to distinguish differences in populations of human sperm or damage to sperm resulting from cryopreservation.

Annually, >1.3 million men are members of couples seeking help because of infertility. Semen from many of these men contains reasonable numbers of motile and normal sperm, but for a subset of individuals, many sperm are deficient in ability to bind to the zona pellucida during in vitro fertilization. Diagnosis of this defect has been hampered by lack of a low-cost test. Molecular similarity exists between the perivitelline membrane of a hen's egg and the mammalian zona pellucida. These facts and some preliminary data led to evaluation of binding of human sperm during incubation for 60 minutes at 37 degrees C to an extract of chicken perivitelline membrane coated in microwell assay plates. The sperm-binding assay had inter- and intraassay plate variations of 21 and 12%, respectively, using washed fresh sperm. All seminal samples were normal, except a few that had 36 to 50% motile sperm with a low rate of sperm movement (if there is a low rate of movement, World Health Organization [WHO] criterion for normalcy is >50% motile). Nevertheless, this sperm-binding assay detected differences among individuals in percentage of sperm bound. Based on data for two to four ejaculates from each of eight occasional sperm donors, the coefficient of variation for ejaculates within donor averaged 31%, and means for the donors differed (P < 0.02). Percentage of sperm bound ranged from <1 to 38% for fresh semen from 57 men and from <1 to 13% for frozen-thawed semen from 34 men. Percentage of motile sperm accounted for <30% of the variation in percentage of sperm bound. In a direct comparison based on 17 ejaculates, aliquots evaluated fresh averaged 13% sperm bound, versus 2% for frozen-thawed aliquots. We concluded that the egg membrane substrate used in these microwell assay plates might serve as the basis for a diagnostic assay. However, it remains to be established whether samples of human semen with a low percentage of sperm binding indeed have relatively low fertilizing potential.

Increased in vitro binding of fresh and frozen-thawed human sperm exposed to a synthetic peptide.

Prosaposin is a well-characterized, approximately 68-kDa protein found in many tissues and as a normal component of human semen. A fragment of prosaposin apparently is involved in primary sperm-egg binding. We hypothesized that binding of sperm from some men to egg investments would be increased by in vitro exposure of their sperm to a synthetic fragment of human prosaposin (FertPlus peptide). Hence, we evaluated samples of washed fresh or frozen-thawed human sperm after a 10-minute exposure to synthetic FertPlus peptide at 0 (control), 80, 160, 320, 640, or 1280 pM, followed by 1:50 dilution for evaluation of binding. The criterion of response was mean percentage of sperm bound to a substrate prepared from chicken egg membranes after sperm were incubated for 60 minutes at 37 degrees C in substrate-coated wells of a sperm-binding assay plate. For each seminal sample, data were normalized against the percentage of sperm bound for control aliquots, providing values for relative binding. With fresh sperm, relative binding was increased (P < 0.01) by exposure of sperm to peptide, and the effect was especially obvious at 1280 pM. Higher doses were not tested. Collectively at three study sites, exposure of fresh sperm to 1280 pM peptide substantially increased (above 99% confidence interval; on the basis of duplicate control samples) percentage of sperm bound for 25 of 74 (34%) samples. For frozen-thawed sperm, exposure to 1280 pM peptide increased binding for 29 of 65 (45%) samples. We concluded that for >30% of men, exposure of their sperm to this synthetic fragment of prosaposin at 1280 pM increased binding of sperm to an egg membrane substrate similar to that offered by the zona pellucida.

Comparison of fresh and cryopreserved human sperm attachment to bovine oviduct (uterine tube) epithelial cells in vitro.

Formation of a prefertilization sperm reservoir in mammals is thought to occur via sperm cell attachment to fallopian tube or oviduct epithelial cells (OEC). Recent data suggests that such an interaction also occurs for human sperm in the fallopian tube. We have previously validated an in vitro sperm-OEC coculture system utilizing bovine OEC monolayers to study postejaculatory human sperm physiology. This study was done to evaluate aspects of human sperm attachment to OEC in coculture and to determine if such attachment and subsequent sperm survival differ between fresh and cryopreserved human sperm. In experiment 1, aliquots of fresh (n = 4) or cryopreserved sperm (n = 3) from normospermic donors were placed into coculture with OEC monolayers at dilutions ranging from 2 x 10(5) to 15 x 10(6) sperm per well. Numbers of each type of sperm attaching to OEC at each concentration were determined. In experiment 2, fresh and cryopreserved sperm from the same donors (n = 4) were put into OEC coculture to observe numbers attaching and subsequent survival time for each sperm type. Sperm attachment to OEC occurred in a linear, dose-dependent manner for fresh and cryopreserved sperm in experiment 1, both as a function of total sperm numbers and as a function of numbers of motile sperm applied (R2 > or = 0.79). However, cryopreserved sperm attached to the OEC at a slower rate than fresh (as a function of the average increase in the number of sperm attaching per unit increase in the number of sperm applied; P < 0.05), with an overall lower percentage of the total and motile sperm applied attaching to OEC (P < 0.01) for cryopreserved versus fresh sperm. Fewer cryopreserved sperm also attached to the OEC, as compared with fresh sperm, in experiment 2 (P < 0.05), even after correcting for motility differences between the sperm types. Sperm survival time in coculture was also decreased for cryopreserved sperm as compared with fresh sperm (P = 0.005). Understanding the kinetics of sperm and OEC interactions may be useful for developing improved cryopreservation protocols or bioassays of sperm function.

Mechanical stretch increases secretion of parathyroid hormone-related protein by cultured bladder smooth muscle cells.

Parathyroid hormone-related protein (PTHrP) immunoreactivity has been detected in the bladder and increases in response to dilatation secondary to obstruction. The hypothesis that PTHrP could be increased solely by stretch rather than other possible in vivo variables was tested by stretching cultured bladder smooth muscle cells and analyzing the culture medium for this protein. In response to mechanical stretch, PTHrP was increased in smooth muscle cell cultures. Immunoradiometric assay revealed maximal rates of secretion for the first eight hours. Comparison of percent change in PTHrP secretion of flexed cells for the various flex parameters revealed a difference (p = .006) when the degree of stretch (i.e. percent elongation) was altered. The protein synthesis inhibitor cycloheximide inhibited basal and stretch-induced PTHrP secretion. PTHrP (1-100 nM) relaxed carbachol-contracted bladder body and base by 15% and 45% respectively. PTHrP did not affect bladder contractions induced by potassium (124 mM) or alpha-beta MeATP (10 microM). Increased PTHrP secretion in response to stretch of smooth muscle raises the possibility of an autocrine action to relax the bladder during filling. PTHrP may also exert a paracrine action on vessels regulating blood flow during bladder filling or it may modulate neural activity.

Pilot study of the immunologic effects of recombinant human growth hormone and recombinant insulin-like growth factor in HIV-infected patients.

OBJECTIVE: To study the immunologic effects of recombinant human growth hormone (rhGH), recombinant human insulin-like growth factor type 1 (rhIGF-1), or the combination, in patients with moderately advanced HIV infection. DESIGN: Randomized but not blinded trial. SETTING: Government medical research center. PATIENTS: Twenty-four HIV-infected patients with CD4 cell counts of 100-400 x 10(6)/l who were receiving nucleoside antiretroviral therapy. INTERVENTIONS: Either rhGH, rhIGF-1, or the combination was administered subcutaneously for 12 weeks. MAIN OUTCOME MEASURES: Immunologic parameters, including T-cell subsets and assays of in vitro interleukin (IL)-2 production in response to antigens and mitogens, and safety profile. RESULTS: Plasma IGF-1 levels were low or low-normal prior to treatment and increased with all three therapies. There were no significant changes in CD4 cell counts, RA/RO CD4 cell subsets, natural killer cell function, immunoglobulin levels, or in vitro IL-2 production in response to mitogen or alloantigens. However, there was an upward trend (and for p18IIIB a statistically significant increase) in the in vitro IL-2 production in response to each of five HIV envelope peptides. Potential toxic effects included fatigue, arthralgia, edema, myalgia, and headache. Patients also were noted to have weight gain averaging 4 kg early in the course of treatment. CONCLUSIONS: These results suggest that treatment with rhGH/rhIGF-1 was reasonably well tolerated and that modest improvement in HIV-specific immune function was attained. Further studies will help clarify the therapeutic potential of rhGH/rhIGF-1 as an immunostimulator in the setting of HIV infection.

Coculture of human sperm with bovine oviduct epithelial cells decreases sperm chromatin structural changes seen during culture in media alone.

OBJECTIVE: To compare sperm chromatin structural changes seen in media only culture or in coculture with bovine oviduct epithelial cells. DESIGN: Three freshly ejaculated and three cryopreserved sperm samples in media culture or in oviduct epithelial cell coculture. Sperm in each treatment were evaluated by the sperm chromatin structure assay during a 72-hour time course. SETTING: An academic research laboratory. PATIENT(S): Normospermic donors with children. INTERVENTION(S): Semen collection through masturbation after 48 hours of abstinence. MAIN OUTCOME MEASURE(S): The sperm chromatin structure assay using flow cytometry to detect the susceptibility of sperm in either treatment to denaturation of DNA in situ. RESULT(S): The sperm chromatin structure assay data differed for sperm type (fresh or cryopreserved), over time, and between treatments within 6 hours of culture. In oviduct epithelial cell coculture, fresh sperm chromatin structure assay values for fresh sperm were stable, whereas in control medium higher chromatin degeneration levels were seen by 10 hours. For cryopreserved sperm, chromatin degeneration had increased by 1 hour postthaw in both treatments, although levels were higher in the control treatment thereafter. CONCLUSION(S): Sperm chromatin structural changes occur over time in culture. Such changes were observed within 2 hours for cryopreserved sperm. Coculture of sperm with oviduct epithelial cells results in a stabilizing effect for sperm against chromatin changes.

Phase II trial with dose titration of paclitaxel for the therapy of human immunodeficiency virus-associated Kaposi's sarcoma.

PURPOSE: To investigate the antitumor activity and safety of paclitaxel in patients with advanced human immunodeficiency virus (HIV)-associated Kaposi's sarcoma (KS). PATIENTS AND METHODS: Twenty-nine patients with advanced HIV-associated KS were enrolled. The patients were overall quite immunosuppressed (median CD4 count, 15 cells/microL). Paclitaxel was initially administered at 135 mg/m2 over 3 hours every 3 weeks without filgrastim support; the dose was increased as tolerated to a maximum of 175 mg/m2. Patients who failed to respond or progressed could then receive filgrastim support or paclitaxel administered over 96 hours. RESULTS: Of 28 assessable patients, 20 had major responses (18 partial responses [PRs], one clinical complete response [CR], and one CR), for a major response rate of 71.4% (95% confidence interval [CI], 51.3% to 86.8%). Each of the five patients with pulmonary KS responded, as did all four assessable patients who had previously received anthracycline therapy for KS. Of six patients who went on to receive a 96-hour infusion of paclitaxel, five had major responses. Neutropenia was the most frequent dose-limiting toxicity; possible novel toxicities included late fevers, late rash, and eosinophilia. Two patients developed an elevated creatinine concentration and one cardiomyopathy. CONCLUSION: Paclitaxel has substantial activity against advanced HIV-associated KS as a single agent, even in patients with pulmonary involvement or who had previously received anthracyclines. Further research is needed to define the optimal treatment schedule and its role vis-a-vis the other available therapies for this disease.

Early prostate-specific antigen failure following radical perineal versus retropubic prostatectomy: the importance of seminal vesicle excision.

OBJECTIVES: Because of renewed interest in the radical perineal prostatectomy, we chose to evaluate factors influencing differences in biochemical failure as measured by prostate-specific antigen (PSA) between radical perineal and the radical retropubic prostatectomies. METHODS: We undertook a retrospective review of 87 men with clinically localized prostate cancer who underwent radical retropubic (64%) or radical perineal (36%) prostatectomy, noting age, race, preoperative PSA, Gleason score, clinical stage, capsular penetration, surgical approach, and completeness of seminal vesicle (SV) excision. The two groups were comparable with respect to tumor factors such as preoperative PSA, Gleason score, clinical stage, and capsular penetration. Time to postoperative PSA failure (0.2 ng/mL or greater) was evaluated with univariate and multivariate analysis of multiple contributing factors. RESULTS: Twenty-eight percent of patients had a PSA level rising to 0.2 ng/mL or greater in the follow-up period. Patients who underwent perineal prostatectomy had a higher PSA failure rate (45%) than those treated by the retropubic approach (18%) and patients with incomplete SV excision had a higher failure rate (69%) than patients with bilateral SV excision (20%). When time to PSA failure was examined by multivariate analysis, completeness of SV excision, clinical stage, and Gleason score had a statistically significant impact on this outcome. In perineal prostatectomy patients, bilateral SV excision had a significantly longer time to PSA failure than in patients with incomplete excision. There was no significant difference in time to PSA failure between patients who underwent radical retropubic prostatectomy and the patients who underwent perineal prostatectomy with bilateral SV excision. CONCLUSIONS: Incomplete excision of SVs during a radical perineal prostatectomy contributes to an earlier postoperative biochemical recurrence as measured by a rising PSA, and may explain the higher disease recurrence rate for radical perineal prostatectomies as opposed to radical retropubic prostatectomies in this study.

Cathepsin D and chromogranin A as predictors of long term disease specific survival after radical prostatectomy for localized carcinoma of the prostate.

BACKGROUND: The accumulation of chromogranin A (Chr A) and cathepsin D (Cath D) gene products may be important in prostate carcinoma progression. This study assessed whether the levels of immunoreactivity for Chr A and Cath D are better predictors of disease specific survival than conventional pathologic parameters of the primary tumor such as Gleason score, capsular penetration, seminal vesicle invasion, and percent tumor in the specimen for patients with clinically localized prostate carcinoma managed by radical prostatectomy. METHODS: Seventy-one patients with modified Jewett clinical stages A1 to B2 adenocarcinoma of the prostate underwent a radical prostatectomy after a negative metastatic workup. No neoadjuvant or adjuvant treatments were given and all disease recurrences and causes of death were recorded. Analysis of prostatectomy specimens was undertaken to determine the conventional pathologic parameters of the primary tumor and Chr A and Cath D immunohistochemical staining. Univariate and multivariate analyses were performed to determine the independent contributions of Chr A and Cath D in predicting survival. RESULTS: On univariate analysis Chr A was the only variable that reached statistical significance for disease specific survival (P = 0.035). Cath D nearly reached significance with a P value of 0.079 for disease specific survival. On multivariate analysis, the only independent factor predicting disease specific survival was the Chr A staining score (P < 0.05). In patients with unequivocal foci of Chr A immunoreactivity, the 14-year disease specific survival was 50% compared with 68% for patients lacking such foci. CONCLUSIONS: The level of Chr A immunohistochemical staining is a strong predictor of disease specific survival and is superior to standard pathologic prognostic factors. Such findings lay the groundwork for future prospective study of the utility of such markers on biopsy specimens to predict patient outcome.

p53, bcl-2 and retinoblastoma proteins as long-term prognostic markers in localized carcinoma of the prostate.

PURPOSE: The accumulation of p53 and bcl-2 gene products as well as the loss of the retinoblastoma (Rb) gene product have been associated with prostate cancer progression. We assessed whether the levels of immunoreactivity for p53, Rb and bcl-2 are better long-term predictors of disease specific survival than conventional pathological parameters of the primary tumor, such as Gleason score, capsular penetration, seminal vesicle invasion and percent tumor in the specimen, in patients with clinically localized prostate cancer treated with radical prostatectomy. MATERIALS AND METHODS: A total of 71 patients with clinical stages A1 to B2 adenocarcinoma of the prostate underwent radical prostatectomy after a negative metastatic evaluation. No neoadjuvant or adjuvant treatments were given and causes of death were recorded. Prostatectomy specimens were analyzed to determine the conventional pathological parameters, and p53, Rb and bcl-2 immunohistochemical staining. Univariate and multivariate analyses were done to determine the independent contributions of p53, Rb and bcl-2 in predicting survival. RESULTS: On multivariate analysis the independent factors predicting disease specific survival were p53 staining score (p < 0.001) and Rb staining score (p < 0.001). In patients with p53 immunoreactive tumors the 15-year disease specific survival was 38% compared to 87% for those with less immunoreactivity. Analysis of Rb immunoreactivity for 15-year disease specific survival yielded 92 and 66% high and low staining levels, respectively. Best subset analysis revealed that the combination of p53 score and Rb score yielded the best predictive value for disease specific survival. CONCLUSIONS: p53 and Rb immunohistochemical staining scores were independent predictors of disease specific survival and were superior to conventional pathological prognostic factors of the primary tumor. These findings lay the groundwork for the prospective study of these markers in patients treated with radical prostatectomy.