The emerging role of hypoxia, HIF-1 and HIF-2 in multiple myeloma.

Hypoxia is an imbalance between oxygen supply and demand, which deprives cells or tissues of sufficient oxygen. It is well-established that hypoxia triggers adaptive responses, which contribute to short- and long-term pathologies such as inflammation, cardiovascular disease and cancer. Induced by both microenvironmental hypoxia and genetic mutations, the elevated expression of the hypoxia-inducible transcription factor-1 (HIF-1) and HIF-2 is a key feature of many human cancers and has been shown to promote cellular processes, which facilitate tumor progression. In this review, we discuss the emerging role of hypoxia and the HIFs in the pathogenesis of multiple myeloma (MM), an incurable hematological malignancy of BM PCs, which reside within the hypoxic BM microenvironment. The need for current and future therapeutic interventions to target HIF-1 and HIF-2 in myeloma will also be discussed.

Genetic Analysis of Soybean Plant Introductions with Resistance to Phytophthora sojae.

ABSTRACT Phytophthora sojae, which causes Phytophthora root and stem rot of soybean, is a serious disease worldwide and is managed primarily by deploying cultivars with resistance. Thirty-two soybean plant introductions (PIs), all but three of which were from South Korea, were proposed as new sources of single-gene resistance to P. sojae. The objective of this study was to characterize the inheritance of resistance to P. sojae in these PIs. Twenty-two soybean populations from crosses of these PIs and the susceptible cv. Williams were inoculated with P. sojae OH17 (vir 1b, 1d, 2, 3a, 3b, 3c, 4, 5, 6, 7), and OH25 (vir 1a, 1b, 1c, 1k, 7). These isolates were selected because they are virulent on soybeans with all known Rps genes and many Rps gene combinations. Thirteen of the twenty-two populations had consistent segregation responses following inoculations between the two generations. In two PIs, resistance was conferred by two genes to OH17 and three genes to OH25. Resistance to both isolates was conferred by a single gene in PI 398440 although the individual families were not resistant to the same isolates. The data suggest that six of the populations have three-Rps gene combinations as previously proposed, while another four may have either a novel Rps gene or a four-Rps gene combination. Based on this phenotypic analysis, novel and uncharacterized Rps genes may be present in this material. More importantly, these PIs may serve as sources of novel Rps genes that can be used to more effectively manage Phytophthora root and stem rot.

Molecular Marker Analysis of Soybean Plant Introductions with Resistance to Phytophthora sojae.

ABSTRACT Molecular analysis of sources of resistance to plant pathogens should expedite and confirm novel gene discovery and consequently the development of disease resistant cultivars. Recently, soybean plant introductions (PIs) were identified that contain putative novel Rps genes for resistance to Phytophthora sojae. The number of resistance genes that confer resistance to P. sojae isolates OH17 (1b,1d,2,3a,3b,3c,4,5,6,7) and OH25 (1a,1b,1c,1k,7) was then determined in several of the PIs. The objective of this study was to determine if the Rps genes present in these PIs were associated with eight described Rps loci that have been mapped on soybean molecular linkage groups F, G, J, and N. Nine F(2:3) soybean populations were genotyped with simple sequence repeat (SSR) markers linked to previously mapped Rps loci. The nine PI populations all had SSR markers associated (P < 0.01) with resistance to P. sojae isolate OH17 in the Rps1 region. Rps1c is a likely candidate in eight PIs but novel genes may also be possible, while novel genes may confer resistance in one PI to P. sojae isolate OHI7. Two or more Rps genes, including some that are potentially novel, confer resistance to P. sojae isolate OH25 in eight of the populations. However, based on the response to these two isolates, virulence already exists for at least some of the novel genes identified in this study.

Assessing Heterodera glycines-Resistant and Susceptible Cultivar Yield Response.

The soybean cyst nematode Heterodera glycines (SCN) is of major economic importance and widely distributed throughout soybean production regions of the United States where different maturity groups with the same sources of SCN resistance are grown. The objective of this study was to assess SCN-resistant and -susceptible soybean yield responses in infested soils across the north-central region. In 1994 and 1995, eight SCN-resistant and eight SCN-susceptible public soybean cultivars representing maturity groups (MG) I to IV were planted in 63 fields, either infested or noninfested, in 10 states in the north-central United States. Soil samples were taken to determine initial SCN population density and race, and soil classification. Data were grouped for analysis by adaptation based on MG zones. Soybean yields were 658 to 3,840 kg/ha across the sites. Soybean cyst nematode-resistant cultivars yielded better at SCN-infested sites but lost this superiority to susceptible soybean cultivars at noninfested sites. Interactions were observed among initial SCN population density, cultivar, and location. This study showed that no region-wide predictive equations could be developed for yield loss based on initial nematode populations in the soil and that yield loss due to SCN in our region was greatly confounded by other stress factors, which included temperature and moisture extremes.

Mass value assignment of total and subclass immunoglobulin G in a human standard anthrax reference serum.

An anti-Anthrax Vaccine Adsorbed (anti-AVA) standard human reference serum pool, AVR414, has been prepared, and the total and protective antigen (PA)-specific immunoglobulin G (IgG) were quantified. AVR414 was prepared by plasmapheresis of healthy adults who had received a minimum of four subcutaneous injections of AVA. Mass values (in milligrams per milliliter) for total IgG and IgG subclasses 1 to 4 were determined by radial immunodiffusion. Anti-PA-specific IgG assignment (in micrograms per milliliter) was done by consensus of two complementary approaches: homologous enzyme-linked immunosorbent assay (ELISA) with affinity-purified anti-PA IgG as a calibrator and summation of mean PA-specific IgG subclass concentrations determined by IgG subclass-specific ELISA using the United States National Reference Preparation for Human Serum Proteins as a standard. The total IgG concentration assigned to AVR414 reference serum was 8.33 mg/ml. IgG subclass concentrations were the following: for IgG1, 4.48 mg/ml; for IgG2, 3.35 mg/ml; for IgG3, 0.37 mg/ml; and for IgG4, 0.30 mg/ml. The assigned mass value for total anti-PA-specific IgG was 141.2 microg/ml. Anti-PA-specific IgG subclass concentrations were the following: for IgG1, 79.6 microg/ml; for IgG2, 35.3 microg/ml; for IgG3, 3.2 microg/ml; and for IgG4, 25.3 microg/ml. Human reference serum pool AVR414 will have direct application in the standardization of anthrax serological assays, in reagent qualification, and as a standard for quantification of PA-specific IgG in humans who have been vaccinated with or otherwise exposed to Bacillus anthracis PA.

Effect of Partial Resistance on Phytophthora Stem Rot Incidence and Yield of Soybean in Ohio.

Phytophthora root and stem rot of soybean commonly causes losses in both stand and yield in Ohio. Environmental conditions which favor the pathogen typically occur in many areas of the state during late spring and summer. This study examined the performance of 12 soybean cultivars with partial resistance, with or without Rps genes, to different populations of Phytophthora sojae and various levels of disease pressure. The soybean cultivars were evaluated in seven field environments with and without metalaxyl over 4 years. There was a highly significant genotype-environment interaction which was due in part to variable disease pressure. The incidence of Phytophthora stem rot in subplots ranged from 0 to 10 plants in the most susceptible cultivar, Sloan, while significantly less stem rot developed in cultivars with high levels of partial resistance or partial resistance combined with an Rps gene in three of the seven environments. Metalaxyl applied in-furrow had a significant effect on early and final plant populations as well as yield (P < 0.001) in two of the seven environments, and for yield (P = 0.05) in one environment. This indicates that at these two environments, 2001 Lakeview and VanBuren, early season Phytophthora disease was controlled with the in-furrow fungicide treatment. When diverse populations of P. sojae were present, yields from soybean cultivars with high levels of partial resistance were significantly higher than those with low levels of partial resistance. Soybean cultivars with specific resistance genes Rps1k, Rps1k + Rps6, or Rps1k +Rps3a had higher yields than plants with only partial resistance in environments where race determination indicated that the populations of P. sojae present were not capable of causing disease on plants with the Rps1k gene. However, in an environment with very low disease pressure, yields of soybean cultivars with partial resistance were not significantly different from those with single Rps genes or Rps gene combinations. These results demonstrate that genetic traits associated with high levels of partial resistance do not have a negative effect on yield. Soybean cultivars that had the most consistent ranking across environments were those with moderate levels of partial resistance in combination with either Rps1k or Rps3a.

A nonspecific nucleoside hydrolase from Leishmania donovani: implications for purine salvage by the parasite.

In contrast to their mammalian hosts, protozoan parasites do not synthesize purines de novo, but depend on preformed nucleotides that they purportedly obtain by salvage pathways. Nucleoside hydrolases may play a crucial role in that salvage process. By screening Leishmania donovani libraries with polyclonal antibodies against promastigote soluble exo-antigens, we have identified a cDNA encoding a protein with significant homology to nonspecific and uridine-inosine-preferring nucleoside hydrolases. Sequence comparison demonstrated that all the residues involved in Ca(2+)-binding and substrate recognition in the active site are conserved among the characterized protozoan nucleoside hydrolases. Genomic analysis suggests that it is a single copy gene in L. donovani, and its homologues are present in members representing other Leishmania species complexes. Both Northern blot and immunoblot analyses indicate that it is constitutively expressed in L. donovani promastigotes. The recombinant enzyme overexpressed in and purified from bacteria showed significant activity with all naturally occurring purine and pyrimidine nucleosides, and efficient utilization of p-nitrophenyl-beta-D-ribofuranoside as a substrate. Altogether, the sequence comparison and substrate specificity data identify this L. donovani nucleoside hydrolase as a nonspecific nucleoside hydrolase. Further, the nucleoside hydrolase was localized to specific foci in L. donovani promastigotes by immunofluorescent assays. Although the conservation of the nucleoside hydrolases among protozoan parasites offers promise for the design of broad-spectrum anti-parasitic drugs, the existence of multiple and distinct nucleoside hydrolases in a single species demands special consideration.

Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens.

An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 microg/ml) was coated with PBS-methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref-ve sera and higher absorbance with Ref+ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref+ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 microg/ml Ld-ESM mixed in PBS-methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n=22 endemic sera from North Africa were evaluated and resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials.

Plasmodium falciparum: differential display analysis of gene expression during gametocytogenesis.

With the Plasmodium falciparum genome sequencing near completion, functional analysis of individual parasite genes has become the major task of the postgenomic era. Understanding the expression patterns of individual genes is the initial step toward this goal. In this report, we have examined gene expression during gametocytogenesis of the malaria parasite, P. falciparum, using a modified differential display (DD) method. The modifications of this method include adjusting the dNTP mix, using upstream primers with higher AT contents, and reducing the extension temperature of the polymerase chain reaction (PCR). With a combination of 16 arbitrary upstream primers and 3 one-base-anchored oligo(dT) primers, we have successfully cloned 80 unique cDNA tags from stage IV-V gametocytes. Further analysis by dot blots and semiquantitative reverse transcriptase-PCR showed that at least 49 cDNAs had induced or elevated levels of expression in gametocytes. These results indicate that this modified DD procedure is suitable for large-scale identification of developmentally regulated genes in the AT-rich Plasmodium genome.

Failure of extraocular light to facilitate circadian rhythm reentrainment in humans.

Although extraocular light can entrain the circadian rhythms of invertebrates and nonmammalian vertebrates, almost all studies show that the mammalian circadian system can only be affected by light to the eyes. The exception is a recent study by Campbell and Murphy that reported phase shifts in humans to bright light applied with fiber-optic pads behind the knees (popliteal region). We tested whether this extraocular light stimulus could accelerate the entrainment of circadian rhythms to a shift of the sleep schedule, as occurs in shift work or jet lag. In experiment 1, the sleep/dark episodes were delayed 8h from baseline for 2 days, and 3h light exposures were timed to occur before the temperature minimum to help delay circadian rhythms. There were three groups: (1) bright (about 13,000 lux) extraocular light from fiber-optic pads, (2) control (dim light, 10-20 lux), and (3) medium-intensity (about 1000 lux) ocular light from light boxes. In experiment 2, the sleep/dark episodes were inverted, and extraocular light was applied either before the temperature minimum to help delay circadian rhythms or after the temperature minimum to help advance rhythms. Circadian phase markers were the salivary dim light melatonin onset (DLMO) and the rectal temperature minimum. There was no evidence that the popliteal extraocular light had a phase-shifting effect in either experiment. Possible reasons for phase shifts in the Campbell and Murphy study and not the current study include the many differences between the protocols. In the current study, there was substantial sleep deprivation before the extraocular light was applied. There was a large shift in the sleep/dark schedule, rather than allowing subjects to sleep each day from midnight to noon, as in the Campbell and Murphy study. Also, when extraocular light was applied in the current protocol, subjects did not experience a change from sleeping to awake, a change in posture (from lying in bed to sitting in a chair), or a change in ocular light (from dark to dim light). Further research is necessary to determine the conditions under which extraocular light might produce phase shifts in human circadian rhythms.

Phylogenetic relationships of human populations in sub-Saharan Africa.

This study utilizes the GM/KM immunoglobulin allotype system to elucidate the phylogenetic relationships of sub-Saharan Africans. The importance of understanding the relatedness of these peoples stems from the sub-Saharan region being the possible birthplace of humans. Haplotype distributions were determined for 19 populations and compared using chi-square analysis. Published data of other sub-Saharan Africans and representative populations worldwide were also added for comparison. Genetic distances between populations were calculated based on haplotype frequencies, and genetic relationships were observed through principal components analysis. Data from the GM/KM system showed a genetic homogeneity of the Bantu populations, with some exceptions, supporting the possibility of a common origin of these peoples. The Malagasy appeared as a divergent population, most likely due to Southeast Asian/Austronesian admixture, as indicated by the presence of the GM*AF B haplotype. The Cape Coloured also showed a divergence, with their genetic structures containing Caucasoid and Khoisan contributions. Finally, the Mbuti Pygmies appeared genetically isolated and had the highest frequency of the GM*A B haplotype out of all studied populations.

Inhibitory factors in breastmilk, maternal and infant sera against in vitro growth of Plasmodium falciparum malaria parasite.

Complications from falciparum malaria are responsible for over one million infant deaths annually. There is as yet no clinically protective vaccine that has been developed against human malaria parasites. While several studies have demonstrated the inhibitory properties of human sera against Plasmodium falciparum, there is no reported investigation that has examined the protective effects of human breastmilk against the malaria parasite. This study demonstrates the presence of significant antibody titers to ring, trophozoite, schizont and gametocyte stages of P. falciparum in 144 Nigerian maternal milk samples and also in paired maternal and infant sera. The study also demonstrates significant in vitro growth inhibition of P. falciparum by maternal and infant sera, but most notably by breastmilk samples and breastmilk constituents, such as lactoferrin and sIgA. The results therefore suggest a protective in vivo role for breastmilk in the possible modulation of malaria frequency, severity and complications.

Nocturnal melatonin secretion is not suppressed by light exposure behind the knee in humans.

Ocular light exposure can phase shift circadian rhythms and suppress nocturnal melatonin production. A recent finding suggests that extraocular light can also produce phase shifts in humans. We investigated whether extraocular light could also suppress melatonin secretion in humans. We assayed the salivary melatonin of 16 subjects during a baseline night and an experimental night in dim light (10-20 lux). The experimental night included either: (1) 3-h ocular light exposure (1000 lux, n = 6); (2) 3-h extraocular light exposure behind the knee (13,000 lux, n = 7) or (3) constant dim light exposure (10-20 lux, n = 3). Melatonin suppression occurred with ocular light but not with extraocular light or constant dim light.

How to use light and dark to produce circadian adaptation to night shift work.

The circadian rhythms of night shift workers do not usually adjust to their unusual work and sleep schedules, reducing their quality of life and producing potentially dangerous health and safety problems. This paper reviews field studies of simulated night work in which shifted light-dark cycles were constructed with artificial bright or medium-intensity light to produce circadian adaptation, ie the shifting of circadian rhythms to align with night work and day sleep schedules. By using these studies we describe fundamental principles of human circadian rhythms relevant to producing circadian adaptation to night shift work at a level designed for the reader with only a basic knowledge of circadian rhythms. These principles should enable the reader to start designing work/sleep-light/dark schedules for producing circadian adaptation in night shift workers. One specific schedule is presented as an example. Finally, we discuss phase-response curves to light and clarify common misconceptions about the production of circadian rhythm phase shifts.

Deoxyribonucleic acid ploidy and serum prostate specific antigen predict outcome following salvage prostatectomy for radiation refractory prostate cancer.

PURPOSE: We assessed clinical and pathological variables for the ability to predict improved outcome following salvage prostatectomy for radiation refractory prostate cancer. We identify factors that might assist in selection of candidates for this procedure. MATERIALS AND METHODS: Between 1966 and 1996, 108 patients (mean age 64.7 years) underwent salvage radical retropubic prostatectomy for radiation refractory prostate cancer. Preoperative serum prostate specific antigen (PSA), available in 70 patients treated since 1987, was less than 4 in 19, 4 to 10 in 31 and greater than 10 ng./ml. in 20. Serum PSA before radiotherapy was available in 37 patients. Serum PSA before radiotherapy and salvage surgery, tumor grade, deoxyribonucleic acid (DNA) ploidy and margin status were analyzed for the ability to predict cancer specific and progression-free survival (local, systemic and PSA 0.2 ng./ml. or greater). Complication rates were compared between early (before 1990) and late (1990 to 1996) salvage prostatectomy groups. RESULTS: Overall cancer specific and progression-free survival at 10 years was 70 and 44%, respectively. The pathological stage was pT2N0 in 39%, pT3-4N0 in 42% and pTxN+ in 19% of cases. DNA ploidy was predominately nondiploid, that is diploid in 25%, tetraploid in 64% and aneuploid in 11% of tumors. Although preoperative serum PSA was not predictive of pathological stage, patients with preoperative PSA less than 10 ng./ml. had better progression-free survival than those with higher levels (p = 0.05). DNA ploidy was the strongest predictor of cancer specific (p = 0.002) and progression-free (p = 0.002) survival. Controlling for grade and PSA using the Cox proportional hazards model, DNA ploidy remained a significant predictor of prostate cancer death (p <0.001) and disease progression (p <0.001). Complication rates improved somewhat in more recently treated patients but incontinence and bladder neck contracture rates remained significant. CONCLUSIONS: DNA ploidy and preoperative serum PSA appear to be the most important predictors of outcome following salvage prostatectomy for radiation refractory prostate cancer. Preoperative consideration of these factors may be helpful in selecting candidates for this procedure.

A diagnostic ELISA for visceral leishmaniasis, based on antigen from media conditioned by Leishmania donovani promastigotes.

Leishmania donovani promastigotes were cultured in a protein-free medium for 3-5 days and the spent medium used to prepare antibody-detection ELISA plates. When the plates were used to test 29 Kenyan and 16 Nepalese patients with visceral leishmaniasis (VL; kala-azar), all the sera collected at diagnosis were found to have high levels of parasite-specific IgG. The levels of these antibodies dropped 6-12 months post-initiation of antileishmanial therapy in all but one of the patients. Although the levels in sera from 59% of the treated patients fell to those measured in sera from healthy controls, those in sera from 17% of the patients did not drop below those seen at diagnosis. The antigen used did not cross-react with sera from patients with parasitological diagnosis of malaria, filariasis, African trypanosomiasis or echinococcosis. Antibodies to antigens in the spent medium were detected, by western blot, in all the sera from Nepalese patients with VL. Promastigote-conditioned media could be the source of cheap antigen for the immunodiagnosis of leishmaniasis.

Plasmodium falciparum: purification of the various gametocyte developmental stages from in vitro-cultivated parasites.

Cultivated Plasmodium falciparum gametocytes reach maturity in vitro in approximately 14-16 days, during which they pass through five morphologically distinct developmental stages. Purification of the earlier developmental stages has not been previously reported. We have modified the standard discontinuous Percoll gradient method for the separation of stage IV and V gametocytes to obtain enriched preparations of those and the earlier P. falciparum gametocyte stages. In contrast to the stages II, III, and IV, the mature stage V gametocytes from our gradient readily transformed into gametes. Such preparations may be useful in research studies on the mechanisms that underlie gametocytogenesis.

Medium-intensity light produces circadian rhythm adaptation to simulated night-shift work.

STUDY OBJECTIVES: To assess the effect of nocturnal light intensity on circadian adaptation to simulated night work. SETTING AND PARTICIPANTS: Normal young men and women, simulated night work, home sleep. DESIGN AND MEASUREMENTS: We compared temperature rhythm phase shifts following timed exposure to high (approximately 5700 lux 3 hours/day), medium (approximately 1230 lux 3 hours/day) or constant low-intensity (< 250 lux) light during consecutive night shifts. Subjects (n = 35) followed a schedule of 7 days baseline, 6 days of 8-hour night shifts (with day sleep delayed 10 hours from baseline sleep), and 4 days of recovery. Subjects wore dark sunglasses while outdoors during daylight. Sleep logs were completed after each 8-hour sleep/dark period. Night work fatigue was rated by questionnaire. RESULTS: During the 3rd through 5th days of night work, most subjects in the high and medium groups (100% and 85%) exhibited phase delays large enough that their body temperature minima occurred within the daytime sleep/dark period. Only 42% of subjects in the low group exhibited phase delays large enough to meet this criterion of circadian adaptation. The phase shifts of the high and medium groups were not significantly different, and were significantly different from the low group. Larger phase shifts were correlated with more sleep and less fatigue. CONCLUSIONS: Extremely "bright" light may not be necessary for circadian adaptation in shift work situations similar to our study protocol (e.g., regular daytime sleep/dark periods, sunglasses).

Retrospective biochemical screening of fatty acid oxidation disorders in postmortem livers of 418 cases of sudden death in the first year of life.

OBJECTIVE: Fatty acid oxidation (FAO) disorders are frequently reported as the cause of sudden and unexpected death, but their postmortem recognition remains difficult. We have devised a biochemical protocol in which informative findings in liver tissue are microvesicular steatosis, elevated concentrations of C8-C16 fatty acids, glucose depletion, and low carnitine concentration. STUDY DESIGN: We analyzed 27 cases representing five FAO disorders and compared the results with those obtained in a retrospective blinded analysis of 418 cases of sudden infant death (313 SIDS, 45 infections, and 34 accidents and abuse). RESULTS: All cases of accidents and abuse correctly tested negative. Among the others, 25 (6%) showed at least two abnormal findings. Of these, 14 closely matched the biochemical profiles seen in specific FAO disorders. These included 2 cases with medium-chain acyl-CoA dehydrogenase deficiency, 4 cases consistent with glutaric acidemia type 2, 4 cases with either very long-chain acylcoenzyme A dehydrogenase deficiency or long-chain 3-hydroxy-acyl-coenzyme A dehydrogenase deficiency, and 4 cases predicted to be affected with carnitine uptake defect. CONCLUSION: The results of this study support the view that approximately 5% of all cases of sudden infant death are likely caused by an FAO disorder.

Pathologic classification of prostate carcinoma: the impact of margin status.

BACKGROUND: A proposed pathologic (pTNM) classification system for prostate carcinoma was analyzed for its impact on survival outcome in the prostate specific antigen (PSA) era. The impact of margin status on the survival outcome of patients with otherwise organ-confined disease (i.e., without extraprostatic extension or seminal vesicle involvement) was assessed. METHODS: Among 5467 patients, the original pathologic classification was T2 in 2094 patients; those with evidence of positive margins, extraprostatic extension, or seminal vesicle involvement were initially classified as having pT3 disease (2920 patients) or pT4 residual disease (211 patients). According to the proposed pTNM system, 1512 patients for whom margin status was considered independent of T classification were reclassified. RESULTS: After reclassification, 803 specimens had been down-classified to pT2, resulting in 2932 (54%) with pT2N0 organ-confined disease and a margin positivity rate of 27%; originally, only 38% of patients had been classified as pT2N0. When the old and new classifications were compared, 5-year progression free survival to the combined endpoint of clinical and/or PSA progression (< or = 0.2 ng/mL) was 86% versus 84% and 70% versus 67% for disease classified as pT2N0 and pT3N0, respectively. Multivariate analysis assessed the effect of margin status on 2334 pT2N0 patients (classified according to the proposed pTNM system) who did not receive adjuvant therapy; adjustments were made for Gleason grade, preoperative PSA, and DNA ploidy. In this analysis, the relative risk (with 95% confidence interval) associated with positive margins was 1.65 (1.24-2.18); this was significant for the combined endpoint of clinical/PSA progression. The 5-year survival, free of clinical/PSA progression, was 86% for those without versus 75% for those with positive margins. CONCLUSIONS: This analysis supports the adoption of the proposed pTNM system, which will allow for uniform reporting of pathologic data on prostate carcinoma. For patients with organ-confined disease, positive margins are associated with higher rates of PSA progression. Accordingly, patients should be stratified based on margin positivity in addition to pT classification.