Transcriptomic and clinical heterogeneity of metastatic disease timing within metastatic castration-sensitive prostate cancer.

BACKGROUND: Metastatic castration-sensitive prostate cancer (mCSPC) is commonly classified into high- and low-volume subgroups which have demonstrated differential biology, prognosis, and response to therapy. Timing of metastasis has similarly demonstrated differences in clinical outcomes; however, less is known about any underlying biologic differences between these disease states. Herein, we aim to compare transcriptomic differences between synchronous and metachronous mCSPC and identify any differential responses to therapy. PATIENTS AND METHODS: We performed an international multi-institutional retrospective review of men with mCSPC who completed RNA expression profiling evaluation of their primary tumor. Patients were stratified according to disease timing (synchronous versus metachronous). The primary endpoint was to identify differences in transcriptomic profiles between disease timing. The median transcriptomic scores between groups were compared with the Mann-Whitney U test. Secondary analyses included determining clinical and transcriptomic variables associated with overall survival (OS) from the time of metastasis. Survival analysis was carried out with the Kaplan-Meier method and multivariable Cox regression. RESULTS: A total of 252 patients were included with a median follow-up of 39.6 months. Patients with synchronous disease experienced worse 5-year OS (39% versus 79%; P < 0.01) and demonstrated lower median androgen receptor (AR) activity (11.78 versus 12.64; P < 0.01) and hallmark androgen response (HAR; 3.15 versus 3.32; P < 0.01). Multivariable Cox regression identified only high-volume disease [hazard ratio (HR) = 4.97, 95% confidence interval (CI) 2.71-9.10; P < 0.01] and HAR score (HR = 0.51, 95% CI 0.28-0.88; P = 0.02) significantly associated with OS. Finally, patients with synchronous (HR = 0.47, 95% CI 0.30-0.72; P < 0.01) but not metachronous (HR = 1.37, 95% CI 0.50-3.92; P = 0.56) disease were found to have better OS with AR and non-AR combination therapy as compared with monotherapy (P value for interaction = 0.05). CONCLUSIONS: We have demonstrated a potential biologic difference between metastatic timing of mCSPC. Specifically, for patients with low-volume disease, those with metachronous low-volume disease have a more hormone-dependent transcriptional profile and exhibit a better prognosis than synchronous low-volume disease.

Extending the Debye scattering equation for diffraction from a cylindrically averaged group of atoms: detecting molecular orientation at an interface.

The Debye scattering equation is now over 100 years old and has been widely used to interpret diffraction patterns from randomly oriented groups of atoms. The present work develops and applies a related equation that calculates diffraction intensity from groups of atoms randomly oriented about a fixed axis, a scenario that occurs when molecules are oriented at an interface by the presentation of a binding motif as in antibody binding. Using an example biomolecule, the high level of sensitivity of the diffraction pattern to the orientation of the molecule and to the direction of the incident beam is shown. The use of the method is proposed not only for determining the orientation of molecules in biosensors and at membrane interfaces, but also for determining molecular conformation without the need for crystallization.

Multidisciplinary total eradication therapy (TET) in men with newly diagnosed oligometastatic prostate cancer.

To evaluate the outcomes of total eradication therapy (TET), designed to eradicate all sites of visible cancer and micrometastases, in men with newly diagnosed oligometastatic prostate cancer (OMPCa). Men with ≤ 5 sites of metastases were enrolled in a prospective registry study, underwent neoadjuvant chemohormonal therapy, followed by radical prostatectomy, adjuvant radiation (RT) to prostate bed/pelvis, stereotactic body radiation therapy (SBRT) to oligometastases, and adjuvant hormonal therapy (HT). When possible, the prostate-specific membrane antigen targeted 18F-DCFPyL PET/CT (18F-DCFPyL) scan was obtained, and abiraterone was added to neoadjuvant HT. Twelve men, median 55 years, ECOG 0, median PSA 14.7 ng/dL, clinical stages M0-1/12 (8%), M1a-3/12 (25%) and M1b-8/12 (67%), were treated. 18F-DCFPyL scan was utilized in 58% of cases. Therapies included prostatectomy 12/12 (100%), neoadjuvant [docetaxel 11/12 (92%), LHRH agonist 12/12 (100%), abiraterone + prednisone 6/12 (50%)], adjuvant radiation [RT 2/12 (17%), RT + SBRT 4/12 (33%), SBRT 6/12 (50%)], and LHRH agonist 12/12 (100%)]. 2/5 (40%) initial patients developed neutropenic fever (NF), while 0/6 (0%) subsequent patients given modified docetaxel dosing developed NF. Otherwise, TET resulted in no additive toxicities. Median follow-up was 48.8 months. Overall survival was 12/12 (100%). 1-, 2-, and 3-year undetectable PSA's were 12/12 (100%), 10/12 (83%) and 8/12 (67%), respectively. Median time to biochemical recurrence was not reached. The outcomes suggest TET in men with newly diagnosed OMPCa is safe, does not appear to cause additive toxicities, and may result in an extended interval of undetectable PSA.

Diffusion of cytokines in live lymph node tissue using microfluidic integrated optical imaging.

Communication and drug efficacy in the immune system rely heavily on diffusion of proteins such as cytokines through the tissue matrix. Available methods to analyze diffusion in tissue require microinjection or saturating the tissue in protein, which may alter local transport properties due to damage or rapid cellular responses. Here, we developed a novel, user-friendly method - Microfluidic Integrated Optical Imaging (micro-IOI) - to quantify the effective diffusion coefficient of bioactive proteins in live tissue samples ex vivo. A microfluidic platform was used to deliver picograms of fluorescently labelled cytokines to microscale regions within slices of murine lymph node, and diffusion was monitored by widefield fluorescence microscopy. Micro-IOI was validated against theory and existing methods. Free diffusion coefficients were within 8% and 24% of Stokes-Einstein predictions for dextrans and cytokines, respectively. Furthermore, diffusion coefficients for dextrans and proteins in a model matrix were within 1.5-fold of reported results from fluorescence recovery after photobleaching (FRAP). We used micro-IOI to quantify the effective diffusion of three cytokines from different structural classes and two different expression systems - tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-2 (IL-2), from human and mouse - through live lymph node tissue. This is the first method to directly measure cytokine transport in live tissue slices, and in the future, it should promote a deeper understanding of the dynamics of cell-cell communication and enable targeted immunotherapy design.

Use of the Prostate Health Index for detection of prostate cancer: results from a large academic practice.

BACKGROUND: The Prostate Health Index (phi) outperforms PSA and other PSA derivatives for the diagnosis of prostate cancer (PCa). The impact of phi testing in the real-world clinical setting has not been previously assessed. METHODS: In a single, large, academic center, phi was tested in 345 patients presenting for diagnostic evaluation for PCa. Findings on prostate biopsy (including Grade Group (GG), defined as GG1: Gleason score (GS) 6, GG2: GS 3+4=7, GG3: GS 4+3=7, GG4: GS 8 and GG5: GS 9-10), magnetic resonance imaging (MRI) and radical prostatectomy (RP) were prospectively recorded. Biopsy rates and outcomes were compared with a contemporary cohort that did not undergo phi testing (n=1318). RESULTS: Overall, 39% of men with phi testing underwent prostate biopsy. No men with phi<19.6 were diagnosed with PCa, and only three men with phi<27 had cancer of GG⩾2. Phi was superior to PSA for the prediction of any PCa (area under the receiver operating characteristic curve (AUC) 0.72 vs 0.47) and GG⩾2 PCa (AUC 0.77 vs 0.53) on prostate biopsy. Among men undergoing MRI and phi, no men with phi<27 and PI-RADS⩽3 had GG⩾2 cancer. For those men proceeding to RP, increasing phi was associated with higher pathologic GG (P=0.002) and stage (P=0.001). Compared with patients who did not undergo phi testing, the use of phi was associated with a 9% reduction in the rate of prostate biopsy (39% vs 48%; P<0.001). Importantly, the reduction in biopsy among the phi population was secondary to decreased incidence of negative (8%) and GG1 (1%) biopsies, whereas the proportion of biopsies detecting GG⩾2 cancers remained unchanged. CONCLUSIONS: In this large, real-time clinical experience, phi outperformed PSA alone, was associated with high-grade PCa, and provided complementary information to MRI. Incorporation of phi into clinical practice reduced the rate of unnecessary biopsies without changing the frequency of detection of higher-grade cancers.

Unscreened older men diagnosed with prostate cancer are at increased risk of aggressive disease.

BACKGROUND: To evaluate the relationship between PSA testing history and high-risk disease among older men diagnosed with prostate cancer. METHODS: Records from 1993 to 2014 were reviewed for men who underwent radiotherapy for prostate cancer at age 75 years or older. Patients were classified into one of four groups based on PSA-testing history: (1) no PSA testing; (2) incomplete/ineffective PSA testing; (3) PSA testing; or (4) cannot be determined. Outcomes of interest were National Comprehensive Cancer Network (NCCN) risk group (that is, low, intermediate or high risk) and biopsy grade at diagnosis. Multivariable logistic regression was used to determine the association between PSA testing history and high-risk cancer. RESULTS: PSA-testing history was available in 274 (94.5%) of 290 subjects meeting study criteria. In total, 148 men (54.0%) underwent PSA testing with follow-up biopsy, 72 (26.3%) underwent PSA testing without appropriate follow-up, and 54 men (19.7%) did not undergo PSA testing. Patients who underwent PSA testing were significantly less likely to be diagnosed with NCCN high-risk cancer (23.0% vs 51.6%, P<0.001). On multivariable analysis, men with no/incomplete PSA testing had more than three-fold increased odds of high-risk disease at diagnosis (odds ratio 3.39, 95% confidence interval 1.96-5.87, P<0.001) as compared to the tested population. CONCLUSIONS: Older men who underwent no PSA testing or incomplete testing were significantly more likely to be diagnosed with high-risk prostate cancer than those who were previously screened. It is reasonable to consider screening in healthy older men likely to benefit from early detection and treatment.

Longitudinal assessment of urinary PCA3 for predicting prostate cancer grade reclassification in favorable-risk men during active surveillance.

BACKGROUND: To assess the utility of urinary prostate cancer antigen 3 (PCA3) as both a one-time and longitudinal measure in men on active surveillance (AS). METHODS: The Johns Hopkins AS program monitors men with favorable-risk prostate cancer with serial PSA, digital rectal examination (DRE), prostate magnetic resonance imaging and prostate biopsy. Since 2007, post-DRE urinary specimens have also been routinely obtained. Men with multiple PCA3 measures obtained over ⩾3 years of monitoring were included. Utility of first PCA3 score (fPCA3), subsequent PCA3 (sPCA3) and change in PCA3 were assessed for prediction of Gleason grade reclassification (GR, Gleason score >6) during follow-up. RESULTS: In total, 260 men met study criteria. Median time from enrollment to fPCA3 was 2 years (interquartile range (IQR) 1-3) and from fPCA3 to sPCA3 was 5 years (IQR 4-6). During median follow-up of 6 years (IQR 5-8), 28 men (11%) underwent GR. Men with GR had higher median fPCA3 (48.0 vs 24.5, P=0.007) and sPCA3 (63.5 vs 36.0, P=0.002) than those without GR, while longitudinal change in PCA3 did not differ by GR status (log-normalized rate 0.07 vs 0.06, P=0.53). In a multivariable model including age, risk classification and PSA density, fPCA3 remained significantly associated with GR (log(fPCA3) odds ratio=1.77, P=0.04). CONCLUSIONS: PCA3 scores obtained during AS were higher in men who underwent GR, but the rate of change in PCA3 over time did not differ by GR status. PCA3 was a significant predictor of GR in a multivariable model including conventional risk factors, suggesting that PCA3 provides incremental prognostic information in the AS setting.

Correlation of B7-H3 with androgen receptor, immune pathways and poor outcome in prostate cancer: an expression-based analysis.

BACKGROUND: B7-H3 (CD276), part of the B7 superfamily of immune checkpoint molecules, has been shown to have an immunomodulatory role. Its regulation, receptor and mechanism of action remain unclear. B7-H3 protein expression correlates with prostate cancer outcomes, and humanized monoclonal antibodies (that is, enoblituzumab) are currently being investigated for therapeutic use. Here we used genomic expression data to examine the relationship between B7-H3 mRNA expression and prostate cancer. METHODS: Prostatectomy tissue from 2781 patients were profiled using the Affymetrix HuEx 1.0 ST microarray. Pairwise comparisons were used to identify significant associations between B7-H3 expression and clinicopathologic variables, and survival analyses were used to evaluate the prognostic significance of B7-H3. Pearson's correlation analyses were performed to assess the relationship of B7-H3 expression with molecular subtypes and individual transcripts. Androgen receptor (AR) occupancy at the B7-H3 locus was determined using chromatin immunoprecipitation (ChIP), and androgen-dependent expression changes in B7-H3 was evaluated by quantitative reverse transcription PCR in LNCaP cell lines. Oncomine was queried to evaluate B7-H3 expression in metastatic disease. RESULTS: B7-H3 mRNA expression was positively associated with higher Gleason score (P<0.001), tumor stage (P<0.001), and castrate resistant metastatic disease (P<0.0001). High B7-H3 expression correlated with the development of metastasis and prostate cancer specific mortality, but this was not significant on multi-variable analysis. B7-H3 expression correlated with ERG-positive disease (r=0.99) and AR expression (r=0.36). ChIP revealed an AR-binding site upstream of B7-H3, and the presence of androgens decreased B7-H3 expression in LNCaP suggesting potential direct AR regulation. Gene set enrichment analysis demonstrated an association of B7-H3 with androgen signaling as well as immune regulatory pathways. CONCLUSIONS: Higher B7-H3 expression correlates with Gleason grade, prostate cancer stage and poor oncologic outcomes in prostatectomy cohorts. B7-H3 expression appears to be related to androgen signaling as well as the immune reactome.

Neuropilin-1 is upregulated in the adaptive response of prostate tumors to androgen-targeted therapies and is prognostic of metastatic progression and patient mortality.

Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.

Metabolism of N-nitrosohexamethyleneimine to give 1,6-hexanediol bound to rat liver nucleic acids.

Rats were gavaged with the liver carcinogen H-nitrosohexamethyleneimine labeled with 3H or 14C and killed 16 hours later. The liver RNA and DNA were isolated and hydrolyzed with 1 M HCl at 100 degrees C. Chromatography of the 3H-labelled RNA hydrolysate on a cation exchange resin (NH4+ form), with water elution, separated five radioactive peaks, with peak "E" containing 27% of the bound 3H. There were no radioactive peaks in the 7-substituted guanine region. Hydrolysis of the 3H-labeled DNA gave a similar profile, but E contained only 5% of the 3H. The major component of E was identified as 1,6-hexanediol by its behavior and/or that of its benzoate derivative on cation exchange, anion exchange, thin-layer and gas-liquid chromatography, and by recrystallization of a mixture of the E and diol benzoates to constant specific radioactivity.

In vitro and in vivo formation of 7-(2'-carboxyethyl)guanine from the liver carcinogen 1-nitroso-5,6-dihydrouracil and its reactions with water and methanol.

Base-catalyzed hydrolysis of the potent liver carcinogen 1-nitroso-5,6-dihydrouracil [(NDHU) CAS: 16813-36-8] afforded 3-hydroxypropionic acid and acrylic acid in 72 and 6% yield, respectively. The base-catalyzed methanolysis of NDHU gave quantitative yields of 3-methoxypropionic acid and methyl carbamate. These results indicate that base-catalyzed NDHU decomposition produces a 2-carboxyethyl carbonium-ion-type intermediate. When 3H-labeled NDHU was reacted at pH 8 with DNA and RNA, 7-(2'-carboxyethyl)guanine (CEG) was detected in the hydrolysate and was identified by its cochromatography with authentic CEG in five (for DNA) or three (for RNA) systems and by a reverse isotope-dilution procedure. Radioactively labeled CEG (identified as before) and five other labeled chromatographic fractions were present in liver DNA and RNA hydrolysates after [3H]NDHU was gavaged into MRC Wistar rats. These fractions persisted in the liver DNA for various times up to 33 days after the gavage. The CEG fraction was 94% of the radioactivity in DNA reacted in vitro, but it reached only 21% of the radioactivity in the liver DNA. The results are related to a study on single-strand breaks produced by NDHU in rat liver DNA.

Predator-prey dynamics stabilised by nonlinearity explain oscillations in dust-forming plasmas.

Dust-forming plasmas are ionised gases that generate particles from a precursor. In nature, dust-forming plasmas are found in flames, the interstellar medium and comet tails. In the laboratory, they are valuable in generating nanoparticles for medicine and electronics. Dust-forming plasmas exhibit a bizarre, even puzzling behaviour in which they oscillate with timescales of seconds to minutes. Here we show how the problem of understanding these oscillations may be cast as a predator-prey problem, with electrons as prey and particles as predators. The addition of a nonlinear loss term to the classic Lotka-Volterra equations used for describing the predator-prey problem in ecology not only stabilises the oscillations in the solutions for the populations of electrons and particles in the plasma but also explains the behaviour in more detail. The model explains the relative phase difference of the two populations, the way in which the frequency of the oscillations varies with the concentration of the precursor gas, and the oscillations of the light emission, determined by the populations of both species. Our results demonstrate the value of adopting an approach to a complex physical science problem that has been found successful in ecology, where complexity is always present.

Which, when and why? Rational use of tissue-based molecular testing in localized prostate cancer.

An increased molecular understanding of localized prostate cancer and the improved ability for molecular testing of pathologic tissue has led to the development of multiple clinical assays. Here we review the relevant molecular biology of localized prostate cancer, currently available tissue-based tests and describe which is best supported for use in various clinical scenarios. Literature regarding testing of human prostate cancer tissue with Ki-67, PTEN (by immunohistochemistry (IHC) or fluroescence in situ hybridization (FISH)), ProMark, Prolaris, OncotypeDX Prostate and Decipher was reviewed to allow for generation of expert opinions. At diagnosis, evaluation of PTEN status, use of ProMark or OncotypeDX Prostate in men with Gleason 6 or 3+4=7 disease may help guide the use of active surveillance. For men with Gleason 7 or above disease considering watchful waiting, Ki-67 and Prolaris add independent prognostic information. For those men who have undergone prostatectomy and have adverse pathology, Decipher testing may aid in the decision to undergo adjuvant radiation. Newly available molecular tests bring opportunities to improve decision making for men with localized prostate cancer. A review of the currently available data suggests clinical scenarios for which each of these tests may have the greatest utility.

PET imaging of prostate-specific membrane antigen in prostate cancer: current state of the art and future challenges.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a cell surface enzyme that is highly expressed in prostate cancer (PCa) and is currently being extensively explored as a promising target for molecular imaging in a variety of clinical contexts. Novel antibody and small-molecule PSMA radiotracers labeled with a variety of radionuclides for positron emission tomography (PET) imaging applications have been developed and explored in recent studies. METHODS: A great deal of progress has been made in defining the clinical utility of this class of PET agents through predominantly small and retrospective clinical studies. The most compelling data to date has been in the setting of biochemically recurrent PCa, where PSMA-targeted radiotracers have been found to be superior to conventional imaging and other molecular imaging agents for the detection of locally recurrent and metastatic PCa. RESULTS: Early data, however, suggest that initial lymph node staging before definitive therapy in high-risk primary PCa patients may be limited, although intraoperative guidance may still hold promise. Other examples of potential promising applications for PSMA PET imaging include non-invasive characterization of primary PCa, staging and treatment planning for PSMA-targeted radiotherapeutics, and guidance of focal therapy for oligometastatic disease. CONCLUSIONS: However, all of these indications and applications for PCa PSMA PET imaging are still lacking and require large, prospective, systematic clinical trials for validation. Such validation trials are needed and hopefully will be forthcoming as the fields of molecular imaging, urology, radiation oncology and medical oncology continue to define and refine the utility of PSMA-targeted PET imaging to improve the management of PCa patients.

Efficacy of post-operative radiation in a prostatectomy cohort adjusted for clinical and genomic risk.

BACKGROUND: To date, there have been no published trials examining the impact of salvage radiation therapy (SRT) in the post-operative setting for prostate cancer (PCa). We conducted a retrospective, comparative study of post-operative radiation following radical prostatectomy (RP) for men with pT3 disease or positive margins (adverse pathological features, APF). METHODS: 422 PCa men treated at four institutions with RP and having APF were analyzed with a primary end point of metastasis. Adjuvant radiation treatment (ART, n=111), minimal residual disease (MRD) SRT (n=70) and SRT (n=83) were defined by PSA levels of <0.2, 0.2-0.49 and ⩾0.5 ng ml(-1), respectively, before radiation therapy (RT) initiation. Remaining 157 men who did not receive additional therapy before metastasis formed the no RT arm. Clinical-genomic risk was assessed by Cancer of the Prostate Risk Assessment Post-Surgical (CAPRA-S) and Decipher. Cox regression was used to evaluate the impact of treatment on outcome. RESULTS: During the study follow-up, 37 men developed metastasis with a median follow-up of 8 years. Both CAPRA-S and Decipher had independent predictive value on multivariable analysis for metastasis (P<0.05). Adjusting for clinical-genomic risk, SRT and no RT had hazard ratios of 4.31 (95% confidence interval, 1.20-15.47) and 5.42 (95% confidence interval, 1.59-18.44) for metastasis compared with ART, respectively. No significant difference was observed between MRD-SRT and ART (P=0.28). Men with low-to-intermediate CAPRA-S and low Decipher value have a low rate of metastatic events regardless of treatment selection. In contrast, men with high CAPRA-S and Decipher benefit from ART, however the cumulative incidence of metastasis remains high. CONCLUSIONS: The decision as to the timing and need for additional local therapy following RP is nuanced and requires providers and patients to balance risks of morbidity with improved oncological outcomes. Post-RP treatment can be safely avoided for men who are low risk by clinical-genomic risk, whereas those at high risk should favor enrollment in clinical trials.

High-throughput transcriptomic analysis nominates proteasomal genes as age-specific biomarkers and therapeutic targets in prostate cancer.

BACKGROUND: Although prostate cancer (PCa) is hypothesized to differ in nature between younger versus older patients, the underlying molecular distinctions are poorly understood. We hypothesized that high-throughput transcriptomic analysis would elucidate biological differences in PCas arising in younger versus older men, and would nominate potential age-specific biomarkers and therapeutic targets. METHODS: The high-density Affymetrix GeneChip platform, encompassing >1 million genomic loci, was utilized to assess gene expression in 1090 radical prostatectomy samples from patients with long-term follow-up. We identified genes associated with metastatic progression by 10 years post-treatment in younger (age<65) versus older (age⩾65) patients, and ranked these genes by their prognostic value. We performed Gene Set Enrichment Analysis (GSEA) to nominate biological concepts that demonstrated age-specific effects, and validated a target by treating with a clinically available drug in three PCa cell lines derived from younger men. RESULTS: Over 80% of the top 1000 prognostic genes in younger and older men were specific to that age group. GSEA nominated the proteasome pathway as the most differentially prognostic in younger versus older patients. High expression of proteasomal genes conferred worse prognosis in younger but not older men on univariate and multivariate analysis. Bortezomib, a Food and Drug Administration approved proteasome inhibitor, decreased proliferation in three PCa cell lines derived from younger patients. CONCLUSIONS: Our data show significant global differences in prognostic genes between older versus younger men. We nominate proteasomeal gene expression as an age-specific biomarker and potential therapeutic target specifically in younger men. Limitations of our study include clinical differences between cohorts, and increased comorbidities and lower survival in older patients. These intriguing findings suggest that current models of PCa biology do not adequately represent genetic heterogeneity of PCa related to age, and future clinical trials would benefit from stratification based on age.

Bloodstream invasion in early Lyme disease: results from a prospective, controlled, blinded study using the polymerase chain reaction.

PURPOSE: The purposes of this study were to determine (1) the optimal techniques for and potential diagnostic usefulness of the polymerase chain reaction (PCR) in early Lyme disease, and (2) the true frequency and clinical correlates of PCR-documented blood-borne infection in the dissemination of Lyme disease. PATIENTS AND METHODS: We performed a prospective, controlled, blinded study of PCR, culture, and serology on fractionated blood samples from 105 patients; 76 with physician-diagnosed erythema migrans and 29 controls. Clinical characteristics of the patients were obtained with a standardized data entry form and correlated with results of the laboratory studies. RESULTS: Only 4 of the 76 (5.3%) patients with erythema migrans were culture positive; however, 14 of 76 (18.4%) had spirochetemia documented by PCR of their plasma. None of 29 controls were PCR or culture positive (P = 0.007, versus patients). PCR-documented spirochetemia correlated with clinical evidence of disseminated disease; 10 of 33 patients (30.3%) with systemic symptom(s) were PCR positive compared to 4 of 43 (9.3%) without such evidence (P = 0.02). PCR positivity was more frequent among patients with each of four specific symptoms: fever, arthralgia, myalgia, and headache (all P < 0.05). A higher total number of symptoms (median 2.5 in PCR-positive patients versus 0 in PCR-negative controls; P < 0.01) and the presence of multiple skin lesions (37.5% of patients with multiple, versus 13.3% of patients with single lesions [P = 0.04] were also correlated with PCR positivity. Patients with both systemic symptoms and multiple skin lesions had a 40% PCR-positivity rate; however, 4 of 42 (9.5%) asympatomatic patients with only single erythema migrans lesions were also PCR positive. In multivariate analysis using logistic regression, the number of systemic symptoms was the strongest independent predictor of PCR positivity (P = 0.004). CONCLUSIONS: PCR detection of Borrelia burgdorferi is at least three times more sensitive than culture for identifying spirochetemia in early Lyme disease and may be useful in rapid diagnosis. PCR positivity significantly correlates with clinical evidence of disease dissemination. Bloodstream invasion is an important and common mechanism for the dissemination of the Lyme disease spirochete.

In vivo binding of N-nitrosopyrrolidine and N-nitrosohexamethyleneimine to non-purine sites on rat liver DNA.

Liver DNA and RNA were isolated from rats treated with the liver carcinogens N-nitrosopyrrolidine (NPYR) and N-nitrosohexamethyleneimine (NHX). After hydrolysis in 70% perchloric acid (100 degrees C, 1.0 h), 70% of the radioactivity in both the DNA and RNA hydrolysates chromatographed as a single peak. The material from both hydrolysates had comparable Rf values on cation exchange and Sephadex G-10 chromatography. Subsequent experiments indicated this material was volatile. After depurination (0.1 M HCl) of the DNA from NPYR- and NHX-treated rats, Sephadex G-10 chromatography separated only a single radioactive peak which co-eluted with the apurinic acid at the void volume. The material which comprised this peak was not volatile. After dialysis of the same 0.1 M HCl hydrolysate from NHX-treated rats, 98% of the radioactivity remained attached to the apurinic acid. These 2 cyclic nitrosamines appear to produce alkylating species that: (1) are capable of extensive, if not exclusive, phosphotriester formation; or (2) have 2 active sites that cross-link to keep purines attached to apurinic acid after 0.1 M HCl hydrolysis.

Occurrence, stability and decomposition of beta-N [gamma-L(+)-glutamyl]-4-hydroxymethylphenylhydrazine (agaritine) from the mushroom Agaricus bisporus.

A chromatographic technique was developed that could clearly separate beta-N [gamma-L(+)-glutamyl]-4-hydroxymethylphenylhydrazine (agaritine) from all other components in 10-500-microliters samples of mushroom extracts. Locally purchased mushrooms were found to contain mean levels of 0.4 - 0.7 mg agaritine g. The agaritine content of the mushrooms had decreased by 2-47% after 1 wk of storage in a domestic refrigerator and by 36-76% after 2 wk of such storage. Canned mushroom soup and canned mushrooms did not contain detectable agaritine; a sample of frozen mushrooms contained a mean level of 0.33 mg/g and a batch of fresh mushrooms lost about 32% of their agaritine content on cooking. In mice given 3 mg agaritine by gavage, agaritine was detected in all parts of the gastro-intestinal tract 15 min after dosing, but none was detectable in the gut after 3 hr. The enzyme gamma-glutamyltranspeptidase derived from pig's kidney was found to be capable of decomposing agaritine to glutamic acid and 4-(hydroxymethyl)phenylhydrazine, and to have nine times such activity as an enzyme isolated from mushrooms.

Office diagnostic procedures.

Tympanocentesis, punch biopsy of the skin, bone marrow aspiration and biopsy, and temporal artery biopsy are procedures discussed in this article. They are principally diagnostic procedures used in the evaluation of skin and serious systemic disease.