Validity of a smartwatch for detecting atrial fibrillation in patients after heart valve surgery: a prospective observational study.

OBJECTIVES: Atrial fibrillation (AF) is a common early arrhythmia after heart valve surgery that limits physical activity. We aimed to evaluate the criterion validity of the Apple Watch Series 5 single-lead electrocardiogram (ECG) for detecting AF in patients after heart valve surgery. DESIGN: We enrolled 105 patients from the University Hospital of North Norway, of whom 93 completed the study. All patients underwent single-lead ECG using the smartwatch three times or more daily on the second to third or third to fourth postoperative day. These results were compared with continuous 2-4 days ECG telemetry monitoring and a 12-lead ECG on the third postoperative day. RESULTS: On comparing the Apple Watch ECGs with the ECG monitoring, the sensitivity and specificity to detect AF were 91% (75, 100) and 96% (91, 99), respectively. The accuracy was 95% (91, 99). On comparing Apple Watch ECG with a 12-lead ECG, the sensitivity was 71% (62, 100) and the specificity was 92% (92, 100). CONCLUSION: The Apple smartwatch single-lead ECG has high sensitivity and specificity, and might be a useful tool for detecting AF in patients after heart valve surgery.

Enduring epigenetic landmarks define the cancer microenvironment.

The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.

Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis.

Membrane-bound proteinase 3 (PR3m) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3m triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3m and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3m expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3m on PLS neutrophils, whereas the total amount of PR3m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3m, cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised.

Mus musculus deficient for secretory antibodies show delayed growth with an altered urinary metabolome.

BACKGROUND: The polymeric immunoglobulin receptor (pIgR) maintains the integrity of epithelial barriers by transporting polymeric antibodies and antigens through the epithelial mucosa into the lumen. In this study, we examined the role of pIgR in maintaining gut barrier integrity, which is important for the normal development in mice. METHODS: Cohorts of pIgR-/- mice and their wildtype controls were housed under Specific Pathogen Free (SPF) conditions and monitored for weight gain as an indicator of development over time. The general physiology of the gastrointestinal tract was analysed using immunohistochemistry in young (8-12 weeks of age) and aged mice (up to 18 months of age), and the observed immunopathology in pIgR-/- mice was further characterised using flow cytometry. Urinary metabolites were analysed using gas chromatography-mass spectrometry (GC-MS), which revealed changes in metabolites that correlated with age-related increase in gut permeability in pIgR-/- mice. RESULTS: We observed that pIgR-/- mice exhibited delayed growth, and this phenomenon is associated with low-grade gut inflammation that increased with ageing. The gross intraepithelial lymphocytic (IEL) infiltration characteristic of pIgR-/- mice was redefined as CD8α+αß+ T cells, the majority of which expressed high levels of CD103 and CD69 consistent with tissue resident memory T cells (TRM). Comparison of the urinary metabolome between pIgR-/- and wild-type mice revealed key changes in urinary biomarkers fucose, glycine and Vitamin B5, suggestive of altered mucosal permeability. A significant increase in gut permeability was confirmed by analysing the site-specific uptake of sugar probes in different parts of the intestine. CONCLUSION: Our data show that loss of the secretory antibody system in mice results in enhanced accumulation of inflammatory IELs in the gut, which likely reflects ongoing inflammation in reaction to gut microbiota or food antigens, leading to delayed growth in pIgR-/- mice. We demonstrate that this leads to the presence of a unique urinary metabolome profile, which may provide a biomarker for altered gut permeability.

Obtaining high quality transcriptome data from formalin-fixed, paraffin-embedded diagnostic prostate tumor specimens.

Prognostic genomic biomarkers that can be measured at diagnosis to aid choice of treatment options are unavailable for most common cancers. This is due in part to the poor quality and quantity of available diagnostic specimens for discovery research and to limitations in genomic technologies. Recent technical advances now enable high-density molecular analyses using suboptimal biological specimens. Here we describe the optimization of a transcriptome-specific protocol for use with formalin-fixed, paraffin-embedded (FFPE) diagnostic prostate cancer (PrCa) specimens. We applied the Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq Kit) to RNA samples extracted from 36 tumor-enriched and 16 adjacent normal tissues (ADJNT) from 37 FFPE PrCa specimens over a series of eight pilot studies, incorporating protocol modifications from Pilots 2 to 5. Data quality were measured by (1) the total number of mapped reads; (2) the percentage of reads that mapped to AmpliSeq target regions (OnTarget%); (3) the percentage of genes on the AmpliSeq panel with a read count ≥10 (TargetsDetected%); and (4) comparing the gene read-count distribution of the prostate tissue samples with the median gene read-count distribution of cell line-derived RNA samples. Modifications incorporated into Pilot study 5 provided gene expression data equivalent to cell line-derived RNA samples. These modifications included the use of freshly cut slides for macrodissection; increased tissue section thickness (8 µm); RNA extraction using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher); 18 target amplification cycles; and processing six samples per Ion PI chip. This protocol will facilitate the discovery of prognostic biomarkers for cancer by allowing researchers to exploit previously underutilized diagnostic FFPE specimens.

Heritable methylation marks associated with breast and prostate cancer risk.

BACKGROUND: DNA methylation can mimic the effects of germline mutations in cancer predisposition genes. Recently, we identified twenty-four heritable methylation marks associated with breast cancer risk. As breast and prostate cancer share genetic risk factors, including rare, high-risk mutations (eg, in BRCA2), we hypothesized that some of these heritable methylation marks might also be associated with the risk of prostate cancer. METHODS: We studied 869 incident prostate cancers (430 aggressive and 439 non-aggressive) and 869 matched controls nested within a prospective cohort study. DNA methylation was measured in pre-diagnostic blood samples using the Illumina Infinium HM450K BeadChip. Conditional logistic regression models, adjusted for prostate cancer risk factors and blood cell composition, were used to estimate odds ratios and 95% confidence intervals for the association between the 24 methylation marks and the risk of prostate cancer. RESULTS: Five methylation marks within the VTRNA2-1 promoter region (cg06536614, cg00124993, cg26328633, cg25340688, and cg26896946), and one in the body of CLGN (cg22901919) were associated with the risk of prostate cancer. In stratified analyses, the five VTRNA2-1 marks were associated with the risk of aggressive prostate cancer. CONCLUSIONS: This work highlights a potentially important new area of investigation for prostate cancer susceptibility and adds to our knowledge about shared risk factors for breast and prostate cancer.

Genome-Wide Measures of Peripheral Blood Dna Methylation and Prostate Cancer Risk in a Prospective Nested Case-Control Study.

BACKGROUND: Global measures of peripheral blood DNA methylation have been associated with risk of some malignancies, including breast, bladder, and gastric cancer. Here, we examined genome-wide measures of peripheral blood DNA methylation in prostate cancer and its non-aggressive and aggressive disease forms. METHODS: We used a matched, case-control study of 687 incident prostate cancer samples, nested within a larger prospective cohort study. DNA methylation was measured in pre-diagnostic, peripheral blood samples using the Illumina Infinium HM450K BeadChip. Genome-wide measures of DNA methylation were computed as the median M-value of all CpG sites and according to CpG site location and regulatory function. We used conditional logistic regression to test for associations between genome-wide measures of DNA methylation and risk of prostate cancer and its subtypes, and by time between blood draw and diagnosis. RESULTS: We observed no associations between the genome-wide measure of DNA methylation based on all CpG sites and risk of prostate cancer or aggressive disease. Risk of non-aggressive disease was associated with higher methylation of CpG islands (OR = 0.80; 95%CI = 0.68-0.94), promoter regions (OR = 0.79; 95%CI = 0.66-0.93), and high density CpG regions (OR = 0.80; 95%CI = 0.68-0.94). Additionally, higher methylation of all CpGs (OR = 0.66; 95%CI = 0.48-0.89), CpG shores (OR = 0.62; 95%CI = 0.45-0.84), and regulatory regions (OR = 0.68; 95% CI = 0.51-0.91) was associated with a reduced risk of overall prostate cancer within 5 years of blood draw but not thereafter. CONCLUSIONS: A reduced risk of overall prostate cancer within 5 years of blood draw and non-aggressive prostate cancer was associated with higher genome-wide methylation of peripheral blood DNA. While these data have no immediate clinical utility, with further work they may provide insight into the early events of prostate carcinogenesis. Prostate 77:471-478, 2017. © 2017 Wiley Periodicals, Inc.

Interleukin-1 receptor antagonist prevents murine bronchopulmonary dysplasia induced by perinatal inflammation and hyperoxia.

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants, with devastating short- and long-term consequences. The pathogenesis of BPD is multifactorial, but all triggers cause pulmonary inflammation. No therapy exists; therefore, we investigated whether the anti-inflammatory interleukin-1 receptor antagonist (IL-1Ra) prevents murine BPD. We precipitated BPD by perinatal inflammation (lipopolysaccharide injection to pregnant dams) and rearing pups in hyperoxia (65% or 85% O2). Pups were treated daily with IL-1Ra or vehicle for up to 28 d. Vehicle-injected animals in both levels of hyperoxia developed a severe BPD-like lung disease (alveolar number and gas exchange area decreased by up to 60%, alveolar size increased up to fourfold). IL-1Ra prevented this structural disintegration at 65%, but not 85% O2. Hyperoxia depleted pulmonary immune cells by 67%; however, extant macrophages and dendritic cells were hyperactivated, with CD11b and GR1 (Ly6G/C) highly expressed. IL-1Ra partially rescued the immune cell population in hyperoxia (doubling the viable cells), reduced the percentage that were activated by up to 63%, and abolished the unexpected persistence of IL-1α and IL-1ß on day 28 in hyperoxia/vehicle-treated lungs. On day 3, perinatal inflammation and hyperoxia each triggered a distinct pulmonary immune response, with some proinflammatory mediators increasing up to 20-fold and some amenable to partial or complete reversal with IL-1Ra. In summary, our analysis reveals a pivotal role for IL-1α/ß in murine BPD and an involvement for MIP (macrophage inflammatory protein)-1α and TREM (triggering receptor expressed on myeloid cells)-1. Because it effectively shields newborn mice from BPD, IL-1Ra emerges as a promising treatment for a currently irremediable disease that may potentially brighten the prognosis of the tiny preterm patients.

Predictors of prostate cancer specific mortality after radical prostatectomy: 10 year oncologic outcomes from the Victorian Radical Prostatectomy Registry.

PURPOSE: To identify the ability of multiple variables to predict prostate cancer specific mortality (PCSM) in a whole of population series of all radical prostatectomies (RP) performed in Victoria, Australia. MATERIALS & METHODS: A total of 2154 open RPs were performed in Victoria between July 1995 and December 2000. Subjects without follow up data, Gleason grade, pathological stage were excluded as were those who had pT4 disease or received neoadjuvant treatment. 1967 cases (91.3% of total) met the inclusion criteria for this study. Tumour characteristics were collated via a central registry. We used competing hazards regression models to investigate associations. RESULTS: At median follow up of 10.3 years pT stage of RP (P < 0.001) and high Gleason score of the RP specimen (P < 0.001 for ≥8 [Subhazard ratio (SHR) 11.19] and 4 + 3 = 7 [SHR 7.10]) compared with Gleason score 6 disease were strong predictors of progression to PCSM. Gleason score 3 + 4 = 7 was not at this time a significant predictor of PCSM (P = 0.08, SHR 1.84). Predictors of PCSM, independent of stage and grade, included rural residency (P = 0.003), primary surgeon contributing less than 40 cases (low-volume) to the VRPR (P = 0.025) and the involvement of a trainee surgeon in the operation (P = 0.031). CONCLUSION: The significant prediction of PCSM by pT cancer stage, Gleason score and primary Gleason pattern at RP in this whole of population study suggests a need to avoid understaging/grading in the process of cancer diagnosis and active surveillance protocols. Multi-modality therapy is likely to have a greater impact on PCSM in higher stage and Gleason grade disease. Identification of increased PCSM with rural residency and with involvement of a trainee urologist, and reduction in PCSM with higher surgeon volume all suggest potential for improved PC outcomes to be achieved with changes to surgical training and service delivery.

A pro-tumourigenic loop at the human prostate tumour interface orchestrated by oestrogen, CXCL12 and mast cell recruitment.

Prostate cancer is hormone-dependent and regulated by androgens as well as oestrogens. The tumour microenvironment also provides regulatory control, but the balance and interplay between androgens and oestrogens at the human prostate tumour interface is unknown. This study reveals a central and dominant role for oestrogen in the microenvironment, fuelling a pro-tumourigenic loop of inflammatory cytokines involving recruitment of mast cells by carcinoma-associated fibroblasts (CAFs). Mast cell numbers were increased in human PCa clinical specimens, specifically within the peritumoural stroma. Human mast cells were also shown to express ERα and ERß, with oestradiol directly stimulating mast cell proliferation and migration as well as altered cytokine/chemokine expression. There was a significant shift in the oestrogen:androgen balance in CAFs versus normal prostatic fibroblasts (NPFs), with a profound increase to ER:AR expression. Androgen signalling is also reduced in CAFs, while ERα and ERß transcriptional activity is not, allowing oestrogen to dictate hormone action in the tumour microenvironment. Gene microarray analyses identified CXCL12 as a major oestrogen-driven target gene in CAFs, and CAFs recruit mast cells via CXCL12 in a CXCR4-dependent manner. Collectively, these data reveal multicellular oestrogen action in the tumour microenvironment and show dominant oestrogen, rather than androgen, signalling at the prostatic tumour interface.

Reducing the risk of false discovery enabling identification of biologically significant genome-wide methylation status using the HumanMethylation450 array.

BACKGROUND: The Illumina HumanMethylation450 BeadChip (HM450K) measures the DNA methylation of 485,512 CpGs in the human genome. The technology relies on hybridization of genomic fragments to probes on the chip. However, certain genomic factors may compromise the ability to measure methylation using the array such as single nucleotide polymorphisms (SNPs), small insertions and deletions (INDELs), repetitive DNA, and regions with reduced genomic complexity. Currently, there is no clear method or pipeline for determining which of the probes on the HM450K bead array should be retained for subsequent analysis in light of these issues. RESULTS: We comprehensively assessed the effects of SNPs, INDELs, repeats and bisulfite induced reduced genomic complexity by comparing HM450K bead array results with whole genome bisulfite sequencing. We determined which CpG probes provided accurate or noisy signals. From this, we derived a set of high-quality probes that provide unadulterated measurements of DNA methylation. CONCLUSIONS: Our method significantly reduces the risk of false discoveries when using the HM450K bead array, while maximising the power of the array to detect methylation status genome-wide. Additionally, we demonstrate the utility of our method through extraction of biologically relevant epigenetic changes in prostate cancer.

Detection of infectious organisms in archival prostate cancer tissues.

BACKGROUND: Seroepidemiological studies have reported associations between exposure to sexually transmitted organisms and prostate cancer risk. This study sought DNA evidence of candidate organisms in archival prostate cancer tissues with the aim of assessing if a subset of these cancers show any association with common genital infections. METHODS: 221 archival paraffin-embedded tissue blocks representing 128 histopathologically confirmed prostate cancers comprising 52 "aggressive" (Gleason score ≥ 7) and 76 "non-aggressive" (Gleason score ≤ 6) TURP or radical prostatectomy specimens were examined, as well as unaffected adjacent tissue when available. Representative tissue sections were subjected to DNA extraction, quality tested and screened by PCR for HSV-1, HSV-2, XMRV, BKV, HPV, Chlamydia trachomatis, Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, and Trichomonas vaginalis. RESULTS: 195 of 221 DNA samples representing 49 "aggressive" and 66 "non-aggressive" prostate cancer cases were suitable for analysis after DNA quality assessment. Overall, 12.2% (6/49) aggressive and 7.6% (5/66) non-aggressive cases were positive for any of the candidate organisms. Mycoplasma genitalium DNA was detected in 4/66 non-aggressive, 5/49 aggressive cancers and in one cancer-unaffected adjacent tissue block of an aggressive case. Ureaplasma urealyticum DNA was detected in 0/66 non-aggressive and 1/49 aggressive cancers and HSV DNA in 1/66 non-aggressive and 0/49 aggressive cancers. This study did not detect BKV, XMRV, T. vaginalis, U. parvum, C. trachomatis or HPV DNA. CONCLUSIONS: The low prevalence of detectable microbial DNA makes it unlikely that persistent infection by the selected candidate microorganisms contribute to prostate cancer risk, regardless of tumour phenotype.

Contribution of secretory antibodies to intestinal mucosal immunity against Helicobacter pylori.

The natural immune response to Helicobacter pylori neither clears infection nor prevents reinfection. However, the ability of secretory antibodies to influence the course of H. pylori infection has not been determined. We compared the natural progression of H. pylori infection in wild-type C57BL/6 mice with that in mice lacking the polymeric immunoglobulin receptor (pIgR) that is essential for the secretion of polymeric antibody across mucosal surfaces. H. pylori SS1-infected wild-type and pIgR knockout (KO) mice were sampled longitudinally for gastrointestinal bacterial load, antibody response, and histological changes. The gastric bacterial loads of wild-type and pIgR KO mice remained constant and comparable at up to 3 months postinfection (mpi) despite SS1-reactive secretory IgA in the intestinal contents of wild-type mice at that time. Conversely, abundant duodenal colonization of pIgR KO animals contrasted with the near-total eradication of H. pylori from the intestine of wild-type animals by 3 mpi. H. pylori was cultured only from the duodenum of those animals in which colonization in the distal gastric antrum was of sufficient density for immunohistological detection. By 6 mpi, the gastric load of H. pylori in wild-type mice was significantly lower than in pIgR KO animals. While there was no corresponding difference between the two mouse strains in gastric pathology results at 6 mpi, reductions in gastric bacterial load correlated with increased gastric inflammation together with an intestinal secretory antibody response in wild-type mice. Together, these results suggest that naturally produced secretory antibodies can modulate the progress of H. pylori infection, particularly in the duodenum.

Influenza-induced inflammation drives pneumococcal otitis media.

Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.

Hedgehog signaling is active in human prostate cancer stroma and regulates proliferation and differentiation of adjacent epithelium.

BACKGROUND: Contribution of stromal Hedgehog (Hh) signaling is evident in the prostate gland in mice, but needs translation to human tissues if Hh therapeutics are to be used effectively. Our goal was to determine if primary human prostate fibroblasts contain cilia, and respond to prostate Hh signaling. METHODS: Primary human prostate cancer-associated (CAFs), and adjacent non-malignant (NPFs) fibroblasts isolated from human tissue specimens were analyzed using immunofluorescence, real-time PCR, and available array data. Cell culture and tissue recombination were used to determine responsiveness of human fibroblasts to Hh pathway manipulation and the paracrine effects of stromal Hh signaling, respectively. RESULTS: Prostatic fibroblasts were capable of forming primary cilia, with the capacity for active Hh signaling as seen by Smo co-localization to the tip of the primary cilium. Expression of genes known to represent a signature of active Hh signaling in the prostate (especially Fgf5 and Igfbp6) were increased in CAFs compared to NPFs. The level of canonical Hh genes and prostate Hh signature genes were rarely synchronous; with lower doses of Purmorphamine/BMS-833923 regulating canonical transcription factors, and higher doses effecting prostate Hh signature genes. Grafts consisting of NPFs with constitutively active Hh signaling induced increased proliferation and dedifferentiation of adjacent non-malignant BPH-1 epithelial cells. CONCLUSIONS: These data show that human prostatic fibroblasts have the capacity for Hh signaling and manipulation. Increased expression of a signature of prostatic Hh genes in the prostate tumor microenvironment suggests a role in the epithelial transformations driving prostate cancer (PCa).

Extraprostatic extension into periprostatic fat is a more important determinant of prostate cancer recurrence than an invasive phenotype.

PURPOSE: Although micrometastasis development correlates closely with the depth of invasion of many tumor types, it is unclear whether invasion into but not through the prostatic pseudocapsule has a negative impact on prognosis, similar to extraprostatic extension. We defined the impact of pseudocapsular invasion on the risk of post-prostatectomy biochemical recurrence. MATERIALS AND METHODS: Patients with pT2-3a prostate cancer were identified from a prospectively recorded database. Those with pT2 disease were categorized according to pseudocapsular invasion presence or absence. The impact of pseudocapsular invasion on biochemical recurrence was determined by univariable and multivariable Cox regression analysis. RESULTS: In a cohort of 1,338 patients we identified 595 with organ confined cancer positive for pseudocapsular invasion. Compared to tumors without evidence of invasion, pseudocapsular invasion was positively associated with higher Gleason grade and tumor volume (1.2 vs 1.9 cc, each p<0.001). On univariable analysis there was no difference in biochemical recurrence-free survival between patients with vs without pseudocapsular invasion, although those with extraprostatic extension had significantly lower biochemical recurrence-free survival (p<0.001). This was confirmed on multivariable analysis, which revealed that extraprostatic extension was a significant independent predictor of biochemical recurrence (HR 1.53, p=0.018). The presence of pseudocapsular invasion had no effect (HR 0.81, p=0.33). CONCLUSIONS: Pseudocapsular invasion is not a pathological feature associated with an adverse outcome after prostatectomy. Thus, the depth of tumor invasion is not a continuum of risk and access to periprostatic adipose tissue is a more important determinant of disease behavior than an invasive phenotype.

Human epithelial basal cells are cells of origin of prostate cancer, independent of CD133 status.

Normal prostatic epithelium is composed of basal and luminal cells. Prostate cancer can be initiated in both benign basal and luminal stem cells, but because basal cell markers are not expressed in patient tumors, the former result was unexpected. Since the cells of origin of prostate cancer are important therapeutic targets, we sought to provide further proof that basal stem cells have tumorigenic potential. Prostatic basal cells were enriched based on α2ß1integrin(hi) expression and further enriched for stem cells using CD133 in nontumorigenic BPH-1 cells. Human embryonic stem cells (hESCs) were also used as a source of normal stem cells. To test their tumorigenicity, we used two alternate stromal-based approaches; (a) recombination with human cancer-associated fibroblasts (CAFs) or (b) recombination with embryonic stroma (urogenital mesenchyme) and treated host mice with testosterone and 17ß-estradiol. Enriched α2ß1integrin(hi) basal cells from BPH-1 cells resulted in malignant tumor formation using both assays of tumorigenicity. Surprisingly, the tumorigenic potential did not reside in the CD133(+) stem cells but was consistently observed in the CD133(-) population. CAFs also failed to induce prostatic tumors from hESCs. These data confirmed that benign human basal cells include cells of origin of prostate cancer and reinforced their importance as therapeutic targets. In addition, our data suggested that the more proliferative CD133(-) basal cells are more susceptible to tumorigenesis compared to the CD133(+)-enriched stem cells. These findings challenge the current dogma that normal stem cells and cells of origin of cancer are the same cell type(s).

No association between XMRV or related gammaretroviruses in Australian prostate cancer patients.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus reported to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS). While the association of XMRV with CFS and PC has recently been discredited, no studies have been performed in Australian patients to investigate the association between PC and XMRV or related murine leukemia virus (MLV) in matched PC and normal tissue. METHODS: Genomic DNA (gDNA) was purified from matched normal and cancer formalin-fixed paraffin-embedded (FFPE) prostate tissue from 35 Australian PC patients with Gleason scores ranging from 7 - 10. The presence of the ribonuclease L (RNase L) polymorphism R462Q was determined by allele specific PCR. Samples were screened for XMRV and related murine leukemia virus (MLV) variants by qPCR. Contaminating mouse DNA was detected using qPCR targeting mouse intracisternal A particle long terminal repeat DNA. RESULTS: gDNA was successfully purified from 94% (66/70) of normal and cancer FFPE prostate tissues. RNase L typing revealed 8% were homozygous (QQ), 60% were heterozygous (RQ) and 32% were wild-type (RR) for the RNase L mutation. None of the 66 samples tested were positive for XMRV or related MLV sequences using broad MLV or XMRV specific primers with detection sensitivities of 1 viral copy of MLV/XMRV and XMRV DNA, respectively. CONCLUSIONS: Using highly sensitive qPCR we found no evidence of XMRV or related gammaretroviruses in prostate tissues from 35 Australian PC patients. Our findings are consistent with other studies demonstrating that XMRV is a laboratory contaminant that has no role in the aetiology of PC.

Presence or absence of a positive pathological margin outperforms any other margin-associated variable in predicting clinically relevant biochemical recurrence in Gleason 7 prostate cancer.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The presence of a positive pathological margin is an independent risk factor for clinically significant disease recurrence only in intermediate-risk disease when the a priori risk of micrometastatic disease is accounted for. The study examines patients with Gleason 7 prostate cancer to assess the relative importance of various margin-related variables (focality, linear length, tumour grade at margin, presence of diathermy artifact and plane of tumour) with regard to biochemical recurrence. We found that the presence or absence of a positive pathological margin outperforms any other margin-associated variable in predicting significant disease recurrence. OBJECTIVE: To determine the influence of pathological margin variables on the risk of clinically significant biochemical recurrence in Gleason 7 prostate cancer. MATERIALS AND METHODS: Patients with Gleason 7 prostate cancer with complete clinical and pathological data and detailed follow-up were identified from a prospectively recorded prostatectomy database. Slides from all patients with positive pathological margins were reviewed by a single expert uropathologist and the following information recorded: multifocality, linear length, predominant Gleason grade at the margin, diathermy artifact and margin plane. Cox regression models were generated to determine the impact of positive pathological margins on the risk of biochemical recurrence (using various definitions thereof). RESULTS: Of 1048 patients with Gleason 7 prostate cancer, 238 (23%) patients had positive margins. With a median follow-up of 11 months, biochemical recurrence occurred in 9.7% of patients with negative surgical margins and 28.4% of patients with positive margins. Positive margins were significantly associated with higher serum prostate-specific antigen (PSA) level, tumour grade, stage and volume. In patients with positive pathological margins, controlling for other factors, no margin-derived variable (focality, linear length, tumour grade at margin, diathermy artifact or plane of tumour) was a consistent predictor of biochemical recurrence, although the presence of Gleason score 4 or tertiary Gleason score 5 tumour at the margin edge was an independent predictor of recurrence with PSA doubling times ≤ 6 and ≤9 months. Similarly, in the cohort as a whole, the pathological margin status was a more important predictor of recurrence than any other margin-derived variable. CONCLUSIONS: In Gleason 7 prostate cancer, positive pathological margin status was the only consistent margin-derived variable determining biochemical failure. The presence of high-grade disease at the margin may also have an impact on the development of clinically significant biochemical recurrence.

Brief report: a bioassay to identify primary human prostate cancer repopulating cells.

Cancer cells are heterogeneous in both their phenotypes and ability to promote tumor growth and spread. Xenografting is used to identify the most highly capable cells of regenerating tumors, referred to as cancer repopulating cells. Because prostate cancers (PCa's) rarely grow as xenografts, indentifying PCa repopulating cells has not been possible. Here, we report improved methods to xenograft localized primary PCa tissues using chimeric grafts with neonatal mouse mesenchyme. Xenograft survival of tumor tissue was significantly increased by neonatal mesenchyme (six of six patients, 66% of grafts, versus four of six patients, 41% of grafts) and doubled the proliferation index of xenografted cancer cells. When applied to isolated PCa cells, neonatal mesenchyme effectively reconstituted PCa's and increased xenograft survival (four of nine patients; 32% of grafts with mesenchyme and 0% without), and supported active cancer cell proliferation. Using this assay, we showed that unfractionated α2ß1integrin(hi) and α2ß1integrin(lo) cells from primary localized PCa's demonstrated tumor formation at comparable rates, similar to previous reports using metastatic specimens. Thus, this new protocol efficiently established tumors and enabled proliferative expansion of both intact tumor tissue and fractionated cancer cells, providing a bioassay to identify and therapeutically target PCa repopulating cells.