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BackgroundLimited studies have functionally evaluated the heterogeneity in early ex vivo immune responses during sepsis. Our aim was to characterize early sepsis ex vivo functional immune response heterogeneity by studying whole blood endotoxin responses and derive a transcriptional metric of ex vivo endotoxin response.MethodsBlood collected within 24 h of hospital presentation from 40 septic patients was divided into two fractions and incubated with media (unstimulated) or endotoxin. Supernatants and cells were isolated, and responses measured using: supernatant cytokines, lung endothelial permeability after supernatant exposure, and RNA expression. A transcriptomic signature was derived in unstimulated cells to predict the ex vivo endotoxin response. The signature was tested in a separate cohort of 191 septic patients to evaluate for association with clinical outcome. Plasma biomarkers were quantified to measure in vivo host inflammation.ResultsEx vivo response to endotoxin varied and was unrelated to immunosuppression, white blood cell count, or the causative pathogen. Thirty-five percent of patients demonstrated a minimal response to endotoxin, suggesting early immunosuppression. High ex vivo cytokine production by stimulated blood cells correlated with increased in vitro pulmonary endothelial cell permeability and was associated with attenuated in vivo host inflammation. A four-gene signature of endotoxin response detectable without the need for a functional assay was identified. When tested in a separate cohort of septic patients, its expression was inversely associated with hospital mortality.ConclusionsAn attenuated ex vivo endotoxin response in early sepsis is associated with greater host in vivo inflammation and a worse clinical outcome.

Purpose of Investigation The purpose of this study was to study the expression of adenosine diphosphate ribosylation factor GTPase-activating protein 3 (ArfGAP3) in the anterior vaginal wall of patients with pelvic organ prolapse (POP). Materials and Methods From July 2016 to July 2018, the anterior vaginal wall of 31 POP patients (pelvic organ prolapse quantification [POP-Q] II-III [n = 10] and POP-Q IV [n = 21]) with pelvic floor dysfunction-related symptoms who underwent vaginal hysterectomy were enrolled in POP group in the Department of Gynecology of Wuhan University People’s Hospital. The anterior vaginal wall of 28 non-POP patients who underwent vaginal hysterectomy was selected as control group. The expression of 3 groups was determined by immunohistochemical staining, Western blotting, and quantitative real-time fluorescence polymerase chain reaction. Results The expression levels of ArfGAP3 of POP-Q II-III and POP-Q IV groups were lower than the control group (P < 0.05), and there were significant differences between POP-Q II-III and POP-Q IV groups (P < 0.05). Conclusions The expression of ArfGAP3 in the anterior vaginal wall of POP patients decreased, which was related to the pathogenesis and clinical grading of POP.

In the era of precision medicine, accurate and reproducible assignment of cell-of-origin (COO) in diffuse large B-cell lymphoma patients has become important. The Lymph2Cx assay is accurately determining COO by analyzing RNA expression of 20 selected genes while the Hans algorithm based on immunohistochemistry is the most popular method for routine daily diagnosis. However, there are discrepancies between the 2 methods, which need to be evaluated for better correlation. We prospectively analyzed 156 cases of diffuse large B-cell lymphoma, not otherwise specified to analyze the characteristics of discrepancy groups of COO determined by Lymph2Cx and Hans algorithm. We investigated the pattern and cause of discrepancy of COO assigned by the 2 methods. Hans algorithm classified 50 cases (32%) as germinal-center B-cell-like (GCB) type and 106 cases (68%) as non-GCB type. Lymph2Cx assay assigned 43 cases (28%) as GCB type, 94 cases (60%) as activated B-cell-like type, and 19 cases (12%) as intermediate/unclassified type. The agreement rate was 86% after exclusion of unclassified type. With regard to the clinicopathologic factors related with discrepancy between Hans algorithm and Lymph2Cx assay, endoscopic biopsy of the gastrointestinal tract (4/11, 36%) showed higher discrepancy rate (P=0.052). Immunophenotypically, CD10/BCL6/MUM1 GCB type and CD10/BCL6//MUM1 (=30%, low level expression) non-GCB type exhibited a significantly higher discrepancy rate (6/13, 46%; 4/13, 31%) (P=0.0001). Activated B-cell-like subgroup via Lymph2Cx assay predicted poor progression-free survival (mean survival duration 28.6 mo, P=0.049) compared with the GCB and unclassified type. Hans algorithm revealed no significant difference in progression-free survival and overall survival (P=0.122 and 0.121). These results suggest that when assigning COO via Hans algorithm, CD10/BCL6/MUM1 GCB type and CD10/BCL6/MUM1 (=30%, low level) non-GCB type require careful interpretation, especially if the MUM1 staining is weak and heterogeneous in the biopsied specimen.

The GPS (Oncotype Dx® Genomic Prostate Score) test is a RNA expression assay which can be performed on prostate biopsies. We sought to determine whether the GPS was associated with an increased risk of adverse pathology findings in men enrolled on active surveillance who later underwent radical prostatectomy.We identified all patients on active surveillance at University of California-San Francisco who had Gleason score 3 + 3 or low volume (33% or fewer positive cores) Gleason score 3 + 4 prostate cancer, GPS testing at diagnostic or confirmatory biopsy, clinical stage T1/T2, prostate specific antigen less than 20 and a clinical CAPRA (Cancer of the Prostate Risk Assessment) score less than 6. The primary outcome was adverse pathology, defined as Gleason score 4 + 3 or greater, stage pT3a or greater, or pN1. The secondary outcome was biochemical recurrence, defined as 2 consecutive prostate specific antigen measurements greater than 0.05 ng/ml following radical prostatectomy.Of the 215 men 179 (83%) were at low risk and 36 (17%) were at intermediate risk by CAPRA scoring. The median GPS was 26.4 (IQR 18.8-34.6). On multivariate analysis a higher GPS was associated with an increased risk of adverse pathology at delayed radical prostatectomy (HR/5 units 1.16, 95% CI 1.06-1.26, p0.01). A higher GPS was also associated with an increased risk of biochemical recurrence (HR/5 units 1.10, 95% CI 1.00-1.21, p=0.04).In patients who undergo radical prostatectomy after a period on active surveillance, as in those who undergo immediate prostatectomy, a higher GPS is associated with an increased risk of adverse pathology. The GPS is also associated with biochemical recurrence following radical prostatectomy in such patients.

Classifying diffuse large B-cell lymphoma (DLBCL) according to the cell-of-origin (COO) was first proposed using gene expression profiling; accordingly, DLBCL is classified into germinal-center B-cell type and activated B-cell type. Immunohistochemistry (IHC)-based classification using different algorithms is used widely due to the ability to use formalin-fixed paraffin-embedded tissue. Recently, newer techniques using RNA expression from formalin-fixed paraffin-embedded were introduced including the nCounter NanoString platform assay. In this brief report, we study the degree of concordance between the NanoString assay and 6 commonly utilized IHC-based algorithms to classify DLBCL cases by COO. Stains for CD10, BCL2, BCL6, FOXP-1, MUM-1, and LOM2 were used to classify a cohort of DLBCL by COO according to the respective IHC-algorithms. Then, RNA was extracted from the same cases for NanoString assay classification. The degree of concordance was calculated between the NanoString classification and each IHC-algorithm as well as among the different IHC-algorithm themselves. The concordance in COO classification of DLBCL between NanonoString assay and IHC-based algorithms is variable depending on the used IHC-algorithm; the highest concordance is seen with the Visco algorithm (κ=0.69; P=0.001). Therefore, discrepancies between the recently introduced NanoString assay and the commonly utilized IHC-algorithms are expected to some extent and should be taken into consideration when interpreting conflicting results.

RNA expression based molecular testing has the potential to improve clinical decision making as an adjunct to histopathological interpretation of prostate cancer biopsies. The GPS (Oncotype Dx Genomic Prostate Score®) assay has been proposed as a predictor of more severe pathology at prostatectomy but its true clinical value is uncertain. We hypothesized that some of the predictive usefulness of this assay relates to its correlation with histopathological features which are apparent but not typically reported on prostate biopsies.In this retrospective, single center cohort we determined an RNA based GPS of prostate biopsies. We retrospectively reviewed the histopathological features of biopsy cores with the score and assessed tumor length, Gleason pattern 4 amount and type, and stromal reaction type. Associations between the GPS and histopathological features were assessed by linear mixed models.From May 2013 to August 2015 a GPS was determined in 319 biopsies in a total of 296 patients. Of the types of Gleason pattern 4 the expansile cribriform, simple cribriform, poorly formed and fused patterns were associated with a higher GPS. The expansile cribriform pattern had the strongest association. The glomerulation pattern was associated with a lower GPS and an increasing stromal reaction also positively correlated with the GPS. A model incorporating these pathological features accounted for 36.9% of the variation in the score.The stromal reaction and the type of Gleason pattern 4 are histopathological features which are not typically reported for prostate biopsies but they correlate with the GPS. These data suggest that more detailed analysis of prostate histopathology might substitute for some of the information gained from this molecular diagnostic assay.

We determined whether prostate multiparametric magnetic resonance imaging and genomic biomarkers might help further define patients with favorable intermediate risk prostate cancer which could safely be considered suitable for active surveillance.From our institutional database we identified 509 patients who underwent radical prostatectomy with preoperative magnetic resonance imaging and a postoperative Decipher® prostate cancer test. According to the NCCN® (National Comprehensive Cancer Network®) risk stratification 125 men had favorable intermediate and 171 had unfavorable intermediate risk disease. Univariable and multivariable binary logistic regression analyses were done to test the utility of different variables in predicting adverse pathology, defined as Gleason Grade Group greater than 2, pT3b or pN1.On univariable analysis favorable intermediate risk, multiparametric magnetic resonance imaging and the prostate cancer test significantly predicted adverse pathology. On multivariable analysis favorable intermediate risk and the prostate cancer test maintained independent predictive value while multiparametric magnetic resonance imaging did not meet statistical significance (p = 0.059). The 19 patients at favorable intermediate risk with high genomic risk had an adverse pathology rate slightly higher than patients at unfavorable intermediate risk (42.1% vs 39.8%, p = 0.56). Those at low genomic risk had an adverse pathology rate slightly lower than patients at very low or low risk (7.5% vs 10.2%, p = 0.84). The 31 patients at favorable intermediate risk but at high multiparametric magnetic resonance imaging and genomic risk had an adverse pathology rate slightly lower than patients at unfavorable intermediate risk (25.8% vs 39.8%, p = 0.14). Those at low multiparametric magnetic resonance imaging and genomic risk had an adverse pathology rate slightly lower than patients at very low or low risk (8.5% vs 10.2%, p = 0.89).Multiparametric magnetic resonance imaging and the Decipher test allowed us to better define the risk of adverse pathology in patients at favorable intermediate risk who were diagnosed with prostate cancer.

INTRODUCTION AND OBJECTIVE:Peripheral blood CD14/CD2 RNA expression ratios have shown associations with prostate biopsy features. The objectives of this study are: 1) To develop a computational imm...

Background: An accurate test to differentiate large artery stroke (LAS) patients from those with cardioembolic stroke (CES) would be of significant clinical utility. Using the Biomarkers of Acute Stroke Etiology (BASE) trial (NCT02014896) dataset, our purpose was to utilize blood gene expression signatures for accurately differentiating LAS from CES acute stroke etiologies. Methods: The BASE trial enrolled suspected stroke patients presenting to 20 hospitals within 24 hrs of symptom onset. Final gold standard diagnosis and stroke etiology were determined by an adjudication committee using all hospital data but blinded to RNA test results. Whole blood, obtained in PAX tubes, was frozen at -20C within 72 hrs and analyzed at a core lab (Ischemia Care, Dayton, OH) using Affymetrix HTA microarrays. Genes on the HTA microarray were filtered to eliminate genes with low expression or high CV (> 10%) when run on replicate samples leaving 9,513 potential signature genes. A two-way random forest classifier was built through cross validation of the training data resulting in a 45 gene diagnostic signature. Results: This is a planned interim cohort study of the 1700 patients enrolled in the BASE trial that does not include lacunar strokes, TIA, or stroke mimics. Overall, 222 patients were enrolled with NIHSS>5, 70 (32%) with LAS and 152 (68%) with CES; 59% were male, and median (IQR) age was 70.7 yrs (62.0, 80.2). Median (IQR) time from symptom onset to blood collection was 1200 (448, 1568) minutes. Coexistent pathology at presentation included atrial fibrillation 90 (48%), hypertension 153 (82%), hyperlipidemia 87 (47%), diabetes 60 (32%), and coronary artery disease 70 (37%). Patients were randomly divided into training (148), early symptom onset (18 hrs) validation (35). The diagnostic gene signature results in the early validation cohort distinguished LAS from CES; C-statistic 0.78 (0.50-1.0, 95% CI), sensitivity 0.90 (0.55-1.0, 95% CI) and specificity of 0.70 (0.43-1.0, 95% CI). Conclusion: Early RNA expression differentiates large artery stroke patients from those with cardioembolic stroke, and may have therapeutic and secondary prevention implications.

Background: Identifying atrial fibrillation in embolic stroke of ischemic stroke populations would be of significant clinical utility. Using the Biomarkers of Acute Stroke Etiology (BASE) trial (NCT02014896) dataset, our goal was to determine if blood gene expression signatures accurately differentiated patients with atrial fibrillation from large artery stroke patients. Methods: The BASE trial enrolled suspected stroke patients presenting to 20 hospitals within 24 hrs of symptom onset. Final gold standard diagnosis and stroke etiology were determined by an adjudication committee using all hospital data but blinded to RNA test results. Whole blood, obtained in PAXgene tubes, was frozen at -20C within 72 hrs and analyzed at a core lab (Ischemia Care, LLC, Dayton, OH) using Affymetrix HTA micro arrays. Approximately 38,000 genes on the HTA microarray were filtered to eliminate genes with low expression or high CV (> 10%) when run on replicate samples leaving 9,513 potential signature genes. A two-way random forest classifier was built through cross validation of the training data resulting in a 23 gene diagnostic signature. Results: There were 58 patients enrolled between 18 and 24 hours of symptom onset, with NIHSS>5, 27 (47%) with atrial fibrillation cause of stroke and 31 (53%) with large artery stroke; 64% were male, and median (IQR) age was 69.7 (62.8, 81.0). Median (IQR) time from symptoms to sample collection was 1323.5 (1208.8, 1381.3) minutes. Coexistent pathology at presentation was high blood pressure 49 (84%), hyperlipidemia 28 (48%), diabetes 9 (16%), and coronary artery disease 15 (26%). The panel was able to distinguish atrial fibrillation from large vessel stroke with a C-statistic 0.92 (0.55-1.0, 95% CI), sensitivity 0.90 (0.51-1.0, 95% CI) and specificity of 0.85. Conclusion: RNA expression differentiates strokes due to atrial fibrillation from large artery stroke and may have therapeutic and outcome implications in ischemic stroke populations.

Background and Purpose: Though there are many biomarker studies of plasma and serum in patients with aneurysmal subarachnoid hemorrhage (SAH), few have examined cells in blood that might contribute to vasospasm and delayed ischemic neurological deficits (DIND). In this study we evaluate inflammatory and prothrombotic pathways by examining RNA expression in whole blood (including leukocytes, platelets) of SAH patients with vasospasm compared to those without vasospasm. Methods: Adult patients with aneurysmal SAH admitted to UCSF from 2003 to 2010 were enrolled. Patients with vasospasm (n=29) and without vasospasm (n=21) were matched for sex, race/ethnicity and aneurysm treatment method. Diagnosis of vasospasm was made by angiography. RNA expression was measured by Affymetrix Human Exon 1.0 ST Arrays. SAH patients with vasospasm were compared to those without vasospasm by ANCOVA to identify differential gene expression, exon expression and alternatively spliced transcript expression. Analyses were adjusted for age, batch, and days after SAH. Results: At the gene level there were 276 differentially expressed between SAH with vasospasm compared to patients without (P

Background: An accurate test to differentiate large artery stroke patients from those with cardioembolic stroke would be of significant utility. Using the Biomarkers of Acute Stroke Etiology (BASE) trial (NCT02014896) dataset, our purpose was to determine if blood gene expression signatures accurately differentiate large artery stroke patients from those with cardioembolic stroke. Methods: The BASE trial enrolled suspected stroke patients presenting to 10 hospitals within 8 hours of symptom onset. Gold standard diagnosis was per local neurologist adjudication blinded to RNA testing. The final gold standard diagnosis was determined by an adjudication committee blinded to RNA test results. Whole blood, obtained in PAX tubes, was frozen at -20C within 72 hours and analyzed at a core lab (Ischemia Care, LLC, Blue Ash, OH) using Affymetrix HTA micro arrays. Significantly differentially expressed genes (p Results: Overall, 32 patients were enrolled, 8 (25%) with large artery stroke and 24 (75%) with cardioembolic stroke; 50% were male, and median (IQR) age was 68.6 (47,88). Median (IQR) time from symptoms to presentation was 102.5 (14, 450) minutes. Coexistent pathology at presentation was atrial fibrillation in 13 (41%), heart failure 7 (22%), prior stroke 7 (22%), and coronary artery disease 8 (25%). The resulting gene signature distinguished large artery stroke from cardioembolic stroke; C-statistic 0.99 (0.94-1.0, 95% CI), sensitivity 0.91 (0.56-1.0, 95% CI), at a fixed specificity of 0.95, as observed in 5-fold cross validation of the training data. Conclusion: RNA expression differentiates large artery stroke patients from those with cardioembolic stroke, and may have therapeutic and outcome implications.

The pS2 trefoil protein has been detected in close association with neuro-endocrine differentiation in prostate cancer and prostatic intraepithelial neoplasia. These preliminary results have suggested that pS2 is a candidate as a specific marker for prostate cancer tissue. To ascertain the specificity of pS2 in prostate cancer tissue, we have used an RT-PCR method from prostate biopsies provided from human malignant and benign prostatic hyperplasia (BPH) tissue.Prostate biopsies were obtained from transrectal biopsies from 153 patients with an abnormal DRE or a PSA more than 4 ng./ml. or symptoms of BPH and a PSA more than 4 ng./ml. Total RNA was extracted from fresh frozen specimens of tissue samples. Detection of pS2 transcript compared with GADPH transcripts was done using RT-PCR.Biopsy results showed that 108 patients had prostate cancer (average Gleason score 6.39+/-0.74) and 45 patients had BPH. PS2 RT-PCR results showed that PS2 RNA expression was negative in 83% of the BPH cases. Conversely, 92% of prostate cancer specimens were positive (Chi-square: 86.09, p0.001). There was no correlation with tumor stage or the Gleason score. Comparing the expression of pS2 in BPH and localized prostate cancer, we found a sensitivity of 92% and a specificity of 82%.On this large sample of prostate biopsies from patients at risk of having prostate cancer, pS2 was demonstrated as an interesting marker significantly associated with prostate cancer. Further work on the expression of pS2 according to differentiation and hormonal status is in progress.

There have been advances in understanding the molecular underpinnings of risk and response to stroke hailing from genetic research. Traditional linkage studies were limited to identifying genetic mutations, representing small fractions of human disease burden. Yet with large-scale genotyping, RNA expression studies and microRNA analyses, modern genetic research has opened the possibility of understanding the greater complexity of variation. Saugstad writes of the nascent understanding of microRNAs in ischemic brain injury.1 MicroRNAs regulate messenger RNA (mRNA), the template …

Molon et al. compared RNA expression profiles in clinically unaffected muscle of patients with hereditary spastic paraplegia (SPG-4 linked) vs normal muscle. They found evidence of disruption of microtubule pathways. They suggest that the study of unaffected tissue may provide important insights into the pathophysiology of neurologic disease. see page 1097 Commentary by Peter Hedera, MD SPG4 is caused by mutations in the SPAST (SPG4) gene, encoding spastin. The normal function of spastin remains poorly understood, even though the analysis of functional domains has suggested several possible roles. Spastin is a member of a diverse AAA (ATPases Associated with various cellular Activities) superfamily of proteins, characterized by the presence of …