PAXgene RNA Tubes Research Articles

Whole blood mRNA expression for prognosis of metastatic clear cell renal carcinoma (mCCRCC).

500 Background: The Memorial Sloan Kettering Cancer Center (MSKCC) prognostic score is based on clinical parameters and has several limitations. We analyzed whole blood mRNA expression in mCCRCC patients (pts) and compared it to MSKCC for predicting overall survival (OS). Methods: A prospective collection of whole blood in PAXgene RNA tubes from mCCRCC pts was performed between 2011 and 2015 with uniform processing. A discovery set (DS) of 19 randomly selected pt samples was used to generate whole transcriptome RNA sequencing and signatures describing differential gene expression patterns associated with OS. We calculated the fragment per kilobase of transcript per million (FPKM) of each transcript and gene to identify correlations between the differentially expressed genes and MSKCC score. Candidate genes were selected based on statistical significance (p<0.05) and associations with OS. In an independent validation set (VS) of 47 pts with mCCRCC, we used the nCounter Analysis System (NanoString Technologies, Seattle, WA, USA) to quantify gene transcript expression selected from the DS. Cox regression and Kaplan-Meier analysis were used for association of candidate mRNAs in the VS with OS (time of metastatic recurrence to death or last follow-up). We compared our gene signature to the MSKCC prognostic score for poor versus good survival. Results: In the DS, at a median follow-up of 4.13 years (yrs) (range: 1.3-4.5), 11 patients had died. We identified eighteen genes in the DS with statistically significant differential gene expression patterns and ability to discriminate patient OS. In the VS at a median follow-up of 4.9 yrs (range: 0.1-11.3), twenty-five patients had died. We confirmed two of the eighteen selected genes that had strong prognostic significance: BAG1 [hazard ratio (HR) 0.31, p=0.007] and NOP56 [HR 0.28, p=0.004]. Comparing OS between good and poor outcome groups for these 2 scores, our two-gene panel had higher prognostic ability (HR 20.07, p <0.0001) than the MSKCC prognostic score (HR 3.19, p 0.04). Conclusions: Prognostic value of using whole blood mRNA gene profiling in mCCRCC is feasible and should be prospectively confirmed in larger studies.

Micro RNAs in Acute Myeloid Leukemia

Acute leukemia is a life-threatening condition that occurs in MDS, MPD or de novo. Although progress has been made with regard to cytogenetics, little is known about the epigenetic biology of leukemia. MicroRNA (miR) plays a role in regulation of gene expression in many cellular processes, including hematopoiesis. About 19- 25 nucleotides in length, miRs target specific mRNAs and influence their stability, degradation, or translation. miR profiling has begun to contribute to the understanding of the complex regulatory events that occur during normal hematopoiesis and are disrupted during the emergence of the leukemic clone. In particular, miR expression may be useful to understanding normal cytogenetic AMLs and developing novel treatments for AML.Our study was approved by the IRB and informed consent was obtained from patients and controls. We collected peripheral blood samples from 10 patients with newly diagnosed AML, prior to induction therapy, and 8 controls. Two ml of whole blood was collected in Paxgene RNA tubes and frozen for later processing. miRNA was purified using standard Trizol method, followed by RNeasy mini column (Qiagen). Quality of the RNA samples was assessed using the Agilent Bioanalyzer prior to library construction using the Illumina TruSeq Small RNA Sample Prep protocol (Illumina; San Diego, California). Multiplexed samples of RNA that exceeded quality control metrics (RIN > 6.0) were run on an Illumina NextSeq500 instrument at a targeted depth of 10 million reads per sample. After filtering and trimming of index and adapter sequences, whole genome alignment of the miR FASTQ reads was performed using the Homo sapiens/hg19 reference genome in the SHRiMPS aligner included in the miRNAs Analysis application available in BaseSpace (Illumina), as well as the sRNA Toolbox application suite. Quantification and normalization of aligned reads to the miRBase 21 database was performed, and differential expression between AML and control groups performed using DESeq2, NOIseq, and EdgeR algorithms. Consensus changes were seen using all three algorithms after correcting for multiple testing using the Benjamni-Hochberg False Discovery Rate (FDR) algorithm and were examined for use as potential diagnostic markers and for evidence of association with medical/demographic measures. Finally, systems-level analysis of consensus miR findings was performed using the proprietary Core Analysis workflow of the Qiagen Ingenuity® Pathway Analysis (IPA) software to identify leukemia-specific pathways and networks, and to predict the activation or inhibition of specific mRNA targets.We sequenced approximately 800 miRs from our 10 patients with AML and 8 control samples. We identified 44 miRs that showed a statistically significant increase in expression in AML patients versus controls and 33 miRs that showed a statistically significant decrease in expression in AML patients versus controls. Among these 77 differentially expressed miRs, only 10 were previously described in leukemia, including miR 181, miR 199b, miR 10a-5p, miR 22-3p, miR 23b-3p, miR 28-3p, miR 34a-5p, miR 409-3p, miR 500a-3p, miR 744-5p, and miR 126-5p. Finding these 10 miRs confirmed the validity of our approach. The remaining 67 of the miRs that showed differential expression in our study have not been described in relation to AML. Most of these have been found in association with colorectal, breast, cervical, NSCLC. Others have been reported in neurodegerative diseases and cardiac pathologies.Four of the patients with AML also had remission samples sequenced. Comparison of this subset of samples before and after treatment revealed statistically significant differences in three additional miRs. We are currently analyzing our data further to determine possible linkage between specific miRs and subtypes of AML. We are continuing to accrue patients to this study and we are following them over time in order to analyze miR expression as a biomarker that may predict relapse prior to usual indicators, i.e. cbc. Our approach of global sequencing of miRs as opposed to microarray analysis provides an unbiased approach regarding which miRs to assay and has demonstrated discovery of new associations of miRs with AML. We are hopeful that our study will provide further information about the molecular changes that lead to evolution of the leukemic clone and offer new targets for treatments.[ DisclosuresNo relevant conflicts of interest to declare.

Methodology of AA CRASH: a prospective observational study evaluating the incidence and pathogenesis of adverse post-traumatic sequelae in African-Americans experiencing motor vehicle collision

IntroductionA motor vehicle collision (MVC) is one of the most common life-threatening events experienced by individuals living in the USA. While most individuals recover following MVC, a significant proportion of...

PD28-04 DETECTION OF ANDROGEN RECEPTOR VARIANT 7 (AR-V7) IN WHOLE BLOOD RNA OF METASTATIC CASTRATION-RESISTANT PROSTATE CANCER (MCRPC) PATIENTS TREATED WITH ABIRATERONE ACETATE (ABI)

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) I1 Apr 2016PD28-04 DETECTION OF ANDROGEN RECEPTOR VARIANT 7 (AR-V7) IN WHOLE BLOOD RNA OF METASTATIC CASTRATION-RESISTANT PROSTATE CANCER (MCRPC) PATIENTS TREATED WITH ABIRATERONE ACETATE (ABI) Tilman Todenhöfer, Arun Azad, Jian Gao, Craig Stewart, Bernie Eigl, Arnulf Stenzl, Miriam Teich, Peter Black, Anthony Joshua, and Kim Chi Tilman TodenhöferTilman Todenhöfer More articles by this author , Arun AzadArun Azad More articles by this author , Jian GaoJian Gao More articles by this author , Craig StewartCraig Stewart More articles by this author , Bernie EiglBernie Eigl More articles by this author , Arnulf StenzlArnulf Stenzl More articles by this author , Miriam TeichMiriam Teich More articles by this author , Peter BlackPeter Black More articles by this author , Anthony JoshuaAnthony Joshua More articles by this author , and Kim ChiKim Chi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.391AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Expression of androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) has been associated with resistance to ABI and enzalutamide (ENZ) (Antonarakis et al. N Engl J Med 2014). We have developed a RT-PCR assay that is neither time sensitive nor requires CTC enrichment for detection of prostate cancer (PCa)-associated transcripts in whole blood. Using this assay, our aim was to correlate AR-V7 expression with outcomes on ABI. METHODS 2.5 ml whole blood was collected into PAXgene RNA tubes from mCRPC patients (pts) commencing ABI. RT-PCR was performed for the following genes: AR-V7, FOXA1, GRHL2, HOXB13, KLK2, KLK3 and TMPRSS2:ERG. For each gene, the highest CT value among 20 normal controls was set as the threshold for a positive (+) test. Clinical endpoints were PSA50 response rate (RR) (PSA decline ≥ 50% confirmed ≥ 3 weeks later), PSA30 RR, time to PSA (PCWG2 criteria) or clinical progression (change in anti-cancer therapy or decline in ECOG performance status (PS) ≥ 2 levels due to PCa), and overall survival (OS). RESULTS Whole blood samples were obtained from 37 pts. Median age was 70. 59% received prior docetaxel. All pts were ABI and ENZ naive. PSA50 RR was 37% and PSA30 RR was 48%. Median progression-free survival (PFS) was 3.8 months (mos) and median OS was 21.0 mos. 11% (4/37) of pts were AR-V7+. AR-V7+ pts were more likely to have high ALP (P=0.04), high LDH (P=0.07) and ECOG PS ≥ 2 (P=0.052). Pts with an AR-V7+ test had lower PSA50 RR (0% vs. 42%, P=0.10) and PSA30 RR (0% vs. 52%, P=0.051) together with shorter median PFS (0.7 mos vs. 4.0 mos, P<0.001; log-rank) and median OS (5.5 mos vs. 22.1 mos, P<0.001). Other factors linked with worse OS were high ALP (P=0.02), liver metastases (P=0.03), ≤ 36 mos on primary hormone therapy (P=0.04) and HOXB13+ (P=0.03). CONCLUSIONS RT-PCR detection of AR-V7 transcripts in whole blood was associated with a 0% PSA RR and significantly inferior PFS and OS in pts treated with ABI. These results reinforce the potential utility of AR-V7 as a prognostic and predictive biomarker for mCRPC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e655-e656 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Tilman Todenhöfer More articles by this author Arun Azad More articles by this author Jian Gao More articles by this author Craig Stewart More articles by this author Bernie Eigl More articles by this author Arnulf Stenzl More articles by this author Miriam Teich More articles by this author Peter Black More articles by this author Anthony Joshua More articles by this author Kim Chi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Correlation of a novel whole blood RT-PCR assay measuring AR-V7 expression with outcomes in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone acetate (ABI).

223 Background: Expression of androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) has been associated with resistance to ABI and enzalutamide (ENZ) (Antonarakis et al. N Engl J Med 2014). We have developed a RT-PCR assay that is neither time sensitive nor requires CTC enrichment for detection of prostate cancer (PCa)-associated transcripts in whole blood. Using this assay, our aim was to correlate AR-V7 expression with outcomes on ABI. Methods: 2.5 ml whole blood was collected into PAXgene RNA tubes from mCRPC patients (pts) commencing ABI. RT-PCR was performed for the following genes: AR-V7, FOXA1, GRHL2, HOXB13, KLK2, KLK3 and TMPRSS2:ERG. For each gene, the highest CT value among 20 normal controls was set as the threshold for a positive (+) test. Clinical endpoints were PSA50 response rate (RR) (PSA decline ≥ 50% confirmed ≥ 3 weeks later), PSA30 RR, time to PSA (PCWG2 criteria) or clinical progression (change in anti-cancer therapy or decline in ECOG performance status (PS) ≥ 2 levels due to PCa), and overall survival (OS). Results: Whole blood samples were obtained from 37 pts. Median age was 70. 59% received prior docetaxel. All pts were ABI and ENZ naïve. PSA50 RR was 37% and PSA30 RR was 48%. Median progression-free survival (PFS) was 3.8 months (mos) and median OS was 21.0 mos. 11% (4/37) of pts were AR-V7+. AR-V7+ pts were more likely to have high ALP (P= 0.04; Χ2), high LDH (P= 0.07) and ECOG PS ≥ 2 (P= 0.052). Pts with an AR-V7+ test had lower PSA50 RR (0% vs. 42%, P= 0.10; Χ2) and PSA30 RR (0% vs. 52%, P= 0.051) together with shorter median PFS (0.7 mos vs. 4.0 mos, P&lt; 0.001; log-rank) and median OS (5.5 mos vs. 22.1 mos, P&lt; 0.001). Other factors linked with worse OS were high ALP (P= 0.02), liver metastases (P= 0.03), ≤ 36 mos on primary hormone therapy (P= 0.04), HOXB13+ (P= 0.03) and KLK2+ (P&lt; 0.001). Conclusions: RT-PCR detection of AR-V7 transcripts in whole blood was associated with a 0% PSA RR and significantly inferior PFS and OS in pts treated with ABI. These results reinforce the potential utility of AR-V7 as a prognostic and predictive biomarker for mCRPC. Validation of the assay in larger datasets is ongoing.

Influence of next-generation sequencing and storage conditions on miRNA patterns generated from PAXgene blood.

Whole blood derived miRNA signatures determined by Next-Generation Sequencing (NGS) offer themselves as future minimally invasive biomarkers for various human diseases. The PAXgene system is a commonly used blood storage system for miRNA analysis. Central to all miRNA analyses that aim to identify disease specific miRNA signatures, is the question of stability and variability of the miRNA profiles that are generated by NGS. We characterized the influence of five different conditions on the genome wide miRNA expression pattern of human blood isolated in PAXgene RNA tubes. In detail, we analyzed 15 miRNomes from three individuals. The blood was subjected to different numbers of freeze/thaw cycles and analyzed for the influence of storage at -80 or 8 °C. We also determined the influence of blood collection and NGS preparations on the miRNA pattern isolated from a single individual, which has been sequenced 10 times. Here, five PAXGene tubes were consecutively collected that have been split in two replicates, representing two experimental batches. All samples were analyzed by Illumina NGS. For each sample, approximately 20 million NGS reads have been generated. Hierarchical clustering and Principal Component Analysis (PCA) showed an influence of the different conditions on the miRNA patterns. The effects of the different conditions on miRNA abundance are, however, smaller than the differences that are due to interindividual variability. We also found evidence for an influence of the NGS measurement on the miRNA pattern. Specifically, hsa-miR-1271-5p and hsa-miR-182-5p showed coefficients of variation above 100% indicating a strong influence of the NGS protocol on the abundance of these miRNAs.

Abstract W P100: Arginase-1 Is A Novel Biomarker Of Post-stroke Immune Suppression

Objective: Arginase-1 (ARG1) is a novel biomarker of vascular and immune responses in ischemic stroke (IS). The objective of this study was to determine the relationship between ARG1 peripheral blood gene expression, history of hypertension and IS recovery. Methods: Peripheral blood samples were collected from IS patients’ ≥18 years of age within 24 hours from symptom onset. Stroke severity was determined by the NIHSS. Diagnosis of hypertension and lymphocyte counts were obtained from medical record. Modified rankin scale (MRS) was used to determine 30 day recovery. RNA was extracted from Paxgene RNA tubes, amplified, and hybridized to Illumina HumanRef-8v2 bead chips. Gene expression was compared in a univariate manner between IS patients with good (MRS 0-1) and bad (MRS 2-6) outcome using t-test in GeneSpring. Inflation of type one error was corrected by Bonferroni. Results: Twenty-three IS patients were recruited. Most patients had cardioembolic (n=14; 41.2%) or large artery stroke (n=5; 14.7%). Median pre stroke MRS was 0; median baseline NIHSS was 3 (range 0-24); and median 30 day MRS was 0 (range 0-6). ARG1 was higher in stroke patients with a history of hypertension (p=0.021); and there was an inverse relationship between ARG1 and lymphocyte count. When controlling for age, NIHSS and hypertension, high baseline expression of ARG1 significantly predicted worse scores on the MRS. We conducted a pilot study in n=3 female rats undergoing middle cerebral artery occlusion. As expected, there is an increase in ARG1 expression in the peripheral blood at 2 hours after ischemia that decreases within 24 hours, similar to that which we see in our human IS population. Discussion: This preliminary data supports the hypothesis that mechanisms of ARG1 induced lymphocytopenia are altered by the presence of pre-existing cardiovascular disease and may play a role in post-stroke immune suppression. Future studies are required to determine the functional relationships between ARG1 and immune dysfunction and determine appropriate clinical interventions.

Immune correlates of varlilumab (CDX-1127) treated cancer patients are consistent with CD27 costimulatory activity

Varlilumab is a human IgG1 agonist anti-CD27 antibody designed to activate T cells through CD27 costimulation. Preclinical studies have shown that Varlilumab efficiently activates human T cells when combined with T cell receptor stimulation, enhances antigen specific CD8 T cell responses in human CD27 transgenic mice when combined with vaccination, and mediates anti-tumor activity in these mice when challenged with syngeneic tumors. A multi-dose, dose-escalation/expansion trial of Varlilumab in patients with advanced solid tumors (n = 56) or lymphoma (n = 24) has demonstrated a good safety profile with no MTD reached through the 10 mg/kg dose level. Evidence of clinical activity includes a complete response in a treatment-refractory Stage IV Hodgkin’s patient (remains in remission at 12.9 months); a renal cell carcinoma (RCC) patient with a partial response (ongoing at 3.8 months); an additional RCC patient with extended stable disease (ongoing at 22.4+ months); and 12 additional patients with stable disease (range: 2.7+ to 14 months). We performed extensive correlative immune monitoring from serum and peripheral blood cells, particularly in melanoma and RCC patients. Differential gene expression analysis was assessed preand post-treatment by hybridizing RNA isolated from whole blood PAXgene RNA tubes onto Illumina HT-12 microarrays. Transcriptomic analysis showed significant impact of Varlilumab on immunological pathways, including T cell receptor signaling that was observed at early time points (day 2 and day 8 post treatment). Interestingly, these changes were most significant at low dose levels (0.1 and 0.3 mg/kg), however, a larger sample size would be required to confirm these results. Varlilumab administration was associated with enhanced T cell activity by evaluation of activation markers and functional analysis using IFN-g Elispot. In particular, evaluation of response to peptides derived from melanoma antigens in selected patients showed evidence that Varlilumab promoted CD8 T cell function against melanoma antigens. Specifically, expanded responses were detected to epitopes from gp100 and MART-1, while de novo responses to epitopes MAGE-A1, NY-ESO-1 and gp100 were evident. These responses were confirmed by MHC-multimer staining. These data, and our previous analyses demonstrating a transient increase in pro-inflammatory cytokines (IP-10, IL-6, MCP-1), up-regulation of HLA-DR expression on T cells, increase in NK cell numbers, and decrease in regulatory T cells in response to Varlilumab treatment, show a pattern consistent with CD27 costimulation. These results, together with our preclinical data, provide the rationale for initiating clinical studies of Varlilumab in combination with therapies such as vaccines, checkpoint inhibitors and targeted therapies.

A genomic profile of the immune response to stroke with implications for stroke recovery.

The objectives of this study were to determine the change in gene expression between two time points following stroke and to identify biomarkers of stroke recovery through gene expression profiling and pathway analysis. Peripheral blood was collected from 34 ischemic stroke patients (confirmed by magnetic resonance imaging) ≥18 years of age, within 24 hr of symptom onset and 24-48 hr later, and from healthy controls. The Modified Rankin Scale (MRS) was used to determine 30-day recovery. Total RNA was extracted from whole blood in Paxgene RNA tubes, amplified, and hybridized to Illumina HumanRef-8v2 bead chips. Gene expression was compared in a univariate manner between stroke patients at both time points and good versus bad outcome using t-test in GeneSpring. Inflation of Type 1 error was corrected by false discovery rate (FDR), and Ingenuity Systems Pathway analysis (IPA) was performed. A secondary validation cohort was recruited from a local hospital. Three genes were significantly downregulated over time (LY96, IL8, and SDPR; FDR corrected p < .05). This finding was confirmed in a validation cohort of stroke patients (n = 8). IPA revealed cytotoxic T-lymphocyte antigen 4 (CTLA4) signaling was the most significant pathway present in the peripheral whole blood of stroke patients 24-48 hr after onset. When controlling for age and National Institutes of Health Stroke Scale score, high baseline expression of TLR2 and TLR4 significantly predicted worse scores on the MRS. CTLA4 signaling is a novel pathway for the study of stroke-induced immune suppression. Markers of immune dysfunction early after stroke may prove useful for identifying patients with increased risk of poor recovery.

Donor verification using Short Tandem Repeat (STR) analysis directly from blood collected in PAXgene RNA tubes.

Biorepository processing includes nucleic acid extractions in batch mode from a large number of blood samples from many different donors. Handling such a large number of biospecimens presents the challenge of ensuring that samples are not switched or mislabeled during processing. One approach for confirming donor identity from DNA samples is the use of multiplexed fluorescent PCR for detecting Short Tandem Repeat (STR) allelic-size polymorphisms for a set of common autosomal loci. While donor identity of DNA extracted directly from blood collected in standard tubes containing anticoagulants can be easily verified by generating STR profiles, RNA from blood collected in PAXgene Blood RNA tubes (PAXgene RNA tubes) is depleted of DNA and is not amenable to STR fingerprinting for donor identity verification. We investigated the feasibility of isolating DNA directly from blood collected in PAXgene RNA tubes for use as template for STR DNA fingerprinting for blood donor identity verification. We determined that DNA extraction can be performed manually with the QIAamp DNA Blood Minikit or on the QIAxtractor instrument with minimal pre-processing protocol additions, and that DNA isolated from blood collected in PAXgene RNA tubes is of sufficient quantity and quality for successful STR fingerprint analysis. Adaptation of quality assurance methods such as the PAXgene RNA tube DNA extraction/STR fingerprinting assay described here is a good practice that ensures that biobanking collections provide scientists with high quality, donor-verified biomaterial.

MicroRNA biomarkers in whole blood for detection of pancreatic cancer.

4052 Background: There is a great need for biomarkers for early diagnosis of patients with pancreatic cancer (PC) to improve their poor prognosis. The aims were (1) to describe differences in miRNA expression in whole blood between patients with PC, healthy subjects (HS) and patients with chronic pancreatitis (CP); and (2) to identify panels of miRNAs for early diagnosis of PC. Methods: Case-control study. 409 patients with PC, 33 patients with other periampullary cancers (PAC) and 25 patients with CP were included prospectively in the Danish BIOPAC biomarker study. 312 blood donors were included as HS. MiRNA expressions in pretreatment 2.5 ml whole blood samples collected in PAXgene RNA tubes were investigated in three independent cohorts: “Discovery Study” (PC n=143, CP n=18, HS n=69); “Training Study” (PC n=180, HS n=199); and “Validation Study” (PC n=86, PAC n=33, CP n=7, HS n=44). TaqMan Human MicroRNA assay was used to screen 754 miRNAs in the “Discovery Study”. Fluidigm BioMark PCR System was used in the “Training Study” (38 miRNAs) and “Validation Study” (13 miRNAs). Results: The “Discovery Study” demonstrated that 38 miRNAs (out of a total of 754 miRNAs) in whole blood were significantly deregulated between patients with PC and controls. These miRNAs were tested in the “Training Study” and two diagnostic indexes were constructed including 4 miRNAs in bPANmiRC index I (= miR-150 + miR-636 – miR-145 – miR-223) or 10 miRNAs in bPANmiRC index II (= 6.9275 – 0.2134xmiR-122 – 0.3560xmiR-34a – 0.8577xmiR-145 + 1.0043xmiR-636 – 0.6725xmiR-223 + 0.7018xmiR-26b – 0.3233xmiR-885.5p + 1.1304xmiR-150 – 0.2204xmiR-126* - 0.1730xmiR-505) (AUC 0.86/0.93, sensitivity 85%/85% and specificity 64%/85%). Conclusions: We identified two diagnostic indexes, using 4 or 10 miRNAs in peripheral whole blood, with a potential clinical value in the evaluation of patients with uncharacteristic symptoms to identify PC at an early stage.

Abstract TP232: A Biomarker Algorithm that Represents Time from Stroke Symptom Onset

Background and Purpose: Time of onset is critical when treating ischemic stroke (IS). The purpose of this project was to investigate the use of our 9 gene profile to develop a biomarker algorithm that represents time from stroke onset for use in the clinical setting to improve utilization of tissue plasminogen activator (tPA) and streamline appropriate secondary prevention. Methods: Peripheral blood samples were collected from n=34 IS patients’ ≥18 years of age within 24 hours from symptom onset and 24-48 hours later. Total RNA was extracted from whole blood in Paxgene RNA tubes, amplified, and hybridized to Illumina HumanRef-8v2 bead chips. Gene expression was compared in a univariate manner between patients at both time points using t -test in GeneSpring. Inflation of type one error was corrected by Bonferroni. A linear regression was used to model the change in gene expression as a linear function of time when controlling for age. Results: The mean age of the sample was 71.9± (14.6 sd ) years. Mean time from symptom onset to acute blood draw was 9:29± (6:2 sd ) hours (range 2:35-23:02); to follow up blood draw was 29:24± (7.1 sd ) hours (range 18:45-43:30); and time between acute and follow up blood draw was 19:55± (3.3 sd ) hours (range 13:30-27:32). CA4 and ARG1 expression significantly decreased &gt;1.5 fold, and LY96 expression by &gt;2-fold between baseline and follow up. This decrease in expression was associated with an increase from time of stroke onset and remained significant for only LY96 expression when controlling for age. ARG1 and CA4 expression were significantly lower in older patients. Conclusions: Our profile provides evidence that the expression of LY96 , CA4 , and ARG1 in the peripheral blood may serve as a surrogate for determining the time of stroke onset. In clinical practice, an algorithm based on this biomarker profile and other clinical covariates could be used when time of onset is unknown. To increase the accuracy of our biomarker algorithm, it will be important to determine the effects of age, stroke severity, and other clinical covariates on the expression of these genes over time.

Abstract TP113: Arginase-1 and S100a12 are Novel Candidates for the Study of Post Stroke Immune Suppression

Objective: Stroke-induced immune suppression is common and clinically observed by the neutrophil- lymphocyte count ratio (NLR). The NLR can predict stroke recovery. The purpose of this study was to identify the relationships between the NLR and our previously established genomic profile for the characterization of immune pathways affecting stroke recovery. Methods: Patients with imaging confirmed ischemic stroke (IS) or transient ischemic attack (TIA) were recruited from West Virginia University Hospital (WVUH) in Morgantown, WV. White blood cell (WBC) differentials were performed on admission then analyzed as a ratio between the neutrophil and lymphocyte counts. Recovery was determined at 90 days for stroke patients only via the Modified Rankin Scale score (MRS). Peripheral blood samples were collected within 24 hours from symptom onset in Paxgene RNA tubes. Total RNA was extracted, amplified, and the expression of ARG1, CA4, CCR7, CSPG2, IQGAP, LY96, MMP9, ORM1, and S100a12 was quantified via Taqman PCR. The gene expression profile was compared to the NLR and MRS via Spearman rho correlation coefficient. Results: A total of 9 IS and 4 TIA patients were included in this study. The mean age of the IS patients was 72 years and TIA patients 70 years. Expression of ARG1 was significantly correlated with elevated NLR at baseline (r=.93, p=.001) for both IS and TIA and poor 90 day MRS (r=.86, p=.01) following IS. Elevated expression of S100A12 was correlated with higher neutrophil count (r=.67, p=.07) and total WBC (r= .78, p=.02) as well as with ARG1 expression (r=0.72; p=0.008) in both patient cohorts. Conclusion: We have identified a significant correlation between the expression of ARG1, S100a12 and the NLR for IS and TIA patients, as well as with ARG1 and outcome following IS. Elevated ARG1 decreases T-cell proliferation resulting in immune suppression. S100A12 modulates neutrophil activity. Our data suggests that ARG1 and S100A12 are interesting candidates for the study of the immune response following cerebral ischemia. Further studies are required to confirm this relationship when controlling for age, disease severity and comorbid conditions.

Abstract TP96: A Genomic Profile of Immune Mediated Mechanisms Associated with Stroke Recovery.

Objective: We recently identified a profile of genomic biomarkers related to the inflammatory and immune signaling mechanisms in human ischemic stroke. The objective of this study was to identify novel biomarkers for ischemic stroke recovery through gene expression profiling and pathway analysis. Methods: Peripheral whole blood samples were collected from n=34 MRI diagnosed ischemic stroke patients’ ≥18 years of age at two time points: within 24 hours from stroke symptom onset and 24-48 hours later. Modified rankin scale (MRS) was used to determine 30 day stroke outcome. Total RNA was extracted from whole blood stabilized in Paxgene RNA tubes, amplified, and hybridized to Illumina HumanRef-8v2 bead chips. Gene expression was compared in a univariate manner between stroke patients at both time points and good versus bad outcome using t-test in GeneSpring. Inflation of type one error was corrected by Bonferroni and Ingenuity Systems Pathway analysis (IPA) was performed. A validation cohort of ischemic stroke patients was recruited from WVU Ruby Memorial Hospital (WVUH). Results: Three genes were significantly downregulated over time (LY96, IL8, and SDPR) ( Bonferroni corrected p&lt;0.05) Pathway analysis revealed cytotoxic t-lymphocyte antigen 4 (CTLA4) and dopamine signaling as highly significant canonical pathways present in the peripheral whole blood of ischemic stroke patients 24-48 post onset of symptoms. When controlling for age and hypertension, high baseline expression of ARG1, MMP9, TLR2 and TLR4 significantly predicted worse scores on the MRS. Validation in a separate cohort of subjects at WVUH confirms these findings. Conclusions: In this study we have demonstrated that ischemic injury to the brain produces an immune response that can be observed in the peripheral blood following stroke. Markers of immune dysfunction in the early post-stroke phase, such as arginase, TLR signaling and T-cell activity may prove useful for identifying patients with increased risk of post-stroke immune suppression and poor recovery.

Gene expression profiling with principal component analysis depicts the biological continuum from essential thrombocythemia over polycythemia vera to myelofibrosis

The recent discovery of the Janus activating kinase 2 V617F mutation in most patients with polycythemia vera (PV) and half of those with essential thrombocythemia (ET) and primary myelofibrosis (PMF) has favored the hypothesis of a biological continuum from ET over PV to PMF. We performed gene expression profiling of whole blood from control subjects (n = 21) and patients with ET (n = 19), PV (n = 41), and PMF (n = 9) using DNA microarrays. Applying an unsupervised method, principal component analysis, to search for patterns in the data, we demonstrated a separation of the four groups with biological relevant overlaps between the different entities. Moreover, the analysis separates Janus activating kinase 2-negative ET patients from Janus activating kinase 2-positive ET patients. Functional annotation analysis demonstrates that clusters of gene ontology terms related to inflammation, immune system, apoptosis, RNA metabolism, and secretory system were the most significantly deregulated terms in the three different disease groups. Our results yield further support for the hypothesis of a biological continuum originating from ET over PV to PMF. Functional analysis suggests an important implication of these gene ontology clusters in the pathogenesis of these neoplasms and in disease evolution from ET over PV to PMF.

Identification of Patients At High Risk for Recurrent Venous Thromboembolism by Whole Blood Gene Expression Analysis

Abstract 2305 Background.Recurrent venous thromboembolism (VTE) occurs in ∼30% of patients with spontaneous VTE after completion of a standard course of anticoagulant therapy. D-dimer levels and selected clinical parameters have been used to identify patients at low risk for recurrent VTE, who may safely discontinue antithrombotic therapy. We have used gene expression profiles to distinguish patients with a single VTE from patients with recurrent VTE. The purpose of this study was to extend this initial report and identify unique gene expression patterns from whole blood that correlate with different risk profiles for VTE recurrence. Methods.Patients with ≥1 prior VTE, with the first event occurring at age 18 years or older and >3 months from the most recent event were recruited for this study. Patients were allocated into 4 groups: (1) ‘low-risk' patients had sustained ≥1 provoked VTE; (2) ‘moderate-risk' patients had sustained 1 unprovoked VTE (with or without provoked VTE); (3) ‘high-risk' patients had sustained ≥2 unprovoked VTE and had no evidence for antiphospholipid antibodies; and (4) antiphospholipid syndrome (APS) patients met established consensus criteria for APS. A similar number of individuals with no prior history of VTE were enrolled as a control population. Citrated plasma, serum and PAXgene RNA tubes were collected, processed and stored at −80°C until shipped to the CDC for analysis. Antiphospholipid testing was performed on all participants to confirm correct group distribution. Total RNA was isolated from whole blood drawn into PAXgene tubes. Following sample labeling and normalization, cRNA samples were hybridized to Illumina HT-12 Beadchips to assay whole genome gene expression with over 47,000 probes against human transcripts. Two hundred and twenty six unique samples passed initial quality control measures. Quality assessment of raw data was done using GenomeStudio. The raw data files were converted to a text file using the IlluminaExpression FileCreator in GenePattern and then log transformed, normalized and median-centered using Cluster. Both unsupervised (hierarchical clustering using Cluster) and supervised analyses (SAM) were used to identify genes that were differentially expressed between the groups. GATHER was used to help understand the biological processes and gene ontology of the gene lists generated by Cluster and SAM. Results.A total of 226 participants were enrolled into the study. Characteristics of the patient groups are summarized in the Table. Demographically, the groups were similar except that patients in the high-risk group tended to be older and were more likely male. The number of events per patient, and the proportion on anticoagulant therapy, increased with the risk group.GroupAge, yrSex, F (%)Race, W (%)Time since last VTE, yrPE, N (%)VTE/Patient N (n; %)AC therapy?Low-risk (n=44)49 (25–90)25 (57%)37 (84%)4 (1–15)15 (34%)1 (37; 84%); 2 (7; 16%)31 (70%)Moderate-risk (n=45)53 (22–86)23 (51%)37 (82%)3 (1–9)34 (76%)1 (28; 62%); 2 (15; 33%); 3 (2; 4%)41 (91%)High-risk (n=46)58 (28–82)15 (33%)37 (80%)6 (1–22)25 (54%)2 (29; 63%); 3 (9; 20%) ≥4 (7; 15%)46 (100%)APS (n=47)48 (20–83)24 (51%)43 (91%)7 (1–26)25 (53%)1 (22; 47%); 2 (19; 40%); ≥3 (3; 6%)44 (94%)Normals (n=44)48 (21–75)26 (59%)39 (89%)–0–2 (5%)Antiphospholipid antibodies were detected in several patients in each of the 3 non-APS VTE patient groups, but in most cases this was a single test positive; antiphospholipid antibodies were present in the majority of patients with APS, typically with more than one test positive (37 of 45 with complete testing, 82%). Preliminary analysis of the gene expression profiles using an unsupervised clustering by gene on the high-risk and low-risk groups identified multiple genes that distinguished the two groups, including 18 immune response genes identified by GATHER. These two patient groups were also distinguished by SAM analysis, and multiple genes in the MAPK signaling pathway that separated the two groups were identified by the KEGG pathways in GATHER. Additional analyses are being performed on all of the groups. Conclusions.Whole blood gene expression profiling can be used to develop profiles that distinguish patients with VTE who differ based on their risk of recurrent events. Individual genes identified in these profiles may provide biological insights into the molecular basis for recurrent VTE. Disclosures:Heit:Daiichi Sankyo: Honoraria; Ortho-McNeil Janssen: Honoraria; Covidien: Honoraria. Manco-Johnson:Octapharma AG: Consultancy; Bayer: Research Funding.

Decreased mRNA expression of CCL5 [RANTES] in Alzheimer's disease blood samples

A recent study reported that an 18-analyte multiplexed plasma panel of signaling proteins differentiated Alzheimer's disease (AD) from controls. This study measured mRNA expression for nine of these promising bio-markers in 23 AD patients and 23 age- and sex-matched controls. Total RNA was isolated from PaxGene RNA tubes. Relative mRNA expression levels of CCL5 [RANTES], CSF1, ICAM1, IGFBP6, IL1A, IL3, IL8, PDGFB and TNF were determined by Q-RT-PCR, with GAPDH as housekeeping gene. A panel of five markers (CCL5, CSF1, ICAM1, IL8, TNF) with detectable expression levels in all individuals differed between AD patients and controls (p interaction <0.10). Especially, the relative expression level of CCL5 was lower in AD patients than in controls (p<0.005). Across groups, levels of both CCL5 and TNF were correlated to CSF levels of τ (r=0.39, r=0.32), pτ-181 (r=0.38, r=0.33), and MMSE (r=-0.31, r=-0.33, all p<0.05). The measured panel, and especially CCL5, could aid in the differentiation of AD from controls.

Genomic biomarkers and cellular pathways of ischemic stroke by RNA gene expression profiling

The objective of this study was to provide insight into the molecular mechanisms of acute ischemic cerebrovascular syndrome (AICS) through gene expression profiling and pathway analysis. Peripheral whole blood samples were collected from 39 MRI-diagnosed patients with AICS and 25 nonstroke control subjects ≥ 18 years of age. Total RNA was extracted from whole blood stabilized in Paxgene RNA tubes, amplified, and hybridized to Illumina HumanRef-8v2 bead chips. Gene expression was compared in a univariate manner between stroke patients and control subjects using t test in GeneSpring. The significant genes were tested in a logistic regression model controlling for age, hypertension, and dyslipidemia. Inflation of type 1 error was corrected by Bonferroni and Ingenuity Systems Pathway analysis was performed. Validation was performed by QRT-PCR using Taqman gene expression assays. A 9-gene profile was identified in the whole blood of ischemic stroke patients using gene expression profiling. Five of these 9 genes were identified in a previously published expression profiling study of stroke and are therefore likely biomarkers of stroke. Pathway analysis revealed toll-like receptor signaling as a highly significant canonical pathway present in the peripheral whole blood of patients with AICS. Our study highlights the relevance of the innate immune system through toll-like receptor signaling as a mediator of response to ischemic stroke and supports the claim that gene expression profiling can be used to identify biomarkers of ischemic stroke. Further studies are needed to validate and refine these biomarkers for their diagnostic potential.

RNA Methods: RNA Extraction from Plasma

RNA Methods: RNA Extraction from Plasma