Fold Enrichment Research Articles

Abstract 4244: Detection and characterization of extrachromosomal circular DNA in human plasma

Abstract Background: eccDNA (extrachromosomal circular DNA) has been reported in most eukaryotes, including in human cells. However, little is known about the eccDNA profiles in human circulating system such as blood. Method: We extracted plasma cell free DNA (cfDNA) from three patients with advanced stage cancers. For each patient, we tested 10ng, 1ng and 0.1ng cfDNA, each with a technical replicate. The cfDNA samples were subjected to ATP-dependent DNase digestion and whole genome amplification (WGA). An additional 2ng cfDNA from each patient skipped the DNase digestion and went directly to WGA step. We sequenced the amplified DNAs using the Illumina 100bp PE read protocol. We analyzed sequencing data using a custom split reads-based strategy to retrieve circular DNA molecules. We further analyzed eccDNAs for their biofeatures using functional annotation tools. Results: We received sequence reads at ~14.8 million (11.4~22.4) for each sample. When read depth≥ 3 as a cutoff, we detected a total number of 2542, 1578, and 57 unique eccDNAs in three patients while negative controls showed <=5 eccDNAs. For non-DNase digested cfDNAs, we detected only 3 eccDNAs. By comparing read numbers mapped to mitochondria and human genome, we found that mitochondrial sequence reads increased significantly from 0.34-1.1% before digestion to 24.1-50.1% after digestion. Numbers of detected eccDNAs were similar between technical replicates and proportional to initial input cfDNA. However, eccDNAs detected also showed few overlaps between technical replicates. Size distribution ranged from 31bp to 19740bp (median 1801-1962bp). To test if eccDNAs were generated in specific genomic regions and if eccDNA-involved genes were enriched in specific pathways, we performed enrichment analysis and observed higher GC content in smaller eccDNAs (<500 bp, 48%) than larger ones (>500bp, 41%) (p<0.001). When compared to random distribution, eccDNAs were more likely located at CpG islands (1.36-1.53 fold enrichment). The eccDNAs were also enriched in exons, 5’UTR, 3’UTR and DNase hypersensitive sites (enrichment folds from 1.39 to 2.3). Using DAVID annotation tool, we observed significant enrichment of the eccDNA-related genes in several categories including the Coiled coil, phosphoprotein, fibronectin, suggesting a potential involvement in gene expression regulation and cell adhesion events. Using GREAT (Genomic Regions Enrichment of Annotations Tool), we found that the eccDNAs were involved in genes associated with invasive cancer cell gene expression regulation (p<0.001). Conclusion: Human plasma contains an abundant number of eccDNAs. Distribution of these eccDNAs is nonrandom event and is likely enriched in genomic regions with known functional consequences and genes that play a role in gene regulations. Further characterization of circulating eccDNAs in peripheral blood will facilitate understanding of their molecular mechanisms and potential clinical utilities. Citation Format: Jing Zhu, Meijun Du, Peng Zhang, Liang Wang. Detection and characterization of extrachromosomal circular DNA in human plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2017-4244

Identification of novel dysregulated key genes in Breast cancer through high throughput ChIP-Seq data analysis

Breast cancer is the most common cancer in women both in the developed and less developed countries, and it imposes a considerable threat to human health. Therefore, in order to develop effective targeted therapies against Breast cancer, a deep understanding of its underlying molecular mechanisms is required. The application of deep transcriptional sequencing has been found to be reported to provide an efficient genomic assay to delve into the insights of the diseases and may prove to be useful in the study of Breast cancer. In this study, ChIP-Seq data for normal samples and Breast cancer were compared, and differential peaks identified, based upon fold enrichment (with P-values obtained via t-tests). The Protein–protein interaction (PPI) network analysis was carried out, following which the highly connected genes were screened and studied, and the most promising ones were selected. Biological pathway involved in the process were then identified. Our findings regarding potential Breast cancer-related genes enhances the understanding of the disease and provides prognostic information in addition to standard tumor prognostic factors for future research.

Analysis of T-cell repertoires in early-stage breast carcinomas to evaluate tumor immunogenicity.

11554 Background: Breast carcinomas (BC) are often considered to be weakly immunogenic and thus poorly sensitive to immunotherapy. Methods: We analyzed the repertoire of tumor-infiltrating T cells (TILs) in 41 early BC by sequencing their T cell receptor β genes (TCRβ). Libraries were built using a digital sequencing approach, barcoding each sequenced molecule to improve accuracy and quantification. T cell repertoires were also obtained from paired blood samples allowing identification of T cell clones enriched in the tumors as compared to blood. For 5 patients, CD8+ TILs were cloned ex-vivo from a tumor sample and screened for recognition of autologous predicted neoepitopes. Results: T cell infiltration differed from one tumor to another. Its amount varied of more than 30 fold and its diversity ranged from < 100 to > 5000 different clonotypes. In 34% of the tumors, there was an important T cell infiltration and we detected several clonotypes with a ≥500 fold enrichment as compared to blood. In 22% of the tumors, an important T cell infiltration was observed but without significantly enriched clonotypes. In 43% of the tumors the T cell infiltration was very limited. For 5 tumors with a high T cell infiltration, we screened ex-vivo isolated CD8+ T cell clones for recognition of predicted neoepitopes. In 4 of these tumors, with no enriched clonotypes, no recognition was observed. In 1 of these tumors, with enriched clonotypes, 6 different CD8+ T cell clones recognized 4 predicted neoepitopes. Three of these clones were > 100 fold more frequent in the tumor as compared to blood. Conclusions: About 30% of early BC were infiltrated by T cell clonotypes significantly enriched relative to blood. In one of these tumors some of the most enriched clonotypes recognized neoepitopes, demonstrating that some primary BC are spontaneously immunogenic. About 20% of the tumors had an important T cell infiltration without enriched clonotypes. None of the TIL clones isolated from 4 such tumors recognized predicted neoepitopes. Our results suggest that the detection of intratumorally enriched T cell clonotypes could identify immunogenic tumors, which may be sensitive to treatment with immunostimulatory antibodies.

An incoherent feed-forward loop mediates robustness and tunability in a plant immune network.

Immune signaling networks must be tunable to alleviate fitness costs associated with immunity and, at the same time, robust against pathogen interferences. How these properties mechanistically emerge in plant immune signaling networks is poorly understood. Here, we discovered a molecular mechanism by which the model plant species Arabidopsis thaliana achieves robust and tunable immunity triggered by the microbe-associated molecular pattern, flg22. Salicylic acid (SA) is a major plant immune signal molecule. Another signal molecule jasmonate (JA) induced expression of a gene essential for SA accumulation, EDS5 Paradoxically, JA inhibited expression of PAD4, a positive regulator of EDS5 expression. This incoherent type-4 feed-forward loop (I4-FFL) enabled JA to mitigate SA accumulation in the intact network but to support it under perturbation of PAD4, thereby minimizing the negative impact of SA on fitness as well as conferring robust SA-mediated immunity. We also present evidence for evolutionary conservation of these gene regulations in the family Brassicaceae Our results highlight an I4-FFL that simultaneously provides the immune network with robustness and tunability in A.thaliana and possibly in its relatives.

M58 - Cross-Tissue EQTL Enrichment of Associations In Schizophrenia

Background In the era of Big Data, genome-wide association studies (GWASs) have been the main instrument to better our understanding of the genetics behind human traits. The schizophrenia GWAS by the psychiatric genomics consortium (PGC) identified many loci plausibly involved in the etiology of the disorder [Ripke et al., 2014]. However, the picture appears to be far from complete [Lichtenstein, 2009; Polderman, 2015; Gejman, 2010; Lee, 2012]. Judging by the number of non-coding variants identified by GWASs of schizophrenia and other complex traits, many causal variants are likely to affect the regulation of the genes involved rather than severely disrupt their function. Expression quantitative trait loci (eQTLs) are genomic loci that contribute to variation in levels of mRNA. It is known [Bacanu, 2014] that many eQTLs are associated with schizophrenia, as well as other phenotypes [Nicolae, 2010]. Methods Given known pleiotropy between schizophrenia and other traits, we looked for association enrichment of eQTLs in tissues not obviously relevant to psychiatric disorders (blood, adipose, LCL, and skin [Buil, 2015]). In order to consolidate any evidence of tissue-specificity, we further looked for differential enrichment in different ENCODE functional annotation categories. We used summary p-value statistics from schizophrenia as well as body mass index, Crohn's disease, height, systolic blood pressure and type II diabetes GWASs. We matched the eQTLs to SNP sets with similar minor allele frequencies (MAF) and distances from their respective transcription start sites (TSS), and no evidence of association with expression levels of any genes. We further subdivided eQTLs into proximal and distal eQTLs, according to their position relative to the TSS or to their affiliation to promoters, and we annotated them according to their ENCODE designation. We visualized the enrichment in fold enrichment plots, screened it using Fisher tests and assessed its extent by regressing the squared association z-score against SNP affiliations and LD-score. Results Significant enrichment was seen in adipose (beta=0.24, p=2.2E-06), LCL (beta=0.27, p=4.0E-08) and skin (beta=0.23, p=2.7E-06) tissues. The enrichment was as widespread as that observed in another highly polygenic trait such as height, and more widespread than in traits such as body mass index (adipose and skin) or blood pressure (adipose). The schizophrenia association extent does not appear to depend on the eQTLs' distance from the TSS nor from their explicit affiliation to specific functional elements, as defined by ENCODE, suggesting that risk alleles for schizophrenia could frequently act in many different tissue contexts. Discussion Our results are in line with the notion that schizophrenia is a disorder involving many tissues and known pleiotropy with cardiovascular and immune diseases back our findings. The statistical power of the underlying GWAS does play a role in the detection of association enrichment of the sort we sought. Polygenicity, however, is also a major factor, as exemplified by the results for height, body mass index and blood pressure. In summary, we find that looking for functional correlates of schizophrenia risk loci in non-obvious tissues is productive, and this could be because of either pleiotropy or increased effectiveness of variants that work in many different environmental contexts. This suggests the utility of large, single tissue eQTL experiments in the study of schizophrenia in addition to smaller, multiple tissue approaches.

The PARP Inhibitor Olaparib Antagonizes Leukemic Growth Induced By TET1 Overexpression in AML1-ETO Positive Acute Myeloid Leukemia

DNA methylation patterns are highly deregulated in human acute myeloid leukemia (AML) cases and stratify AML patient samples into different subgroup. AML1-ETO is the most commonly occurring fusion gene in AML and these AML cases exhibit an aberrant and distinct methylation pattern. So far, the underlying mechanisms for this are only poorly understood. The TET1 dioxygenase has recently emerged as an important epigenetic modifier: by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) TET1 plays an important role in active demethylation, thereby regulating a variety of biological processes. It was linked to tumorigenesis based on the observation that its expression is frequently deregulated in solid cancer. However, the role of TET1 in AML1-ETO+ (AE+)human AML cases is yet unexplored. Using quantitative real time (qRT)- PCR we now show that AE+ AML is characterized by high and aberrant expression of TET1: the gene was significantly higher expressed in the majority of AE+ patients (n=7, p<0.01) compared to other AML subtypes such as inv(16) (n=11), PML-RARα+ (n=31), cytogenetically normal (CN)-AML patients (n=33) and CD34+ normal BM cells (n=4). This observation was consistent with published cDNA microarray data on large patient cohorts (Haferlach et al., JCO 2010, p<0.008 t-test, p<0.01 Anova) and recently published transcriptome data (TCGA) of AML patients. In contrast to TET1, TET2 and TET3 did not show significant higher expression in AE+ patients compared to other AML subtypes. In line with patient data, TET1 was highest expressed in the AE+ AML cell line KASUMI-1 and SKNO-1 compared to other AML cell lines (p<0.05 and n=3). Compared to normal CD34+ and myeloid (CD33+, CD15+ and CD14+) cells (n=3), TET1 was 10-fold and 16-fold higher expressed in AE+ patient samples (n= 7). Aberrant expression of TET1 in AE+ leukemic cells was associated with hypomethylation of its promoter and enrichment for H3K4me3 euchromatic marks at its promoter as determined by LC/MS and ChIP-qPCR respectively. Knockdown (KD) of TET1 mRNA using two short hairpin RNAs (shRNAs) in AE+ AML cell lines impaired their cell growth and clonogenicity by over 50% in vitro (n=3 and p<0.01). shRNA mediated depletion of TET1 did not impact the cell growth and clonogenicity of the TET1 negative cell line RAJI, ruling out off target effects of the shRNAs (n=3). In mice, KD of Tet1 in leukemic bone marrow cells expressing the truncated leukemogenic AML1-ETO9a (AE9a) fusion, dramatically inhibited cell growth (>60% compared to scrambled, n=3, p<0.01), clonogenicity (>50-70% reduction in primary CFCs, p<0.01, n=3) and importantly delayed onset of leukemia in vivo (median survival 35 days for scr vs 80 days for shRNA mice, n=4/arm, p<0.03). Tet1-knock-out c-kit+ hematopoietic stem and progenitor cells (HSPCs) transduced with AE9a showed reduced primary colony formation and impaired serial replanting capacity in vitro compared to AE9a transduced Tet1-wild-type HSPCs (>50% and >70%, respectively; p<0.001, n=3). Global analysis of 5hmC and 5mC levels using hMeDIP/MeDIP-seq performed on TET1 depleted KASUMI-1 cells revealed lower global 5hmC levels and increase in 5mC as compared to cells transduced with scrambled control (n=2). 3324 promoter regions lost 5hmC and gained 5mC upon TET1 depletion (-5kTSS, Fold enrichment cut off <2-fold, q-value<1e5). Recent studies have shown that PARP activity induces TET1 expression by regulating its promoter epigenetically. We could show that aberrant TET1 expression could be antagonized by the PARP inhibitor olaparib in AE+ leukemic cell lines. Furthermore, olaparib treatment decreased 5hmC levels and reduced cell growth and clonogenicity of human AE+ cell lines and of the murine AE9a+ leukemic cell line in vitro (n=3, p<0.01). In conclusion, our data indicates that aberrant TET1 expression contributes to the growth of AE+ AML by maintaining the 5-hydroxymethylome and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic effect of TET in AML. DisclosuresMulaw:NuGEN: Honoraria. Buske:Celltrion, Inc.: Consultancy, Honoraria.

Abstract P219: Exercise Training Normalizes Vascular Changes in Aging Hypertension Involving microRNAs Profile and Target Genes

MiRNAs profile and target genes involved in hypertension-associated vascular changes were evaluated. In addition, we checked the therapeutic role of exercise training (ET) on these parameters. Spontaneously hypertensive rats (SHR) aged 6 months and their controls Wistar Kyoto (WKY) were divided into 4 groups: SHR, trained SHR (SHR-T), WKY and trained WKY (WKY-T). Swimming ET consisted of 60 min of duration, 1x/day/10 weeks, with 4% caudal body weight workload. SHR showed an increased in systolic blood pressure (207 ± 5.5 mmHg) compared to WKY (133 ± 3.9 mmHg) analyzed by tail-cuff system, with no changes in baseline heart rate. We observed a reduction in VO 2 peak (WKY: 62 ± 1.5; SHR: 53 ± 2.5 mL.kg -1 .min -1 ) accompanied by soleus muscle atrophy (fiber type I - WKY: 4039 ± 195; SHR: 2658 ± 53; type IIa - WKY: 2903 ± 182, SHR: 2050 ± 68; Intermediate - WKY: 2663 ± 136, SHR: 1967 ± 95 μm 2 ) in SHR. Vascular function of femoral artery was similar in SHR and WKY, however, wall-to-lumen ratio was increased in femoral artery (WKY: 0.17±0.01, SHR: 0.27±0.01 a.u.) and muscular arteriole (WKY: 0.54±0.02, SHR: 1.14±0.03 a.u.) accompanied by capillary rarefaction (WKY: 1.2 ± 0.05, SHR: 0.6 ± 0.03 capillary-to-fiber ratio) in soleus muscle from SHR vs. WKY. In contrast, ET promoted reduction in blood pressure and resting bradycardia in trained animals. ET corrected the VO 2 peak reduction, muscle wasting, microvascular remodeling in SHR-T toward control levels. ET downregulated 8 miRNAs (-96, -205, -182, -146b-5p, -140, -328a, -665, -1) and upregulated 3 miRNAs (-499, -208b and -99b) in SHR-T when compared to SHR. Bioinformatics study for functional analysis of the predicted targets genes for the 11 miRNAs restored by ET demonstrated enrichment of different signaling pathways including cell death (p value &lt;0.001; Fold enrichment 16.78) and vascular development (p value &lt; 0.001; Fold enrichment 10.62). Thus, targets involved in angiogenesis and vascular integrity by the VEGF/VEGFR2/AKT/eNOS/Bcl-2 pathway were impaired in hypertension and corrected by ET. The results support the hypothesis that the structural changes arising from the progression of hypertension may be regulated by a set of miRNAs and target genes; and ET participates in restoring the vascular remodeling.

Abstract 1928: Comprehensive identification of miR-122 targetome in the liver and HCC by HITS-CLIP analysis reveals conserved regulation of AMPK signaling in hepatocarcinogenesis

Abstract miR-122, a conserved liver-specific miRNA, is critical for maintaining liver homeostasis. Loss of miR-122 is associated with loss of hepatic phenotype, metastasis and poor prognosis. Recent in vivo work has established miR-122 as a bona fide tumor suppressor, with poorly differentiated, Afp-positive, high grade, spontaneous HCC observed in liver-specific (LKO) and germ-line (KO) miR-122 knockout mice. We hypothesized that the pathophysiology exhibited by KO mice would be mediated through de-repressed targets in KO livers and therefore, dysfunctional regulation of specific pathways involved in liver homeostasis and carcinogenesis. Although the role of miR-122 in the maintenance of liver homeostasis is well established, systematic and direct biochemical elucidation of the miR-122 target network remains incomplete. To this end, we performed Ago-dHITS-CLIP analysis in livers of 6 week-old wild type (WT) and KO mice to identify miRNA targetome in vivo, which revealed ∼1800 miR-122 binding sites, with over 30 fold enrichment for the miR-122 seed sequence sites in WT compared to KO livers. Surprisingly, a majority of miR-122 binding sites were observed on coding exons (40%) followed by 3’-UTRs, introns and 5’-UTRs. Motif analysis of miR-122 dependent binding sites lacking the seed sequence revealed a novel G-bulged motif in addition to the canonical miR-122 seed sequences. Moreover, the hepatic gene expression profile of these mice measured by RNA-seq showed up regulation of ∼2500 genes and down regulation of ∼1200 genes in KO livers compared to WT livers. A comparison of HITS-CLIP and RNA-seq data revealed that majority of miR-122 targets harboring canonical binding sites at their 3’UTRs as well as some coding regions, are significantly upregulated in miR-122 KO livers without notable alteration in the expression of or G-bulged targets at the RNA level. HITS-CLIP analysis of several human HCC and matching benign liver tissues also identified both canonical and G-bulged targets; where the majority of both are located in the coding regions followed by 3’UTRs. Association of miR-122 targets with Argonaute was precipitously reduced in HCCs expressing lower miR-122 levels than respective benign livers. Notably, though miR-122 is completely conserved and highly expressed in human and mouse livers, a large number of identified targets seem to be species-specific. Ingenuity Pathway Analysis of enriched targets revealed conserved pathway specific regulation in mouse and human livers. Pathways commonly associated with critical roles in liver carcinogenesis and liver metabolism, such as AMPK signaling and PI3/AKT pathways, were the major targets of miR-122-mediated regulation. This study suggests that miR-122 functionality is conserved across species. Citation Format: Juan Barajas. Comprehensive identification of miR-122 targetome in the liver and HCC by HITS-CLIP analysis reveals conserved regulation of AMPK signaling in hepatocarcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1928.

Clonal hematopoiesis as determined by the HUMARA assay is a marker for acquired mutations in epigenetic regulators in older women.

Recent large cohort studies revealed that healthy older individuals harbor somatic mutations that increase their risk for hematologic malignancy and all-cause cardiovascular deaths. The majority of these mutations are in chromatin and epigenetic regulatory genes (CERGs). CERGs play a key role in regulation of DNA methylation (DNMT3A and TET2) and histone function (ASXL1) and in clonal proliferation of hematopoietic stem cells. We hypothesize that older women manifesting clonal hematopoiesis, defined here as a functional phenomenon in which a hematopoietic stem cell has acquired a survival and proliferative advantage, harbor a higher frequency of somatic mutations in CERGs. The human androgen receptor gene (HUMARA) assay was used in our study to detect the presence of nonrandom X inactivation in women, a marker for clonal hematopoiesis. In our pilot study, we tested 127 blood samples from women ≥65years old without a history of invasive cancer or hematologic malignancies. Applying stringent qualitative criteria, we found that 26% displayed clonal hematopoiesis; 52.8% displayed polyclonal hematopoiesis; and 21.3% had indeterminate patterns (too close to call by qualitative assessment). Using Illumina MiSeq next-generation sequencing, we identified somatic mutations in CERGs in 15.2% of subjects displaying clonal hematopoiesis (three ASXL1 and two DNMT3A mutations with an average variant allele frequency of 15.7%, range: 6.3%-23.3%). In a more limited sequencing analysis, we evaluated the frequency of ASXL1 mutations by Sanger sequencing and found mutations in 9.7% of the clonal samples and 0% of the polyclonal samples. By comparing several recent studies (with some caveats as described), we determined the fold enrichment of detecting CERG mutations by using the HUMARA assay as a functional screen for clonal hematopoiesis. We conclude that a functional assay of clonal hematopoiesis is enriching for older women with somatic mutations in CERGs, particularly for ASXL1 and TET2 mutations and less so for DNMT3A mutations.

44. Novel Combination of megaTAL Nuclease-Driven Genome Engineering with a Drug Selection Cassette Increases Efficiency of HIV Gene Therapy

Human Immunodeficiency Virus (HIV) infection remains a substantial health problem worldwide. The C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV entry into CD4+ T cells and represent an alternative therapeutic target. Early clinical trials using CCR5-targeting zinc finger nucleases demonstrated transient control of HIV infection in the course of antiretroviral treatment interruption (Tebas, NEJM, 2014). Our current work improves on these advances by combining high level of CCR5 gene disruption with preferential selection of gene modified cells. The CCR5-targeting megaTAL combines a LAGLIDADG homing endonuclease scaffold with an eleven repeat transcription activator-like (TAL) effector array to achieve site specific DNA cleavage. This nuclease produces highly efficient CCR5 targeting in primary human CD4+ T cells in vitro (70-90% disruption). To test the protective effects of megaTAL treatment, primary human CD4+ T cells treated with CCR5-megaTAL were transplanted into NOD/SCID/γc-null (NSG) ‘humanized’ mice and challenged with HIV-1. We observed a 100-fold increase of megaTAL-treated cells compared to untreated controls during an active in vivo infection demonstrating the functionality of this approach. Based on the decline of CCR5 modified cells in the clinical trials to date, we hypothesized that we could improve maintenance of HIV resistant cells by expanding them either ex vivo or in vivo. We propose that coupling megaTAL nuclease treatment with drug selection will help us achieve therapeutically relevant levels of HIV-protected cells by enabling efficient selection only of CCR5-modified T-cells. The mutant human dihydrofolate reductase (mDHFR) chemoselection system has been used to render cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX). We tested our experimental approach by transducing cells with lentiviral vector encoding a mDHFR cassette followed by chemoselection in MTX at 0.02uM. This approach resulted in a six fold enrichment of gene modified primary CD4+ T cells ex vivo. Previously we have shown that combining megaTAL treatment with adeno-associated virus (AAV) transduction produces very high rates of homology-driven repair (HDR) in primary human T cells. Hence, we combined megaTAL/AAV treatment to integrate the mutant DHFR into the CCR5 locus, producing a population of MTX-resistant CD4+ cells that also lack CCR5. Primary human CD4+ T cells were transfected with CCR5-megaTAL mRNA and transduced with AAV6 containing a mutant DHFR donor template flanked by 0.6kb CCR5 homology arms. They demonstrated a greater than five-fold enrichment in MTX compared to untreated controls ex vivo. Next, we have transplanted NSG mice with 1×106 gene modified cells/mouse to assess the therapeutic potential of our approach. Mice that engraft effectively will be treated with daily injections of 0, 0.5 and 2 mg/kg of MTX to monitor preferential selection and enrichment of our target cell population. Subsequent studies will assess the long term control of viremia in these mice following HIV challenge. In conclusion, the CCR5-megaTAL nuclease platform produces very high levels of gene-modified CD4+ T-cells and protects these cells from subsequent HIV infection in vivo. Furthermore, combining targeted integration and chemical selection results in the specific selection of gene modified primary human T cells. To our knowledge we are the first group to report MTX-mediated chemoselection and expansion of CD4+ T cells following targeted integration at the CCR5 locus.

A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome.

BackgroundThe initiating nucleotide found at the 5’ end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. The currently available methods do not use targeted enrichment for the 5′end of primary transcripts, but rather attempt to deplete non-targeted RNA.ResultsWe developed a method, Cappable-seq, for directly enriching for the 5' end of primary transcripts and enabling determination of transcription start sites at single base resolution. This is achieved by enzymatically modifying the 5′ triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli, achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and identified TSS in a microbiome.ConclusionsCappable-seq allows for the first time the capture of the 5′ end of primary transcripts. This enables a unique robust TSS determination in bacteria and microbiomes. In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2539-z) contains supplementary material, which is available to authorized users.

Optimized procedure of extraction, purification and proteomic analysis of nuclear proteins from mouse brain

BackgroundThe cell nucleus is a highly dynamic subcellular compartment performing crucial processes for functioning and administration of the cell. Proteomic analysis of isolated nuclear fraction enables in-depth insight into these processes leading to better understanding of physiological and pathological state of the brain. There is no universal method for nuclear proteome investigation and each biological material needs individual optimization. An additional difficulty is the large amount of nucleic acids, which impairs isoelectrofocusing of proteins and shotgun mass spectrometry analysis of complex peptide samples. New methodWe performed the first comprehensive optimization of intact nuclei isolation from mouse brain in combination with nuclear protein purification prior to two-dimensional gel electrophoresis (2DE) and gel-free proteomic analysis. ResultsApplication of sonication, digestion with nuclease and protein precipitation with acetone allowed to obtain high quality 2DE gels. Shotgun mass spectrometry analysis of isolated proteins proved an enrichment in nuclear proteins. The 66.4% of 265 identified proteins had assigned nucleus localization in UniProt database. Gene Ontology analysis using DAVID platform revealed the highest fold enrichment in spliceosome (24.5), nuclear periphery (12.4) and nuclear matrix (11.3). Comparison with existing methodsThe proposed procedure is tailored to mouse brain tissue nuclear subproteome investigation. The quality of isolated nuclei, the effectiveness of the protein purification, efficiency of protein recovery after precipitation and overall method reproducibility was taken into detailed consideration. ConclusionsThe elaborated procedure could be further applied for in-depth proteomic analysis of molecular processes occurring in the mouse brain nucleus.

Effect of Lower Hematocrits on Buffy Coat Collection during Extracorporeal Photopheresis in Patients with Graft-Versus-Host Disease

Introduction: Extracorporeal photopheresis (ECP) is an effective therapy for patients with acute or chronic graft-versus-host disease (GVHD) who have significant skin involvement. During ECP, buffy coat is collected from the peripheral blood (PBL), mixed with 8-methoxypsoralen (8-MOP), and exposed to ultraviolet-A (UVA). Photo-activated 8-MOP crosslinks DNA in nucleated cells, which are infused back to the patient to induce an immune modulatory effect. The exact mechanism of ECP and the optimal cellular dose to treat are unknown. We previously demonstrated a linear relationship between lymphocyte counts in the PBL and the buffy coat, with the collection efficiency affected primarily by the type of ECP machine used (UVAR or CELLEX; Therakos, West Chester, PA). It is therefore feasible to track the cellular dose of ECP by measuring the lymphocyte count in the PBL prior to each procedure. Another outstanding question is whether ECP can be successfully performed at a decreased hematocrit, which is a common finding among patients with GVHD. Due to concerns over the collection efficiency and patient safety, our protocol recommends transfusion of red cells prior to ECP when a patient's hematocrit is in the range of 28-30%. However, there is no evidence from human subjects supporting the relevance of this specific threshold. In this study, we compared the collection efficiency during ECP in GVHD patients with hematocrits between 24% and 29% and those with hematocrits of 29% or higher.Methods: Patients with GVHD and hematocrits between 24% and 29% were prospectively recruited for this study which was approved by the institutional review board (n=26). The hematocrit of 24% approximated the red cell transfusion threshold for our transplant patient population. One ECP procedure was performed for each patient using the CELLEX with 1500 mL of whole blood processed. PBL was drawn within 24 hours prior to the procedure for a complete cell count with automated differentials. At the end of cell collection but prior to 8-MOP treatment, 1 mL of well-mixed buffy coat was drawn for the complete cell count. Spearman correlation coefficient was calculated for the counts of each nucleated cell type, including neutrophils, lymphocytes and monocytes, in the PBL versus the buffy coat. The collection efficiency, or fold enrichment, was evaluated using the ratio of lymphocyte count in the buffy coat to that in the PBL. The ratio was then compared among the current cohort (lower-hct-CELLEX group) and two historical control groups with hematocrits of 29% or higher who were treated with either UVAR (higher-hct-UVAR group n=6) or CELLEX (higher-hct-CELLEX group, n=16). Mann-Whitney test was performed with a double-sided p value of 0.05. Data visualization and statistical analysis were performed using Prism 6.Results: Consistent with our previous findings in patients with higher hematocrits, there was a significant correlation between lymphocyte counts in the PBL and the buffy coat in the lower-hematocrit-CELLEX group (r=0.66, p=0.0002) (Figure 1A). A significant correlation was also observed for monocytes (r=0.46, p=0.02) but not neutrophils (r=0.26, p=0.20). The distribution of lymphocyte counts was fit into a linear regression model with a slope of 4.22 (95% CI: 3.06-5.37, p < 0.0001). The fold enrichment for lymphocytes was higher in the lower-hct-CELLEX group than in the higher-hct-UVAR group (medians: 4.94 versus 1.66, p=0.008). The fold enrichment for lymphocytes was not significantly different between the lower-hct-CELLEX group and the higher-hct-CELLEX group (medians: 4.94 versus 5.22, p=0.27) (Figure 1B). According to previous protocols, approximately 1-2 units of red blood cells would have been transfused for each patient in the lower-hct-CELLEX group prior to each procedure. These transfusions were completely avoided without any adverse events observed throughout the study.Conclusions: The fold enrichment of lymphocytes during ECP using the CELLEX is comparable between GVHD patients with hematocrits between 24% and 29% and patients with hematocrits of 29% or higher. Unnecessary red blood cell transfusion may be avoided without significant increase in adverse events. The efficacy and safety of this practice warrant further investigation in a larger cohort. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Emergence of Sub-Clonal Drug Resistance Mutations during CML Therapy

▪Tyrosine kinase inhibitors (TKIÕs) vastly improve survival in chronic myeloid leukemia (CML). However resistance is a common occurrence, and is most frequently mediated by point mutations in the Abl kinase. There have been conflicting reports regarding the frequency of resistance mutations in the Abl kinase at the time of CML diagnosis, with some studies indicating that a large number of resistance mutations are pre-existing in many patients. However, a high burden of pre-existing resistance mutations is seemingly at odds with the ability of TKIÕs to initially control CML for months to years in the large majority of patients.Nearly all prior studies have used dozens of cycles of PCR amplification to enrich the Abl gene prior to mutation detection. PCR amplification introduces thousands of errors throughout the gene, which makes accurate identification of mutations challenging. Methods of mutation detection such as next-generation sequencing (NGS) are also error prone, with a background error rate near 1%. These high rates of background mutation render accurate detection of minority variants difficult or impossible.We have developed an alternate DNA mutation detection method, termed Duplex Sequencing (Schmitt MW et al, Nature Methods 2015;12:423-425), which improves on the accuracy of DNA sequencing by >100,000 fold. In Duplex Sequencing, the two strands of single DNA molecules are individually tagged and sequenced. True mutations are present at the same position in both strands and are complementary, whereas artifacts arising from amplification or sequencing errors are seen in only one strand and are not scored. We have additionally developed methods for >1,000,000 fold enrichment of the Abl gene from human cells. This enrichment protocol allows for unbiased detection of all possible mutations throughout the Abl gene, with an accuracy exceeding 99.9999%.We applied Duplex Sequencing to 18 patients with newly diagnosed chronic phase CML (CP-CML). We sequenced the active site exons of Abl from tens of thousands of individual CML genomes; no sub-clonal resistance mutations were seen in any patient. We demonstrate that prior reports indicating frequent resistance mutations at the time of CML diagnosis may be attributable to artifactual mutations introduced by reverse transcription and PCR amplification.We are now studying patients from the PACE trial (Cortes JE et al. N Engl J Med. 2013;369:1783-1796); these patients were heavily pre-treated, with the majority having received 3 or more prior TKIÕs. We identified 29 patients from the trial with a treatment-emergent mutation not detectable at baseline by conventional methods (NGS or Sanger), and have performed Duplex Sequencing on 9 of these patients to date. In contrast to treatment-naïve patients, we find that these heavily pre-treated patients commonly harbor multiple co-existing resistant sub-clones, most of which fall below the detection limit of conventional methods but are readily detected by Duplex Sequencing. In addition, in 44% of patients, mutations that emerged during ponatinib treatment were found as rare sub-clones at baseline (average detection limit of 1 in 11,412 cells), indicating that the mutant sub-clones pre-existed prior to ponatinib therapy. We are now sequencing the remainder of the cohort, and are increasing our detection limit through interrogation of a larger number of cells per patient.Our results indicate the feasibility of detecting sub-clonal resistance mutations at the time of initiation of next-line TKI therapy in order to guide treatment, and argue that high-sensitivity methods for mutation detection are required to optimally stratify patients to the most appropriate TKI. Moreover, our approach can detect sub-clonal mutations in any gene, and could be broadly applicable for identification of sub-clonal drug resistance mutations relevant to other targeted therapies. [Display omitted] DisclosuresPritchard:ARIAD: Employment, Equity Ownership. Hodgson:ARIAD Pharmaceuticals: Employment, Equity Ownership. Rivera:ARIAD: Employment, Equity Ownership.

The Novel Leukemia Stem Cell Marker GPR56 Discriminates Leukemic Subclones with Divergent Stem Cell Properties in Human Acute Myeloid Leukemia

Insights into the complex clonal architecture of acute myeloid leukemia (AML) unravelled by deep sequencing technologies have challenged the concept of AML as a hierarchically organised disease initiated and driven by rare self-renewing leukemic stem cells (LSCs). In contrast to normal human hematopoietic stem cells (HSCs), which are highly enriched in the CD34+ CD38- population, LSCs have also been found in the CD34- and the CD38+ fractions questioning the existence of a consistent LSC surface marker profile for AML. Besides, low LSC frequencies in primary samples, rapid onset of differentiation upon ex vivo culture, and genetic inter-specimen heterogeneity hamper the dissection of the molecular machinery that drives LSC self-renewal.We performed RNA-Sequencing of primary human AML samples and assessed LSC frequencies by limiting dilution analyses for 56 of these in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. By comparing gene expression profiles between high vs low LSC frequency leukemias, we identified the G-protein coupled receptor 56 (GPR56) has significantly more expressed in high LSC frequency leukemias. We validated the RNA-seq data with protein expression by FACS and found an excellent correlation. To determine whether GPR56 positive cells overlapped with the known LSC-associated phenotype CD34+ CD38-, we stained 45 AML samples with CD34, CD38, GPR56, and antibodies against other described LSC markers. Although CD34+ GPR56+ and CD34+ CD38- compartments identified the same population in some samples, we found in the majority of samples that GPR56 further subdivided the CD34+ CD38- compartment. Accordingly, not only the proportions of total GPR56+ and CD34+ GPR56+ cells were significantly higher in LSChigh versus LSClow samples, but also the proportion of GPR56+ cells within the CD34+ CD38- compartment was significantly different between the groups indicating that GPR56 might be of additional value to what is currently considered the best described LSC phenotype. The percentage of total CD34 positive cells did not correlate with LSC frequency clearly distinguishing GPR56 from CD34 or CD38, which are only suitable LSC markers when used in combination. We analysed other potential LSC markers (TIM3, CD96, CD44, CD123, CLL1 and CD47) in our RNA-Seq dataset and by FACS analysis in combination with CD34 as we did for GPR56 and none of them correlated with LSC frequency in our sample collection.To determine whether GPR56 discriminates engrafting LSCs from non-LSCs, we sorted GPR56+ and GPR56- cells within the CD34-positive and -negative compartments from selected specimens with known engraftment potential. We found that GPR56 identified the engrafting fraction in CD34positive AML samples, with a >50 fold enrichment in LSC in the CD34+GRP56+ fraction vs the CD34+GPR56- fraction within the same sample, demonstrating that GPR56 is a good LSC marker.Specimens with high molecular or cytogenetic risk such as chromosome 5 or 7 anomalies and EVI1- rearrangementexpressed high levels of both, GPR56 and CD34, while samples with coexistent FLT3 -ITD, DNMT3A, and NPM1 mutations displayed a unique CD34low GPR56high profile. Moreover, we found a divergent distribution of variant allele frequencies in GPR56+ versus GPR56- fractions identifying GPR56 as a discriminator of leukemic sub-clones with high and low NSG engrafting capacity. Analysis of engrafted cells re-sorted based on GPR56 after being harvested from mouse bone marrow revealed reduced complexity of the clonal composition. Most importantly, GPR56 positive cells differentiated to GPR56 negative cells in mice, which did not happen in the human niche, in which GPR56 positive and negative fractions represented two independently evolved subclones.In summary our work identifies GPR56 as a novel LSC marker in AML and also shows that GPR56 readily identifies a functionally distinct LSC-rich subclone in the majority of human AML patients and reveals hitherto unforeseen complexity in the interaction between human LSCs and the NSG mouse environment. DisclosuresNo relevant conflicts of interest to declare.

Identification of Erythroid Specific Enhancers By Analyzing Genome Wide Association of Transcriptional Co-Activators CBP and P300

Enhancers are cruicial for gene regulation and demonstrate tissue, cell and developmental stage specificity. Recent studies have shown that analyzing genome wide association of CBP and p300 predicts the tissue spcific transcriptional enhancers in various tissues. Our aim was to study the transcriptional enhancers in haematopoietic stem cells and erythroid cells to identify the role of these genomic regions in determining transcriptional regulation of haematopoietic stem cell maintenance and erythroid differentiation. We obtained CD34+ cells from normal healthy donors after magnetic activated cell sorting from peripheral blood mononuclear cells. The purified CD34+ cells were differentiated by ex vivo erythropoiesis using a serum-free two-phase liquid culture system. Cells were collected at 2 different time points, before (day 5 in culture) and after differentiation (day13 in culture). ChIP assays were carried out using antibodies against CBP and p300 and two independent libraries were created using Illumina TrueSeq library kit as per the manufacturer's instruction. The libraries were sequenced in Illumina HiSeq producing 50 bp single end reads. A control sample (without antibody) was run as input as a background control. Sequenced reads were mapped to the human genome (UCSC Genome Browser hg19). To identify ChIP-Seq peaks, Model-based Alignment of ChIP-Seq (MACS) program was used with a p value of <10e-5 and enrichment factor > 5. Localization of the binding sites relative to the annotated genes and co-localization of CBP/p300 were determined using the ChIP seek analyzer. Validation of co-occupancy at selected regions was performed by quantitative-PCR analysis.Genome wide analysis of CBP and p300 enrichment showed these co-factors are highly enriched in introns and intergenic regions than the gene promoters suggesting that these co-activators efficiently mark enhancers in the haematopoietic stem cells and erythroid cells. Co-localization of CBP/p300 with erythroid transcription factors (GATA1, KLF1, NFE2 and TAL1) was performed. The results showed that CBP/p300 are highly associated with erythroid transcription factors during differentiation indicating that transcriptional activator complexes consisting of these transcription factors and CBP/p300 in enhancer mediated transcriptional regulation in erythropoiesis. The sites of CBP/p300 occupancy were correlated with the transcriptome data and it was found that most of the top regulated genes were enriched with CBP and p300 within the intronic region in erythroid cells. We then explored whether CBP and p300 are enriched in the regions associated with DNA polymorphisms relevant to erythroid cell traits and observed that CBP/p300occupy at HBD (5'UTR), BCL11A (intron 2)and HBS1L-MYB intergenic region which contains polymorphisms linked with levels of fetal haemoglobin (HbF). We also found enrichment of these co-activators in previously mapped erythroid specific enhancers such as IKZF1, ANK1 and ABO gene loci.Gene ontology (GO) was performed using GREAT for regions associated with CBP and p300 (binomial fold enrichment >4) and the results indicated that the genes associated with CBP were involved in erythrocyte development whereas the genes associated with p300 were found to regulate erythroid differentiation. These erythroid specific genes delineated in our study also showed conservation in mouse and were found to be associated with erythroid cell phenotypes. We also found significant enrichment of CBP and p300 in erythroid cells compared to haematopoietic stem cells for several genes that have not been previously characterized for erythroid differentiation. Taken together, our findings elucidated the roles of co-activators CBP and p300 in erythroid differentiation and further we identified these factors are enriched in previously known and new erythroid specific enhancers in association with cell specific transcription factors. Functional evaluation of the newly idenfied regulatory elements bound by CBP and p300 in the erythroid cells will provide insights in to erythroid cell development and differentiation. DisclosuresNo relevant conflicts of interest to declare.

Controlled Lateral Positioning of Microparticles Inside Droplets Using Acoustophoresis.

In this paper, we utilize bulk acoustic waves to control the position of microparticles inside droplets in two-phase microfluidic systems and demonstrate a method to enrich the microparticles. In droplet microfluidics, different unit operations are combined and integrated on-chip to miniaturize complex biochemical assays. We present a droplet unit operation capable of controlling the position of microparticles during a trident shaped droplet split. An acoustic standing wave field is generated in the microchannel, and the acoustic forces direct the encapsulated microparticles to the center of the droplets. The method is generic, requires no labeling of the microparticles, and is operated in a noncontact fashion. It was possible to achieve 2+-fold enrichment of polystyrene beads (5 μm in diameter) in the center daughter droplet with an average recovery of 89% of the beads. Red blood cells were also successfully manipulated inside droplets. These results show the possibility to use acoustophoresis in two-phase systems to enrich microparticles and open up the possibility for new droplet-based assays that are not performed today.

Dclk1+ small intestinal epithelial tuft cells display the hallmarks of quiescence and self-renewal.

To date, no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. In order to elucidate the potential function of Dclk1+ cells, we FACS-sorted Dclk1+ cells from mouse small intestinal epithelium using transgenic mice expressing YFP under the control of the Dclk1 promoter (Dclk1-CreER;Rosa26-YFP). Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFP- cells. Dclk1+ population display ~6 fold enrichment for the putative quiescent stem cell marker Bmi1. We observed significantly greater expression of pluripotency genes, pro-survival genes, and quiescence markers in the Dclk1+ population. A significant increase in self-renewal capability (14-fold) was observed in in vitro isolated Dclk1+ cells. The unique genetic report presented in this manuscript suggests that Dclk1+ cells may maintain quiescence, pluripotency, and metabolic activity for survival/longevity. Functionally, these reserve characteristics manifest in vitro, with Dclk1+ cells exhibiting greater ability to self-renew. These findings indicate that quiescent stem-like functionality is a feature of Dclk1-expressing tuft cells.

Abstract 1053: Potential utility of DNA methytlation as a biomarker for prediction of ulcerative colitis associated colitic cancer (UCAC)

Abstract Background: Patients with Ulcerative colitis (UC) have more increased risk for the development of colorectal cancers compared with the average-risk population. Methods or strategies other than intensive colonoscopic surveillance to diagnose ulcerative colitis-associated colorectal carcinoma (UCAC) at early stage are limited. DNA methylation is deeply involved in carcinogenesis in many tumor types and its potential utility as a biomarker has been suggested. Aim: To evaluate potential utility of DNA methylation as the risk marker for UCAC, we quantified DNA methylation of inflamed rectal mucosa, neoplastic tissue and adjacent colonic mucosa of UC patients. Method: Inflamed rectal mucosa (n = 84) and normal appealing proximal mucosa (n = 10) of 84 cancer-free UC patients were examined. Neoplastic tissue (n = 33) and adjacent colonic mucosa (n = 26) from 16 UC patients with neoplasia (UCAC or DALM) were also studied. Bisulfite pyro sequencing was performed for the quantification of 45 cancer or age-related candidate panels and LINE1 repetitive element. Illumina Infinium Human Methylation 450 platform was also used to determine 470,000 CpG sites distributed across the genome. Results: Inflamed mucosa in UC was characterized as hyper methylation in candidate panels using the helical unsupervised clustering. In several panels, hyper methylation was associated with severe clinical phenotypes including steroid dependent, refractory, longstanding severe inflammation etc. (DPYS and miR-1247: p&amp;lt;0.05. GSTP1: p&amp;lt;0.01. mean of three: p&amp;lt;0.0001). Notably, in many panels, hyper methylation was significantly associated with longer duration of disease in cancer-free patients. Among them, some panels showed hyper methyhlation in both neoplastic and adjacent tissue compared to rectal mucosa of cancer-free cases (DPYS and N33, both p values &amp;lt;0.0001). Genome scale analysis revealed that UC with severe inflammation or neoplasia was characterized as hyper methylation at CpG sites rather than outside CpG sites. Gene ontology analysis demonstrated frequently methylated promoter CpG sites in these cases frequently encoded protein related to biosynthetic process (Fold enrichment: 2.07. p value using Benjamini correction: 5.69E-05). Conclusion: Hyper methylation at CpG sites of non-neoplastic colonic mucosa in UC with severe inflammation and neoplasia suggested the potential utility of DNA methytlation as a biomarker for prediction of UCAC. Moreover, enrichment of specific pathway in methylated genes suggests the possibility of targeting therapy for treating UCAC. Citation Format: Tomomitsu Tahara, Naoko Nakano, Mitsuo Nagasaka, Yoshihito Nakagawa, Tomoyuki Shibata, Naoki Ohmiya, Ichiro Hirata. Potential utility of DNA methytlation as a biomarker for prediction of ulcerative colitis associated colitic cancer (UCAC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1053. doi:10.1158/1538-7445.AM2015-1053

Abstract 1997: Label-free concentration of viable breast cancer CTCs for single cell Western blotting

Abstract The isolation of circulating tumor cells (CTCs) in blood can aid in cancer prognosis, characterize genetic mutations for targeted therapies, and elucidate the biological mechanisms of metastasis. While CTCs provide clinical utility, there are still several technical difficulties with CTC isolation and analysis. CTCs are very rare (1-10 cells/mL of blood) and current isolation methods rely on immunocapture, require long sample preparation, and fail to deliver viable cells. Further, recent advances in single cell analysis of CTCs have been limited to genomic studies, in which DNA/mRNA does not provide a complete picture of CTCs at the functional level, as mRNA expression may only account for a portion of protein expression. The ability to study and measure the protein expression of CTCs could provide insight into the mechanisms driving cancer progression and signaling dysregulation. Unfortunately, conventional proteomic tools (e.g. immunocytochemistry and Western blot) either do not provide sufficient specificity or sensitivity toward single cell levels. Thus, there is a lack of tools that can provide quantitative protein analysis for rare CTCs. To address this gap, we enable rare cell Western blotting by combining isolation and proteomic technologies in a unique workflow: (i) Label-free Vortex enrichment of rare larger cells from blood samples. Larger cancer cells are trapped within an array of microscale fluid vortices that form in simple rectangular reservoirs, while smaller blood cells pass through. The cancer cells are stably maintained in the vortices, allowing for solution exchange, followed by release upon lowering the flow rate. With this device, we were able to release a small volume (∼500 μL) of concentrated, viable cancer cells (MCF7) from healthy blood, at high throughput, capture efficiency (35%), purity (&amp;gt;80%), and ∼10,000 fold enrichment. CTCs were also successfully extracted and enumerated from the blood of patients with breast cancer (N = 18, from 0.75 to 23.25 CTCs/mL). (ii) High-specificity and multiplexed protein analysis of rare cancer cells with single cell Western blotting (scWB). Enriched CTCs are placed into microwells patterned into a polyacrylamide gel for the scWB assay (in-situ chemical lysis in the microwells, electrophoresis into bulk gel layer, photo-immobilization, and subsequent antibody-probing of immobilized proteins). Here, we demonstrate scWB compatibility with rare cells by spiking MCF7s into normal blood and performing protein separations. The scWB is multiplex and can assay several target proteins simultaneously. Cells stained for surface markers (anti-EpCAM) are also compatible, and the scWB can resolve complete immunocomplexes without disrupting the location of the intracellular targets (GAPDH, ERK). With the integration of these technologies, we push the limits of quantitative protein analysis of CTCs, an essential step to better understand these rare populations. Citation Format: Elly Sinkala, Elodie Sollier, Corinne Renier, James Che, Stefanie S. Jeffrey, Amy E. Herr. Label-free concentration of viable breast cancer CTCs for single cell Western blotting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1997. doi:10.1158/1538-7445.AM2015-1997