Investigation of genomic and transcriptomic risk factors in clopidogrel response in African Americans.

Clopidogrel, an anti-platelet drug, used to prevent thrombosis after percutaneous coronary intervention. Clopidogrel resistance results in recurring ischemic episodes, with African Americans suffering disproportionately. The aim of this study was to identify biomarkers of clopidogrel resistance in African American patients. We conducted a genome-wide association study, including local ancestry adjustment, in 141 African Americans on clopidogrel to identify associations with high on-treatment platelet reactivity (HTPR). We validated genome-wide and suggestive hits in an independent cohort of African American clopidogrel patients (N = 823) from the Million Veteran's Program (MVP) along with in vitro functional follow up. We performed differential gene expression (DGE) analysis in whole blood with functional follow-up in MEG-01 cells. We identified rs7807369, within thrombospondin 7A (THSD7A), as significantly associated with increasing risk of HTPR (p = 4.56 × 10-9). Higher THSD7A expression was associated with HTPR in an independent gene expression cohort of clopidogrel treated patients (p = 0.004) and supported by increased gene expression on THSD7A in primary human endothelial cells carrying the risk haplotype. Two SNPs (rs1149515 and rs191786) were validated in the MVP cohort. DGE analysis identified an association with decreased LAIR1 expression to HTPR. LAIR1 knockdown in a MEG-01 cells resulted in increased expression of SYK and AKT1, suggesting an inhibitory role of LAIR1 in the Glycoprotein VI pathway. Notably, the CYP2C19 variants showed no association with clopidogrel response in the discovery or MVP cohorts. In summary, these finding suggest that other variants outside of CYP2C19 star alleles play an important role in clopidogrel response in African Americans.

Prostate cancer cell-platelet bidirectional signaling promotes calcium mobilization, invasion and apoptotic resistance via distinct receptor-ligand pairs.

Platelets play a crucial role in cancer and thrombosis. However, the receptor-ligand repertoire mediating prostate cancer (PCa) cell-platelet interactions and ensuing consequences have not been fully elucidated. Microvilli emanating from the plasma membrane of PCa cell lines (RC77 T/E, MDA PCa 2b) directly contacted individual platelets and platelet aggregates. PCa cell-platelet interactions were associated with calcium mobilization in platelets, and translocation of P-selectin and integrin αIIbß3 onto the platelet surface. PCa cell-platelet interactions reciprocally promoted PCa cell invasion and apoptotic resistance, and these events were insensitive to androgen receptor blockade by bicalutamide. PCa cells were exceedingly sensitive to activation by platelets in vitro, occurring at a PCa cell:platelet coculture ratio as low as 1:10 (whereas PCa patient blood contains 1:2,000,000 per ml). Conditioned medium from cocultures stimulated PCa cell invasion but not apoptotic resistance nor platelet aggregation. Candidate transmembrane signaling proteins responsible for PCa cell-platelet oncogenic events were identified by RNA-Seq and broadly divided into 4 major categories: (1) integrin-ligand, (2) EPH receptor-ephrin, (3) immune checkpoint receptor-ligand, and (4) miscellaneous receptor-ligand interactions. Based on antibody neutralization and small molecule inhibitor assays, PCa cell-stimulated calcium mobilization in platelets was found to be mediated by a fibronectin1 (FN1)-αIIbß3 signaling axis. Platelet-stimulated PCa cell invasion was facilitated by a CD55-adhesion G protein coupled receptor E5 (ADGRE5) axis, with contribution from platelet cytokines CCL3L1 and IL32. Platelet-stimulated PCa cell apoptotic resistance relied on ephrin-EPH receptor and lysophosphatidic acid (LPA)-LPA receptor (LPAR) signaling. Of participating signaling partners, FN1 and LPAR3 overexpression was observed in PCa specimens compared to normal prostate, while high expression of CCR1 (CCL3L1 receptor), EPHA1 and LPAR5 in PCa was associated with poor patient survival. These findings emphasize that non-overlapping receptor-ligand pairs participate in oncogenesis and thrombosis, highlighting the complexity of any contemplated clinical intervention strategy.

Selective disruption of trigeminal sensory neurogenesis and differentiation in a mouse model of 22q11.2 deletion syndrome.

22q11.2 Deletion Syndrome (22q11DS) is a neurodevelopmental disorder associated with cranial nerve anomalies and disordered oropharyngeal function, including pediatric dysphagia. Using the LgDel 22q11DS mouse model, we investigated whether sensory neuron differentiation in the trigeminal ganglion (CNgV), which is essential for normal orofacial function, is disrupted. We did not detect changes in cranial placode cell translocation or neural crest migration at early stages of LgDel CNgV development. However, as the ganglion coalesces, proportions of placode-derived LgDel CNgV cells increase relative to neural crest cells. In addition, local aggregation of placode-derived cells increases and aggregation of neural crest-derived cells decreases in LgDel CNgV. This change in cell-cell relationships was accompanied by altered proliferation of placode-derived cells at embryonic day (E)9.5, and premature neurogenesis from neural crest-derived precursors, reflected by an increased frequency of asymmetric neurogenic divisions for neural crest-derived precursors by E10.5. These early differences in LgDel CNgV genesis prefigure changes in sensory neuron differentiation and gene expression by postnatal day 8, when early signs of cranial nerve dysfunction associated with pediatric dysphagia are observed in LgDel mice. Apparently, 22q11 deletion destabilizes CNgV sensory neuron genesis and differentiation by increasing variability in cell-cell interaction, proliferation and sensory neuron differentiation. This early developmental divergence and its consequences may contribute to oropharyngeal dysfunction, including suckling, feeding and swallowing disruptions at birth, and additional orofacial sensory/motor deficits throughout life.

Expanding the prostate cancer cell line repertoire with ACRJ-PC28, an AR-negative neuroendocrine cell line derived from an African-Caribbean patient.

Prostate cell lines from diverse backgrounds are important to addressing disparities in prostate cancer (PCa) incidence and mortality rates among Black men. ACRJ-PC28 was developed from a transrectal needle biopsy and established via inactivation of the CDKN2A locus and simultaneous expression of human telomerase. Characterization assays included growth curve analysis, immunoblots, IHC, 3D cultures, immunofluorescence imaging, confocal microscopy, flow cytometry, WGS, and RNA-Seq. ACRJ-PC28 has been passaged more than 40 times in vitro over 10 months with a doubling time of 45 hours. STR profiling confirmed the novelty and human origin of the cell line. RNA-Seq confirmed the expression of prostate specific genes alpha-methylacyl-CoA racemase (AMACR) and NKX3.1 and Neuroendocrine specific markers synaptophysin (SYP) and enolase 2 (ENO2) and IHC confirmed the presence of AMACR. Immunoblots indicated the cell line is of basal-luminal type; expresses p53 and pRB and is AR negative. WGS confirmed the absence of exonic mutations and the presence of intronic variants that appear to not affect function of AR, p53, and pRB. RNA-Seq data revealed numerous TP53 and RB1 mRNA splice variants and the lack of AR mRNA expression. This is consistent with retention of p53 function in response to DNA damage and pRB function in response to contact inhibition. Soft agar anchorage-independent analysis indicated that the cells are transformed, confirmed by principal component analysis (PCA) where ACRJ-PC28 cells cluster alongside other PCa tumor tissues, yet was distinct. The novel methodology described should advance prostate cell line development, addressing the disparity in PCa among Black men.

Differences in the Platelet mRNA Landscape Portend Racial Disparities in Platelet Function and Suggest Novel Therapeutic Targets.

The African American (AA) population displays a 1.6 to 3-fold higher incidence of thrombosis and stroke mortality compared with European Americans (EAs). Current antiplatelet therapies target the ADP-mediated signaling pathway, which displays significant pharmacogenetic variation for platelet reactivity. The focus of this study was to define underlying population differences in platelet function in an effort to identify novel molecular targets for future antiplatelet therapy. We performed deep coverage RNA-Seq to compare gene expression levels in platelets derived from a cohort of healthy volunteers defined by ancestry determination. We identified > 13,000 expressed platelet genes of which 480 were significantly differentially expressed genes (DEGs) between AAs and EAs. DEGs encoding proteins known or predicted to modulate platelet aggregation, morphology, or platelet count were upregulated in AA platelets. Numerous G-protein coupled receptors, ion channels, and pro-inflammatory cytokines not previously associated with platelet function were likewise differentially expressed. Many of the signaling proteins represent potential pharmacologic targets of intervention. Notably, we confirmed the differential expression of cytokines IL32 and PROK2 in an independent cohort by quantitative real-time polymerase chain reaction, and provide functional validation of the opposing actions of these two cytokines on collagen-induced AA platelet aggregation. Using Genotype-Tissue Expression whole blood data, we identified 516 expression quantitative trait locuses with Fst values > 0.25, suggesting that population-differentiated alleles may contribute to differences in gene expression. This study identifies gene expression differences at the population level that may affect platelet function and serve as potential biomarkers to identify cardiovascular disease risk. Additionally, our analysis uncovers candidate novel druggable targets for future antiplatelet therapies.

Genomics of Black American colon cancer disparities: An RNA sequencing (RNA-Seq) study from an academic, tertiary referral center.

BACKGROUND: Black Americans have a higher incidence and mortality rate from colorectal cancer compared to their non-Hispanic White American counterparts. Even when controlling for sociodemographic differences between these 2 populations, Black Americans remain disproportionately affected by colorectal cancer. The purpose of our study was to determine if differences in gene expression between Black American and non-Hispanic White American colon cancer specimens could help explain differences in the incidence and mortality rate between these 2 populations. METHODS: Black Americans and non-Hispanic White Americans undergoing colon resection for stages I, II, or III colon cancer at a single institution were identified. Black American and non-Hispanic White American patients were matched for age, sex, and colon cancer stage to minimize the risk of confounding variables. Tissue samples were obtained at the time of colon resection and were analyzed using RNA sequencing to determine if there were differences in the expression of genes and biologic processes between the 2 groups. RESULTS: A total of 17 colon cancer specimens were analyzed; 8 (47.1%) patients were Black Americans. A total of 456 genes were identified as being expressed differently (ie, up or downregulated) in Black American compared to non-Hispanic White American colon cancer specimens. Moreover, 500 different genetic pathways were noted to be significantly over-represented with differentially expressed genes in our comparison of Black American and non-Hispanic White American colon cancer specimens, the majority of which plays a role in inflammation and immune cell function. CONCLUSION: Significant differences in gene expression and genetic pathways exist between Black Americans and non-Hispanic White Americans. Additional and multi-institutional and registry-based studies are needed to validate our findings and to further elucidate the contribution that these differences have to the overall incidence and mortality rate from colon cancer in these 2 patient populations.

Prostate Cancer Diagnosis in the Clinic Using an 8-Protein Biomarker Panel.

The inability to distinguish aggressive from indolent prostate cancer is a longstanding clinical problem. Prostate specific antigen (PSA) tests and digital rectal exams cannot differentiate these forms. Because only ∼10% of diagnosed prostate cancer cases are aggressive, existing practice often results in overtreatment including unnecessary surgeries that degrade patients' quality of life. Here, we describe a fast microfluidic immunoarray optimized to determine 8-proteins simultaneously in 5 µL of blood serum for prostate cancer diagnostics. Using polymeric horseradish peroxidase (poly-HRP, 400 HRPs) labels to provide large signal amplification and limits of detection in the sub-fg mL-1 range, a protocol was devised for the optimization of the fast, accurate assays of 100-fold diluted serum samples. Analysis of 130 prostate cancer patient serum samples revealed that some members of the protein panel can distinguish aggressive from indolent cancers. Logistic regression was used to identify a subset of the panel, combining biomarker proteins ETS-related gene protein (ERG), insulin-like growth factor-1 (IGF-1), pigment epithelial-derived factor (PEDF), and serum monocyte differentiation antigen (CD-14) to predict whether a given patient should be referred for biopsy, which gave a much better predictive accuracy than PSA alone. This represents the first prostate cancer blood test that can predict which patients will have a high biopsy Gleason score, a standard pathology score used to grade tumors.

Transcriptional dysregulation in developing trigeminal sensory neurons in the LgDel mouse model of DiGeorge 22q11.2 deletion syndrome.

LgDel mice, which model the heterozygous deletion of genes at human chromosome 22q11.2 associated with DiGeorge/22q11.2 deletion syndrome (22q11DS), have cranial nerve and craniofacial dysfunction as well as disrupted suckling, feeding and swallowing, similar to key 22q11DS phenotypes. Divergent trigeminal nerve (CN V) differentiation and altered trigeminal ganglion (CNgV) cellular composition prefigure these disruptions in LgDel embryos. We therefore asked whether a distinct transcriptional state in a specific population of early differentiating LgDel cranial sensory neurons, those in CNgV, a major source of innervation for appropriate oropharyngeal function, underlies this departure from typical development. LgDel versus wild-type (WT) CNgV transcriptomes differ significantly at E10.5 just after the ganglion has coalesced. Some changes parallel altered proportions of cranial placode versus cranial neural crest-derived CNgV cells. Others are consistent with a shift in anterior-posterior patterning associated with divergent LgDel cranial nerve differentiation. The most robust quantitative distinction, however, is statistically verifiable increased variability of expression levels for most of the over 17 000 genes expressed in common in LgDel versus WT CNgV. Thus, quantitative expression changes of functionally relevant genes and increased stochastic variation across the entire CNgV transcriptome at the onset of CN V differentiation prefigure subsequent disruption of cranial nerve differentiation and oropharyngeal function in LgDel mice.

Role of Alternative Splicing in Prostate Cancer Aggressiveness and Drug Resistance in African Americans.

Alternative splicing, the process of removing introns and joining exons of pre-mRNA, is critical for growth, development, tissue homeostasis, and species diversity. Dysregulation of alternative splicing can initiate and drive disease. Aberrant alternative splicing has been shown to promote the "hallmarks of cancer" in both hematological and solid cancers. Of interest, recent work has focused on the role of alternative splicing in prostate cancer and prostate cancer health disparities. We will provide a review of prostate cancer health disparities involving the African American population, alternative RNA splicing, and alternative splicing in prostate cancer. Lastly, we will summarize our work on differential alternative splicing in prostate cancer disparities and its implications for disparate health outcomes and therapeutic targets.

Author Correction: Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Novel FGFR3 Splice Variant Preferentially Expressed in African American Prostate Cancer Drives Aggressive Phenotypes and Docetaxel Resistance.

Alternative splicing (AS) has been shown to participate in prostate cancer development and progression; however, a link between AS and prostate cancer health disparities has been largely unexplored. Here we report on the cloning of a novel splice variant of FGFR3 that is preferentially expressed in African American (AA) prostate cancer. This novel variant (FGFR3-S) omits exon 14, comprising 123 nucleotides that encode the activation loop in the intracellular split kinase domain. Ectopic overexpression of FGFR3-S in European American (EA) prostate cancer cell lines (PC-3 and LNCaP) led to enhanced receptor autophosphorylation and increased activation of the downstream signaling effectors AKT, STAT3, and ribosomal S6 compared with FGFR3-L (retains exon 14). The increased oncogenic signaling imparted by FGFR3-S was associated with a substantial gain in proliferative and antiapoptotic activities, as well as a modest but significant gain in cell motility. Moreover, the FGFR3-S-conferred proliferative and motility gains were highly resistant to the pan-FGFR small-molecule inhibitor dovitinib and the antiapoptotic gain was insensitive to the cytotoxic drug docetaxel, which stands in marked contrast with dovitinib- and docetaxel-sensitive FGFR3-L. In an in vivo xenograft model, mice injected with PC-3 cells overexpressing FGFR3-S exhibited significantly increased tumor growth and resistance to dovitinib treatment compared with cells overexpressing FGFR3-L. In agreement with our in vitro and in vivo findings, a high FGFR3-S/FGFR3-L expression ratio in prostate cancer specimens was associated with poor patient prognosis. IMPLICATIONS: This work identifies a novel FGFR3 splice variant and supports the hypothesis that differential AS participates in prostate cancer health disparities.

Accessible Telemedicine Diagnostics with ELISA in a 3D Printed Pipette Tip.

We report herein a novel pipet-based "ELISA in a tip" as a new versatile diagnostic tool featuring better sensitivity, shorter incubation time, accessibility, and low sample and reagent volumes compared to traditional ELISA. Capture and analysis of data by a cell phone facilitates electronic delivery of results to health care providers. Pipette tips were designed and 3D printed as adapters to fit most commercial 50-200 µL pipettes. Capture antibodies (Ab1) are immobilized on the inner walls of the pipet tip, which serves as the assay compartment where samples and reagents are moved in and out by pipetting. Signals are generated using colorimetric or chemiluminescent (CL) reagents and can be quantified using a cell phone, CCD camera, or plate reader. We utilized pipet-tip ELISA to detect four cancer biomarker proteins with detection limits similar to or lower than microplate ELISAs at 25% assay cost and time. Recoveries of these proteins from spiked human serum were 85-115% or better, depending slightly on detection mode. Using CCD camera quantification of CL with femto-luminol reagent gave limits of detection (LOD) as low as 0.5 pg/mL. Patient samples (13) were assayed for 3 biomarker proteins with results well correlated to conventional ELISA and an established microfluidic electrochemical immunoassay.

Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells.

Histone deacetylases (HDACs) are involved in diverse cellular regulatory mechanisms including non-canonical functions outside the chromatin environment. Several publications have demonstrated that selective HDAC inhibitors (HDACi) can influence tumor immunogenicity and the functional activity of specific immune cells. In particular, the selective inhibition of HDAC6 has been reported to decrease tumor growth in several malignancies. However, there is still no clarity about the cellular components mediating this effect. In this study, we evaluated the HDAC6i Nexturastat A as a priming agent to facilitate the transition of the tumor microenvironment from "cold" to "hot", and potentially augment immune check-point blockade therapies. This combination modality demonstrated to significantly reduce tumor growth in syngeneic melanoma tumor models. Additionally, we observed a complete neutralization of the up-regulation of PD-L1 and other immunosuppressive pathways induced by the treatment with anti-PD-1 blockade. This combination also showed profound changes in the tumor microenvironment such as enhanced infiltration of immune cells, increased central and effector T cell memory, and a significant reduction of pro-tumorigenic M2 macrophages. The evaluation of individual components of the tumor microenvironment suggested that the in vivo anti-tumor activity of HDAC6i is mediated by its effect on tumor cells and tumor-associated macrophages, and not directly over T cells. Overall, our results indicate that selective HDAC6i could be used as immunological priming agents to sensitize immunologically "cold" tumors and subsequently improve ongoing immune check-point blockade therapies.

The ACCOuNT Consortium: A Model for the Discovery, Translation, and Implementation of Precision Medicine in African Americans.

The majority of pharmacogenomic (PGx) studies have been conducted on European ancestry populations, thereby excluding minority populations and impeding the discovery and translation of African American-specific genetic variation into precision medicine. Without accounting for variants found in African Americans, clinical recommendations based solely on genetic biomarkers found in European populations could result in misclassification of drug response in African American patients. To address these challenges, we formed the Transdisciplinary Collaborative Center (TCC), African American Cardiovascular Pharmacogenetic Consortium (ACCOuNT), to discover novel genetic variants in African Americans related to clinically actionable cardiovascular phenotypes and to incorporate African American-specific sequence variations into clinical recommendations at the point of care. The TCC consists of two research projects focused on discovery and translation of genetic findings and four cores that support the projects. In addition, the largest repository of PGx information on African Americans is being established as well as lasting infrastructure that can be utilized to spur continued research in this understudied population.

Aberrant RNA Splicing in Cancer and Drug Resistance.

More than 95% of the 20,000 to 25,000 transcribed human genes undergo alternative RNA splicing, which increases the diversity of the proteome. Isoforms derived from the same gene can have distinct and, in some cases, opposing functions. Accumulating evidence suggests that aberrant RNA splicing is a common and driving event in cancer development and progression. Moreover, aberrant splicing events conferring drug/therapy resistance in cancer is far more common than previously envisioned. In this review, aberrant splicing events in cancer-associated genes, namely BCL2L1, FAS, HRAS, CD44, Cyclin D1, CASP2, TMPRSS2-ERG, FGFR2, VEGF, AR and KLF6, will be discussed. Also highlighted are the functional consequences of aberrant splice variants (BCR-Abl35INS, BIM-γ, IK6, p61 BRAF V600E, CD19-∆2, AR-V7 and PIK3CD-S) in promoting resistance to cancer targeted therapy or immunotherapy. To overcome drug resistance, we discuss opportunities for developing novel strategies to specifically target the aberrant splice variants or splicing machinery that generates the splice variants. Therapeutic approaches include the development of splice variant-specific siRNAs, splice switching antisense oligonucleotides, and small molecule inhibitors targeting splicing factors, splicing factor kinases or the aberrant oncogenic protein isoforms.

MicroRNA and mRNA expression associated with ectopic germinal centers in thymus of myasthenia gravis.

BACKGROUND: A characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic germinal centers (GC). However, the mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Dysregulation of small, non-coding microRNAs (miRNAs) and their target genes has been identified in the pathology of several autoimmune diseases. We assessed the miRNA and mRNA profiles of the MG thymus and have investigated their role in GC formation and maintenance. METHODS: MG thymus samples were assessed by histology and grouped based upon the appearance of GC; GC positive and GC negative. A systems biology approach was used to study the differences between the groups. Our study included miRNA and mRNA profiling, quantitative real-time PCR validation, miRNA target identification, pathway analysis, miRNA-mRNA reciprocal expression pairing and interaction. RESULTS: Thirty-eight mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>1.5 fold change, ANOVA p<0.05). The miRNAs were found to be involved in immune response pathways and identified in other autoimmune diseases. The cellular and molecular functions of the mRNAs showed involvement in cell death and cell survival, cellular proliferation, cytokine signaling and extra-cellular matrix reorganization. Eleven miRNA and mRNA pairs were reciprocally regulated. The Regulator of G protein Signalling 13 (RGS13), known to be involved in GC regulation, was identified in specimens with GC and was paired with downregulation of miR-452-5p and miR-139-5p. MiRNA target sites were validated by dual luciferase assay. Transfection of miRNA mimics led to down regulation of RGS13 expression in Raji cells. CONCLUSION: Our study indicates a distinct miRNA and mRNA expression pattern in ectopic GC in MG thymus. These miRNAs and mRNAs are involved in regulatory pathways common to inflammation and immune response, cell cycle regulation and anti-apoptotic pathways suggesting their involvement in support of GC formation in the thymus. We demonstrate for the first time that miR-139-5p and miR-452-5p negatively regulate RGS13 expression.

Automated 3D-Printed Microfluidic Array for Rapid Nanomaterial-Enhanced Detection of Multiple Proteins.

We report here the fabrication and validation of a novel 3D-printed, automated immunoarray to detect multiple proteins with ultralow detection limits. This low cost, miniature immunoarray employs electrochemiluminescent (ECL) detection measured with a CCD camera and employs touch-screen control of a micropump to facilitate automated use. The miniaturized array features prefilled reservoirs to deliver sample and reagents to a paper-thin pyrolytic graphite microwell detection chip to complete sandwich immunoassays. The detection chip achieves high sensitivity by using single-wall carbon nanotube-antibody conjugates in the microwells and employing massively labeled antibody-decorated RuBPY-silica nanoparticles to generate ECL. The total cost of an array is $0.65, and an eight-protein assay can be done in duplicate for $0.14 per protein with limits of detection (LOD) as low as 78-110 fg mL-1 in diluted serum. The electronic control system costs $210 in components. Utility of the automated immunoarray was demonstrated by detecting an eight-protein prostate cancer biomarker panel in human serum samples in 25 min. The system is well suited to future clinical and point-of-care diagnostic testing and could be used in resource-limited environments.

Single-nucleotide polymorphisms of stemness genes predicted to regulate RNA splicing, microRNA and oncogenic signaling are associated with prostate cancer survival.

Prostate cancer (PCa) is a clinically and molecularly heterogeneous disease, with variation in outcomes only partially predicted by grade and stage. Additional tools to distinguish indolent from aggressive disease are needed. Phenotypic characteristics of stemness correlate with poor cancer prognosis. Given this correlation, we identified single-nucleotide polymorphisms (SNPs) of stemness-related genes and examined their associations with PCa survival. SNPs within stemness-related genes were analyzed for association with overall survival of PCa in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. Significant SNPs predicted to be functional were selected for linkage disequilibrium analysis and combined and stratified analyses. Identified SNPs were evaluated for association with gene expression. SNPs of CD44 (rs9666607), ABCC1 (rs35605 and rs212091) and GDF15 (rs1058587) were associated with PCa survival and predicted to be functional. A role for rs9666607 of CD44 and rs35605 of ABCC1 in RNA splicing regulation, rs212091 of ABCC1 in miRNA binding site activity and rs1058587 of GDF15 in causing an amino acid change was predicted. These SNPs represent potential novel prognostic markers for overall survival of PCa and support a contribution of the stemness pathway to PCa patient outcome.

Automated 4-Sample Protein Immunoassays using 3D-Printed Microfluidics.

Low cost, miniaturized assay platforms that work with small sample volumes, high sensitivity and rapid detection will have high value in future biomolecular diagnostics. Herein we report an automated, 3D printed electrochemiluminescent (ECL) immunoarray integrated with a nanostructured pyrolytic graphite sheet (PGS) microwell chip configured to detect 2 proteins simultaneously from complex liquid samples with high sensitivity and selectivity. Assays are done in 18 min at cost of < $1.00 using 1-2 microliters of sample. 3D printed microfluidic array design integrates reagent and sample chambers with rapid ECL detection. A commercial programmable syringe pump used with a preset program allows pump to pause and resume reagent delivery as required for completion of the sandwich immunoassays. Nanostructured surfaces feature antibody-decorated single wall carbon nanotube forests on PGS chip microwells, and sensitivity is amplified via massively labeled RuBPY-silica nanoparticles for detection. Prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) were measured simultaneously from human serum on the immunoarray with detection limits 150 fg mL-1 for PSA and 230 fg mL-1 for PSMA, with dynamic ranges up to 5 ng mL-1. Validation of the immunoarray by measuring these proteins in human serum showed good correlation with single protein ELISA. These 3D printed platforms can be easily adapted to multiple applications and configurable CAD files for the immunoarray can be downloaded from our lab's website.