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Supplementary Data from Common Trajectories of Highly Effective CD19-Specific CAR T Cells Identified by Endogenous T-cell Receptor Lineages

Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27−). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products. Significance: Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients. This article is highlighted in the In This Issue feature, p. 2007

Although mRNA vaccine efficacy against severe coronavirus disease 2019 remains high, variant emergence has prompted booster immunizations. However, the effects of repeated exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens on memory T cells are poorly understood. Here, we utilize major histocompatibility complex multimers with single-cell RNA sequencing to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two or three antigen exposures, including vaccination, primary infection and breakthrough infection. Exposure order determined the distribution between spike-specific and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7

SummaryBacterial cell wall, a universal pathogen-associated molecular pattern (PAMP), crosses the placenta into the fetal brain. We determined that PAMPs interact with TLR2/6 on murine fetal neural progenitor cells (NPCs) to induce overexpansion of all neocortical layers leading to a larger, folded cortex and abnormal postnatal behavior. The NPC overexpansion originated at E10 and targeted ventricular radial glia (vRG), the primary NPC, by shortening cell cycle and increasing self-renewal. The mechanism involved two novel signaling pathways in NPCs mediated by recognition of bacterial PAMPs by TLR2/6 including: a) loss of primary cilia, activation of hedgehog signaling, and increased FOXG1 and b) increased PI3K/AKT activity. These findings reveal PAMP/TLR2/6 acts as a morphogen in fetal neurodevelopment. In addition, the loss ofTlr2orTlr6without pathogenic challenge, increased the number of neurons, establishing the requirement for an endogenous TLR2 signal for normal neurodevelopment in the embryo.

Fibrolamellar carcinoma (FLC) is a rare liver malignancy that has no known cure. Novel therapies for FLC are urgently needed to improve patient outcomes. The key genetic event in FLC tumorigenesis is a highly conserved gene fusion between the first exon of DNAJB1 and the last nine exons of PRKACA. This fusion gene is present in all FLC tumors, and more than 90% of patients express fusion proteins with identical amino acid sequences. Genetic mutations like this gene fusion can serve as important therapeutic vulnerabilities, in part because they may be processed and presented on the surface of tumor cells as neoantigens that can activate an anti-tumor T cell response. The purpose of our study was to test the hypothesis that DNAJB1-PRKACA fusion neoantigens could elicit T cell responses against FLC and serve as targets for novel immunotherapies. Spatial transcriptomic analyses of human FLC tumor samples indicated that CD8+ T cells could infiltrate these tumors, but often expressed markers of dysfunction and exhaustion, such as TOX. Nevertheless, we successfully expanded tumor-infiltrating T cells from FLC ex vivo and used intracellular cytokine staining (ICS) to identify a subset of FLC patient T cells that produced IFNγ and TNFα in response to stimulation with a fusion neoantigen. We used both peptide-MHC tetramer staining and functional response after stimulation to identify T cells in FLC patients and healthy donors that recognize fusion neoantigens. We then determined the paired T cell receptor (TCR) sequences using single-cell sequencing and expressed the TCRs in primary human T cells to test their potential utility in TCR-engineered immunotherapies. We validated the specificity and functionality of these TCRs using peptide-MHC tetramer staining, ICS, and in vitro cytotoxicity assays on the xCelligence and Berkeley Lights Lightning platforms. Primary T cells expressing fusion-specific TCRs bound to their cognate peptide-MHC tetramer, produced multiple cytokines in response to stimulation with their cognate fusion peptide, and specifically killed fusion-presenting target cells in vitro. Ongoing experiments will test whether T cells expressing these fusion-specific TCRs can control growth of fusion-presenting tumors in vivo. Collectively, these studies have defined the first reported fusion-specific T cell response in FLC, as well as fusion-specific TCRs that hold promise for use in adoptive T cell therapies. Our spatial transcriptomic analyses have also begun to illuminate the immune microenvironment in FLC and will inform future efforts to develop immunotherapies for this disease. Citation Format: Allison M. Kirk, Ching-Heng Chou, Jeremy Chase Crawford, Walid Awad, E. Kaitlynn Allen, Xiaoyu Zhang, Anthony E. Zamora, Scott E. Strome, Paul G. Thomas. Characterization of CD8 T cell responses to DNAJB1-PRKACA fusion neoantigens in fibrolamellar carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1383.

BackgroundChimeric antigen receptor modified (CAR) T cells have revolutionized the treatment of blood cancers, though some patients still show a poor response in either CAR expansion, effector response, or persistence.1 In this study, we determined the features of pre-infusion CAR-transduced T cells that generated optimally functional responses after infusion.MethodsUsing both the pre-infusion product and PBMCs isolated at weeks 1–4, 8, and 3-months post-infusion from 15 patients undergoing experimental anti-CD19 CAR T cell treatment for refractory or relapsed B-ALL, we generated a comprehensive single cell gene expression and T cell receptor (TCR) sequencing dataset on over 180,000 CAR T cells (figure 1).ResultsAs expected, pre-infusion CAR T cells tend to highly express genes associated with proliferation, while post-infusion CARs show signs of either cytotoxic effector differentiation or dysfunctional terminal differentiation. Sequencing of the endogenous TCR, at the single cell level, allows us to track the trajectories of clonally and transcriptionally related cells (figure 2). Post-infusion cells with significant cytotoxic effector function share TCRs with a statistically defined subset of CARs in the pre-infusion sample (figure 3). Using a machine learning approach, we found that potent effector precursor CAR T cells have a specific transcriptional profile distinct from the other pre-infusion CAR T cells, including markers of early effector function such as increased EOMES, GNLY, GZMH, GZMK, KLRD1, and IFNγ. Formalizing this signature, we have developed a robust classifier that can predict with 82.8% accuracy whether a CAR T is likely to become a favorable effector based on its pre-infusion profile (figure 4). This prediction model can be used to evaluate the extent to which a patient‘s generated CAR product will be able to mount a robust response after encountering its target. Additionally, there are a number of genes, as a part of this signature, that are expressed on the cell surface and can be utilized as a method to differentiate the effector precursor pre-infusion CAR T cells from other pre-infusion CARs, including CD52, CD74, CD86, and LAG3, among others.Abstract 152 Figure 1Clustering of 184, 791 CAR-transduced T cells based on gene expressionAbstract 152 Figure 2Alluvial plot depicting CAR T cell lineage tracing using the endogenous T cell receptorAbstract 152 Figure 3Visualization of CAR T cell clusters with arrows indicating the shared TCRs between pre-infusion and post-infusion cellsAbstract 152 Figure 4Machine learning classifier of pre-infusion, early effector CAR T cell phenotypeConclusionsOur findings suggest a therapeutic approach that enriches these cells prior to infusion resulting in superior per cell CAR effector activity.ReferenceXu X, Huang S, Xiao X, Sun Q, Liang X, Chen S, et al. Challenges and Clinical Strategies of CAR T-cell Therapy for Acute Lymphoblastic Leukemia: Overview and Developments. Front Immunol 2020;11:569117.Ethics ApprovalThis study was approved by St. Jude Children’s Research Hospital’s Institutional Review Board (IRB); IRB number Pro00007661. All patients consented to the use of materials for the research study.

Fibrolamellar carcinoma (FLC) is an often-lethal liver malignancy of adolescents and young adults. No systemic therapies are currently approved for FLC, so novel treatments are needed to improve patient outcomes. A promising therapeutic vulnerability in FLC is a highly-conserved gene fusion between DNAJB1 and PRKACA. The resultant fusion protein is identical in more than 90% of patients, making it a potentially ideal neoantigen target for T cell-based immunotherapy. The goal of our study was to test if DNAJB1-PRKACA neoantigens could elicit T cell responses and serve as targets for immunotherapy. Spatial transcriptomic analyses of FLC tumor tissue indicated that CD8 T cells could infiltrate FLC tumors, but expressed exhaustion markers such as TOX. Nevertheless, we expanded T cells from an FLC patient tumor ex vivo and detected T cells that produced IFNγ and TNFα in response to stimulation with a fusion neoantigen. We then used functional response after peptide stimulation or peptide-MHC tetramer staining to identify fusion-specific T cell receptors (TCRs) in both FLC patients and healthy donors. We expressed these TCRs in primary human T cells and found that cells expressing fusion-specific TCRs bound to their cognate tetramer and produced IFNγ, TNFα, and IL-2 in response to stimulation with their cognate peptide. TCR-expressing cells also specifically killed target cells presenting their cognate fusion peptide in vitro. Ongoing experiments will test if cells expressing these TCRs can control growth of fusion-expressing tumors in vivo. Together, these studies have defined the first reported fusion-specific T cell response in an FLC patient, as well as fusion-specific TCRs that hold promise for development in adoptive T cell therapies. Supported by grants from NIH (R01 AI136514, F31 CA254423) and The Mark Foundation for Cancer Research (Aspire Award)

SARS-CoV-2 mRNA vaccines are effective for COVID-19 prevention, eliciting both robust antibody responses in naive individuals and boosting antibody levels in convalescent donors. However, the effect of repeated antigen exposures, such as vaccination following infection or breakthrough infections, on the magnitude, repertoire, and phenotype of pre-existing memory T cells, is still poorly understood. Thus, we compared epitope-specific CD8 T cells elicited after SARS-CoV-2 infection, vaccination of both naive and recovered individuals, and breakthrough infection cases. We used pools of 18 DNA-barcoded MHC-class I multimers, combined with scRNAseq and scTCRseq, to characterize T cell responses as defined by magnitude, specificity, T cell receptor (TCR) repertoire, and gene expression profile to both spike-derived and non-spike derived epitopes. In-depth analysis of over 4000 unique epitope-specific TCR sequences demonstrates that both vaccination and infection, including breakthrough cases, elicit identical repertoires as measured by dominant TCR motifs and repertoire diversity, indicating that BNT162b2 vaccination largely recapitulates spike-specific T cell repertoire generation by infection. Importantly, in COVID-19-recovered individuals receiving the vaccine, pre-existing spike-specific memory cells showed both clonal expansion and a phenotypic shift towards more differentiated CCR7-CD45RA+ effector cells, demonstrating the potency of this vaccine to recall spike-specific CD8 memory T cells. Importantly, in breakthrough infections, we observed a high proportion of T cells targeting non-spike epitopes, showing that new immune memory could be formed during SARS-CoV-2 infection after vaccination. The work was funded by 75N93019C00052, HHSN272201400006C, 3U01AI144616-02S1, R01AI136514

BackgroundClinically similar older adults demonstrate variable responses to health stressors, heterogeneity attributable to differences in physical resilience. However, molecular mechanisms underlying physical resilience are unknown. We previously derived a measure of physical resilience after hip fracture—the expected recovery differential (ERD)—that captures the difference between actual recovery and predicted recovery. Starting with biomarkers associated with physical performance, morbidity, mortality, and hip fracture, we evaluated associations with the ERD to identify biomarkers of physical resilience after hip fracture.MethodsIn the Baltimore Hip Studies (N = 304) sera, we quantified biomarkers of inflammation (TNFR-I, TNFR-II, sVCAM-1, and IL-6), metabolic and mitochondrial function (non-esterified fatty acids, lactate, ketones, acylcarnitines, free amino acids, and IGF-1), and epigenetic dysregulation (circulating microRNAs). We used principal component analysis, canonical correlation, and least absolute shrinkage and selection operator regression (LASSO) to identify biomarker associations with better-than-expected recovery (greater ERD) after hip fracture.ResultsParticipants with greater ERD were more likely to be women and less disabled at baseline. The complete biomarker set explained 37% of the variance in ERD (p < .001) by canonical correlation. LASSO regression identified a biomarker subset that accounted for 27% of the total variance in the ERD and included a metabolic factor (aspartate/asparagine, C22, C5:1, lactate, glutamate/mine), TNFR-I, miR-376a-3p, and miR-16-5p.ConclusionsWe identified a set of biomarkers that explained 27% of the variance in ERD—a measure of physical resilience after hip fracture. These ERD-associated biomarkers may be useful in predicting physical resilience in older adults facing hip fracture and other acute health stressors.

We elucidated the molecular cross-talk between cartilage and synovium in osteoarthritis, the most widespread arthritis in the world, using the powerful tool of single-cell RNA-sequencing. Multiple cell types were identified based on profiling of 10,640 synoviocytes and 26,192 chondrocytes: 12 distinct synovial cell types and 7 distinct articular chondrocyte phenotypes from matched tissues. Intact cartilage was enriched for homeostatic and hypertrophic chondrocytes, while damaged cartilage was enriched for prefibro- and fibro-, regulatory, reparative and prehypertrophic chondrocytes. A total of 61 cytokines and growth factors were predicted to regulate the 7 chondrocyte cell phenotypes. Based on production by > 1% of cells, 55% of the cytokines were produced by synovial cells (39% exclusive to synoviocytes and not expressed by chondrocytes) and their presence in osteoarthritic synovial fluid confirmed. The synoviocytes producing IL-1beta (a classic pathogenic cytokine in osteoarthritis), mainly inflammatory macrophages and dendritic cells, were characterized by co-expression of surface proteins corresponding to HLA-DQA1, HLA-DQA2, OLR1 or TLR2. Strategies to deplete these pathogenic intra-articular cell subpopulations could be a therapeutic option for human osteoarthritis.

Background. The majority of patients develop posttraumatic osteoarthritis within 15 years of anterior cruciate ligament (ACL) injury. Inflammatory and chondrodegenerative biomarkers have been associated with both pain and the progression of osteoarthritis; however, it remains unclear if preoperative biomarkers differ for patients with inferior postoperative outcomes. Hypothesis/Purpose. The purpose of this pilot study was to compare biomarkers collected on the day of ACL reconstruction between patients with “good” or “poor” 2-year postoperative outcomes. We hypothesized that inflammatory cytokines and chondrodegenerative biomarker concentrations would be significantly greater in patients with poorer outcomes. Study Design. Prospective cohort design. Methods. 22 patients (9 females, 13 males; age = 19.5±4.1 years; BMI = 24.1±3.6 kg/m2) previously enrolled in a randomized trial evaluating early anti-inflammatory treatment after ACL injury. Biomarkers of chondrodegeneration and inflammation were assessed from synovial fluid (sf) samples collected on the day of ACL reconstruction. Participants completed Knee Injury and Osteoarthritis Outcome Score (KOOS) and International Knee Documentation Committee (IKDC) questionnaires two years following surgery. Patients were then categorized based on whether their KOOS Quality of Life (QOL) score surpassed the Patient Acceptable Symptom State (PASS) threshold of 62.5 points or the IKDC PASS threshold of 75.9 points. Results. Patients that failed to reach the QOL PASS threshold after surgery (n = 6, 27%) had significantly greater sf interleukin-1 alpha (IL-1α; p = 0.004), IL-1 receptor antagonist (IL-1ra; p = 0.03), and matrix metalloproteinase-9 (MMP-9; p = 0.01) concentrations on the day of surgery. Patients that failed to reach the IKDC PASS threshold (n = 9, 41%) had significantly greater sf IL-1α (p = 0.02). Conclusion. These pilot data suggest that initial biochemical changes after injury may be an indicator of poor outcomes that are not mitigated by surgical stabilization alone. Biological adjuvant treatment in addition to ACL reconstruction may be beneficial; however, these data should be used for hypothesis generation and more definitive randomized clinical trials are necessary.

CBFA2T3-GLIS2 is an oncogenic fusion gene that acts as the driver mutation in ~18% of pediatric patients with acute megakaryoblastic leukemia (AMKL) that do not have Down syndrome. CBFA2T3-GLIS2 driven leukemia carries the worst prognosis among all pediatric acute myeloid (AML) subtypes, with event free and overall survival probabilities of 8% and 14% respectively. Chemotherapy and stem cell transplantation fail to cure this disease, thus innovative treatment approaches are needed. While monoclonal antibody and chimeric antigen receptor T-cell (CAR-T) therapies have proven successful in acute lymphoblastic leukemia (ALL), similar successes haven't been realized in AML due to the lack of targetable antigens that eradicate leukemia cells while minimizing off target toxicities such as depletion of normal myeloid cells which can lead to prolonged neutropenia. An alternative is T cell receptor engineered T cell (TCR-T) immunotherapy which use heterodimers consisting of alpha and beta peptide chains to recognize polypeptide fragments presented by MHC molecules on the tumor cells. An advantage of this approach is the ability to recognize intracellular tumor specific and tumor associated antigen fragments in addition to extracellular proteins which results in a wider range of targets. To investigate the immunogenicity of CBFA2T3-GLIS2 positive AMKL cells, we established a humanized patient derived xenograft (PDX) murine model by conditioning 15 immunodeficient NSG-SGM3 mice with 200 rads 24 hours prior to transplantation of 3.5 million CBFA2T3-GLIS2 positive PDX cells derived from an individual female patient. After 20 days of engraftment, 2.5 million HLA class I matched PBMCs from an unrelated female donor were introduced. Experimental mice (engrafted with both PDX cells and PBMCs), leukemia only control mice, and PBMC only control mice were sacrificed at days 30, 35, and 40. Flow cytometry analysis at day 40 showed dramatic reduction of leukemic blasts (huCD45lo, huCD34+) when exposed to PBMCs compared to the leukemia only control (1.48% and 93.7%, respectively). Circulating human Granzyme A was elevated at day 40 in the PDX mice with PBMCs compared to the PBMC only control (9665.4 pg/ml vs. 789.7 pg/ml, p Disclosures Gruber: Kura Oncology: Consultancy.

ObjectivesEmerging evidence suggests that the microbiome plays an important role in the pathogenesis of osteoarthritis (OA). We aimed to test the two-hit model of OA pathogenesis and potentiation in which one ‘hit’ is provided by an adverse gut microbiome that activates innate immunity; the other ‘hit’ is underlying joint damage.MethodsMedical history, faecal and blood samples were collected from human healthy controls (OA-METS-, n=4), knee OA without metabolic syndrome (OA+METS-, n=7) and knee OA with metabolic syndrome (OA+METS+, n=9). Each group of human faecal samples, whose microbial composition was identified by 16S rRNA sequencing, was pooled and transplanted into germ-free mice 2 weeks prior to meniscal/ligamentous injury (MLI) (n≥6 per group). Eight weeks after MLI, mice were evaluated for histological OA severity and synovitis, systemic inflammation and gut permeability.ResultsHistological OA severity following MLI was minimal in germ-free mice. Compared with the other groups, transplantation with the OA+METS+ microbiome was associated with higher mean systemic concentrations of inflammatory biomarkers (interleukin-1β, interleukin-6 and macrophage inflammatory protein-1α), higher gut permeability and worse OA severity. A greater abundance of Fusobacterium and Faecalibaterium and lesser abundance of Ruminococcaceae in transplanted mice were consistently correlated with OA severity and systemic biomarkers concentrations.ConclusionThe study clearly establishes a direct gut microbiome-OA connection that sets the stage for a new means of exploring OA pathogenesis and potentially new OA therapeutics. Alterations of Fusobacterium, Faecalibaterium and Ruminococcaceae suggest a role of these particular microbes in exacerbating OA.

To explore mechanisms underlying the association of TSG-6 with osteoarthritis (OA) progression.TSG-6-mediated heavy chain (HC) transfer (TSG-6 activity) and its association with inflammatory mediators were quantified in knee OA (n=25) synovial fluids (SFs). Paired intact and damaged cartilages from the same individuals (20 tibial and 12 meniscal) were analyzed by qRT-PCR and immunohistochemistry (IHC) for gene and protein expression of TSG-6 and components of Inter-alpha-Inhibitor (IαI) and TSG-6 activity ± spiked in IαI. Primary chondrocyte cultures (n=5) ± IL1β or TNFα were evaluated for gene expression. The effects of TSG-6 activity on cartilage extracellular matrix (ECM) assembly were explored using quantitative hyaluronan (HA)-aggrecan binding assays.TSG-6 activity was significantly associated (R 0.683, P 0.0002) with inflammatory mediators including TIMP-1, A2M, MMP3, VEGF, VCAM-1, ICAM-1 and IL-6. Although TSG-6 protein and mRNA were highly expressed in damaged articular and meniscal cartilage and cytokine-treated chondrocytes, there was little or no cartilage expression of components of the IαI complex (containing HC1). By IHC, TSG-6 was present throughout lesioned cartilage but HC1 only at lesioned surfaces. TSG-6 impaired HA-aggrecan assembly, but TSG-6 mediated HA-HC formation reduced this negative effect.TSG-6 activity is a global inflammatory biomarker in knee OA SF. IαI, supplied from outside cartilage, only penetrates the cartilage surface, restricting TSG-6 activity (HC transfer) to this region. Therefore, unopposed TSG-6 in intermediate and deep regions of OA cartilage could possibly block matrix assembly, leading to futile synthesis and account for increased risk of OA progression.

Osteoarthritis (OA) is a common disease with a genetic component for its etiology. Recently, a genetic association of a single nucleotide polymorphism (SNP), rs17039192 in HIF-2α with knee OA has been reported in a Japanese population; however, controversy exits for its replication and a role of HIF-2α in OA. This study aimed to evaluate the association of the SNP by a large-scale replication study. A total of 8,457 subjects (3,129 OA cases and 5,328 controls) from seven independent cohorts from six countries (Japan, China, Taiwan, Korea, Greece, and Australia) were recruited and genotyped. The association of rs17039192 with knee OA was evaluated by meta-analyses. The association of the HIF-2α SNP was not replicated in any of the populations. Contrary to the previous report, the odds ratios (ORs) of the risk allele frequency were all less than 1. A combined analysis for the seven populations also showed no replication of the association (OR = 0.91, 95% confidence interval = 0.81-1.03). Our large-scale meta-analysis showed that the association of rs17039192 in HIF-2α with knee OA is negative. The significance of HIF-2α in human OA (idiopathic OA as a common disease) should be further evaluated carefully.

We performed a genome-wide linkage analysis to identify susceptibility loci in a large six-generation extended family previously reported with early-onset osteoarthritis (OA) DNA sequencing was performed to investigate involvement of the COMP (Cartilage oligomeric matrix protein) gene in this family. The region covering D19S884, D19S226, and D19S414 on chromosome 19p following genome-wide scan from 70 individuals of this kindred showed significant linkage, with a maximum point LOD (logarithm of the odds ratio) score of 2.51 at D19S226. Direct sequencing of the COMP gene, the most plausible candidate gene in the region, identified a c.2152C>T substitution in exon 18 which resulted in a substitution of tryptophan for arginine at position 718 located in the C terminal globular domain of the gene product. A total of 26 individuals were identified with this mutation of which 21 affected individuals had the mutation, and the other five younger individuals (18.6 ± 11.3 years of age) carried the mutation without symptoms. The results indicate that COMP is the disease susceptibility gene and the c.2152C>T mutation in exon 18 could cause early-onset OA phenotypes in this kindred, which is compatible with a previous report that this mutation also causes a mild form of multiple epiphyseal dysplasia (MED).

Deficiency of the collagen receptor discoidin domain receptor tyrosine kinase (DDR2) in mice and humans results in dwarfism and short limbs, of which the mechanism remains unknown. Here we report that DDR2 is a key regulator of osteoblast differentiation. DDR2 mRNA expression was increased at an early stage of induced osteoblast differentiation. In the subchondral bone of human osteoarthritic knee, DDR2 was detected in osteoblastic cells. In mouse embryos, DDR2 expression was found from E11 to E15, preceding osteocalcin (OCN) and coinciding with Runx2 expression. Activating transcription factor 4 (ATF4) enhanced DDR2 mRNA expression, and knockdown of ATF4 expression delayed DDR2 induction during osteoblast differentiation. A CCAAT/enhancer binding protein (C/EBP) binding site at −1150 bp in the DDR2 promoter was required for ATF4-mediated DDR2 activation. C/EBPβ bound to and cooperated with ATF4 in stimulating DDR2 transcription; accordingly, the ATF4 mutants deficient of C/EBPβ binding were incapable of transactivating DDR2. Overexpression of DDR2 increased osteoblast-specific gene expression. Conversely, knockdown of DDR2 suppressed osteogenic marker gene expression and matrix mineralization during the induced osteogenesis. The stimulation of p38 MAPK by DDR2 was required for DDR2-induced activation of Runx2 and OCN promoters. Together our findings uncover a pathway in which ATF4, by binding to C/EBPβ transcriptionally upregulates DDR2 expression, and DDR2, in turn, activates Runx2 through p38 MAPK to promote osteoblast differentiation. © 2010 American Society for Bone and Mineral Research.

Cleidocranial dysplasia (CCD; MIM 119600) is a rare autosomal dominant disorder characterized by facial, dental, and skeletal malformations. To date, rearrangement and mutations involving RUNX2, which encodes a transcription factor required for osteoblast differentiation on 6p21, has been the only known molecular etiology for CCD. However, only 70% patients were found to have point mutations, 13% large/contiguous deletion but the rest of 17% remains unknown. We ascertained a family consisted of eight affected individuals with CCD phenotypes. Direct sequencing analysis revealed no mutations in the RUNX2. Real time quantitative PCR were performed which revealed an exon 2 to exon 6 intragenic deletion in RUNX2. Our patients not only demonstrated a unique gene change as a novel mechanism for CCD, but also highlight the importance of considering “deletion” and “duplication” in suspected familial cases before extensive effort of gene hunting be carried.

s / Osteoarthritis and Cartilage 20 (2012) S54–S296 S199 Therefore, a very sensitive ELISA was established but again no differential binding to the two different binding sites could be observed using ETS-1 antibodies in competitive displacement experiments. ChIP assays using the ETS-1 antibodies also showed no divers binding to the two variants. Conclusions: As all assays showed no differential binding of ETS-1 to the two variants, it can be excluded that ETS-1 is the responsible factor for the increased amount of N-cadherin in OASF. Now zinc finger transcription factors, especially ZNF35, and their potential effect on N-cadherin expression levels in OASF are analyzed. Furthermore, the function of high N-cadherin levels expressed by OASF in contrast to the protective effect of even higher N-cadherin levels in patients carrying the minor SNP variant has to be examined. In summary, these results show that the influence of N-cadherin on the aggressiveness of OASF is complex and requires further investigation. 401 IDENTIFICATION OF GENES INVOLVED IN THE INITIATION OF OSTEOARTHRITIS C.-H. Chou , C. Wu , L.-S. Lu , J.-Y. Wu , Y.-T. Chen , M. Lee . Academia sinica, Taipei, Taiwan; Dept. of Orthopaedic Surgery Tri-Service Gen. Hosp., Natl. Defense Med. Ctr., Taipei, Taiwan Objective: Osteoarthritis (OA) is the most prevalent form of arthritis and is characterized primarily by the degeneration of articular cartilage. Several gene expression studies have been performed to identify genes involve in the pathogenesis of OA. However, in order to obtain sufficient quantity and quality RNA for gene expression study, large regions of cartilage are often required. Cartilage is a very heterogeneous tissue, the cartilage used in prior studies may appear normal but they might consist of OA at different stages (different severity). The aim of this study is to select small areas from osteoarthritic cartilages which represent different severity to provide a more complete picture of the molecular alternations in OA pathogenesis as well as to identify genes involved in the initiation of OA. Methods: Joint tissues were collected from the knee tibia plateau from primary OA and non-OA patients undergoing total knee arthroplasty. Severity of destructionwas estimated based on histopathology assessment (OARSI grading system). Each tibia plateau was divided into three parts: outer lateral tibia (oLT) regions defined as undamaged stage (OARSI score: OA1⁄45.23 1.95, n1⁄467; Normal1⁄4 2 2, n1⁄45), inner lateral tibia (iLT) regions defined as intermediate stage (OARSI score: OA1⁄45.23 1.95, n1⁄471; Normal1⁄4 4, n1⁄45), and medial tibia (MT) regions defined as damage stage (OARSI score: OA1⁄416.8 2.56, n1⁄452; Normal1⁄4 4.8 1.09, n1⁄45). Expression profiling analysis was performed using Agilent microarray (OA: n1⁄417 at oLT, n1⁄413 at iLT, n1⁄412 at MT; and n1⁄4 4 from non-OA at the three regions) and real-time quantitative PCR using a second cohort of patients were performed for replication. Results:Our results revealed that 958 transcripts were significantly up or down regulated at least 2-fold between these three stages. These genes were related to the cell matrix interaction, extracellular matrix remodeling, bone development, inflammation, cytokine, cell proliferation, WNT signaling. Conclusion: This study revealed some novel genes which have not been reported in cartilage to play a role in the pathology of OA. These results identify molecular targets that can be further investigated in the search for therapy or as biomarker for OA. 402 GENETIC POLYMORPHISM IN GDF-5 GENE AS RISK FACTOR FOR DEVELOPMENT AND PROGRESSION OF OSTEOARTHRITIS KNEE A. Mishra, Jr., D. Sanghi, Jr., S. Avasthi, Jr., R.N. Srivastava, Sr.. C.S.M.Med. Univ. (Upgraded K.G's.Med. Coll.) Lucknow, India Purpose: In a case-control study, investigate the association of SNP in GDF5 gene with osteoarthritis knee Methods: In a case-control study, 300 cases with knee osteoarthritis and an equal number of age, gender matched healthy controls were included. Cases were diagnosed using the ACR Guidelines of knee osteoarthritis (KOA). Clinical symptoms were assessed with the knee specific WOMAC index and VAS for knee pain. The severity of disease was determined by radiological KL grades (Kellgren Lawren). The informed consent of the patients was obtained for the participation in this study. The study was approved by the Institutional Ethics Committee. The genomic DNA samples were isolated from blood and polymorphic study was done by polymerase chain reaction (PCR) with restriction fragments length polymorphism (RFLP). All statistical analysis was performed with the SPSS software package (version 16.0 for windows; SPSS Chicago, IL). Results: The GDF-5 (BSiE1) genotypes were found to be present at significantly higher frequency in cases than in controls, resulting in about 1.62 fold increase of OA risk (P Value1⁄40.040). OA knee was found to be significantly associated with BMI (P Value1⁄40.00). A significant association was found with clinical score of knee OA VAS with poor and good index (P value1⁄40.010 and 0.026 respectively) and in WOMAC with poor index only (P value1⁄40.0040). On stratifying all osteoarthritis subjects into 3 groups according to severity (KL grade 2 minimal, grade 3 moderate and grade 4 severe OA), no significant association was found. Conclusions: GDF5, are now known to be consistently associated with the risk of knee OA, in different population. An association between the +104T/ C GDF5 polymorphismwith knee OA in Indian population further confirms a strong genetic influence of this SNP in KOA. This can serve as a potential biomarker and a risk factor for KOA. It may become a gateway for further research into epigenetic of this SNP, highlighting potential pathways for prevention and therapeutic intervention of knee osteoarthritis Imaging & Joint Morphometry 403 JOINT SHAPE AS A PREDICTOR OF END-STAGE OSTEOARTHRITIS OF THE HIP: A 19 YEAR RETROSPECTIVE ANALYSIS OF THE CHINGFORD STUDY. J.E. Jeffrey , R.J. Barr , C.P. Arden , D.J. Hart , G.E. Thomas , S. Garden , T.D. Spector , R.M. Aspden , N.K. Arden , J.S. Gregory . Univ. of Aberdeen, Aberdeen, United Kingdom; Univ. of Oxford, Oxford, United Kingdom; King's Coll., London, United Kingdom Purpose: Abnormalities in the shape of the hip joint are thought to be important factors in the development of osteoarthritis (OA) of the hip. Quantifying these changes in shape may help us to understand their role in the progression of the disease. Active Shape Modelling (ASM) of the hip enables sensitive quantification of changes in hip morphology. In this study ASM was used to examine the relationship of hip shape and the risk of end-stage OA in a group of women who have undergone total hip arthroplasty (THA). The results were compared with a previous study by Nicholls et al. where morphological parameters of the hip were measured in the same subjects with Hip Morf 2.0 software. Methods: 44 women (aged 45 65) were selected from 1003 participants in the Chingford Study. The subjects belonged to 2 groups: 22 who had undergone THA by year 20 of the study (THA group) and 22 randomly selected controls who were THA free in both hips by year 20 (control group). Pelvic radiographs, taken at year 2 of the study, were examined. A 66 point ASM template was applied to one hip joint from each radiograph that included the proximal femur, osteophytes and part of the pelvis. The first 10 scores of shape variance, or mode scores, were calculated for each subject. T-tests and logistic regression were used to compare differences between the control and THA groups. Pearson correlation was used to compare the results of the ASM and Hip Morf outputs (SPSS v19). Results:Mode 6 score was significantly higher in the THA group compared with the control group, (P1⁄40.02), as seen in the figure. This was still significant after adjusting for age, height, weight and Kellgren Lawrence Grade, (P1⁄40.04), odds ratio 3.3 (95% CI 1.1-10.2). A higher Mode 6 scorewas significantly correlated with several morphological parameters from the previous study (P

Deficiency of the collagen receptor discoidin domain receptor tyrosine kinase (DDR2) in mice and humans results in dwarfism and short limbs, of which the mechanism remains unknown. Here we report that DDR2 is a key regulator of osteoblast differentiation. DDR2 mRNA expression was increased at an early stage of induced osteoblast differentiation. In the subchondral bone of human osteoarthritic knee, DDR2 was detected in osteoblastic cells. In mouse embryos, DDR2 expression was found from E11 to E15, preceding osteocalcin (OCN) and coinciding with Runx2 expression. Activating transcription factor 4 (ATF4) enhanced DDR2 mRNA expression, and knockdown of ATF4 expression delayed DDR2 induction during osteoblast differentiation. A CCAAT/enhancer binding protein (C/EBP) binding site at -1150 bp in the DDR2 promoter was required for ATF4-mediated DDR2 activation. C/EBPβ bound to and cooperated with ATF4 in stimulating DDR2 transcription; accordingly, the ATF4 mutants deficient of C/EBPβ binding were incapable of transactivating DDR2. Overexpression of DDR2 increased osteoblast-specific gene expression. Conversely, knockdown of DDR2 suppressed osteogenic marker gene expression and matrix mineralization during the induced osteogenesis. The stimulation of p38 MAPK by DDR2 was required for DDR2-induced activation of Runx2 and OCN promoters. Together our findings uncover a pathway in which ATF4, by binding to C/EBPβ transcriptionally upregulates DDR2 expression, and DDR2, in turn, activates Runx2 through p38 MAPK to promote osteoblast differentiation.

We report the first genome-wide association study in 1000 bipolar I patients and 1000 controls, with a replication of the top hits in another 409 cases and 1000 controls in the Han Chinese population. Four regions with most strongly associated single-nucleotide polymorphisms (SNPs) were detected, of which three were not found in previous GWA studies in the Caucasian populations. Among them, SNPs close to specificity protein 8 (SP8) and ST8 α-N-acetyl- neuraminide α-2,8-sialyltransferase (ST8SIA2) are associated with Bipolar I, with P-values of 4.87 × 10(-7) (rs2709736) and 6.05 × 10(-6) (rs8040009), respectively. We have also identified SNPs in potassium channel tetramerization domain containing 12 gene (KCTD12) (rs2073831, P=9.74 × 10(-6)) and in CACNB2 (Calcium channel, voltage-dependent, β-2 subunit) gene (rs11013860, P=5.15 × 10(-5)), One SNP nearby the rs1938526 SNP of ANK3 gene and another SNP nearby the SNP rs11720452 in chromosome 3 reported in previous GWA studies also showed suggestive association in this study (P=6.55 × 10(-5) and P=1.48 × 10(-5), respectively). This may suggest that there are common and population-specific susceptibility genes for bipolar I disorder.

Warfarin, a widely prescribed oral anticoagulant, is used for the prevention of thromboembolism. Polymorphisms in CYP2C9 and VKORC1 have been shown to be associated with warfarin dose requirements. However, it is likely that other genes could also affect warfarin dose. Aims: In this study, we aimed to identify additional genes influencing warfarin dosing in the Han-Chinese population. Materials & methods: In this study, we screened for SNPs in 13 genes (VKORC1, CYP2C9, CYP2C18, PROC, APOE, EPHX1, CALU, GGCX, ORM1, ORM2, factor II, factor VII and CYP4F2) and tested their associations with warfarin dosing with univariate and multiple regression analysis. Results: Polymorphisms in the VKORC1 gene have the strongest effects on warfarin dose, followed by CYP2C9*3. In addition, our results showed that CYP2C18, PROC and EPHX1 have small but significant associations with warfarin dose. In multiple regression analysis, PROC and EPHX1 explained 3% of the dose variation. The incorporation of these two genes into warfarin dosing algorithms could improve the accuracy of prediction in the Han-Chinese population.

Aims: Warfarin, a widely prescribed oral anticoagulant, is used for the prevention of thromboembolism. Several polymorphisms in VKORC1 have been shown to be associated with warfarin dose requirements. The frequencies of these VKORC1 polymorphisms display population differences; however, this has not been examined in many populations. In this study, we examined VKORC1 polymorphisms in five East-Asian populations (Han Chinese, Indonesian, Filipino, Thai and Vietnamese) and Indians. Materials & methods: A total of six SNPs in the VKORC1 gene (-1639G>A, 497T>G, 1173C>T, 1542T>G, 2255C>T and 3730G>A) were genotyped. Frequencies, linkage disequilibrium and haplotype structures of these six VKORC1 SNPs were analyzed. Results: Our data showed that 497T>G is only polymorphic in the Indian population. The 497G allele is very rare in the East-Asian populations (frequency < 1%). The remaining SNPs demonstrated high linkage disequilibrium and had similar frequencies and haplotype structures in all but the Indian population. The Indian population is mostly made up of the H7 haplotype (76%) while the rest of the recruited populations consisted of the H1 haplotype (>80%).

The difficulties in identifying the precise defect location and real leakage path is increasing as the integrated circuit design and process have become more and more complicated in nano scale technology node. Most of the defects causing chip leakage are detectable with only one of the FA (Failure Analysis) tools such as LCD (Liquid Crystal Detection) or PEM (Photon Emission Microscope). However, due to marginality of process-design interaction some defects are often not detectable with only one FA tool [1][2]. This paper present an example of an abnormal power consumption process-design interaction related defect which could only be detected with more advanced FA tools.

Summary Objective To evaluate the interaction of articular cartilage (AC) and subchondral bone (SB) through analysis of osteoarthritis (OA)-related genes of site-matched tissue. Design We developed a novel method for isolating site-matched overlying AC and underlying SB from three and four regions of interest respectively from the human knee tibial plateau ( n = 50). For each site, the severity of cartilage changes of OA were assessed histologically, and the severity of bone abnormalities were assessed by microcomputed tomography. An RNA isolation procedure was optimized that yielded high quality RNA from site-matched AC and SB tibial regions. Quantitative polymerase chain reaction (Q-PCR) analysis was performed to evaluate gene expression of 61 OA-associated genes for correlation with cartilage integrity and bone structure parameters. Results A total of 27 (44%) genes were coordinately up- or down-regulated in both tissues. The expression levels of 19 genes were statistically significantly correlated with the severity of AC degeneration and changes of SB structure; these included: ADAMTS1 , ASPN , BMP6 , BMPER , CCL2 , CCL8 , COL5A1 , COL6A3 , COL7A1 , COL16A1 , FRZB , GDF10 , MMP3 , OGN , OMD , POSTN , PTGES , TNFSF11 and WNT1 . Conclusions These results provide a strategy for identifying targets whose modification may have the potential to ameliorate pathological alterations and progression of disease in both AC and SB simultaneously. In addition, this is the first study, to our knowledge, to overcome the major difficulties related to isolation of high quality RNA from site-matched joint tissues. We expect this method to facilitate advances in our understanding of the coordinated molecular responses of the whole joint organ.

Summary Objective To identify disease relevant genes and pathways associated with knee Osteoarthritis (OA) progression in human subjects using medial and lateral compartment dominant OA knee tissue. Design Gene expression of knee cartilage was comprehensively assessed for three regions of interest from human medial dominant OA ( n = 10) and non-OA ( n = 6) specimens. Histology and gene expression were compared for the regions with minimal degeneration, moderate degeneration and significant degeneration. Agilent whole-genome microarray was performed and data were analyzed using Agilent GeneSpring GX11.5. Significant differentially regulated genes were further investigated by Ingenuity Pathway Analysis (IPA) to identify functional categories. To confirm their association with disease severity as opposed to site within the knee, 30 differentially expressed genes, identified by microarray, were analyzed by quantitative reverse-transcription polymerase chain reaction on additional medial ( n = 16) and lateral ( n = 10) compartment dominant knee OA samples. Results A total of 767 genes were differentially expressed ≥two-fold ( P ≤ 0.05) in lesion compared to relatively intact regions. Analysis of these data by IPA predicted biological functions related to an imbalance of anabolism and catabolism of cartilage matrix components. Up-regulated expression of IL11, POSTN, TNFAIP6, and down-regulated expression of CHRDL2, MATN4, SPOCK3, VIT, PDE3B were significantly associated with OA progression and validated in both medial and lateral compartment dominant OA samples. Conclusions Our study provides a strategy for identifying targets whose modification may have the potential to ameliorate pathological alternations and progression of disease in cartilage and to serve as biomarkers for identifying individuals susceptible to progression.

Introduction The aim of this study was to evaluate, for the first time, the differences in gene expression profiles of normal and osteoarthritic (OA) subchondral bone in human subjects. Methods Following histological assessment of the integrity of overlying cartilage and the severity of bone abnormality by micro-computed tomography, we isolated total RNA from regions of interest from human OA (n = 20) and non-OA (n = 5) knee lateral tibial (LT) and medial tibial (MT) plateaus. A whole-genome profiling study was performed on an Agilent microarray platform and analyzed using Agilent GeneSpring GX11.5. Confirmatory quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis was performed on samples from 9 OA individuals to confirm differential expression of 85 genes identified by microarray. Ingenuity Pathway Analysis (IPA) was used to investigate canonical pathways and immunohistochemical staining was performed to validate protein expression levels in samples. Results A total of 972 differentially expressed genes were identified (fold change ≥ ± 2, P ≤0.05) between LT (minimal degeneration) and MT (significant degeneration) regions from OA samples; these data implicated 279 canonical pathways in IPA. The qRT-PCR data strongly confirmed the accuracy of microarray results (R2 = 0.58, P