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SummaryMonoclonal antibodies that bind and inhibit nerve growth factor (NGF) have demonstrated both, good analgesic efficacy and improvement in function in patients with osteoarthritis (OA). Despite initial promising data, trials in OA had been suspended by the Federal Food and Drug Administration (FDA) due to concerns over accelerated rates of OA progression. Imaging will play a crucial role in future clinical trials to define eligibility of potential participants and to monitor safety during the course of these studies. This will require baseline and frequent follow-up radiographs of both, the index joints and other large weight bearing joints to identify subjects at risk prior inclusion and on study so treatment can be discontinued.This imaging overview in the form of an atlas describes and illustrates potential exclusionary joint imaging findings at eligibility and potential adverse joint events on radiography and magnetic resonance imaging (MRI) in studies investigating a-NGF compounds. The overarching goal of this atlas is to facilitate trial design and to promote a common language and understanding between potential expert readers. This first section of the atlas will focus on knee joint specific findings that are relevant to a-NGF studies.

s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A360 eligibility and safety findings relevant for aNGF clinical trials. More than 400 images were reviewed to define the most relevant and characteristic imaging findings. Diagnoses of exclusion for eligibility due to potentially increased risk of RPOA are subchondral insufficiency fractures (SIF), atrophic OA and severe malalignment of the knee in the medial-lateral direction. Diagnoses relevant for safety after enrolment (i.e. joint safety findings), i.e. RPOA, SIF, osteonecrosis and pathologic fractures will also be discussed. Several of these diagnoses have non-specific findings on the radiograph or cannot be detected radiographically in early stages. Thus, in cases of inconclusive or suspicious radiography, an additional MRI examination may be acquired to rule out or confirm some of these diagnoses. Results: Early and late signs of diagnoses relevant for eligibility will be presented. Fig 3A. Radiograph of the hip joint shows superior JSN (arrows) without relevant osteophytes present. Fig 3B. Follow up image 12 months later shows near-complete destruction of the femoral head (arrows) and subsequent migration of the femur superiorly consistent with RPOA. Conclusions: Expert readers in aNGF programs need to be aware of relevant imaging findings. These include early signs of diagnoses relevant for RPOA on radiography, and other potential X-ray and MRI diagnoses relevant for eligibility and safety during aNGF studies. 591 PROLONGED JOINT RESIDENCY OF TRIAMCINOLONE ACETONIDE AFTER AN INTRA-ARTICULAR INJECTION OF FX006, A SUSTAINED RELEASE FORMULATION FOR THE TREATMENT OF OSTEOARTHRITIS N. Bodick y, J. Lufkin y, C. Willwerth y, J. Hauben y, A. Kumar y, P. Boen y, J. Bolognese z, C. Schoonmaker z, M. Clayman y. y Flexion Therapeutics, Burlington, MA, USA; zCytel Inc, Cambridge, MA, USA Purpose: Although available intra-articular (IA) corticosteroids are well established in the treatment of painful osteoarthritis (OA), limitations include short duration of pain relief (1-4 weeks), an effect consistent with the transient residency of these compounds in the joint and high plasma corticosteroid concentrations immediately following injection. FX006 is a novel IA sustained-release injectable formulation of 25% triamcinolone acetonide (TCA) in poly(lactic-co-glycolic acid) microspheres intended to prolong therapeutic concentrations of TCA in the joint and limit systemic exposure. In a previously described 6-week long clinical study, FX006 at 40 mg demonstrated maintenance of synovial concentrations of TCA consistent with pharmacologic activity for a period of at least 6 weeks, when levels of TCA produced by an equivalent dose of TCA IR were below the limit of quantitation. The current study extended these data on joint residency by measuring synovial fluid levels of TCA at later time points. Methods: In this multi-center, open-label study, patients with OA of the knee received a single IA injection of FX006 containing 10 or 40mg TCA, or 40mg of a standard, immediate-release suspension of TCA (TCA IR). A total of 50 patients were enrolled (FX006 10 mg: 10 patients, FX006 40 mg: 30 patients, TCA IR: 10 patients). Synovial fluid and plasma samples were obtained at baseline (pre-dose) and at the planned Final Visit at either Week 12, 16, or 20, depending on cohort assignment and availability of fluid in the knee. Concentrations of TCA were measured in plasma or in filtered synovial fluid samples using High Performance Liquid Chromatographic methods with Tandem Mass Spectrometry Detection and Automated Extraction, validated for the determination of TCA in human plasma and synovial fluid respectively. The quantitation limits of both assays were 50 to 50000 pg/mL. Results: In the FX006 10 mg group, 8 patients had synovial fluid available for analysis at Week 12. In the FX006 40 mg group, 6 patients had synovial fluid available for analysis at Week 12, 8 patients at Week 16 and 11 patients at Week 20. In the TCA IR group, 5 patients had synovial fluid available for analysis at Week 12. The concentrations of TCA achieved by 40mg FX006 in the synovial fluid atWeek 12 were 924 pg/mL, at Week 16, the IA concentrations of TCAwere 224 pg/mL and by Week 20 they were below the lower limit of quantitation (Figure; geometric means from the earlier 6-week study and the current study are merged). The IA concentration of TCA in patients receiving 40 mg of TCA IR at Week 12 was below the lower limit of quantitation. FX006 maintained a gradient between synovial and systemic plasma concentrations of TCA for 16 weeks.

Membrane-associated guanylate kinase homologs (MAGUKs) may play a role in cellular functions preventing tumorigenesis as indicated by the neoplastic phenotype caused by genetic loss of the MAGUK Dlg in Drosophila . To test this possibility, we examined the expression and subcellular localization of the tight junction MAGUK ZO-1, as well as the cell adhesion molecule E-cadherin, in paraffin-embedded breast cancer samples, using immunohistochemistry and confocal microscopy. As expected, normal tissue showed intense staining for ZO-1 at the position of the epithelial tight junctions, but this staining was reduced or lost in 69% of breast cancers analyzed ( n = 48). In infiltrating ductal carcinomas ( n = 38) there was a reduction in staining in 42% of well differentiated, in 83% of moderately differentiated and 93% of poorly differentiated tumors. ZO-1 staining was positively correlated with tumor differentiation ( P =.011) and more specifically with the glandular differentiation of tumors ( P =.0019). Reduction in ZO-1 staining was strongly correlated with reduced E-cadherin staining ( P = 4.9 × 10 −5 ). The results suggest that down-regulation of ZO-1 expression and its failure to accumulate at cell junctions may be causally related to cancer progression. To detect loss of heterozygosity, the ZO-1 gene tjp-1 was mapped relative to other markers in 15q13 and polymorphic markers flanking tjp-1 were identified. The marker D15S1019 showed loss of heterozygosity in 23% of informative tumors ( n = 13). Loss of a tjp-1 -linked marker suggests that genetic loss may, in some cases, be responsible for the reduction in ZO-1 expression in breast cancer.

Proteins of the 4.1 family play a key role in the integrity of the cytoskeleton and in epithelial tissue movement, as shown by the disruption of the actin cytoskeleton in human erythrocytes caused by genetic loss of protein 4.1, and the failure of epithelial tissue migration during Drosophila embryogenesis caused by genetic loss of the 4.1 homolog Coracle. Here we report the genetic characterization of Yurt, a novel protein 4.1 family member in Drosophila that is associated with the plasma membrane of epithelial cells. Homozygous loss-of-function mutations in the yurt gene cause failure of germ-band retraction, dorsal closure, and head involution, associated with degeneration of the amnioserosa and followed by embryonic lethality. A mammalian homolog of Yurt is up-regulated in metastatic melanoma cells. These novel cytoskeletal proteins appear to play important roles in epithelial cell movements and in the morphogenetic tissue changes that depend on them.

We have determined the cDNA sequence, expression pattern, and chromosomal location of the human gene DLG4, encoding the postsynaptic density-95 (PSD95) protein. hPSD95 is a 723-amino-acid protein that is 99% identical to its rodent counterparts. This is the fourth human protein identified as showing significant similarity to the Drosophila tumor suppressor Dlg. These proteins constitute the DLG subfamily of the membrane-associated guanylate kinase protein family. The expression of DLG4 in neural tissue is consistent with the pattern observed for its rat homolog. However, DLG4 is also expressed in a wide range of nonneural tissues, suggesting that the protein may have additional roles in humans. Using radiation-hybrid mapping panels, we mapped the DLG4 locus to 17p13.1, a region associated with several diseases, the phenotypes of which are consistent with loss of PSD95 function.