Abstract Background and Aims Fabry disease (FD) is a rare, X-linked, genetic disorder caused by mutations in the gene for the lysosomal enzyme alpha-galactosidase A (αGal-A). The progressive accumulation of glycosphingolipids, most notably globotriaosylceramide (GL-3), in lysosomes of a variety of organs and cell types leads, over decades, to renal, cardiovascular, and other clinical manifestations. In a phase 2 study, glucosylceramide synthase inhibition with venglustat led to a progressive reduction in plasma GL-3 to low-normal levels. Scoring by light microscopy showed a reduction in lysosomal GL-3 inclusions in skin capillary endothelial cells (EC) after 3 years of therapy, although not after 6 months. To further characterize the effect of venglustat using more precise methods, we applied unbiased stereological methods to quantitate GL-3 inclusions in skin ECs. Methods Skin biopsies were obtained from classic male Fabry disease patients (N = 11) at baseline and during daily treatment with venglustat in a phase 2 study (NCT02228460, NCT02489344). Images from at least 50 superficial skin capillaries were obtained using transmission electron microscopy at 7,500 X magnification. The fraction of the volume (Vv) of cytoplasm occupied by GL-3 inclusions [Vv(Inc/Endo)] in all ECs in these capillaries was estimated using point counting by a masked reader. Two-sided paired t tests were used to evaluate differences between baseline and post-treatment values at each time point. Results Venglustat therapy led to a significant reduction from baseline in Vv(Inc/Endo) of 21.1% after 6 months and 38.7% after 3 years of treatment (Figure). Conclusions Treatment with venglustat led to a reduction in GL-3 inclusion fractional volume in skin capillary ECs which was detectable after 6 months using precise quantitative methods. This was followed by further reduction over the next 2 1/2 years. We posit that, in the absence of αGal-A activity, inhibition of GL-3 production with venglustat allowed other enzymatic or non-enzymatic mechanisms to progressively reduce EC lysosomal GL-3 content, and that long-term venglustat therapy may therefore prevent or reverse progressive tissue injury in Fabry disease.

Abstract Background and Aims Fabry disease (FD) is a rare, X-linked, genetic disorder caused by pathogenic variants in the gene for the lysosomal enzyme alpha-galactosidase A (αGal-A). The progressive accumulation of glycosphingolipids, most notably globotriaosylceramide (GL-3), leads to renal, cardiovascular, and other clinical manifestations over decades. In a single-arm phase 2 study, glucosylceramide synthase inhibition with venglustat led to a reduction in lysosomal GL-3 inclusions in skin capillary endothelial cells, as well as a progressive reduction in plasma GL-3 to normal levels by 26 weeks, with a continued decrease to low-normal levels after 3 years of therapy. We used patient data from a previously completed placebo-controlled phase 3 study of agalsidase beta (Fabrazyme) as historical control to compare the effect of venglustat treatment with placebo over 6 months and with agalsidase beta over 3 years. Methods In the venglustat phase 2 study, previously untreated classic FD patients were treated with daily venglustat for up to 3 years (NCT02228460, NCT02489344). For comparison to placebo, change in plasma GL-3 levels after 26 weeks of venglustat treatment was compared with matched historical control data from previously untreated classic FD patients who were treated for 20 weeks with placebo in the phase 3 agalsidase beta study (Eng et al, N Engl J Med 345:9, 2001). For comparison to agalsidase beta, the historical control arm was matched patients treated with agalsidase beta in the phase 3 study; change in plasma GL-3 was compared at multiple time points up to 3 years. Entry criteria and baseline characteristics were similar between the two studies. Due to the much larger numbers of patients in the phase 3 agalsidase beta study, venglustat patients were matched 1:X (variable match) with control (placebo or agalsidase beta) patients based on propensity scores using baseline variables of age, plasma GL-3, gender, UPCR (< 500 vs 500-1000 vs >1000 mg/g), and eGFR (<80 vs >= 80 mL/min/1.73m2) as matching variables. Plasma GL-3 was measured at Sanofi Genzyme by LC/MS/MS, using the same method for the two studies, with controls in place to assure assay consistency. Results Venglustat patients (N=11) were matched to 19 patients for the placebo comparison and to 28 patients for comparison to Fabrazyme. All patients in all 3 groups were male and had elevated plasma GL-3, UPCR <500 mg/g, and eGFR ≥80 mL/min/1.73m2 at baseline. Mean ages in the 3 groups were 26.6, 25.7, and 26.5 years after matching, respectively. Venglustat treatment for ∼6 months led to a highly significant reduction in plasma GL-3 compared to placebo of 3.62 vs 1.06 µg/mL (p <0.0001). Long-term treatment with venglustat yielded similar reductions in plasma GL-3 compared to agalsidase beta for the first year of treatment, but while GL-3 levels reached a plateau with agalsidase beta, the reduction with venglustat continued with longer treatment and was significantly greater than that of agalsidase beta after 2 years (p=0.0351) or 3 years (p=0.0081) (Figure). Plasma GL-3 levels after 3 years of treatment were 1.90 and 4.44 µg/mL for venglustat and agalsidase beta, respectively (normal range ≤7.02 µg/mL). Conclusions Glucosylceramide synthase inhibition with venglustat led to a significantly greater reduction in plasma GL-3 compared to placebo after 6 months, and treatment for 2-3 years led to a significantly greater reduction in plasma GL-3 compared with agalsidase beta. These results highlight the potential of long-term treatment venglustat to reverse the accumulation of GL-3 in classic FD.

Abstract This abstract was withdrawn by the authors. Citation Format: Hamilton EP, Barve M, Bardia A, Beeram M, Edenfield WJ, Noonan A, Tolcher A, Bendell J, Mosher R, Xu J, Hailman E, Burris III H, Soliman HH. Withdrawn [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-35.

3010 Background: XMT-1536 is a Dolaflexin ADC targeting the sodium-phosphate cotransporter NaPi2b, expressed in ovarian, non-squamous lung, papillary thyroid, endometrial, papillary renal and salivary duct cancers. Methods: In this ongoing Phase 1 study, pts with solid tumors likely to express NaPi2b, who progressed on standard therapy, are treated with intravenous XMT-1536 using a 3+3 design with a modified Fibonacci escalation. NaPi2b expression by IHC is being examined retrospectively in archived tumors. Primary objectives in dose escalation are safety and tolerability and determination of maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D). (ClinicalTrials.gov NCT03319628). Results: As of Jan. 28, 2019, 36 pts (22 ovarian, 7 endometrial, 4 NSCLC, 3 other) have received treatment with XMT-1536. Treatment was initially given every 3 weeks (q3w); 20 pts were treated in dose cohorts from 3 to 40 mg/m2. There was one DLT of reversible AST elevation at 40 mg/m2. The dosing interval was then changed to every 4 weeks (q4w), and dose escalation was restarted at 20 mg/m2. There was one DLT of reversible AST elevation at 30 mg/m2 on the q4w schedule. Further followup and dose escalation are ongoing. The most common (≥10% of patients) treatment-related adverse events (TRAEs) have been nausea, fatigue, headache, increased AST, anorexia, increased alkaline phosphatase, fever, increased GGT, myalgia, and vomiting. Grade 3 TRAEs were reversible AST increases in 3 patients and increased GGT, decreased lymphocytes, and systolic congestive heart failure in 1 patient each. Treatment-related serious AEs of fever and systolic congestive heart failure occurred in 1 patient each. Among patients dosed at 20 mg/m2 or higher who had restaging scans (n=20), there were 2 PR, in ovarian cancer pts at 30 mg/m2 q3w and 20 mg/m2 q4w, and 11 SD, with disease control maintained for up to 24 weeks. Patient-level results for NaPi2b expression will be presented. The systemic exposure of total payload showed approximately dose-proportional increase. Plasma concentration of free drug payload and its active metabolite were low. Conclusions: XMT-1536 has been well-tolerated up to the 30 mg/m2 dose level with early signs of anti-tumor activity. Dose escalation continues in pts with advanced solid tumors likely to express NaPi2b. Clinical trial information: NCT03319628.

NaPi2b is a sodium-dependent phosphate transporter expressed in lung, ovarian, and thyroid cancers. Prior studies have suggested an enrichment of expression in lung adenocarcinoma (ACA). XMT-1536 is a NaPi2b targeting ADC (Antibody Drug Conjugate) comprised of a humanized antibody (XMT-1535) conjugated with 10-15 auristatin F-HPA (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is capable of controlled bystander-effect killing, resulting in efficacy in models with heterogeneous antigen expression, and is metabolized intra-tumorally to an active non-permeable metabolite to enable greater systemic tolerability. Previously, we demonstrated pre-clinical activity of XMT-1536 in human primary xenograft models of non-small cell lung cancer (NSCLC). MERS67 is a human-rabbit chimeric antibody derived from XMT-1535. MERS67 has been formatted for use as an immunohistochemical reagent by multiple methods and expression has been shown to correlate with response in an unselected series of primary ovarian cancer xenografts. (AACR-EORTC, 2017) We evaluated MERS67 to see if it would preferentially stain lung adenocarcinoma (ACA), as has been demonstrated using other NaPi2b antibodies. An immunohistochemical assay for MERS67 was established on a Leica BondRx Instrument. The assay was performed on tissue microarrays (TMA), including NSCLC and small cell lung cancer (SCLC) cell line arrays, and a NSCLC human tumor array. Tumors in the NSCLC array had previously been classified based on morphologic features only. All arrays were scored based on the H-score method. To characterize the primary tumors further, the tumor TMA was stained with TTF-1 and p40, markers of ACA and squamous cell carcinoma (SqCC), respectively. Results of this staining were compared to MERS67 staining patterns. H-Scores in the NSCLC cell line TMA ranged from 0-260, and from 0-100 in the SCLC TMA. Within the tissue microarray, 99 individual cases were evaluable. By morphologic classification 63 cases were SqCC, and 23 cases were ACA. Using an arbitrary cut point of H=50, there was a statistically significant difference in the number of NaPi2b positive ACA cases (19/23) vs SqCC (3/63). Among 43 cases where p40 and TTF-1 were evaluable and were in agreement with morphologic diagnosis, 7/7 cases of ACA were positive for NaPi2b, while 0/36 SqCC were positive. MERS67 is an anti-NaPi2b antibody that frequently demonstrates immunoreactivity in lung ACA. MERS67 is a chimeric antibody related to XMT-1536, a proprietary anti-NaPi2b ADC. Target expression using MERS67 is being evaluated in an ongoing XMT-1536 Phase 1 clinical trial enrolling non-squamous NSCLC patients.

2546 Background: XMT-1522 is comprised of 10-15 molecules of the payload AF-HPA, an auristatin-derivative with two-step intra-tumor metabolism intended to optimize therapeutic index, conjugated to a novel anti-HER2 monoclonal antibody via the Dolaflexin ADC platform. Methods: In this ongoing Phase 1 study, pts with advanced HER2-expressing (IHC ≥1+) breast cancer (BC), gastric cancer (GC) and non-small cell lung cancer (NSCLC) progressing on standard therapy or for whom no standard therapy exists are treated with XMT-1522 administered intravenously every 3 weeks. Dose escalation uses a 3+3 design with a modified Fibonacci escalation and a 3 week dose limiting toxicity (DLT) evaluation period. Primary objectives in dose escalation are safety and tolerability and determination of maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D). (ClinicalTrials.gov NCT02952729) Results: As of February 1, 2018 19 pts have completed the DLT evaluation period across 6 dose levels (2 to 21.3 mg/m2). There have been no DLTs nor serious adverse events (SAEs) attributed to study drug. Treatment-related adverse events (TRAEs) have been Grade 1 or 2; the most common TRAEs included increased liver enzymes, fatigue, nausea, vomiting, headache, and anorexia. 18 patients have had post-treatment restaging scans. Disease control rate (DCR) was 5/6 (83%) for patients dosed at 16 or 21.3 mg/m2 with 1 PR and 4 SD. The ongoing PR occurred at first assessment in a pt with HER2-positive BC previously treated with ado-trastuzumab emtansine. Two SD were seen in HER2-positive BC pts (ongoing 13+ and 12+ wks duration) and SD was seen in two GC pts (12 weeks and ongoing 6+ weeks duration). DCR in patients treated at doses less than 16 mg/m2 was 3/12 (25%) with 3 SD (duration 23, 13 and 9 wks). The systemic exposure of total payload showed approximately dose-proportional increase. Plasma concentration of free drug payload and its active metabolite were low. Conclusions: XMT-1522 has been well-tolerated up to the 21.3 mg/m2 dose level with early signs of anti-tumor activity. Neither MTD nor RP2D has been identified. Dose escalation continues in pts with advanced HER2-expressing BC, GC and NSCLC. Clinical trial information: NCT02952729.

BACKGROUND:The promise of targeted therapies in molecularly defined subsets of cancer has led to a transformation of the process of drug development in oncology. To target cancer successfully and precisely requires high-quality translational data. Such data can be generated by the use of biomarkers that answer key questions in drug development.CONTENT:Translational data for aiding in decision-making and driving cancer drug development can be generated by systematic assessments with biomarkers. Types of biomarkers that support decisions include: pharmacodynamic assessments for selecting the best compound or dosage; assessment of early tumor response with tissue biomarkers and imaging, mutation, and other assessment strategies for patient selection; and the use of markers of organ injury to detect toxicity and improve safety. Tactics used to generate biomarker data include fit-for-purpose assay validation and real-time biomarker assessments. Successfully translated and clinically informative biomarkers can mature into novel companion diagnostic tests that expand the practice of laboratory medicine.SUMMARY:Systematic biomarker assessments are a key component of the clinical development of targeted therapies for cancer. The success of these biomarker assessments requires applying basic principles of laboratory medicine to generate the data required to make informed decisions. Successful biomarkers can transition into diagnostic tests that expand the laboratory medicine armamentarium.

OBJECTIVES A prospective, multicenter (18)fluorine-fluorodeoxyglucose (F-18-FDG) positron emission tomography (PET)/computed tomography (CT) imaging study was performed to estimate the correlations among arterial FDG uptake and atherosclerotic plaque biomarkers in patients with peripheral artery disease.BACKGROUND Inflammation within atherosclerotic plaques is associated with instability of the plaque and future cardiovascular events. Previous studies have shown that F-18-FDG-PET/CT is able to quantify inflammation within carotid artery atherosclerotic plaques, but no studies to date have investigated this correlation in peripheral arteries with immunohistochemical confirmation.METHODS Thirty patients across 5 study sites underwent F-18-FDG-PET/CT imaging before Silver-Hawk atherectomy (FoxHollow Technologies, Redwood City, California) for symptomatic common or superficial femoral arterial disease. Vascular FDG uptake (expressed as target-to-background ratio) was measured in the carotid arteries and aorta and femoral arteries, including the region of atherectomy. Immunohistochemistry was performed on the excised atherosclerotic plaque extracts, and cluster of differentiation 68 (CD68) level as a measure of macrophage content was determined. Correlations between target-to-background ratio of excised lesions, as well as entire arterial regions, and CD68 levels were determined. Imaging was performed during the 2 weeks before surgery in all cases.RESULTS Twenty-one patients had adequate-quality F-18-FDG-PET/CT peripheral artery images, and 34 plaque specimens were obtained. No significant correlation between lesion target-to-background ratio and CD68 level was observed.CONCLUSIONS There were no significant correlations between CD68 level (as a measure of macrophage content) and FDG uptake in the peripheral arteries in this multicenter study. Differences in lesion extraction technique, lesion size, the degree of inflammation, and imaging coregistration techniques may have been responsible for the failure to observe the strong correlations with vascular FDG uptake observed in previous studies of the carotid artery and in several animal models of atherosclerosis. (J Am Coll Cardiol Img 2012; 5: 38-45) (C) 2012 by the American College of Cardiology Foundation

Background— Atherosclerosis is a complex disease requiring improvements in diagnostic techniques and therapeutic treatments. Both improvements will be facilitated by greater exploration of the biology of atherosclerotic plaque. To this end, we carried out large-scale gene expression analysis of human atherosclerotic lesions. Methods and Results— Whole genome expression analysis of 101 plaques from patients with peripheral artery disease identified a robust gene signature (1514 genes) that is dominated by processes related to Toll-like receptor signaling, T-cell activation, cholesterol efflux, oxidative stress response, inflammatory cytokine production, vasoconstriction, and lysosomal activity. Further analysis of gene expression in microdissected carotid plaque samples revealed that this signature is differentially expressed in macrophage-rich and smooth muscle cell–containing regions. A quantitative PCR gene expression panel and inflammatory composite score were developed on the basis of the atherosclerotic plaque gene signature. When applied to serial sections of carotid plaque, the inflammatory composite score was observed to correlate with histological and morphological features related to plaque vulnerability. Conclusions— The robust mRNA expression signature identified in the present report is associated with pathological features of vulnerable atherosclerotic plaque and may be useful as a source of biomarkers and targets of novel antiatherosclerotic therapies.

BACKGROUNDWith expanding biomarker discovery efforts and increasing costs of drug development, it is critical to maximize the value of mass-limited clinical samples. The main limitation of available methods is the inability to isolate and analyze, from a single sample, molecules requiring incompatible extraction methods. Thus, we developed a novel semiautomated method for tissue processing and tissue milling and division (TMAD).METHODSWe used a SilverHawk atherectomy catheter to collect atherosclerotic plaques from patients requiring peripheral atherectomy. Tissue preservation by flash freezing was compared with immersion in RNAlater®, and tissue grinding by traditional mortar and pestle was compared with TMAD. Comparators were protein, RNA, and lipid yield and quality. Reproducibility of analyte yield from aliquots of the same tissue sample processed by TMAD was also measured.RESULTSThe quantity and quality of biomarkers extracted from tissue prepared by TMAD was at least as good as that extracted from tissue stored and prepared by traditional means. TMAD enabled parallel analysis of gene expression (quantitative reverse-transcription PCR, microarray), protein composition (ELISA), and lipid content (biochemical assay) from as little as 20 mg of tissue. The mean correlation was r = 0.97 in molecular composition (RNA, protein, or lipid) between aliquots of individual samples generated by TMAD. We also demonstrated that it is feasible to use TMAD in a large-scale clinical study setting.CONCLUSIONSThe TMAD methodology described here enables semiautomated, high-throughput sampling of small amounts of heterogeneous tissue specimens by multiple analytical techniques with generally improved quality of recovered biomolecules.

Background Cholesterol deposition in arterial wall drives atherosclerosis. The key goal of this study was to examine the relationship between plaque cholesterol content and patient characteristics that typically associate with disease state and lesion vulnerability. Quantitative assays for free cholesterol, cholesteryl ester, triglyceride, and protein markers in atherosclerotic plaque were established and applied to plaque samples from multiple patients and arterial beds (Carotid and peripheral arteries; 98 lesions in total). Results We observed a lower cholesterol level in restenotic than primary peripheral plaque. We observed a trend toward a higher level in symptomatic than asymptomatic carotid plaque. Peripheral plaque from a group of well-managed diabetic patients displayed a weak trend of more free cholesterol deposition than plaque from non-diabetic patients. Plaque triglyceride content exhibited less difference in the same comparisons. We also measured cholesterol in multiple segments within one carotid plaque sample, and found that cholesterol content positively correlated with markers of plaque vulnerability, and negatively correlated with stability markers. Conclusions Our results offer important biological validation of cholesterol as a key lipid marker for plaque severity. Results also suggest cholesterol is a more sensitive plaque marker than routine histological staining for neutral lipids.

CD4+CD8+ double-positive (DP) thymocytes express a lower level of surface TCR than do mature T cells or single-positive (SP) thymocytes. Regulation of the TCR on DP thymocytes appears to result from intrathymic signaling, as in vitro culture of these cells results in spontaneous TCR up-regulation. In this study, we examined cell spreading and cytoskeletal polarization responses that have been shown to occur in response to TCR engagement in mature T cells. Using DP thymocytes stimulated on lipid bilayers or nontransgenic thymocytes added to anti-CD3-coated surfaces, we found that cell spreading and polarization of the microtubule organizing center and the actin cytoskeleton were inefficient in freshly isolated DP thymocytes, but were dramatically enhanced after overnight culture. SP (CD4+) thymocytes showed efficient responses to TCR engagement, suggesting that releasing DP thymocytes from the thymic environment mimics some aspects of positive selection. The poor translation of a TCR signal to cytoskeletal responses could limit the ability of DP thymocytes to form stable contacts with APCs and may thereby regulate thymocyte selection during T cell development.

handful of agonist peptide–MHC complexes can evoke a sustained signal from the T cell receptor. A combination of mathematical models and imaging experiments suggests a key function for CD4 and endogenous (self) peptides in the generation of this sensitive response.

The immunological synapse formed during mature T cell activation consists of a central cluster of TCR and MHC molecules surrounded by a ring of LFA-1 and ICAM-1. We examined synapse formation in thymocytes undergoing activation in a lipid bilayer system by following the movement of fluorescent MHC and ICAM-1 molecules. Immature CD4(+)CD8(+) thymocytes formed a decentralized synapse with multiple foci of MHC accumulation corresponding to areas of exclusion of ICAM-1. The MHC clusters and ICAM-1 holes were mobile and transient and correlated with active and sustained signaling, as shown by staining with antibodies against phosphotyrosine and activated Lck. Our findings show that signaling in immature thymocytes can result from a novel, multifocal pattern of receptor accumulation.

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

Lipopolysaccharide binding protein (LBP) is a plasma protein known to facilitate the diffusion of bacterial LPS (endotoxin). LBP catalyzes movement of LPS monomers from LPS aggregates to HDL particles, to phospholipid bilayers, and to a binding site on a second plasma protein, soluble CD14 (sCD14). sCD14 can hasten transfer by receiving an LPS monomer from an LPS aggregate, and then surrendering it to an HDL particle, thus acting as a soluble "shuttle" for an insoluble lipid. Here we show that LBP and sCD14 shuttle not only LPS, but also phospholipids. Phosphatidylinositol (PI), phosphatidylcholine, and a fluorescently labeled derivative of phosphatidylethanolamine (R-PE) are each transferred by LBP from membranes to HDL particles. The transfer could be observed using recombinant LBP and sCD14 or whole human plasma, and the plasma-mediated transfer of PI could be blocked by anti-LBP and partially inhibited by anti-CD14. sCD14 appears to act as a soluble shuttle for phospholipids since direct binding of PI and R-PE to sCD14 was observed and because addition of sCD14 accelerated transfer of these lipids. These studies define a new function for LBP and sCD14 and describe a novel mechanism for the transfer of phospholipids in blood. In further studies, we show evidence suggesting that LBP transfers LPS and phospholipids by reciprocal exchange: LBP-catalyzed binding of R-PE to LPS x sCD14 complexes was accompanied by the exit of LPS from sCD14, and LBP-catalyzed binding of R-PE to sCD14 was accelerated by prior binding of LPS to sCD14. Binding of one lipid is thus functionally coupled with the release of a second. These results suggest that LBP acts as a lipid exchange protein.