Jill Miller

Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.We document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model.These novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.

Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (> 600 kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

Abstract Inflammation is a key mediator in the development of malignant mesothelioma, which has a dismal prognosis and poor therapeutic strategies. Curcumin, a naturally occurring polyphenol in turmeric, has been shown to possess anticarcinogenic properties through its anti-inflammatory effects. Inflammasomes, a component of inflammation, control the activation of caspase-1 leading to pyroptosis and processing of proinflammatory cytokines, interleukin (IL)-1β and IL-18. In the present study, we investigate the role of curcumin in pyroptotic cell death of malignant mesothelioma cells. Using in vitro models with mouse and human malignant mesothelioma cells, curcumin is shown to induce pyroptosis through activation of caspase-1 and increased release of high-mobility group box 1 (HMGB1) without processing of IL-1β and IL-18. Absence of IL-1β processing in response to curcumin-mediated caspase-1 activation is attributed to blockade of pro-IL-1β priming through inhibition of the NF-κB pathway. Furthermore, curcumin's cytotoxicity in malignant mesothelioma cells is demonstrated to be dependent on pyroptosis as inhibition of caspase-1 resulted in protection against curcumin-induced cell death. We also demonstrate that curcumin-mediated caspase-1 activation is oxidant dependent by using N-acetyl-L-cysteine (NAC) to inhibit pyroptosis. PCR array analysis using the human inflammasome template revealed that curcumin significantly downregulated levels of inflammasome-related gene expression involved in inflammation, e.g., NF-κB, toll-like receptors (TLR), and IL-1β. Our data indicate that curcumin has a double effect on malignant mesothelioma cells through induction of pyroptosis while subsequently protecting against inflammation. Cancer Prev Res; 7(3); 330–40. ©2014 AACR.

Malignant mesothelioma is a devastating disease with a need for new treatment strategies. In the present study, we showed the importance of extracellular signal-regulated kinase 5 (ERK5) in malignant mesothelioma tumor growth and treatment.ERK5 as a target for malignant mesothelioma therapy was verified using mesothelial and mesothelioma cell lines as well as by xenograft severe combined immunodeficient (SCID) mouse models.We first showed that crocidolite asbestos activated ERK5 in LP9 cells and mesothelioma cell lines exhibit constitutive activation of ERK5. Addition of doxorubicin resulted in further activation of ERK5 in malignant mesothelioma cells. ERK5 silencing increased doxorubicin-induced cell death and doxorubicin retention in malignant mesothelioma cells. In addition, shERK5 malignant mesothelioma lines exhibited both attenuated colony formation on soft agar and invasion of malignant mesothelioma cells in vitro that could be related to modulation of gene expression linked to cell proliferation, apoptosis, migration/invasion, and drug resistance as shown by microarray analysis. Most importantly, injection of shERK5 malignant mesothelioma cell lines into SCID mice showed significant reduction in tumor growth using both subcutaneous and intraperitoneal models. Assessment of selected human cytokine profiles in peritoneal lavage fluid from intraperitoneal shERK5 and control tumor-bearing mice showed that ERK5 was critical in regulation of various proinflammatory (RANTES/CCL5, MCP-1) and angiogenesis-related (interleukin-8, VEGF) cytokines. Finally, use of doxorubicin and cisplatin in combination with ERK5 inhibition showed further reduction in tumor weight and volume in the intraperitoneal model of tumor growth.ERK5 inhibition in combination with chemotherapeutic drugs is a beneficial strategy for combination therapy in patients with malignant mesothelioma.

Malignant mesothelioma (MM) is an aggressive tumor with no treatment regimen. Previously we have demonstrated that cyclic AMP response element binding protein (CREB) is constitutively activated in MM tumor cells and tissues and plays an important role in MM pathogenesis. To understand the role of CREB in MM tumor growth, we generated CREB-inhibited MM cell lines and performed in vitro and in vivo experiments. In vitro experiments demonstrated that CREB inhibition results in significant attenuation of proliferation and drug resistance of MM cells. CREB-silenced MM cells were then injected into severe combined immunodeficiency mice, and tumor growth in s.c. and i.p. models of MM was followed. We observed significant inhibition in MM tumor growth in both s.c. and i.p. models and the presence of a chemotherapeutic drug, doxorubicin, further inhibited MM tumor growth in the i.p. model. Peritoneal lavage fluids from CREB-inhibited tumor-bearing mice showed a significantly reduced total cell number, differential cell counts, and pro-inflammatory cytokines and chemokines (IL-6, IL-8, regulated on activation normal T cell expressed and secreted, monocyte chemotactic protein-1, and vascular endothelial growth factor). In vitro studies showed that asbestos-induced inflammasome/inflammation activation in mesothelial cells was CREB dependent, further supporting the role of CREB in inflammation-induced MM pathogenesis. In conclusion, our data demonstrate the involvement of CREB in the regulation of MM pathogenesis by regulation of inflammation.

Malignant mesothelioma is an aggressive cancer in desperate need of treatment. We have previously shown that extracellular signaling regulated kinase 5 (ERK5) plays an important role in mesothelioma pathogenesis using ERK5 silenced human mesothelioma cells exhibiting significantly reduced tumor growth in immunocompromised mice. Here, we used a specific ERK 5 inhibitor, XMD8-92 in various in vitro and in vivo models to demonstrate that inhibition of ERK5 can slow down mesothelioma tumorigenesis. First, we show a dose dependent toxicity of XMD8-92 to 2 human mesothelioma cell lines growing as a monolayer. We also demonstrate the inhibition of ERK5 phosphorylation in various human mesothelioma cell lines by XMD8-92. We further confirmed the toxicity of XMD8-92 towards mesothelioma cell lines grown as spheroids in a 3-D model as well as in intraperitoneal (immune-competent) and intrapleural (immune-deficient) mouse models with and without chemotherapeutic drugs. To ascertain the mechanism, we explored the role of the nod-like receptor family member containing a pyrin domain 3 (NLRP3) inflammasome in the process. We found XMD8-92 attenuated naïve and chemotherapeutic-induced inflammasome priming and activation in mesothelioma cells. It can thus be concluded that ERK5 inhibition attenuates mesothelioma tumor growth and this phenomenon in part is regulated by the inflammasome.

Malignant mesothelioma (MM), lung cancers, and asbestosis are hyperproliferative diseases associated with exposures to asbestos.All have a poor prognosis; thus, the need to develop novel and effective therapies is urgent.Vandetanib (Van) (ZD6474, ZACTIMA) is a tyrosine kinase inhibitor that has shown equivocal results in clinical trials for advanced non-small cell lung cancer.However, tyrosine kinase inhibitors alone have shown no significant clinical activity in phase II trials of patients with unresectable MM.Using epithelioid (HMESO) and sarcomatoid (H2373) human MM lines, the efficacy of tumor cell killing and signaling pathways modulated by Van with and without doxorubicin (Dox) was examined.Van alone reduced total cell numbers in HMESO MM and synergistically increased the toxicity of Dox in HMESO and H2373 cells.Most importantly, we identified two novel cell survival/resistance pathways, ERK5 and cyclic AMP response element binding protein (CREB), that were inhibited by Van and Dox.After silencing of either ERK5 or CREB, significant decreases in cell numbers in the Dox-resistant sarcomatoid H2373 line were observed.Results suggest that a plethora of cell signaling pathways associated with cell survival are induced by Dox but inhibited by the addition of Van in MM.Data from our study support the combined efficacy of Van and Dox as a novel approach in the treatment of MM that is further enhanced by blocking ERK5 or CREB signaling cascades.

Abstract Inflammation plays an important role in development of various cancers including malignant mesothelioma (MM). We have shown that asbestos activates NOD-like receptor protein 3 (NLRP3), a component of the inflammasome in human macrophages. As chronic asbestos exposure is a key risk factor for the development of MM, we hypothesized that inflammasome-mediated inflammation might underlie the pathogenesis of this cancer. To show the involvement of NLRP3 in asbestos-induced mesothelioma, we demonstrated that exposure of asbestos to immortalized human mesothelial cells (LP9/hTERT), a cell type responsible for origin of MM, caused mRNA increase and activation of NLRP3 as measured by caspase-1 activation and IL-1β release. Inhibition of NLRP3 by siRNA caused significant decreases in NLRP3 mRNA levels as well as asbestos-induced IL-1β release in medium. On the other hand, human MM lines and tumor tissues showed significantly decreased levels of NLRP3 and caspase-1 as compared to LP9 or matching normal tissues respectively. Our findings suggest that initial exposure to asbestos causes increased mRNA levels and activity of NLRP3, which may help in MM development by promoting mesothelial cell transformation. However, tumor development culminates in MM with decreased NLRP3 protein and increased drug resistance which may be due to caspase-1 inactivation. Thus NLRP3 may be an appropriate target for therapy of MM, especially in combination with cytotoxic chemotherapeutic drugs and IL-1 receptor antagonists. This study is supported by a Meothelioma Applied Research Foundation (MARF) grant (AS) and by NIEHS grants T32 ES07122 (BM). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5461. doi:1538-7445.AM2012-5461

Anaplastic lymphoma kinase (ALK) fusion oncogenes are present in multiple cancer types. The inversion of echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes on chromosome 2 is present in a subset of patients with non-small-cell lung cancer (NSCLC). ALK-rearranged lung cancers demonstrate a significantly higher incidence of signet ring cell histology than do ALK-negative tumors. Based on the histologic similarities of ALK-rearranged NSCLC and signet ring cell carcinomas (SRCCs) of the gastrointestinal tract, we hypothesized that SRCC of the upper gastrointestinal (GI) tract may also harbor ALK translocations.Thirty-five formalin-fixed, paraffin-embedded (FFPE) diagnostic tissue specimens of SRCC or poorly differentiated adenocarcinoma with greater than 10% signet ring cell features originating from the upper GI tract were obtained and confirmed by a board-certified, GI pathologist. SRCC specimens were analyzed by fluorescence in situ hybridization (FISH) analysis, with an ALK (2p23) break-apart probe.The FISH analysis revealed no evidence of ALK translocation. All 35 (100%) SRCC specimens showed intact ALK FISH signals.These data indicate that, despite histologic similarities between SRCC of the upper GI tract and ALK-positive NSCLC, ALK translocations are unlikely to be a significant contributor to the molecular etiology of SRCC. Further genomic investigations are ongoing.

The College of American Pathologists published guideline recommending bone marrow synoptic reporting for hematologic neoplasms.To evaluate the impact of pathology-driven algorithmic testing (PDAT) with integrated reporting for bone marrow examination on test utilization, ability to render a specific World Health Organization diagnosis, and clinician satisfaction 1 year after implementation.We reviewed the hematopathology reports, integrated synoptic reports, and ancillary test results generated during a 12-month period. The initial diagnosis from the hematopathology report was compared with the final diagnosis on the integrated synoptic reports. Test utilization data were compared with a previous year in which ancillary testing was ordered at clinician discretion. Clinicians were anonymously surveyed to assess their satisfaction with PDAT and integrated reporting.Integrated reporting resulted in a World Health Organization diagnosis for 80 of 85 cases (94%) compared with 54 (64%) for the hematopathology report alone. Unnecessary testing decreased from 45% pre-PDAT (124 of 274 cases) to 0.7% PDAT (2 of 268 cases), and PDAT resulted in fewer omissions of necessary tests. Clinicians preferred PDAT and valued integrated reporting for a variety of reasons, including the ease of finding relevant prognostic information.Pathology-driven algorithmic testing with integrated reporting improves the pathologist's ability to render a specific World Health Organization diagnosis and improves test utilization. Clinicians prefer PDAT to clinician-ordered testing. This is the first study to examine how synoptic reporting can modify hematologic diagnoses.

ABP 501 AMJEVITA®; adalimumab) is an approved biosimilar to adalimumab (Humira®), a fully human recombinant monoclonal antibody. Amgen validated separate assays to independently detect ADA against ABP 501 and adalimumab; this testing strategy was applied to both binding and neutralising ADA detection to evaluate immunogenicity of ABP 501 compared with adalimumab. The goal of the present analyses is to assess the extent of ADA cross-reactivity between the biosimilar and reference product among patients with rheumatoid arthritis and psoriasis in two pivotal Phase 3 studies. All protocol-defined antibody samples collected from drug-exposed subjects, irrespective of treatment group, were tested for binding antibodies against ABP 501 and adalimumab using a validated electrochemiluminescence-based assay. Binding ADA magnitude was expressed as signal-to-noise (S/N), defined as the mean signal of the study sample divided by the mean signal of the negative control analysed on the same plate. Samples positive for binding ADAs were then tested in a TNFα-target binding assay for neutralising activity. Neutralising antibody titer was reported as the highest dilution that tested positive in the assay. Validated assay parameters for both binding and neutralising assays such as assay cut point, sensitivity, precision, and drug tolerance for each assay were highly similar. Correlation of binding and neutralising antibody results (S/N or titer) between ABP 501 or and adalimumab assays was evaluated using the Pearson’s correlation coefficient. Concordance of antibody results (positive/negative) was evaluated using the kappa statistic. Irrespective of treatment group, the binding ADA results for ABP 501 and adalimumab assays correlated well, with a Pearson correlation coefficient >0.950 and concordance measure of (kappa > 0.860). In a small subset of samples that were not concordant, the distribution of sample reactivity was detected equally across all assays. The magnitude of ADAs was at or near the assay cut point, indicating low probability of clinical impact. Furthermore, neutralising antibody titer results correlated well between ABP 501 and adalimumab assays with a Pearson correlation coefficient > 0.780 and strong concordance (kappa > 0.884). No meaningful differences were observed in the detection of binding and neutralising ADAs in the different ADA assays, providing evidence of high similarity between ABP 501 and adalimumab.

Amphetamine intoxication in dogs referred to the Veterinary Diagnostic Laboratory or the Veterinary Hospital of the University of Minnesota was characterized by excitement, agitation, hyperthermia, and convulsive episodes that could be confused with other convulsant poisonings. Extraction procedures on stomach contents or urine enabled indentification of the drug, using ultraviolet spectrophotometry.

Abstract Mechanisms involved in the tumorigenesis of the devastating cancer, malignant mesothelioma (MM) are poorly understood. We have recently shown that interleukin-1β (IL-1β), an inflammatory cytokine is upregulated by asbestos via the activation of the inflammasome (a molecular platform that assembles for the activation of caspase-1) in mesothelial cells. Furthermore we have demonstrated that IL-1β secretion may lead to the activation of downstream signaling cascades involved in malignant transformation of mesothelial cells. Preliminary data from our lab indicate that in addition to IL-1β, asbestos exposure upregulated the secretion of basic fibroblast growth factor (bFGF/FGF2) and tissue factor pathway inhibitor 2 ((TFPI2) a kunitz type protease inhibitor). These factors were also regulated by the inflammasome and have never before been implicated in asbestos-induced mesothelial to fibroblastic transition (MFT). Based on our preliminary data, we hypothesized that upregulation of IL-1β by asbestos-induced inflammasome activation increases FGF2 secretion and signaling. Furthermore, we hypothesize that FGF2 together with increased TFPI2 secretion induces transition of mesothelial cells to a fibroblastic phenotype that facilitates MM carcinogenesis. In the proposed study we will delineate the role of TFPI2 (siRNA) and FGF2 (pan FGFR inhibitor, BGJ398) in the process of asbestos-induced MFT. Data from this study will provide added insight into the mechanisms involved in the initiation of MM and indicate whether TFPI2 and FGF2 can serve as drugable targets for combination therapy against MM. This work is supported by NIH grant, RO1 ES021110. Citation Format: Joyce K. Thompson, Jill Miller, Maximilian B. MacPherson, Arti Shukla. The role of TFPI2 and FGF2 in asbestos-induced mesothelial to fibroblastic transition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4046.

Abstract Malignant mesothelioma (MM) is a neoplastic disease of the pleural, peritoneal or pericardial cavity. Currently, there is no therapy for MM as it is resistant to most of the common chemotherapies and therefore, there is a need for new treatment strategies. In the present study we demonstrated that ERK5 is important in MM tumor growth and can potentially be targeted in combination with the chemotherapeutic drugs for more effective treatment. Human MM cell lines exhibit constitutive activation of ERK5. Addition of doxorubicin to these MM cell lines resulted in further activation of ERK5. ERK5 silencing by small hairpin RNA (shERK5) increased DOX-induced cell death and DOX retention in human MM cells. In addition, shERK5 MM lines exhibited both attenuated colony formation on soft agar and invasion of MM cells in vitro. Most importantly, injection of shERK5 MM cell lines into SCID mice showed significant reduction in tumor growth using both subcutaneous and intraperitoneal models. Finally, use of doxorubicin and cisplatin in combination with ERK5 inhibition showed further reduction in tumor weight and volume in the IP model of tumor growth, proving our hypothesis that ERK5 inhibition in combination with chemotherapeutic drugs is a beneficial strategy for combination therapy in MM patients. This work is supported by Mesothelioma Applied Research Foundation (AS), NIEHS-RO1ES021110 (AS) and T32 ES07122 (BM) grants. Citation Format: Arti Shukla, Jill Miller, Christopher Cason, Mutlay Sayan, Maximilian MacPherson, Stacie Beuschel, Brooke Mossman. Extracellular signal regulated kinase 5 inpathogenesis of malignant mesothelioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4315. doi:10.1158/1538-7445.AM2013-4315

e15106 Background: Anaplastic lymphoma kinase (ALK) fusion oncogenes are present in multiple cancer types. The inversion of echinoderm microtubule associated protein like 4 (EML4) and ALK genes on chromosome 2 is present in a subset of non-small cell lung cancer (NSCLC) patients. ALK mutated lung cancers demonstrate a significantly higher incidence of signet ring cell histology than compared to ALK-negative tumors. Based on the histological similarities of ALK positive NSCLC and signet ring cell carcinomas (SRCC) of the GI tract, we hypothesized that gastric and/or esophageal SRCC may also harbor ALK translocations. Methods: Thirty-five formalin-fixed, paraffin-embedded (FFPE) tissue specimens of SRCC originating from esophageal, GE junction or gastric locations were obtained from the Fletcher Allen Healthcare (Burlington, Vermont) tissue bank following Internal Review Board guidelines. Confirmation of SRCC or adenocarcinoma with signet ring cell features was confirmed by a board certified, gastrointestinal pathologist. SRCC specimens were analyzed by fluorescence in situ hybridization (FISH) analysis using an ALK (2p23) break-apart probe (Kreatech Diagnostics). Results: The FISH analysis revealed no evidence of ALK translocation: all thirty-five (100%) SRCC specimens showed intact (yellow) ALK FISH signals. Conclusions: These data indicate that despite histological similarities between SRCC of the GI tract and ALK positive NSCLC, ALK translocations are unlikely to be a significant contributor to gastric and esophageal SRCC molecular etiology. Further genomic investigations are on-going. This study was performed with funding received from the Lake Champlain Cancer Research Organization.

Background Despite the improved 5‐year survival rate, colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States and is sub‐classified according to mucin content as mucin‐rich CRC (MCRC, ≥50% mucin content) or conventional colorectal adenocarcinoma (CCRA, <50% mucin content). Ten percent of CRCs are considered mucin‐rich and are reported to be less sensitive to chemotherapy treatment than CCRAs. The unfolded protein response (UPR) is an adaptive response activated when unfolded proteins accumulate in the endoplasmic reticulum (ER). When the UPR is incapable of dealing with an increased protein load, it leads to apoptosis. Despite the increased protein load and UPR activation, tumor cells are known to maintain protein homeostasis and avoid apoptosis. Objectives We hypothesized that the higher protein load in MCRC cells, initiated by the high demand of producing mucins, induces greater ER stress and activation of the UPR. Here, we aimed to identify UPR markers uniquely upregulated in their expression in MCRC versus CCRA patient biopsies. We also utilized CRC cell lines with similar mucin and UPR profiles as the biopsies, to target differentially regulated UPR markers for inhibition as potential targets for CRC therapy. Results Our data showed that when compared to CCRA samples, MCRC biopsies express significantly higher mRNAs for several UPR markers such as IRE1α, XBP1, and CHOP, which significantly correlate (p<0.0001, p<0.0001, p=0.0003, respectively) with MUC6 expression. In contrast, CCRA samples expressed higher levels of several protein disulfide isomerases (PDIs) including PDIA1 (p=0.0001), PDIA3 (p<0.0001), PDIA5 (p<0.0001), and PDIA17 (p=0.0025), that are known to be the downstream of canonical transducers of the UPR. We also identified CCRA (Caco2) and MCRC (LS174T) cell lines with similar UPR profiles to the biopsies. Our data indicate that cells from the MCRC cell line were less responsive to LOC14, a PDI inhibitor, whereas the CCRA cell line, which expressed higher levels of several PDIs, were less proliferative and had a greater decrease in total cell numbers following 6 days of treatment with LOC14. Accordingly, treatment with LOC14 inhibited one of the PDIs, PDIA3 in the CCRA cell, but not the MCRC cell line. Conclusions These novel results suggest that CCRAs and MCRCs have different signatures of UPR markers and CCRAs may be more responsive to PDI inhibitors of the UPR than MCRCs. Further characterization of the UPR profiles of CCRAs and MCRCs could aid in the development of future anti‐cancer therapies and targeting of UPR transducers and their downstream mediators. Support or Funding Information NIH R01 HL122383 and Department of Pathology and Laboratory Medicine (UVM) Startup Fund (VA) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Sustained clinical response to biologic therapies is predicated on adequate drug exposure, which can be impacted by development of anti-drug antibodies (ADA). The objective was to compare the serum trough concentrations of ABP 501 (AMGEVITA®; adalimumab) with adalimumab (Humira®) reference product (RP), and correlate these with incidence of developing anti-drug antibodies (ADAs). We descriptively analysed serum drug trough concentration data and binding ADAs from baseline to primary analysis time point in two randomised controlled trials of ABP 501 compared with adalimumab—a 52-week study among patients with plaque psoriasis (PsO) and 26-week study among patients with moderate-to-severe rheumatoid arthritis (RA) on background therapy with methotrexate. The proportion of subjects with binding ADA positive results increased from 18.1% to 33.5% from Week 4 to Week 26 in the RA study and 29.0–57.0% from Week 4 to Week 16 in the PsO study. Mean serum trough concentrations of ABP 501 and adalimumab RP was lower among those who tested positive for binding ADA time in both studies due to binding ADAs. However, trough concentration over the duration of the study remained similar between ABP 501 and adalimumab RP treatment arm in binding ADA-negative and binding ADA-positive subgroups. The formation of binding ADAs in both RA and PsO studies impacts the exposure of both ABP 501 and adalimumab RP similarly over time. Furthermore, this reflects on the importance of using sensitive assays for understanding the immunogenicity of these products.

Poster presentations are obtained from inflamed and uninflamed tissue from the colon.Mucosal TNF-α expression is detected by confocal microscopy after immunofluorescent staining.Serum, mucosal and faecal anti TNF-α and anti-drug antibody levels is determined by ELISA assay.Kendalltau correlation coefficient was used to assess bivariate correlations.Results: Data of 19 patients have been analysed.Our preliminary measurements confirmed the higher number of TNF-α positive cell in the mucosal samples of active vs. inactive part of the bowel.However, no association could be detected between TNF-α-positive cell and mucosal anti TNF-α concentration.We were unable to find evidence against the hypothesis of no association between serum drug levels and mucosal anti TNF-α concentration, between serum drug levels and clinical or endoscopic response to anti-TNF therapy.Conclusions: Our study would be the first that simultaneously examine serum, tissue and faecal concentrations of anti TNF-α comparing with clinical and endoscopic activities.Monitoring of serum drug levels is a widely accepted approach during biological therapy.However, the sample size is currently very low, and more data are needed to be collected in order to better investigate the association between serum and tissue drug levels or disease activity.Nevertheless, it is hoped that these measurements will allow a better overview of the drug distribution and clearance and may help to identify a useful surrogate marker of clinical and endoscopic activity.

Immunomodulatory therapy with agents such as methotrexate and corticosteroids can impact the development of anti-drug antibodies (ADAs) to monoclonal antibodies such as adalimumab used to treat inflammatory bowel disease. ABP 501 (AMGEVITA®; adalimumab) is an approved biosimilar to adalimumab. Here we report results from a Phase 3 study in patients with rheumatoid arthritis (RA) comparing the incidence of ADAs and the relative magnitude of the ADA response between ABP 501 and adalimumab reference product (RP) on background methotrexate (MTX). We analysed data from a randomised, double-blind, 26 week, 1:1 active-controlled study designed to show clinical equivalence between ABP 501 40 mg and adalimumab 40 mg subcutaneously every two weeks among adalimumab-naive adult patients with moderate-to-severe RA who had an inadequate response to MTX. Patients were required to receive a stable dose of MTX (range 7.5 to 25 mg/week) for the duration of the study. Patients were also allowed to remain on oral corticosteroids at a dose of ≤10 mg/day of prednisone, or equivalent. Validated electrochemiluminescent assays were developed and used for detection of binding ADAs. ADA magnitude was expressed as signal-to-noise (S/N), defined as the mean signal of the study sample divided by the mean signal of the negative control analysed on the same plate. Samples positive for binding ADAs were then tested in a TNFα-target binding assay for neutralising activity. Neutralising antibody titre was reported as the highest dilution that tested positive in the assay. For the ABP 501 and adalimumab (RP) groups, 5 (1.9%) and 6 (2.3%) patients, respectively, tested positive for pre-existing binding antibodies and no patients tested positive for pre-existing neutralising antibodies. Overall, 201 (38.2%) of all patients tested positive for binding antibodies at weeks 4, 12, or 26, with similar percentages in each treatment group (ABP 501, n = 101, 38.3%; adalimumab, n = 100, 38.2%) across all time points. A total of 53 (10.1%) of all randomised patients tested positive for neutralising antibodies at weeks 4, 12, or 26, which was also similar in each treatment group (ABP 501, n = 24, 9.1%; adalimumab, n = 29, 11.1%). The rate of seroconversion over time for both treatment groups was similar, progressively increasing throughout the study. For subjects testing ADA positive, the magnitude of both the binding and neutralising ADAs across the treatment groups were evenly distributed, with similar median S/N or titre values at each time point. Similar immunogenicity rates were observed and relative magnitude of the ADAs was similar between the ABP 501 and adalimumab RP treated patients.

<h3>Background</h3> Adalimumab and its biosimilars are anti-tumour necrosis factor (TNF)- monoclonal antibodies that are approved in Europe as treatment for various autoimmune-related indications, including Crohn’s disease and ulcerative colitis. Despite the clinical success of adalimumab, some patients show a diminished response after prolonged treatment. It is known that serum adalimumab trough levels are correlated with clinical response and the development of anti-adalimumab antibodies (AAA) may negatively impact trough levels. Currently, therapeutic drug monitoring (TDM) is used to measure adalimumab concentration and/or AAA allowing individualized optimization of treatment regimens. This then facilitates better clinical outcomes by avoiding delay in treatment decisions. Therefore, having a single assay that provides reliable TDM for both adalimumab and adalimumab biosimilars is important for physicians and patients. ABP 501, the first adalimumab biosimilar, is approved for the same indications as adalimumab (except those protected by regulatory exclusivity). <h3>Objectives</h3> The aim of this study was to evaluate the Promonitor® TDM kits for measurement of drug levels and AAA in serum samples from a selected representative subset of subjects, treated with ABP 501 or adalimumab reference product (RP) in the phase 3 study (NCT01970475). <h3>Methods</h3> A total of 30 subjects (15 ADA-positive; 15 ADA-negative) served as a representative subset in this evaluation. AAA positive control antibody and serum samples from subjects treated with either ABP 501 or adalimumab RP in the Phase 3 study were used to assess the suitability of the TDM (Promonitor®) kits for AAA (Promonitor® ANTI-ADL) and quantitative drug detection (Promonitor® ADL). <h3>Results</h3> The Promonitor®-ANTI-ADL TDM kit was able to detect a low level (10 ng/ml) of AAA positive control antibodies for ABP 501. In subjects whose serum was evaluated (18 treated with ABP 501 adalimumab biosimilar and 10 treated with adalimumab RP), the TDM kit produced 100% concordant positive or negative AAA results when compared to the assay that had been used in the phase 3 study. The quantitative drug assessment in subjects whose serum was evaluated (11 treated with ABP 501 and 9 treated with adalimumab RP) using the Promonitor® ADL TDM kit displayed a high degree of correlation (average Pearson’s r = 0.987) compared with results obtained in the phase 3 study. <h3>Conclusion</h3> This evaluation indicates that the Promonitor® kits are suitable for use in routine detection of AAA and in quantitating serum levels of the ABP 501 adalimumab biosimilar in patients. <h3>Acknowledgement</h3> Monica Ramchandani <h3>Disclosure of Interests</h3> Dan Mytych Shareholder of: Amgen, Employee of: Amgen, Marta Starcevic Manning Shareholder of: Amgen, Employee of: Amgen, Alexander Colbert Shareholder of: Amgen, Employee of: Amgen, Sarah Hoofring Shareholder of: Amgen, Employee of: Amgen, Jill Miller Shareholder of: Amgen, Employee of: Amgen, Melissa Gessner Shareholder of: Amgen, Employee of: Amgen, Vincent Chow Shareholder of: Amgen, Employee of: Amgen, Gary Fanjiang Shareholder of: Amgen, Employee of: Amgen