Tumor necrosis factor-mediated disposition of infliximab in ulcerative colitis patients.

Ulcerative Colitis (UC) is an inflammatory bowel disease typically affecting the colon. Patients with active UC have elevated tumor necrosis factor (TNF) concentrations in serum and colonic tissue. Infliximab is a monoclonal antibody directed against TNF and binds with high affinity. Target-mediated drug disposition (TMDD) is reported for monoclonal antibodies meaning that their pharmacokinetics are affected by high target affinity. Here, a TMDD model is proposed to describe the interaction between infliximab and TNF in UC patients. Data from 20 patients with moderate to severe UC was used. Patients received standard infliximab induction therapy (5 mg kg-1) at week 0, followed by infusions at week 2 and 6. IFX, anti-drug antibodies and TNF serum concentrations were measured at day 0 (1 h after infusion), 1, 4, 7, 11, 14, 18, 21, 28 and 42. A binding model, TMDD model, and a quasi-steady state (QSS) approximation were evaluated using nonlinear mixed effects modeling (NONMEM). A two-compartment model best described the concentration-time profiles of infliximab. Typical clearance of infliximab was 0.404 L day-1 and increased with the presence of anti-drug antibodies and with lower albumin concentrations. The TMDD-QSS model best described the pharmacokinetic and pharmacodynamics data. Estimate for TNF baseline (Bmax was 19.8 pg mL-1 and the dissociation constant (Kss) was 13.6 nM. This model could eventually be used to investigate the relationship between suppression of TNF and the response to IFX therapy.

Commensal-Specific CD4(+) Cells From Patients With Crohn's Disease Have a T-Helper 17 Inflammatory Profile.

BACKGROUND & AIMS: Crohn's disease (CD) has been associated with an altered immune response to commensal microbiota, mostly based on increased seroreactivity to microbial proteins. Although T cells are believed to contribute to the development of CD, little is known about the antigens involved. We investigated the antigen-specificity of T cells isolated from patients with CD. METHODS: We isolated peripheral blood mononuclear cells from 65 patients with CD and 45 healthy individuals (controls). We investigated T-cell reactivity to commensal microbial antigens using proliferation assays (based on thymidine incorporation and carboxyfluorescein succinimidyl ester dilution). Gene expression patterns were determined using microarray and real-time polymerase chain reaction analyses. Cytokines, chemokines, and antibodies were measured by enzyme-linked immunosorbent assay, flow cytometry, or multiplex cytokine assays. Intestinal crypts were obtained from surgical resection specimens of 7 individuals without inflammatory bowel disease. We examined the effects of commensal-specific CD4(+) T cells on primary intestinal epithelial cells from these samples. RESULTS: The bacterial proteins FlaX, A4-fla2, and YidX increased proliferation of CD4(+) T cells isolated from peripheral blood of patients with CD compared with controls. In blood samples from controls, CD4(+) T cells specific for FlaX, A4-fla2, or YidX had a T-helper (Th)1 phenotype; a larger proportion of CD4(+) T cells specific for these proteins in patients with CD had a Th17 phenotype or produced Th1 and Th17 cytokines. When supernatants collected from commensal-specific CD4(+) T cells from patients with CD were applied to healthy intestinal epithelial cells, the epithelial cells increased the expression of the chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL8 and the CC chemokine ligand 20 (CCL20). CONCLUSIONS: A larger proportion of commensal-specific CD4(+) T cells from patients with CD have a Th17 phenotype or produce Th1 and Th17 cytokines, compared with T cells from controls; this might contribute to intestinal inflammation in patients with CD. These cells might be targeted for treatment of CD. The transcriptional data of commensal-specific CD4(+) T cells from healthy individuals and CD patients have been deposited in the Gene Expression Omnibus at the National Center for Biotechnology Information (accession no: GSE70469).

Evolution of cytokines and inflammatory biomarkers during infliximab induction therapy and the impact of inflammatory burden on primary response in patients with Crohn's disease.

OBJECTIVE: Primary non-response to infliximab in Crohn's disease is still incompletely understood. Our aim was to further characterize the role of inflammatory burden during infliximab induction therapy. MATERIALS AND METHODS: We studied a well-characterized cohort of 201 anti-TNF naive Crohn's disease patients treated with infliximab 5mg/kg at week 0, 2, 6 and 14 who had serum samples drawn just before every infusion. All serum samples were analyzed for CRP, albumin, TNF, IFN-γ, IL-6, IL-8, IL-10, infliximab trough concentrations (in-house-developed ELISA) and antibodies to infliximab (HMSA, Prometheus Laboratories Inc., San Diego, CA). Primary non-response was defined as the absence of clinical improvement at week 14. RESULTS: The incidence of primary non-response to infliximab was 8% (n = 16). IL-8 concentrations at baseline were higher (p = .01) and albumin at week 6 was lower in primary non-responders (p = .01) compared to responders. During induction, IFN-γ and IL-6 concentrations decreased significantly at week 2 and week 6 in responders compared to primary non-responders (p < .05). Serum TNF increased significantly after each infliximab infusion and this increase from week 0 to week 14 was more pronounced in responders (p = .03). Multiple logistic regression identified TNF/CRP ratio at baseline as predictive for primary non-response to infliximab at week 14 (OR 2.8 (95% CI 1.4-5.5; p = .003)). CONCLUSIONS: In this intensively sampled cohort of Crohn's disease patients, we demonstrate that inflammatory burden is more determining for primary non-response than drug exposure or immunogenicity. Our findings furthermore suggest that the contribution of TNF in inflammation might be higher in primary non-response, contradicting the non-TNF-driven concept.

The association of tissue anti-TNF drug levels with serological and endoscopic disease activity in inflammatory bowel disease: the ATLAS study.

OBJECTIVE: The aim of this study was to assess the correlation between serum and intestinal anti-tumour necrosis factor (TNF) levels, and their relationship to endoscopic disease activity and levels of TNF. DESIGN: Cross-sectional study of 30 patients receiving treatment with infliximab or adalimumab for Crohn's disease or UC. For each patient, a sample of serum was matched to tissue biopsies. Endoscopic and histological disease activity was recorded for each tissue sample. RESULTS: There was a significant positive correlation between anti-TNF in serum and tissue (r=0.3920, p=0.002), especially in uninflamed tissue (r=0.50, p<0.001), but not with those samples that had inflammation (r=0.19, p=0.54). Anti-TNF concentration in tissue correlated with degree of endoscopic inflammation, except for tissue with severe inflammation in which anti-TNF levels were again lower (mean normalised anti-TNF in tissue: uninflamed=0.93, mild=2.17, moderate=13.71, severe=2.2 inflammation (p=0.0042)). The ratio of anti-TNF-to-TNF in tissue was highest in uninflamed areas and lowest in severely inflamed areas. Patients with active mucosal disease had a higher rate of serum to tissue drug level mismatch when compared to those in remission (73.3% vs 33.3%, respectively; p=0.03). CONCLUSIONS: Our data suggest that local tissue inflammation characterised by high levels of TNF serves as a sink for anti-TNF. We further postulate that some patients with high serum anti-TNF levels have active disease because tissue levels of anti-TNF are insufficient to neutralise local TNF production.

Antibodies to adalimumab are associated with future inflammation in Crohn's patients receiving maintenance adalimumab therapy: a post hoc analysis of the Karmiris trial.

INTRODUCTION: Data on immunogenicity to adalimumab (ADL) therapy in patients with IBD is limited. We performed additional analyses on the Karmiris cohort using the homogeneous mobility shift assay (HMSA) focusing on the inter-relationship of serum ADL concentration, antibodies-to-adalimumab (ATA), inflammatory markers and sustained response. METHODS: 536 prospectively collected serum samples were available for analysis of ADL concentration and ATA using HMSA. We studied the role of week 4 serum ADL concentration and immunomodulator (IMM) use on ATA formation with a Cox proportional hazards model. Mixed model repeated measures analysis was performed to assess the independent effects of serum ADL concentration and ATA on C-reactive protein (CRP) and response. RESULTS: ATA was detected in 20% of patients after a median of 34 (12.4-60.5) weeks. ATA-positive samples correlated with lower serum ADL concentration (p<0.001). Cox regression modelling showed that week 4 ADL concentration of <5 µg/mL significantly increased the future risk of ATA formation (HR=25.1; 95% CI 5.6 to 111.9; p=0.0002) and that IMM co-treatment prevented ATA formation (HR=0.23; 95% CI 0.06 to 0.86; p=0.0293). Regression modelling showed a negative correlation between CRP and ADL concentration (p=0.0001) and a positive one with ATA (p=0.0186). The model revealed that both lower serum ADL concentration and ATA were independently associated with future CRP (p=0.0213 and p=0.0013 respectively). ATA positivity was associated with discontinuation of ADL because of loss or response (OR=3.04; 95% CI 1.039 to 9.093; p=0.034). CONCLUSIONS: ATA were detected in 20% of patients. Risk of ATA formation increased with lower early serum ADL concentration and in patients not on IMM. ATA and ADL were strongly associated with higher future CRP level and discontinuation of ADL.

Pharmacokinetic Features and Presence of Antidrug Antibodies Associate With Response to Infliximab Induction Therapy in Patients With Moderate to Severe Ulcerative Colitis.

BACKGROUND & AIMS: The pharmacokinetics of infliximab during induction treatment for ulcerative colitis (UC) have not been studied. We investigated serum concentrations of infliximab and the early appearance of antibodies to infliximab (ATI) during induction treatment in patients with moderate-to-severe UC. METHODS: We performed a prospective analysis of 19 consecutive patients with moderate-severe UC (endoscopic Mayo ≥ 2) receiving induction therapy with infliximab (5 mg/kg at weeks 0, 2, and 6) at 2 centers in Amsterdam, The Netherlands, from July 2012 through March 2014. Serial serum and fecal samples were collected for 6 weeks and concentrations of infliximab, ATI, c-reactive protein (CRP), albumin, and fecal calprotectin were measured. Treatment success was defined as endoscopic response (≥ 1 point reduction in the endoscopic Mayo score) at week 8. RESULTS: Eleven patients (58%) had an endoscopic response. The median serum concentrations of infliximab at week 6 were 8.1 µg/mL in responders (interquartile range, 3.0-13.7 µg/mL) and 2.9 µg/mL in nonresponders (interquartile range, 0.01-5.8 µg/mL) (P = .03). ATIs were detected in 7 patients as early as day 18 (median, 28 d; interquartile range, 18-42 d). Six of the 8 nonresponders tested positive for ATIs vs 1 of 11 responders (P < .01; odds ratio, 30.0; 95% CI, 2.2-406.2). Patients with a baseline concentration of CRP greater than 50 mg/L had lower drug exposure from weeks 0 to 6 (587 mg/L/d in patients with high levels of CRP vs 1361 mg/L/day in patients with low CRP; P = .001). The median area under the curve for serum concentration of infliximab during induction therapy was 1230 mg/L/d in nonresponders vs 1352 mg/L/d in responders (P = .65). CONCLUSIONS: There is a significant difference in serum concentration of infliximab at week 6 of treatment between responders and nonresponders. Early development of ATIs during induction therapy reduces the serum concentration of infliximab and is associated with nonresponse to treatment. Patients with high baseline serum levels of CRP had lower serum concentrations of infliximab. CLINICAL TRIAL NUMBER: NL39626.018.12.

The relationship between infliximab concentrations, antibodies to infliximab and disease activity in Crohn's disease.

OBJECTIVE: Although low infliximab trough concentrations and antibodies to infliximab (ATI) are associated with poor outcomes in patients with Crohn's disease (CD), the clinical relevance of ATI in patients with adequate infliximab concentrations is uncertain. We evaluated this question using an assay sensitive for identification of ATI in the presence of infliximab. DESIGN: In an observational study, 1487 trough serum samples from 483 patients with CD who participated in four clinical studies of maintenance infliximab therapy were analysed using a fluid phase mobility shift assay. Infliximab and ATI concentrations most discriminant for remission, defined as a C-reactive protein concentration of ≤ 5 mg/L, were determined by receiver operating characteristic curves. A multivariable regression model evaluated these factors as independent predictors of remission. RESULTS: Based upon analysis of 1487 samples, 77.1% of patients had detectable and 22.9% had undetectable infliximab concentrations, of which 9.5% and 71.8%, respectively, were positive for ATI. An infliximab concentration of > 2.79 µg/mL (area under the curve (AUC) = 0.681; 95% CI 0.632 to 0.731) and ATI concentration of < 3.15 U/mL (AUC = 0.632; 95% CI 0.589 to 0.676) were associated with remission. Multivariable analysis showed that concentrations of both infliximab trough (OR 1.8; 95% CI 1.3 to 2.5; p < 0.001) and ATI (OR 0.57; 95% CI 0.39 to 0.81; p = 0.002) were independent predictors of remission. CONCLUSIONS: The development of ATI increases the probability of active disease even at low concentrations and in the presence of a therapeutic concentration of drug during infliximab maintenance therapy. Evaluation of strategies to prevent ATI formation, including therapeutic drug monitoring with selective infliximab dose intensification, is needed.

Long-term outcome of patients with Crohn's disease who discontinued infliximab therapy upon clinical remission.

BACKGROUND & AIMS: There are limited data on the effects of discontinuing infliximab therapy for Crohn's disease (CD). We investigated the long-term outcome of patients with CD who discontinued infliximab while in clinical remission, and searched for prognostic markers of continued remission after infliximab cessation. METHODS: We performed a retrospective, single-center study of 100 patients with CD who discontinued infliximab upon achieving clinical remission; 84 patients continued immunomodulator therapy. Clinical and endoscopic data were retrieved from a medical database in Belgium, and patients were followed up through April 2013 (median, 9.7 y; interquartile range, 8-11.5 y). Sustained clinical remission (SCR) was defined as maintenance of disease remission, without escalation in medical therapy or CD-related surgeries, until the end of the follow-up period. We measured trough concentrations of infliximab, antibodies to microbial antigens, and circulating inflammatory markers in serum samples collected before treatment and at the time of infliximab discontinuation. RESULTS: At the end of the follow-up period, 52 patients had SCR. Univariate (log-rank) analysis associated SCR with patient age at diagnosis (≥25 y; P = .012) and disease duration (<1 y; P = .017). Among factors evaluated at the time of infliximab discontinuation, infliximab trough concentrations (<6 µg/mL; P = .031), complete mucosal healing (P = .046), and serum positivity for vascular cell adhesion molecule-1 (>0.67 µg/mL; P = .024) were associated with SCR. In multiple Cox proportional hazards regression analysis, only age at diagnosis of 25 years and older was associated independently with SCR (hazard ratio, 1.83; 95% confidence interval, 1.03-3.25; P = .04). CONCLUSIONS: In a large, real-life study, 52% of patients with CD who discontinued infliximab upon achieving clinical remission remained in SCR after a median period of approximately 10 years; Most patients remained on immunomodulator therapy. Although patients with CD have variable responses to infliximab, a subgroup achieved long-term remission after infliximab discontinuation.

Concentrations of 6-thioguanine nucleotide correlate with trough levels of infliximab in patients with inflammatory bowel disease on combination therapy.

BACKGROUND & AIMS: In patients with inflammatory bowel diseases, the combination of infliximab and thiopurines (such as 6-thioguanine) is more effective treatment than monotherapy. We assessed the correlation between serum levels of 6-thioguanine (6-TGN) and infliximab levels or antibodies to infliximab (ATI). METHODS: We performed a cross-sectional study of 72 patients receiving maintenance therapy with infliximab and a thiopurine for inflammatory bowel disease at the Crohn's and Colitis Center of the University of Miami, FL. We collected clinical, endoscopic, and biochemical data, and levels of thiopurine metabolites. The primary outcomes were trough level of infliximab and the presence of ATI. RESULTS: Levels of 6-TGN correlated with those of infliximab (ρ, 0.53; P < .0001). The cut-off point of 6-TGN that best predicted a higher level of infliximab was 125 pmol/8 × 10(8) red blood cells (RBCs) (area under receiver operating characteristic, 0.86; P < .001). Patients in the lowest quartile of 6-TGN had infliximab levels that were similar to patients on no thiopurines (4.3 vs. 4.8 mcg/mL, respectively; P = .8). An infliximab level of 8.3 mcg/mL or greater was associated with mucosal healing. Only 8 patients (11%) had detectable ATI. Patients with 6-TGN levels less than 125 pmol/8 × 10(8) RBCs were significantly more likely to have ATI (odds ratio, 1.3; 95% confidence interval, 2.3-72.5; P < .01). CONCLUSIONS: Although 6-TGN levels of greater than 230 pmol/8 × 10(8) RBCs have been associated with improved outcomes in patients on monotherapy, a level of 6-thioguanine of 125 pmol/8 × 10(8) RBCs or greater may be adequate to achieve therapeutic levels of infliximab. In the long term, this may minimize the toxicity for patients on combination therapy.

Early trough levels and antibodies to infliximab predict safety and success of reinitiation of infliximab therapy.

BACKGROUND & AIMS: Few agents are available for the treatment of inflammatory bowel diseases, and patients frequently become unresponsive to biologics. We investigated the feasibility of reinitiating infliximab therapy for patients who previously received only episodic therapy with, lost response to, or had infusion reactions to infliximab. We also aimed to identify factors associated with the success and safety of restarting infliximab, such as antibodies to infliximab and trough levels of the drug. METHODS: From the inflammatory bowel disease biobank, we identified 128 consecutive patients (105 patients with Crohn's disease, 23 patients with ulcerative colitis) who restarted infliximab after a median 15-month discontinuation (range, 6-125 mo; 28 patients for loss of response or infusion reactions, 100 patients for remission or pregnancy). We also analyzed serum samples that had been collected during the first period of infliximab therapy (T-1), when therapy was reinitiated (T0), and at later time points (T+1, T+2) for trough levels and antibodies to infliximab. We investigated correlations among response to treatment, infusion reactions, treatment modalities, trough levels, and antibodies to infliximab. RESULTS: Reinitiation of infliximab therapy produced a response in 84.5% of patients at week 14, 70% of patients at 1 year, and in 61% of patients at more than 4 years. Fifteen patients had acute infusion reactions and 10 patients had delayed infusion reactions. The absence of antibodies to infliximab at T+1 (hazard ratio [HR], 0.14; 95% confidence interval [CI], 0.026-0.74; P = .021) and reinitiation with concomitant immunomodulator therapy were associated with short-term responses (HR, 6.0; 95% CI, 1.3-27; P = .019). Pregnancy or remission as reason for discontinuation (HR, 2.70; 95% CI, 1.09-6.67; P = .033) and higher trough levels at T+1 (HR, 2.94; 95% CI, 1.18-7.69; P = .021) were associated with long-term response. Undetectable antibodies to infliximab at T+1 were associated with the safety of reinitiating therapy (HR for infusion reaction with detectable antibodies to infliximab, 7.7; 95% CI, 1.88-31.3; P = .004). CONCLUSIONS: Reinitiating infliximab therapy can be safe and effective for patients with Crohn's disease or ulcerative colitis after a median 15-month discontinuation period.

Antibody response to infliximab and its impact on pharmacokinetics can be transient.

OBJECTIVES: Infliximab (IFX) is successfully used in the treatment of inflammatory bowel diseases but some patients generate antibodies to IFX (ATI) jeopardizing the pharmacokinetics of the drug. Little is known about the factors influencing ATI formation and whether or not this immune reaction is permanent. Our aim was to investigate the kinetics of ATI formation and drug levels in relation to inflammatory markers and the clinical evolution of the patients. METHODS: IFX trough and ATI levels were measured retrospectively in 1,232 consecutive serum samples of 90 (64 Crohn's disease and 26 ulcerative colitis) patients, 57 with previously detected and 33 without antibodies with a new homogenous mobility shift assay. RESULTS: Testing with new assay confirmed ATI in 53/90 patients (59%) and 37/90 patients (41%) were ATI negative. In 15/53 patients (28%), ATI disappeared over time whereas in 38/53 patients (72%) ATI persisted. The 26/38 (68%) patients with sustained ATI needed to discontinue IFX treatment compared with 2/15 (13%) patients with transient ATI (relative risk 5.1; 95% confidence interval 1.4-19.0; P=0.0005). An IFX trough level at week 14<2.2 µg/ml predicted IFX discontinuation due to persistent loss of response (LOR) or hypersensitivity reactions with 74% specificity and 82% sensitivity (likelihood ratio 3.1; P=0.0026). CONCLUSIONS: ATI may be transient and do not always lead to a worse clinical outcome. Sustained high levels of ATI, however, lead to permanent LOR. Patients with low IFX trough levels at week 14 are at risk for ATI formation and IFX discontinuation. Therefore, we recommend to measure IFX trough levels at week 14 and at time of LOR. When undetectable or low, ATI should be determined and if positive followed up on consecutive time points to rule out sustained ATI.

Serum and colonic mucosal immune markers in irritable bowel syndrome.

OBJECTIVES: Low-grade colonic mucosal inflammation has been postulated to have an important role in the pathophysiology of irritable bowel syndrome (IBS). The objectives of this study were (i) to identify serum and tissue-based immunological and neuroendocrine markers associated with mucosal inflammation in male (M) and female (F) patients with non-post-infectious IBS (non-PI-IBS) compared with healthy controls and (ii) to assess possible correlations of such markers with IBS symptoms. METHODS: Sigmoid mucosal biopsies were obtained from 45 Rome II positive IBS patients without a history of PI-IBS (26 F, 35.5% IBS-C, 33.3% IBS-D, 31.1% IBS-A/M) and 41 healthy controls (22 F) in order to measure immunological markers (serum cytokine levels, colonic mucosal mRNA levels of cytokines, mucosal immune cell counts) and neuroendocrine markers associated with mucosal inflammation (corticotropin releasing factor- and neurokinin (NK)-related ligands and receptors, enterochromaffin cells). Symptoms were measured using validated questionnaires. RESULTS: Of all the serum and mucosal cytokines measured, only interleukin-10 (IL-10) mRNA expression showed a group difference, with female, but not male, patients showing lower levels compared with female controls (18.0±2.9 vs. 29.5±4.0, P=0.006). Mucosal mRNA expression of NK-1 receptor was significantly lower (1.15±0.19 vs. 2.66±0.56, P=0.008) in female, but not male, patients compared with healthy controls. No other significant differences were observed. CONCLUSIONS: Immune cell counts and levels of cytokines and neuropeptides that are associated with inflammation were not significantly elevated in the colonic mucosa of non-PI-IBS patients, and did not correlate with symptoms. Thus, these findings do not support that colonic mucosal inflammation consistently has a primary role in these patients. However, the finding of decreased IL-10 mRNA expression may be a possible biomarker of IBS and warrants further investigation.

PI3K independent activation of mTORC1 as a target in lapatinib-resistant ERBB2+ breast cancer cells.

Therapies targeting the ERBB2 receptor, including the kinase inhibitor lapatinib (Tykerb, GlaxoSmithKline), have improved clinical outcome for women with ERBB2-amplified breast cancer. However, acquired resistance to lapatinib remains a significant clinical problem, and the mechanisms governing resistance remain poorly understood. We sought to define molecular alterations that confer an acquired lapatinib resistance phenotype in ER-/ERBB2+ human breast cancer cells. ERBB2-amplified SKBR3 breast cancer cells were rendered resistant to lapatinib via culture in increasing concentrations of the drug, and molecular changes associated with a resistant phenotype were interrogated using a collaborative enzyme-enhanced immunoassay platform and immunoblotting techniques for detection of phosphorylated signaling cascade proteins. Interestingly, despite apparent inactivation of the PI3K/AKT signaling pathway, resistant cells exhibited constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) and were highly sensitive to mTOR inhibition with rapamycin and the dual PI3K/mTOR inhibitor NVP-BEZ235. These data demonstrate a role for downstream activation of mTORC1 in the absence of molecular alterations leading to PI3K/AKT hyperactivation as a potential mechanism of lapatinib resistance in this model of ERBB2+ breast cancer and support the rationale of combination or sequential therapy using ERBB2 and mTOR-targeting molecules to prevent or target resistance to lapatinib. Moreover, our data suggest that assessment of mTOR substrate phosphorylation (i.e., S6) may serve as a more robust biomarker to predict sensitivity to mTOR inhibitors in the context of lapatinib resistance than PI3K mutations, loss of PTEN and p-AKT levels.

Highly sensitive proximity mediated immunoassay reveals HER2 status conversion in the circulating tumor cells of metastatic breast cancer patients.

BACKGROUND: The clinical benefits associated with targeted oncology agents are generally limited to subsets of patients. Even with favorable biomarker profiles, many patients do not respond or acquire resistance. Existing technologies are ineffective for treatment monitoring as they provide only static and limited information and require substantial amounts of tissue. Therefore, there is an urgent need to develop methods that can profile potential therapeutic targets with limited clinical specimens during the course of treatment. METHODS: We have developed a novel proteomics-based assay, Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) that can be used for analyzing clinical samples. CEER utilizes the formation of unique immuno-complex between capture-antibodies and two additional detector-Abs on a microarray surface. One of the detector-Abs is conjugated to glucose oxidase (GO), and the other is conjugated to Horse Radish Peroxidase (HRP). Target detection requires the presence of both detector-Abs because the enzyme channeling event between GO and HRP will not occur unless both Abs are in close proximity. RESULTS: CEER was able to detect single-cell level expression and phosphorylation of human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 1 (HER1) in breast cancer (BCa) systems. The shift in phosphorylation profiles of receptor tyrosine kinases (RTKs) and other signal transduction proteins upon differential ligand stimulation further demonstrated extreme assay specificity in a multiplexed array format. HER2 analysis by CEER in 227 BCa tissues showed superior accuracy when compared to the outcome from immunohistochemistry (IHC) (83% vs. 96%). A significant incidence of HER2 status alteration with recurrent disease was observed via circulating tumor cell (CTC) analysis, suggesting an evolving and dynamic disease progression. HER2-positive CTCs were found in 41% (7/17) while CTCs with significant HER2-activation without apparent over-expression were found in 18% (3/17) of relapsed BCa patients with HER2-negative primary tumors. The apparent 'HER2 status conversion' observed in recurrent BCa may have significant implications on understanding breast cancer metastasis and associated therapeutic development. CONCLUSION: CEER can be multiplexed to analyze pathway proteins in a comprehensive manner with extreme specificity and sensitivity. This format is ideal for analyzing clinical samples with limited availability.

Current Trends in IBD-Development of Mucosal-Based Biomarkers and a Novel Minimally Invasive Recoverable Sampling System.

Despite recent developments in therapy for inflammatory bowel diseases (IBDs), there have been limited advances in diagnostic tools available to aid in disease management. A growing body of evidence suggests that there are important host-microbe interactions at the mucosal interface that modulate the inflammatory response in patients with IBD. Additionally, the importance of mucosal integrity and its disruption appears to be important in the pathophysiology and perpetuation of the disease. The ability to characterize this interface may provide valuable information for both disease monitoring and identification of new treatment targets. Endoscopy remains the primary tool for disease monitoring, and mucosal healing is the primary therapeutic target in IBD treatment. However, establishing mucosal healing requires repetitive endoscopic procedures, and endoscopy is limited by factors such as invasiveness, cost, and risk of adverse events. Moreover, the use of a bowel preparation for colonoscopies alters the mucus layer and thus perturbs evaluation of the host-microbe interaction. Stool sampling may also be inaccurate because it reflects the end state of metabolites and proteins, failing to take into account the degradation or alteration of substrates of interest by bacterial proteases and other enzymes during passage through the colon. A novel sampling capsule, called the Recoverable Sampling System (RSS), is being developed as a complementary tool to colonoscopy. The RSS is intended to be a platform for noninvasive autonomous sampling, preservation, handling, and storage of analytes of interest found in the gastrointestinal fluids. A proprietary preservative contained within the chambers of the capsule has been developed to stabilize DNA and proteins for ex vivo microbiome and metabolomics analyses. Surrogate markers such as SPP24 and GUCA2a have been identified to correlate with gut health, intestinal permeability, and inflammation and could be locally sampled by the RSS. The potential clinical utility of an RSS device is broad and would likely be able to guide therapy by allowing for more frequent disease monitoring, aiding in disease characterization, and facilitating in the identification of novel therapeutic targets.

A new technology is being developed, Recoverable Sampling System (RSS), that may allow sampling without a colonoscopy. This may lead to new biomarkers and even drug targets which may ultimately improve the care of these patients.

A novel class of polymeric fluorescent dyes assembled using a DNA synthesizer.

In the pursuit of a novel class of fluorescent dyes we have developed a programmable polymer system that enables the rational design and control of macromolecular constructs through simple control of polymer primary sequence. These polymers are assembled using standard phosphoramidite chemistry on a DNA synthesizer which allows for extremely rapid prototyping and enables many permutations due to the large selection of phosphoramidite monomers presently available on the market. This programmability to some extent allows us to control the interactions/spacing of payload molecules distributed along the designed polymeric backbone. Control of molecular architecture using this technology has allowed us to address the long-standing technical issue of contact quenching between fluorescent dyes offering new possibilities in the life sciences arena. Much like peptidic sequences coding for enzymes, cofactors, and receptors (all needing control of tertiary structure for proper function via primary sequence) our programmable system approaches a similar endpoint using a phosphate based polymeric backbone assembled in a completely automated fashion. Using this novel technology, we have efficiently synthesized several types of fluorescent dyes and demonstrated the programmability in molecule design, including the increases in brightness of the fluorescence emission.

An in vivo platform for translational drug development in pancreatic cancer.

Effective development of targeted anticancer agents includes the definition of the optimal biological dose and biomarkers of drug activity. Currently available preclinical models are not optimal to this end. We aimed at generating a model for translational drug development using pancreatic cancer as a prototype. Resected pancreatic cancers from 14 patients were xenografted and expanded in successive groups of nude mice to develop cohorts of tumor-bearing mice suitable for drug therapy in simulated early clinical trials. The xenografted tumors maintain their fundamental genotypic features despite serial passages and recapitulate the genetic heterogeneity of pancreatic cancer. The in vivo platform is useful for integrating drug screening with biomarker discovery. Passages of tumors in successive cohorts of mice do not change their susceptibility to anticancer agents and represent a perpetual live bank, facilitating the application of new technologies that will result in the creation of an integrated stable database of tumor-drug response data and biomarkers.

Feedback activation of HER3 attenuates response to EGFR inhibitors in colon cancer cells.

The EGFR inhibitor cetuximab is approved for the treatment of colorectal cancer. However, both innate and acquired resistance mechanisms, including compensatory feedback loops, limit its efficacy. Nevertheless, the emergence of these feedback loops has remained largely unexplored to date. Here, we showed feedback upregulation of HER3 and induction of HER3 phosphorylation after cetuximab treatment in colon cancer cells. We also showed that this upregulation occurs, at least partly, through AKT inhibition. Together with this, we observed increased HER2:HER3 dimerization upon cetuximab treatment. Interestingly, lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, blocked the increase of cetuximab-induced HER3 phosphorylation. Additionally, we showed that upon HER3 knockdown, cetuximab combined with lapatinib was able to decrease cell viability compared to HER3 expressing cells. These results suggest the existence of a cetuximab-induced feedback HER3 activation that could potentially result in reduced cetuximab efficacy in colorectal cancer patients. Taken together, we provide evidence of the limited effectiveness of cetuximab monotherapy compared to rational combinations.

A clinically feasible multiplex proteomic immunoassay as a novel functional diagnostic for pancreatic ductal adenocarcinoma.

To date, targeted therapy for pancreatic ductal adenocarcinoma (PDAC) remains largely unsuccessful in the clinic. Current genomics-based technologies are unable to reflect the quantitative, dynamic signaling changes in the tumor, and require larger tumor samples that are difficult to obtain in PDAC patients. Therefore, a highly sensitive functional tool that can reliably and comprehensively inform intra-tumoral signaling events is direly needed to guide treatment decision. We tested the utility of a highly sensitive proteomics-based functional diagnostic platform, Collaborative Enzyme Enhanced Reactive-immunoassay (CEERTM), on fine-needle aspiration (FNA) samples obtained from 102 patients with radiographically-evident pancreatic tumors. Two FNA passes were collected from each patient, hybridized to customized chips coated with an array of capture antibodies, and detected using two enzyme-conjugated antibodies which emit quantifiable signals. We demonstrate that this technique is highly sensitive in detecting total and phosphorylated forms of multiple signaling molecules in FNA specimens, with reasonable correlation of marker intensities between two different FNA passes. Notably, signals of several markers were significantly higher in PDAC compared to non-cancerous samples. In PDAC samples, we found high total c-Met signal to be associated with poor survival, and confirmed this finding using an independent PDAC tissue microarray.