Richard Kirkland

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 Crohns 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.

Markers of neuroinflammation associated with Alzheimer's disease pathology in older adults

BACKGROUND In vitro and animal studies have linked neuroinflammation to Alzheimers disease (AD) pathology. Studies on markers of inflammation in subjects with mild cognitive impairment or AD dementia provided inconsistent results. We hypothesized that distinct blood and cerebrospinal fluid (CSF) inflammatory markers are associated with biomarkers of amyloid and tau pathology in older adults without cognitive impairment or with beginning cognitive decline. OBJECTIVE To identify blood-based and CSF neuroinflammation marker signatures associated with AD pathology (i.e. an AD CSF biomarker profile) and to investigate associations of inflammation markers with CSF biomarkers of amyloid, tau pathology, and neuronal injury. DESIGN/METHODS Cross-sectional analysis was performed on data from 120 older community-dwelling adults with normal cognition (n=48) or with cognitive impairment (n=72). CSF Aβ1-42, tau and p-tau181, and a panel of 37 neuroinflammatory markers in both CSF and serum were quantified. Least absolute shrinkage and selection operator (LASSO) regression was applied to determine a reference model that best predicts an AD CSF biomarker profile defined a priori as p-tau181/Aβ1-42 ratio >0.0779. It was then compared to a second model that included the inflammatory markers from either serum or CSF. In addition, the correlations between inflammatory markers and CSF Aβ1-42, tau and p-tau181 levels were assessed. RESULTS Forty-two subjects met criteria for having an AD CSF biomarker profile. The best predictive models included 8 serum or 3 CSF neuroinflammatory markers related to cytokine mediated inflammation, vascular injury, and angiogenesis. Both models improved the accuracy to predict an AD biomarker profile when compared to the reference model. In analyses separately performed in the subgroup of participants with cognitive impairment, adding the serum or the CSF neuroinflammation markers also improved the accuracy of the diagnosis of AD pathology. None of the inflammatory markers correlated with the CSF Aβ1-42 levels. Six CSF markers (IL-15, MCP-1, VEGFR-1, sICAM1, sVCAM-1, and VEGF-D) correlated with the CSF tau and p-tau181 levels, and these associations remained significant after controlling for age, sex, cognitive impairment, and APOEε4 status. CONCLUSIONS The identified serum and CSF neuroinflammation biomarker signatures improve the accuracy of classification for AD pathology in older adults. Our results suggest that inflammation, vascular injury, and angiogenesis as reflected by CSF markers are closely related to cerebral tau pathology.

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.

Sa1237 Prediction of Primary Response to Infliximab in Crohn's Disease: A Matrix-Based Prediction Model

Background Primary non-response (PNR) to TNF antagonists in IBD still holds a challenge for clinicians. Furthermore, with the advent of anti-integrin molecules, selecting the right therapeutic class for a given patient would be welcomed. Current predictors are insufficiently identifying patients at risk for PNR and are therefore not used in clinical practice. We designed a matrix-based prediction model, which may avoid exposure in patients who are unlikely to have benefit of the drug. Methods 201 anti-TNF Naive Crohns disease patients started on infliximab (IFX) induction 0-2-6 were used. For each patient, clinical information and biological markers (CRP, albumin,...) were collected prior to IFX start. Baseline serum TNF load and the IBD SGI serological panel (Prometheus Laboratories Inc.) were also available. PNR was defined as no clinical benefit after induction regimen. Univariate and correlation analyses were performed to select possible predictors. A combination of final predictors was selected through multiple regression based on the Bayesian information criterion (BIC). Finally these were categorized according to a clinically relevant threshold and were used to develop a matrix-based prediction model. Predicted probabilities were calculated and were organized into a matrix. Results: The incidence of PNR was 8% (16/ 201). Duration of disease, age at first IFX, BMI, previous surgery, serum albumin, ASCA IgA, ASCA IgG and serum TNF load differed substantially between responders and nonresponders in univariate analysis (all p<0.2). The parsimonious BIC in a multiple regression analysis withheld three independent significant final predictors (p<0.05): age at first IFX, BMI and previous surgery. The ROC-AUC for this final model was 0.79 and the predictors remained significant after bootstrapping. BMI was categorized into ≤20 (28% of patients), 21-25 (45%) and ≥ 26 (27%) and age at first IFX was categorized into ≤ 25 years (27%) , 26-64 years (68%) and ≥65 years (5%). Previous surgery was the strongest associated predictor of PNR with OR of 5.18 [1.51-23.96]. The matrix model using these final predictors showed a good spread of primary response rates for the different categories with a matching color code (see figure). Discussion We developed a matrix-based prediction tool to aid physicians in optimizing therapeutic decisions. After stringent selection of predictors only age at first IFX, BMI and previous surgery were withheld. A younger age has already been associated with primary response in several independent cohorts. BMI is also known to influence response and this is assigned to the weight based dosing of IFX. Previous surgery has also been associated with PNR and might reflect more refractory disease. The next step is to validate this matrix in an independent cohort and to construct a matrix-prediction tool for secondary loss of response.

Prevalence of Activated & Total p95HER2 and Other Receptor Tyrosine Kinases in Breast Cancer.

Background: HER2-overexpressing breast cancer has poor prognosis and is often resistant to HER2 targeted monoclonal antibody therapy. One of the mechanisms of de novo or acquired resistance is expression of p95HER2, various forms of truncated HER2 receptors with missing amino-terminal extra cellular domains. Methods for profiling various forms of HER2 receptors and other receptor tyrosine kinases (RTKs) with transactivation potential in primary and metastatic tumors may provide valuable insight into the shifting disease pathogenesis.Methods: A novel technology capable of specifically detecting phosphorylation events in ErbB family RTKs has been developed. This multiplexed protein microarray platform utilizes the formation of a unique immuno-complex requiring the co-localization of two detector enzyme-conjugated-antibodies once target proteins are captured on the microarray-surface. The channeling events between two detector enzymes (glucose oxidase & horse radish peroxidase) in proximity enabled the profiling of the RTK with extreme sensitivity. The analytical specificity is greatly enhanced given the requirement for simultaneous binding of three different antibodies. Different configuration of detector antibodies allowed differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., HER2). Here we report successful profiling of signal transduction pathway molecules for their expression and activation using 240 FNA samples collected from breast cancer patients (stage II to IV) with various ER/PR/HER2 status.Results: The FNA samples collected using G23 gauge needles were analyzed for expression and activation status for various RTKs and downstream signal transduction molecules including p95HER2, HER2, HER1, HER3, IGF-1R, PI3K and Shc. We found:O 100% concordance between primary HER2-IHC status and HER2 expression analysis based on proximity mediated immuno-microarray system O presence of varying degree of p95HER2 in over 40% of HER2-positive (HER2: 3+ and 2+ with FISH+) patients O ∼50% of p95HER2 expressors had activated p95HER2 O 25% of HER2-positive samples also had some levels of HER1 and HER3 activation O HER2-negative (HER2: 2+ with FISH-, 1+ and 0) samples also had 100% concordant HER2 expression, but number of them showed low but significant levels of HER2, HER3 and HER 1 activation.These findings may have implication on selection of appropriate targeted therapeutics.Discussion: The multiplexed-immuno microarray was utilized to detect the expression and phosphorylation of p95HER2, HER2, other RTKs (including HER3, HER1, IGF-1R, Shc, and PI3K) in 240 FNA samples collected from unique breast cancer patients with various ER/PR/HER2 status. Having the ability to profile tumors at different metastatic sites with an expanded pathway panel could provide information on their differential metastatic potentials; hence minimally invasive single-passage-mFNA samples may be utilized to tailor therapy options as disease-profile changes. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3053.

Blood-brain barrier breakdown, neuroinflammation, and cognitive decline in older adults

Blood‐brain barrier (BBB) breakdown is observed in older versus younger adults and in late‐onset Alzheimers disease versus age‐matched controls, but its causes and consequences in aging are unclear. We tested the hypothesis that BBB breakdown is associated with cognitive decline and inflammation in nondemented elders.

MARKERS OF NEUROINFLAMMATION ASSOCIATED WITH ALZHEIMER’S DISEASE PATHOLOGY IN OLDER ADULTS

BACKGROUND: In vitro and animal studies have linked neuroinflammation to Alzheimer’s disease (AD) pathology. Studies on markers of inflammation in subjects with mild cognitive impairment or AD dementia provided inconsistent results. We hypothesized that distinct blood and cerebrospinal fluid (CSF) inflammatory markers are associated with biomarkers of amyloid and tau pathology in older adults without cognitive impairment or with beginning cognitive decline. OBJECTIVE: To identify blood-based and CSF neuroinflammation marker signatures associated with AD pathology (i.e. an AD CSF biomarker profile) and to investigate associations of inflammation markers with CSF biomarkers of amyloid, tau pathology, and neuronal injury. DESIGN/METHODS: Cross-sectional analysis was performed on data from 120 older community-dwelling adults with normal cognition (n = 48) or with cognitive impairment (n = 72). CSF Aβ1-42, tau and p-tau181, and a panel of 37 neuroinflammatory markers in both CSF and serum were quantified. Least absolute shrinkage and selection operator (LASSO) regression was applied to determine a reference model that best predicts an AD CSF biomarker profile defined a priori as p-tau181/Aβ1-42 ratio >0.0779. It was then compared to a second model that included the inflammatory markers from either serum or CSF. In addition, the correlations between inflammatory markers and CSF Aβ1-42, tau and p-tau181 levels were assessed. RESULTS: Forty-two subjects met criteria for having an AD CSF biomarker profile. The best predictive models included 8 serum or 3 CSF neuroinflammatory markers related to cytokine mediated inflammation, vascular injury, and angiogenesis. Both models improved the accuracy to predict an AD biomarker profile when compared to the reference model. In analyses separately performed in the subgroup of participants with cognitive impairment, adding the serum or the CSF neuroinflammation markers also improved the accuracy of the diagnosis of AD pathology. None of the inflammatory markers correlated with the CSF Aβ1-42 levels. Six CSF markers (IL-15, MCP-1, VEGFR-1, sICAM1, sVCAM-1, and VEGF-D) correlated with the CSF tau and p-tau181 levels, and these associations remained significant after controlling for age, sex, cognitive impairment, and APOEε4 status. CONCLUSIONS: The identified serum and CSF neuroinflammation biomarker signatures improve the accuracy of classification for AD pathology in older adults. Our results suggest that inflammation, vascular injury, and angiogenesis as reflected by CSF markers are closely related to cerebral tau pathology.

Su1292 Biomarker Panel for Prediction of Mucosal Healing in Patients With Crohn's Disease Under Infliximab Therapy

Introduction. Infliximab has led to new therapeutic goals in Crohns disease (CD) such as complete mucosal healing and improvement of quality of life. The current standard for assessing mucosal healing is endoscopy. However, frequent assessments are costly and uncomfortable to the patient. Non-invasive, accurate surrogate serum markers would therefore be welcomed to aid clinicians in predicting mucosal healing.Methods. In a retrospective study, 119 CD patients who started infliximab and who underwent serial endoscopies (before and during infliximab) were included. Serum samples were available at the time of endoscopy and levels of markers were correlated with the degree of healing (not healed, marked improvement or complete healing). Thirty-five biomarkers were measured in 181 serum samples with the use of CEER, a proprietary highly sensitive protein micro-array, or homogenous mobility shift assays (Prometheus Laboratories Inc., San Diego, CA). These markers included growth and repair factors, pro -and anti-inflammatory markers and the IBD SGI serology panel. Infliximab and antibodies to infliximab were also measured. Clinical information regarding age at sampling, gender, age at diagnosis, location of disease, anal involvement and previous surgery was included. For statistical analysis SPSS and R were used. Results. From the 119 CD patients, 64 CD patients showed complete healing with no relapse, whereas 55 CD patients never showed mucosal healing. Univariate analyses indicated that age and 12 serum markers (HGF, BTC, TWEAK, CRP, ICAM, SAA, VCAM, IL-2, IL-8, IFN-γ, IL-6 and IL-10) were significantly associated with mucosal healing (p 11.44 CU/ml, TWEAK>20.62 CU/ml and VCAM<4200 μg/ml) which resulted in a significant and gradual increased prediction of mucosal healing (Figure 1, linear-by-linear p<0.001). Conclusion. We have identified 1 clinical parameter and 4 serum markers as independent predictors of mucosal healing. All serum markers play a role in the pathogenic mechanisms of CD. HGF has a central role in angiogenesis and tissue regeneration, BTC is a member of the EGF family of growth factors, TWEAK is a cytokine that has overlapping signaling functions with TNF and VCAM is a cell-adhesion molecule. The combined biomarker panel could facilitate prediction of mucosal healing in the future.

Abstract 4273: FGFR inhibition modulates ErbB receptor tyrosine phosphorylation in FGFR amplified cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) are currently under investigation as therapeutic targets for the treatment of breast cancer. FGFR1 amplifications range from 7.5-17% in breast cancers and are associated with a shorter overall survival. FGFR1 amplified cell lines drive activation of AKT and ERK pathways and are highly sensitive to FGFR1 inhibition, suggesting an oncogenic addiction to FGFR1. FGFR2 has been reported to be activated in lapatinib-resistant, HER2-positive cells. Likewise, FGFR3 expression levels are often elevated in tamoxifen-resistant, ER positive patients. FGFR 4 overexpression correlated with poor response to chemotherapy due to activation of MAPK and increase in B-cell lymphoma extra large (BCL-XL) levels. Using a highly sensitive, novel proximity mediated immuno-microarray, Collaborative Enzyme Enhanced Reactive-immunoassay (CEERTM), this study seeks to determine whether FGFR signal transduction pathway inhibition modulate ErbB receptor tyrosine kinases and AKT/ERK signal transduction pathway in cell lines expressing varying levels of FGFR and ErbB receptors. FGFR 1, 2, 3, and 4 amplified cell lines (MDA-MB-134VI (KRAS), SNU16, RT112, and MDA-MB-453) were individually treated with varying dosages (1, 10, 100, and 1000nM) of pan FGFR inhibitors (AZD4547 and Ponatinib-AP24534) in presence of corresponding FGF ligand (FGF1, FGF7, FGF9, and FGF19). Expression and activation of FGFR 1, 2, 3, 4 and EGFR, HER2, HER3, HER4 and downstream signaling proteins FRS2, AKT, ERK, MEK, and RSK were measured utilizing CEERTM. Both FGFR inhibitors sufficiently inhibited phosphorylation of FGFRs in each of the four FGFR amplified cell line. In MDA-MB-453, FGFR4 inhibition increased phosphorylation of HER2, and HER3 along with AKT. In MDA-MB-134, FGFR1 inhibition decreased levels of HER3 phosphorylation along with reduction in phosphorylated ERK, MEK, and RSK. In SNU16, FGFR2 inhibition led to a decrease in EGFR, HER2, and HER3 phosphorylation along with reduction in phosphorylated MEK, ERK, and RSK. Analysis of RT112 and additional cell lines with varying levels of ErbB and FGFR receptor tyrosine kinases are currently in progress. Our data suggests that FGFR inhibition may modulate parallel / compensatory ErbB and other signaling pathway as observed in this study. Utilizing CEERTM, comprehensive profiling may provide unique insight into potential feedback mechanisms in each patient and evidence for rational selection of appropriate combinational therapies. Citation Format: Nicholas Hoe, Kelly Jin, Yating Ma, Crystal Kuy, Michael Mateling, JinYao Zhou, Richard Kirkland, Saswati Hazara, Phillip Kim, Xinjun Liu, Sharat Singh. FGFR inhibition modulates ErbB receptor tyrosine phosphorylation in FGFR amplified cell lines. [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 4273. doi:10.1158/1538-7445.AM2013-4273

Abstract 5632: MEK inhibition leads to elevated HER3:PI3K in EGFR or HER2 driven cancer through feedback induction of HER1:3 and HER2:3 dimers.

In wide range of human cancer, the phosphoinositide 3-kinase (PI3K)/AKT and RAF/MEK/ERK signaling pathways are activated, regulating cell growth, metabolism, survival, and proliferation. Inhibition of either PI3K/AKT or MEK/ERK signaling pathway as a single-agent targeted therapy is often ineffective due to feedback mechanisms in which inhibition of one signaling pathway leads to activation of another. Treatment with a single-agent MEK inhibitor substantially increases ERBB3/PI3K/AKT phospho levels by relieving an ERK mediated negative feedback on threonine phosphorylation on the juxtamembrane domains of EGFR (T669) and HER2 (T677). Loss of inhibitory threonine phosphorylation in EGFR and HER2 suggests that increase in phospho HER3 levels may be due to formation of heterodimers (EGFR:HER3 and HER2:HER3) driving trans-phosphorylation of ERBB3. Herein we report, utilizing Collaborative Enzyme Enhanced Reactive (CEERTM) immunoassay, comprehensive analysis of key receptor tyrosine kinases (HER1, HER2, HER3, cMET, IGF1R, and others) and their downstream signal proteins (PI3K, Shc, AKT, MEK, ERK, PRAS40, RPS6, P70S6K, RSK), and dimerization partners (HER1:HER2, HER2:HER3, HER1:HER3, and HER3:PI3K) in MDA-MB-468, and BT474 treated with either MEK inhibitor alone (AZD6244) or in combination with PI3K inhibitor (GDC0941). CEER platform utilizes the formation of an unique “triple-antibody-enzyme-channeling” immuno-complex, requiring minimal sample amount. At MEK inhibitor concentration of 1μmol/L, both MDA-MB-468 and BT474 cells sufficiently inhibited ERK and RSK phosphorylation. In each cell line, we observed increase in phospho-HER3 and activated HER3:PI3K complex, a phospho-tyrosine signaling cascades that directly activate AKT. Levels of HER1:3 and HER2:3 dimers were elevated in MDA-MB-468 and BT474, respectively post MEK inhibitor treatment. Feedback activation of AKT with AZD6244 was suppressed when combined with GDC0941 at concentration of 1μmol/L. With combination of MEK and PI3K inhibitors, levels of HER1:3 and HER2:3 dimers were unaffected in their respective cell line, but HER3:PI3K complex was completely suppressed in both cell lines. The data suggests that loss of negative feedback with MEK inhibition alone promoted alternate oncogenic AKT signaling. As often the case, single-targeted therapies are ineffective, since the pathway it is targeted to inhibit, leads to feedback contributing to therapeutic resistance. Utilizing CEERTM, clinical specimens with limited availability can be profiled in a comprehensive manner for potential feedback mechanisms and selection of combinational therapies suppressing both the oncoprotein and the feedback program. Citation Format: Nicholas Hoe, Michael Mateling, Yating Ma, Kelly Jin, JinYao Zhou, Richard Kirkland, Crystal Kuy, Xinjun Liu, Phillip Kim, Sharat Singh. MEK inhibition leads to elevated HER3:PI3K in EGFR or HER2 driven cancer through feedback induction of HER1:3 and HER2:3 dimers. [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 5632. doi:10.1158/1538-7445.AM2013-5632