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Net Change of Mutational Load

Emergent Mutational Profiles

Plasma-Select Sequence Mutations Gene List

Mean RAS Mutant Allele Frequency by Response Category

Cox PH Model of OS Versus Baseline RAS Mutant Allele Frequency (with cfDNA concentration normalized or unnormalized, and with WT samples included or excluded)

Supplementary Table S3. Response and OS for RAS Mutants

Example Plot of Prognostic Risk Profile and Cut-Point for EGFR Pathway Genes

Mutation Prevalence at SFU

Mutation Prevalence at Baseline

Purpose:Mutations in EGFR pathway genes are poor prognostic indicators in patients with metastatic colorectal cancer. Plasma analysis of cell-free DNA is a minimally invasive and highly sensitive method to detect somatic mutations in tumors.Experimental Design:Plasma samples collected from panitumumab-treated patients in the ASPECCT study at baseline and safety follow-up (SFU) were analyzed by a next-generation sequencing–based approach for extended RAS mutant allele frequency as a continuous variable and their association with clinical outcomes and the mutational prevalence of 63 cancer-related genes. The correlation between patient outcome and baseline mutational status of EGFR pathway genes was also examined.Results:Overall, 261 patients in the panitumumab arm had evaluable plasma samples. Patients with a higher RAS mutant allele frequency at baseline had worse clinical outcomes than those with a lower frequency (P < 0.001, Cox PH model); however, RAS mutations did not necessarily preclude patients from deriving benefits. The objective response rate (complete or partial response) was 10.8% for patients with baseline RAS mutations and 21.7% for those with BRAF mutations. The 63-gene panel analysis revealed an increase in tumor mutational burden from baseline to SFU (P < 0.001, Wilcoxon signed rank test). Baseline mutations in EGFR pathway genes, when analyzed both categorically and continuously, were associated with shorter survival.Conclusions:When mutations in EGFR pathway genes were analyzed continuously, higher mutant allele frequency correlated with poorer outcomes. However, extended RAS mutation, by itself, did not preclude clinical responses to panitumumab in a monotherapy setting.

Patient Ascertainment for Extended RAS Analysis

Background: Although estimates of suboptimal adherence to oral corticosteroids in asthma range from 30% to 50%, no ideal method for measurement exists; the impact of poor adherence in severe asthma is likely to be particularly high. Research Questions: What is the prevalence of suboptimal adherence detected by self-reporting and direct measures? Is suboptimal adherence associated with disease activity? Study Design and Methods: Data were included from individuals with severe asthma taking part in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study and prescribed daily oral corticosteroids. Participants completed the Medication Adherence Report Scale, a five-item questionnaire used to grade adherence on a scale from 1 to 5, and provided a urine sample for analysis of prednisolone and metabolites by liquid chromatography-mass spectrometry. Results: Data from 166 participants were included in this study: mean (SD) age, 54.2 (± 11.9) years; FEV 1, 65.1% (± 20.5%) predicted; female, 58%; 37% completing the Medication Adherence Report Scale reported suboptimal adherence; and 43% with urinary corticosteroid data did not have detectable prednisolone or metabolites in their urine. Good adherence by both methods was detected in 49 of the 142 (35%) of participants in whom both methods were performed; adherence detection did not match between methods in 53%. Self-reported high adherers had better asthma control and quality of life, whereas directly measured high adherers had lower blood eosinophil levels. Interpretation: Low adherence is a common problem in severe asthma, whether measured directly or self-reported. We report poor agreement between the two methods, suggesting some disassociation between self-assessment of medication adherence and regular oral corticosteroid use, which suggests that each approach may provide complementary information in clinical practice.

Low grade, chronic inflammation is a critical risk factor for immunologic dysfunction including autoimmune diseases. However, the multiplicity of complex mechanisms and lack of relevant murine models limit our understanding of the precise role of chronic inflammation. To address these hurdles, we took advantage of multi-omics data and a unique murine model with a low but chronic expression of IFN-γ, generated by replacement of the AU-rich element (ARE) in the 3' UTR region of IFN-γ mRNA with random nucleotides. Herein, we demonstrate that low but differential expression of IFN-γ in mice by homozygous or heterozygous ARE replacement triggers distinctive gut microbial alterations, of which alteration is female-biased with autoimmune-associated microbiota. Metabolomics data indicates that gut microbiota-dependent metabolites have more robust sex-differences than microbiome profiling, particularly those involved in fatty acid oxidation and nuclear receptor signaling. More importantly, homozygous ARE-Del mice have dramatic changes in tryptophan metabolism, bile acid and long-chain lipid metabolism, which interact with gut microbiota and nuclear receptor signaling similarly with sex-dependent metabolites. Consistent with these findings, nuclear receptor signaling, encompassing molecules such as PPARs, FXR, and LXRs, was detectable as a top canonical pathway in comparison of blood and tissue-specific gene expression between female homozygous vs heterozygous ARE-Del mice. Further analysis implies that dysregulated autophagy in macrophages is critical for breaking self-tolerance and gut homeostasis, while pathways interact with nuclear receptor signaling to regulate inflammatory responses. Overall, pathway-based integration of multi-omics data provides systemic and cellular insights about how chronic inflammation driven by IFN-γ results in the development of autoimmune diseases with specific etiopathological features.

Background Asthma prevalence is 339 million globally. ‘Severe asthma’ (SA) comprises subjects with uncontrolled asthma despite proper management. Objectives To compare asthma from diverse ethnicities and environments. Methods A cross-sectional analysis of two adult cohorts, a Brazilian (ProAR) and a European (U-BIOPRED). U-BIOPRED comprised of 311 non-smoking with Severe Asthma (SAn), 110 smokers or ex-smokers with SA (SAs) and 88 mild to moderate asthmatics (MMA) while ProAR included 544 SA and 452 MMA. Although these projects were independent, there were similarities in objectives and methodology, with ProAR adopting operating procedures of U-BIOPRED. Results Among SA subjects, age, weight, proportion of former smokers and FEV1 pre-bronchodilator were similar. The proportion of SA with a positive skin prick tests (SPT) to aeroallergens, the scores of sino-nasal symptoms and quality of life were comparable. In addition, blood eosinophil counts (EOS) and the % of subjects with EOS > 300 cells/μl were not different. The Europeans with SA however, were more severe with a greater proportion of continuous oral corticosteroids (OCS), worse symptoms and more frequent exacerbations. FEV1/FVC pre- and post-bronchodilator were lower among the Europeans. The MMA cohorts were less comparable in control and treatment, but similar in the proportion of allergic rhinitis, gastroesophageal reflux disease and EOS >3%. Conclusions ProAR and U-BIOPRED cohorts, with varying severity, ethnicity and environment have similarities, which provide the basis for global external validation of asthma phenotypes. This should stimulate collaboration between asthma consortia with the aim of understanding SA, which will lead to better management.

Background: To investigate tumour biomarker status and efficacy of first-line panitumumab+FOLFIRI for metastatic colorectal carcinoma (mCRC). Methods: 154 patients received first-line panitumumab + FOLFIRI every 14 days. Primary end point was objective response rate (ORR). Data were analysed by tumour RAS (KRAS/NRAS) and BRAF status, and baseline amphiregulin (AREG) expression. Results: Objective responses occurred more frequently in RAS wild type (WT) (59%) vs RAS mutant (MT) (41%) mCRC and in RAS WT/BRAF WT (68%) vs RAS or BRAF MT (37%) disease. Median response duration was longer in RAS WT (13.0 months) vs RAS MT (5.8 months) (hazard ratio (HR): 0.16). Median progression-free survival was longer in RAS WT vs MT (11.2 vs 7.3 months; HR, 0.37) and was also longer in RAS WT/BRAF WT vs RAS or BRAF MT (13.2 vs 6.9 months; HR, 0.25). Incidence of adverse events was similar regardless of RAS/BRAF status, and no new safety signals were noted. Among patients with RAS WT tumours, ORR was 67% with high AREG expression and 38% with low AREG expression. Conclusions: First-line panitumumab+FOLFIRI was associated with favourable efficacy in patients with RAS WT and RAS WT/BRAF WT vs MT mCRC tumours and was well tolerated.

Stratified medicine requires discretisation of disease populations for targeted treatments. We have developed and applied a discrete Morse theory clustering algorithm to a Topological Data Analysis (TDA) network model of 498 gene expression profiles of peripheral blood from asthma and healthy participants. The Morse clustering algorithm defined nine clusters, BC1-9, representing molecular phenotypes with discrete phenotypes including Type-1, 2 & 17 cytokine inflammatory pathways. The TDA network model and clusters were also characterised by activity of glucocorticoid receptor signalling associated with different expression profiles of glucocorticoid receptor (GR), according to microarray probesets targeted to the start or end of the GR mRNA’s 3’ UTR; suggesting differential GR mRNA processing as a possible driver of asthma phenotypes including steroid insensitivity.

Introduction: The U-BIOPRED cohort presents an exceptional opportunity to longitudinally monitor disease variation in severe asthma. Aims: To determine the variability of severe asthma over 12-24 months in the U-BIOPRED cohort by initially focusing on clinical parameters; lung function, exacerbations and asthma symptom control. Methods: Lung function, exacerbation history and asthma control questionnaire (ACQ-5) were determined at baseline (Shaw et al.) and longitudinally. Results: 321 severe asthma patients were present in the longitudinal cohort with a mean of 454 days in the study. The longitudinal set of participants (321) were a good representation of the whole of severe asthma cohort at baseline (421). Most clinical and biomarker measurements including the FEV1 actual, exacerbation and ACQ means were not significantly different between the baseline and longitudinal visits- Table 1. There was no difference between the smoking and non-smoking cohorts. However at an individual level there was variation and some participants deteriorated, and some improved. Summary: Longitudinal U-BIOPRED data quality is valid. Further methods of analysis will move away from characterisation of group means towards understanding individual factors relating to disease progression in the U-BIOPRED cohort.

Background: Molecular stratification of childhood asthma could enable targeted therapy.Aims: Unbiased analysis of gene expression in paediatric severe (SA) and moderate/mild asthma (MA) blood samples to identify sub-phenotypes. Methods: Transcriptomic profiling by microarray analysis of blood from the U-BIOPRED paediatric cohort (Fleming ERJ 2015), pre- and school-age children, (SApre, n=62; MApre, n=42; SAsc, n=75 and MAsc, n=37). Topological data analysis (TDA) was used for unbiased clustering. Results: Sub-phenotypes, P1, P2, P3 and P4 were identified and are highlighted in the TDA network in the figure and a heatmap of selected variables. P1 (38% of the cohort, median 11 yrs) was characterised by low expression of glucocorticoid receptor (GR) mRNA splice variant with a long 3' UTR (q = 2.43E-17), but no significant difference in the expression of glucocorticoid receptor (GR) mRNA splice variant with a short 3' UTR. In P1, COX2 expression was up (q = 1.89E-06) and IFN-g was down (q = 5.61E-06), characteristics of a decreased steroid response. Conclusion: Unbiased analysis of U-BIOPRED paediatric peripheral blood gene expression identified a sub-phenotype, P1, with an inhibited steroid response. P1 is associated with low expression of a splice variant of GR with a long 3' UTR.

Purpose: Mutations in EGFR pathway genes are poor prognostic indicators in patients with metastatic colorectal cancer. Plasma analysis of cell-free DNA is a minimally invasive and highly sensitive method to detect somatic mutations in tumors. Experimental Design: Plasma samples collected from panitumumab-treated patients in the ASPECCT study at baseline and safety follow-up (SFU) were analyzed by a next-generation sequencing–based approach for extended RAS mutant allele frequency as a continuous variable and their association with clinical outcomes and the mutational prevalence of 63 cancer-related genes. The correlation between patient outcome and baseline mutational status of EGFR pathway genes was also examined. Results: Overall, 261 patients in the panitumumab arm had evaluable plasma samples. Patients with a higher RAS mutant allele frequency at baseline had worse clinical outcomes than those with a lower frequency (P < 0.001, Cox PH model); however, RAS mutations did not necessarily preclude patients from deriving benefits. The objective response rate (complete or partial response) was 10.8% for patients with baseline RAS mutations and 21.7% for those with BRAF mutations. The 63-gene panel analysis revealed an increase in tumor mutational burden from baseline to SFU (P < 0.001, Wilcoxon signed rank test). Baseline mutations in EGFR pathway genes, when analyzed both categorically and continuously, were associated with shorter survival. Conclusions: When mutations in EGFR pathway genes were analyzed continuously, higher mutant allele frequency correlated with poorer outcomes. However, extended RAS mutation, by itself, did not preclude clinical responses to panitumumab in a monotherapy setting.

Background Low-dose interleukin-2 (IL-2) therapy expands regulatory T cells (Treg) and provides clinical benefit for inflammatory diseases. AMG 592 is an investigational IL-2 mutein designed to expand Treg more selectively than recombinant IL-2 (aldesleukin). In a phase 1, double-blind, placebo (PBO)-controlled first-in-human (FIH) study, we investigated the safety and tolerability of AMG 592 and pharmacodynamic (PD) effects on Treg. Objectives We recently presented FIH study results including summary of safety, PK and PD.1 Here we extend those findings by exploring phenotypes of AMG 592 expanded Foxp3+ Treg subsets using flow cytometry. We compared both analysis using predefined gates and unsupervised gating. Potential implications for dose selection and mechanism of action will be discussed. Methods In the FIH study, healthy subjects in multiple ascending dose cohorts received a single subcutaneous dose of AMG 592 (n=6 per cohort) or placebo (n=2 per cohort). Pharmacodynamic response was evaluated for 28 days after treatment. In addition to enumerating CD4+ Foxp3+ Treg we evaluated changes in Treg subsets after AMG 592 treatment. Changes from baseline were analysed with linear mixed effects models with visit, dose level, and baseline result as main effects. To identify cell subsets, independent of predefined gates the same data were also evaluated with unsupervised gating tools using raw data from days 1, 8, 15 and 22. Results We observed a robust, dose-dependent expansion of Tregs that peaked at day 8 (~4–5 fold increase) and remained elevated above baseline up to day 29 for the highest doses. Expanded Tregs had increased levels of CD25 and Foxp3, and were enriched for CD31+recent thymic emigrants (RTE). Both naive and memory Treg increases peaked at day 8, however, naive Treg including RTE persisted at elevated levels through day 29 while memory Treg returned to normal levels earlier. The majority of Treg expressed the transcription factor Helios. Furthermore, expanded Tregs expressed higher proportions of PD-1. Unsupervised gating analysis identified several primary clusters of expanded Treg, one including naive and RTE Treg and another including memory Treg with elevated levels of HLA-DR expression. Evaluation of these clusters over time suggests that both increase initially at day 8 followed by preferential persistence of cells in the naive over memory Treg cluster by day 22. Conclusions Foxp3+ Treg were expanded in a dose dependent fashion in healthy subjects treated with AMG 592. The phenotype of expanded Treg included elevation of CD25 and Foxp3 as well as enrichment for PD-1 positive subsets. Taken together the increase in Treg with an RTE phenotype and persistence of naive Treg suggests that AMG 592 may increase diversity of the Treg pool as a possible mechanism of action in addition to effects on memory Treg. Reference [1] ASH 2017, AMG 592 Is an Investigational IL-2 Mutein That Induces Highly Selective Expansion of Regulatory T Cells, Nadia Tchao, Kevin S Gorski, Theresa Yuraszeck, Sue J Sohn, Katsuhiko Ishida, Hansen Wong and Kyong Park Disclosure of Interest K. Gorski Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., J. Stern Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., Y.-H. Hsu Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., A. Anderson Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., M. Boedigheimer Shareholder of: Amgen, Inc., Employee of: Amgen, Inc., N. Tchao Shareholder of: Amgen, Inc., Employee of: Amgen, Inc.

BACKGROUND & AIMS: The α4β7 integrin is a validated target in inflammatory bowel disease. This randomized, phase 2b, placebo-controlled, double-blind study evaluated the efficacy and safety of the anti-α4β7 antibody abrilumab in patients with moderate-to-severe ulcerative colitis despite treatment with conventional therapies. METHODS: Patients (total Mayo Score 6-12, recto-sigmoidoscopy score ≥2) with inadequate response or intolerance to conventional therapies were randomized to receive subcutaneous abrilumab (7, 21, or 70 mg) on day 1, weeks 2 and 4, and every 4 weeks; abrilumab 210 mg on day 1; or placebo. The primary end point was remission (total Mayo Score ≤2 points, no individual sub-score >1 point) for the 2 highest dosages at week 8. Key secondary end points were response and mucosal healing (centrally read) at week 8. RESULTS: For 354 patients who received ≥1 dose of investigational product (placebo, n = 116; 7 mg, n = 21; 21 mg, n = 40; 70 mg, n = 98; 210 mg, n = 79), non-adjusted remission rates at week 8 were 4.3%, 13.3%, and 12.7% for the placebo and abrilumab 70-mg and 210-mg groups, respectively (P < .05 for 70 and 210 mg vs placebo); odds of achieving remission were significantly greater with abrilumab 70 mg (odds ratio 3.35; 90% CI 1.41-7.95; P = .021) and 210 mg (odds ratio 3.33; 90% confidence interval 1.34-8.26; P = .030) than with placebo. Response and mucosal healing rates with these dosages also were significantly greater than with placebo. Higher baseline α4β7 levels on naïve CD4+ T cells were a prognostic indicator for overall outcome, but not a predictive biomarker of abrilumab response. There were no cases of progressive multifocal leukoencephalopathy or deaths. CONCLUSIONS: Abrilumab treatment for 8 weeks induced remission, clinical response, and mucosal healing in patients with moderate-to-severe ulcerative colitis. ClinicalTrials.gov, number NCT01694485. ispartof: GASTROENTEROLOGY vol:156 issue:4 pages:946-+ ispartof: location:United States status: published

Objective To assess the safety and immunologic impact of inhibiting interferon-γ (IFNγ) with AMG 811, a human IgG1 monoclonal antibody against IFNγ, in patients with systemic lupus erythematosus (SLE). Methods Twenty-six patients with mild-to-moderate, stable SLE were administered placebo or a single dose of AMG 811, ranging from 2 mg to 180 mg subcutaneously or 60 mg intravenously. Results Similar to results previously reported following inhibition of type I IFNs, treatment of SLE patients with AMG 811 led to a dose-dependent modulation of the expression of genes associated with IFN signaling, as assessed by microarray analysis of the whole blood. The list of impacted genes overlapped with that identified by stimulating human whole blood with IFNγ and with those gene sets reported in the literature to be differentially expressed in SLE patients. Serum levels of IFNγ-induced chemokines, including IFNγ-inducible protein 10 (IP-10), were found to be elevated at baseline in SLE patients as compared to healthy volunteers. In contrast to previously reported results from studies using type I IFN–blocking agents, treatment with AMG 811 led to dose-related reductions in the serum levels of CXCL10 (IP-10). Conclusion The scope and nature of the biomarkers impacted by AMG 811 support targeting of IFNγ as a therapeutic strategy for SLE.

Background: We have previously reported altered gene expression in adults with asthma compared to healthy controls from the U-BIOPRED study (Bigler, 2016). Altered transcripts may define dysregulated biological pathways and identify novel therapeutic targets. We hypothesised that similar dysregulation would be seen in children with asthma. Aim: To compare blood transcriptomic profiles of children and adults with asthma. Methods: Affymetrix blood transcriptomic profiles of severe asthmatic adult non-smokers, n=152, were compared to mild moderate asthmatics, n=50 (Shaw, 2015; Fleming, 2015). Profiles of school-aged children with severe asthma, n=75, were compared to mild moderate asthmatics, n=37, and in the preschool age group severe wheeze, n=62, was compared to mild moderate wheeze, n=42. Differentially expressed genes (DEG) were identified as probe sets with maximum median group intensity >log2 5, with a significant (raw P≤0.01) change ≥ 20%. Overlapping genes were determined and pathway analysis performed. Results: We found 1887 DEG comparing severe and mild moderate asthmatic adults. Only 28 DEG were found between the severe wheeze and mild pre-school age children, with a larger signature (569 DEG) in the school aged children. 480 genes were specific to school-aged children and 1801 specific to adults, with 89 DEG in common between the adults and school-aged children. Conclusions: Preschool age children were poorly defined in terms of blood transcriptomics by the clinical definitions used. While the school-aged children showed some DEG overlap with the adults, they were distinct in many DEG and pathways indicating that childhood and adult asthma may be mechanistically different.

238 Background: RAS mutations are negative predictors of response to anti-EGFR therapies such as panitumumab in mCRC. Mutations at baseline (BL) and follow-up (FU) during randomized phase 3 studies of first line treatment (1L; study 20050203 [‘203]; panitumumab + fluorouracil, leucovorin and oxaliplatin [FOLFOX4] vs FOLFOX4) were compared with those in third line treatment (3L; study 20100007 [‘0007]; panitumumab + best supportive care [BSC] vs BSC) to assess tumor heterogeneity via ctDNA analysis. Methods: Biomarker analysis was conducted for pts with plasma samples at BL and FU. Samples were analyzed using the Plasma Select-R 63-gene panel (Personal Genome Diagnostics, Inc.), with a limit of detection of 0.1%. Mutations were defined at the amino acid level. The Cox hazard ratio (HR) by sum of RAS mutant allele frequency (MAF) was determined, as were event-free survival (EFS) and best response by RAS mutation status. Results: For all pts with available samples (‘203, n = 120; ‘0007, n = 90), fewer mutations and fewer mutations/gene were observed in the 1L vs 3L setting at BL ( KRAS 2 vs 3 maximum mutations/gene; EGFR 1 vs 4 maximum mutations/gene). In 3L the Cox HR increased continuously with RAS MAF; while this was not found in 1L. In the 1L setting, emergent RAS mutations were not predictive of EFS for FOLFOX4, but were predictive of shorter EFS for panitumumab + FOLFOX4. More panitumumab-treated pts in the 3L setting had detectable RAS mutations emerging from BL to FU (28.6%, 57.1%) vs pts treated in 1L (24.5%, 26.5%). For pts who achieved a partial response, more treated in 1L maintained a higher frequency of wild-type RAS from BL to FU (84.4%, 78.1%) vs pts treated in 3L (95.7%, 43.5%). Conclusions: The overall mutational landscape differs between the 1L vs 3L setting in anti-EGFR–treated mCRC pts. Panitumumab monotherapy in the 3L setting appears to induce greater RAS-specific selective pressure than panitumumab FOLFOX4 combination therapy in 1L, resulting in increased RAS mutations at FU in the 3L setting. The combination of panitumumab + FOLFOX in 1L is associated with delayed emergence of expansion of RAS mutations compared to later line single agent panitumumab.

To investigate the relationships between AMG 811 exposure, concentration changes in serum IFN-γ, and IFN-γ-induced protein 10 (CXCL10), and to identify important contributions of baseline covariates to these relationships. A mechanism based pharmacokinetic (PK)-pharmacodynamic (PD) model was developed. A target mediated disposition model was used to describe AMG 811 and target IFN-γ interaction. CXCL10 was predicted to be driven by estimated free IFN-γ levels. For an average systemic lupus erythematosus (SLE) subject, the linear clearance (CL) of AMG 811 was 0.176 L/day, and the central (Vc) and peripheral (Vp) volumes of distribution were 1.48 and 2.12 L, respectively. Body weight was found to correlate with CL, Vc, Vp, and inter compartment clearance (Q); and age was found to correlate with Vc. The relationship between estimated free serum IFN-γ concentration levels and serum CXCL10 in logarithmic scales was best described by a linear model with slope and intercept estimated to be 0.197 and -0.3, respectively. The largest observed reduction of serum CXCL10 concentration was achieved at the highest AMG 811 dose tested (180 mg SC). This model enables simulations of AMG 811 PK-PD profiles under various dosing regimens to support future clinical studies.

Background Mutations in rat sarcoma (RAS) genes may be a mechanism of secondary resistance in epidermal growth factor receptor inhibitor-treated patients. Tumor-tissue biopsy testing has been the standard for evaluating mutational status; however, plasma testing of cell-free DNA has been shown to be a more sensitive method for detecting clonal evolution. Materials and methods Archival pre- and post-treatment tumor biopsy samples from a phase II study of panitumumab in combination with irinotecan in patients with metastatic colorectal cancer (mCRC) that also collected plasma samples before, during, and after treatment were analyzed for emergence of mutations during/post-treatment by next-generation sequencing and BEAMing. Results The rate of emergence of tumor tissue RAS mutations was 9.5% by next-generation sequencing (n= 21) and 6.3% by BEAMing (n = 16). Plasma testing of cell-free DNA by BEAMing revealed a mutant RAS emergence rate of 36.7% (n = 39). Exploratory outcomes analysis of plasma samples indicated that patients who had emergent RAS mutations at progression had similar median progression-free survival to those patients who remained wild-type at progression. Serial analysis of plasma samples showed that the first detected emergence of RAS mutations preceded progression by a median of 3.6 months (range, -0.3 to 7.5 months) and that there did not appear to be a mutant RAS allele frequency threshold that could predict near-term outcomes. Conclusions This first prospective analysis in mCRC showed that serial plasma biopsies are more inclusive than tissue biopsies for evaluating global tumor heterogeneity; however, the clinical utility of plasma testing in mCRC remains to be further explored. ClinicalTrials.gov Identifier NCT00891930

BACKGROUND: Asthma is a heterogeneous disease in which there is a differential response to asthma treatments. This heterogeneity needs to be evaluated so that a personalised management approach can be provided.OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinico-physiological parameters and performed an -omics analysis of sputum.METHODS: Partition-around-medoid clustering was applied to a training set of 266 asthma participants from the European U-BIOPRED adult cohort using 8 pre-specified clinic-physiological variables. This was repeated in a separate validation set of 152 asthmatics. The clusters were compared based on sputum proteomic and transcriptomic data.RESULTS: Four reproducible and stable clusters of asthmatics were identified. The training set cluster T1 consists of well-controlled moderate-to-severe asthmatics, while cluster T2 is a group of late-onset severe asthmatics with history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of non-smokers. Cluster T4 is predominantly composed of obese female uncontrolled severe asthmatics with increased exacerbations, but with normal lung function. The validation set exhibited similar clusters, demonstrating reproducibility of the classification. There were significant differences in sputum proteomics and transcriptomics between the clusters. The severe asthma clusters, T2, T3 and T4, had higher sputum eosinophilia than T1 with no differences in sputum neutrophil counts, exhaled nitric oxide and serum IgE levels.CONCLUSION: Clustering based on clinico-physiological parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.CLINICAL IMPLICATIONS: The definition of four distinct clusters of asthma linked to different pathobiological pathways provides a better template for the phenotyping and personalised treatment of severe asthma, where high unmet needs remain.CAPSULE SUMMARY: Unsupervised clustering of asthma on clinical features alone has led to the definition of four phenotypes. Sputum 'omics' analysis has revealed different biological pathways pointing towards potential new treatments.

Background Epidermal growth factor receptor (EGFR)-targeted agents have demonstrated clinical benefit in patients with cancer. Identifying tissue-of-origin-independent predictive biomarkers is important to optimally treat patients. We sought to identify a gene array profile that could predict responsiveness to panitumumab, a fully human EGFR-binding antibody, using preclinical models of human cancer. Methods Mice bearing 25 different xenograft models were treated twice weekly with panitumumab or immunoglobulin G2 control to determine their responsiveness to panitumumab. Samples from these xenografts and untreated xenografts were arrayed on the Affymetrix human U133A gene chip to identify gene sets predicting responsiveness to panitumumab using univariate and multivariate analyses. The predictive models were validated using the leave-one-group-out (LOO) method. Results Of the 25 xenograft models tested, 12 were responsive and 13 were resistant to panitumumab. Unsupervised analysis demonstrated that the xenograft models clustered by tissue type rather than responsiveness to panitumumab. After normalizing for tissue effects, samples clustered by responsiveness using an unsupervised multidimensional scaling. A multivariate selection algorithm was used to select 13 genes that could stratify xenograft models based on responsiveness after adjustment for tissue effects. The method was validated using the LOO method on a training set of 22 models and confirmed independently on three new models. In contrast, a univariate gene selection method resulted in higher misclassification rates. Conclusion A model was constructed from microarray data that prospectively predict responsiveness to panitumumab in xenograft models. This approach may help identify patients, independent of disease origin, likely to benefit from panitumumab.

Stratification of asthma at the molecular level, especially using accessible biospecimens, could greatly enable patient selection for targeted therapy. To determine the value of blood analysis to identify transcriptional differences between clinically defined asthma and nonasthma groups, identify potential patient subgroups based on gene expression, and explore biological pathways associated with identified differences. Transcriptomic profiles were generated by microarray analysis of blood from 610 patients with asthma and control participants in the U-BIOPRED (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes) study. Differentially expressed genes (DEGs) were identified by analysis of variance, including covariates for RNA quality, sex, and clinical site, and Ingenuity Pathway Analysis was applied. Patient subgroups based on DEGs were created by hierarchical clustering and topological data analysis. A total of 1,693 genes were differentially expressed between patients with severe asthma and participants without asthma. The differences from participants without asthma in the nonsmoking severe asthma and mild/moderate asthma subgroups were significantly related (r = 0.76), with a larger effect size in the severe asthma group. The majority of, but not all, differences were explained by differences in circulating immune cell populations. Pathway analysis showed an increase in chemotaxis, migration, and myeloid cell trafficking in patients with severe asthma, decreased B-lymphocyte development and hematopoietic progenitor cells, and lymphoid organ hypoplasia. Cluster analysis of DEGs led to the creation of subgroups among the patients with severe asthma who differed in molecular responses to oral corticosteroids. Blood gene expression differences between clinically defined subgroups of patients with asthma and individuals without asthma, as well as subgroups of patients with severe asthma defined by transcript profiles, show the value of blood analysis in stratifying patients with asthma and identifying molecular pathways for further study. Clinical trial registered with www.clinicaltrials.gov (NCT01982162)

Objectives To evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of single-dose and multiple-dose administration of AMG 557, a human anti-inducible T cell co-stimulator ligand (ICOSL) monoclonal antibody, in subjects with systemic lupus erythematosus (SLE). Methods Patients with mild, stable SLE (n=112) were enrolled in two clinical trials to evaluate the effects of single (1.8–210 mg subcutaneous or 18 mg intravenous) and multiple (6 –210 mg subcutaneous every other week (Q2W)×7) doses of AMG 557. Subjects received two 1 mg intradermal injections 28 days apart of keyhole limpet haemocyanin (KLH), a neoantigen, to assess PD effects of AMG 557. Safety, PK, target occupancy, anti-KLH antibody responses, lymphocyte subset analyses and SLE-associated biomarkers and clinical outcomes were assessed. Results AMG 557 demonstrated an acceptable safety profile. The PK properties were consistent with an antibody directed against a cell surface target, with non-linear PK observed at lower concentrations and linear PK at higher concentrations. Target occupancy by AMG 557 was dose dependent and reversible, and maximal occupancy was achieved in the setting of this trial. Anti-AMG 557 antibodies were observed, but none were neutralising and without impact on drug levels. A significant reduction in the anti-KLH IgG response was observed with AMG 557 administration without discernible changes in the anti-KLH IgM response or on the overall IgG levels. No discernible changes were seen in lymphocyte subsets or in SLE-related biomarkers and clinical measures. Conclusions The selective reduction in anti-KLH IgG demonstrates a PD effect of AMG 557 in subjects with SLE consistent with the biology of the ICOS pathway and supports further studies of AMG 557 as a potential therapeutic for autoimmune diseases. Trial registration numbers NCT02391259 and NCT00774943.

Objective Interferon-γ (IFNγ) is implicated in the pathogenesis of discoid lupus erythematosus (DLE). This study sought to evaluate a single dose of AMG 811, an anti-IFNγ antibody, in patients with DLE. Methods The study was designed as a phase I randomized, double-blind, placebo-controlled crossover study of the pharmacodynamics, safety, and clinical efficacy of AMG 811 in patients with DLE. Patients received a single subcutaneous dose of AMG 811 (180 mg) or placebo. The patients in sequence 1 received AMG 811 followed by placebo, while those in sequence 2 received placebo followed by AMG 811. Pharmacodynamic end points included global transcriptional analyses of lesional and nonlesional skin, IFNγ blockade signature (IGBS) transcriptional scores in the skin and blood, keratinocyte IFNγ RNA scores, and serum levels of CXCL10 protein. Additional end points were efficacy outcome measures, including the Cutaneous Lupus Erythematosus Disease Area and Severity Index, and safety outcome measures. Results Sixteen patients with DLE were enrolled in the study (9 in sequence 1 and 7 in sequence 2). AMG 811 treatment reduced the IGBS score (which was elevated in DLE patients at baseline) in both the blood and lesional skin. The keratinocyte IFNγ RNA score was not affected by administration of AMG 811. Serum CXCL10 protein levels (which were elevated in the blood of DLE patients) were reduced with AMG 811 treatment. The AMG 811 treatment was well tolerated but did not lead to statistically significant improvements in any of the efficacy outcome measures. Conclusion AMG 811 treatment led to changes in IFNγ-associated biomarkers and was well tolerated, but no significant clinical benefit was observed in patients with DLE.

Introduction: Triplet regimens incorporating a proteasome inhibitor and immunomodulatory drug are standards of care for the treatment of patients with newly diagnosed multiple myeloma (NDMM). The combinations of carfilzomib-lenalidomide-dexamethasone (KRd) and bortezomib-lenalidomide-dexamethasone (VRd) are recommended regimens for the treatment of NDMM by the National Comprehensive Care Network. However, there are limited data directly comparing the relative effectiveness and tolerability of these two regimens in patients with NDMM. Here, we report prospective evaluation of efficacy and preliminary tolerability data for patients who received KRd or VRd as frontline therapy in the Multiple Myeloma Research Foundation (MMRF) Clinical Outcomes in MM to Personal Assessment of Genetic Profile (CoMMpass, NCT01454297) study. Methods: CoMMpass is a prospective observational study conducted since 2011 by the MMRF that has enrolled over 1100 patients from Multiple Myeloma Research Consortium sites. Eligible adults with NDMM and symptomatic disease were enrolled within 30 days of initiating frontline therapy. Frontline therapy was chosen at the discretion of the investigator but must have included a proteasome inhibitor and/or immunomodulatory drug. In the current analysis, we evaluated the effectiveness and reasons for treatment discontinuation among enrolled patients who received KRd or VRd as first-line therapy. KRd patients were matched to VRd patients based on propensity score matching including age, gender, ISS and renal insufficiency as covariates. Treatment response was assessed by investigators and defined by International Myeloma Working Group Uniform Response Criteria. Event-free survival (EFS) was the pre-specified primary endpoint and defined as the time from the start of treatment until disease progression, death, or the initiation of new therapy. EFS was compared between treatment groups using a Cox proportional hazards model. Results: A total of 609 evaluable patients received first-line KRd (n=149) or VRd (n=460). Of these, 149 KRd patients and 149 VRd patients were matched according to baseline co-variates. Patient demographics and disease characteristics were balanced between treatment arms for the matched set of patients (KRd vs VRd) including by median age (years, 58 vs 59), gender (male, 63% vs 62%), and ISS (stage I, 46% vs 53%; stage II, 41% vs 40%; stage III, 13% vs 7%). With median follow up of 11.5 months for KRd and 41.9 months for VRd, 12-month EFS rates (95% CI) were 95% (90-99%) for KRd vs 84% (78-90%) for VRd (12-month HR, 0.28; 95% CI, 0.10-0.75; p=0.0043; Figure 1). By 12 months, 87% (95% CI, 81-93%) of KRd patients vs 68% (95% CI, 60-76%) of VRd patients had a partial response or better (p=0.0029) and 35% (95% CI, 25-45%) of KRd patients vs 14% (95% CI, 8-20%) of VRd patients achieved a complete response or better (p=0.0054; Figure 2). The treatment discontinuation rate due to adverse events was 3.4% for each arm. Conclusions: In the CoMMpass study, KRd demonstrated significant improvements in 12-month EFS compared with VRd in patients with NDMM (HR, 0.28; 95% CI, 0.10-0.75; p=0.0043). By 12 months, patients treated with KRd also achieved significantly higher response rates and complete response rates or better compared with VRd treated patients. Discontinuation rates due to AEs were similar between KRd and VRd. With limitations of non-randomized evaluation and relatively short median follow-up in the KRd arm, these results are consistent with previous single arm studies that KRd is not only effective but potentially a superior treatment option compared with VRd for patients with NDMM. Updated results with extended follow-up will be presented. Disclosures Landgren: Karyopharm: Consultancy; Pfizer: Consultancy; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees. Siegel:Novartis: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Merck: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau. Chari:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Array Biopharma: Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Consultancy; Bristol Myers Squibb: Consultancy. Boedigheimer:Amgen Inc.: Employment, Equity Ownership. Welliver:Amgen: Employment, Equity Ownership. Mezzi:Amgen: Employment, Equity Ownership. Iskander:Amgen: Employment, Equity Ownership. Jakubowiak:Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; SkylineDx: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

Vitamin D sterol administration, a traditional treatment for secondary hyperparathyroidism, may increase serum calcium and phosphorus, and has been associated with increased vascular calcification (VC). In vitro studies suggest that in the presence of uremic concentrations of phosphorus, vitamin D sterols regulate gene expression associated with trans-differentiation of smooth muscle cells (SMCs) to a chondro/osteoblastic cell type. This study examined effects of vitamin D sterols on gene expression profiles associated with phosphate-enhanced human coronary artery SMC (CASMC) calcification. Cultured CASMCs were exposed to phosphate-containing differentiation medium (DM) with and without calcitriol, paricalcitol, or the calcimimetic R-568 (10−11–10−7 M) for 7 days. Calcification of CASMCs, determined using colorimetry following acid extraction, was dose dependently increased (1.6- to 1.9-fold) by vitamin D sterols + DM. In contrast, R-568 did not increase calcification. Microarray analysis demonstrated that, compared with DM, calcitriol (10−8 M) + DM or paricalcitol (10−8 M) + DM similarly and significantly (P < 0.05) regulated genes of various pathways including: metabolism, CYP24A1; mineralization, ENPP1; apoptosis, GIP3; osteo/chondrogenesis, OPG, TGFB2, Dkk1, BMP4, BMP6; cardiovascular, HGF, DSP1, TNC; cell cycle, MAPK13; and ion channels, SLC22A3 KCNK3. R-568 had no effect on CASMC gene expression. Thus, SMC calcification observed in response to vitamin D sterol + DM may be partially mediated through targeting mineralization, apoptotic, osteo/chondrocytic, and cardiovascular pathway genes, although some gene changes may protect against calcification. Further studies to determine precise roles of these genes in development of, or protection against VC and cardiovascular disease are required. J. Cell. Biochem. 111: 911–921, 2010. © 2010 Wiley-Liss, Inc.

Flow Cytometry has become a mainstay technique for measuring fluorescent and physical attributes of single cells in a suspended mixture. These data are reduced during analysis using a manual or semiautomated process of gating. Despite the need to gate data for traditional analyses, it is well recognized that analyst-to-analyst variability can impact the dataset. Moreover, cells of interest can be inadvertently excluded from the gate, and relationships between collected variables may go unappreciated because they were not included in the original analysis plan. A multivariate non-gating technique was developed and implemented that accomplished the same goal as traditional gating while eliminating many weaknesses. The procedure was validated against traditional gating for analysis of circulating B cells in normal donors (n = 20) and persons with Systemic Lupus Erythematosus (n = 42). The method recapitulated relationships in the dataset while providing for an automated and objective assessment of the data. Flow cytometry analyses are amenable to automated analytical techniques that are not predicated on discrete operator-generated gates. Such alternative approaches can remove subjectivity in data analysis, improve efficiency and may ultimately enable construction of large bioinformatics data systems for more sophisticated approaches to hypothesis testing. © 2008 International Society for Advancement of Cytometry

Background Autosomal dominant polycystic kidney disease (ADPKD) causes progressive loss of renal function in adults as a consequence of the accumulation of cysts. ADPKD is the most common genetic cause of end-stage renal disease. Mutations in polycystin-1 occur in 87% of cases of ADPKD and mutations in polycystin-2 are found in 12% of ADPKD patients. The complexity of ADPKD has hampered efforts to identify the mechanisms underlying its pathogenesis. No current FDA (Federal Drug Administration)-approved therapies ameliorate ADPKD progression. Results We used the de Almeida laboratory’s sensitive new transcriptogram method for whole-genome gene expression data analysis to analyze microarray data from cell lines developed from cell isolates of normal kidney and of both non-cystic nephrons and cysts from the kidney of a patient with ADPKD. We compared results obtained using standard Ingenuity Volcano plot analysis, Gene Set Enrichment Analysis (GSEA) and transcriptogram analysis. Transcriptogram analysis confirmed the findings of Ingenuity, GSEA, and published analysis of ADPKD kidney data and also identified multiple new expression changes in KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways related to cell growth, cell death, genetic information processing, nucleotide metabolism, signal transduction, immune response, response to stimulus, cellular processes, ion homeostasis and transport and cofactors, vitamins, amino acids, energy, carbohydrates, drugs, lipids, and glycans. Transcriptogram analysis also provides significance metrics which allow us to prioritize further study of these pathways. Conclusions Transcriptogram analysis identifies novel pathways altered in ADPKD, providing new avenues to identify both ADPKD’s mechanisms of pathogenesis and pharmaceutical targets to ameliorate the progression of the disease. Electronic supplementary material The online version of this article (doi:10.1186/s40246-016-0095-x) contains supplementary material, which is available to authorized users.

Administration of active vitamin D sterols to treat secondary hyperparathyroidism in patients with chronic kidney disease receiving dialysis has been associated with elevated serum calcium and phosphorus levels, which may lead to increased risk of vascular calcification. However, calcimimetics, by binding to the parathyroid gland calcium-sensing receptors, reduce serum parathyroid hormone, calcium, phosphorus, and the calcium-phosphorus product. Using cultured bovine aorta vascular smooth muscle cells (BASMCs), an in vitro model of vascular calcification, we compared calcification levels and gene expression profiles after exposure to the phosphate source ss-glycerolphosphate (BGP), the active vitamin D sterols calcitriol and paricalcitol, the calcimimetic R-568, or BGP with the active vitamin D sterols or R-568. Cells exposed to BGP (10 mM) alone or with calcitriol or paricalcitol showed dose-dependent BASMC calcification. No change in calcification was observed in cultures exposed to BGP with R-568, consistent with the observed lack of calcium-sensing receptor expression. Microarray analysis using total cellular RNA from cultures exposed to vehicle or BGP in the absence and presence of 10(-8) M calcitriol or paricalcitol for 7 days showed that cells exposed to BGP with calcitriol or BGP with paricalcitol had virtually identical gene expression profiles, which differed from those of cells treated with BGP or vehicle alone. Several osteoblast- and chondrocyte-associated genes were modulated by BGP and vitamin D exposure. In this study, exposure of BASMCs to phosphate and active vitamin D sterols induced calcification and changes in expression of genes associated with mineralized tissue.

Background: Mutations in RAS family genes are a negative predictor for response to anti-EGFR therapy. ASPECCT was the first prospective study to show that panitumumab was noninferior to cetuximab for overall survival (OS) in chemorefractory wild-type (WT) KRAS exon 2 mCRC. This analysis used next-generation sequencing of ctDNA before and after panitumumab therapy to explore mutations in EGFR pathway genes and their association with outcomes. Methods: Plasma samples collected at baseline (BL) and safety follow-up (SFU) were analyzed for ctDNA mutations using the PlasmaSelect-R™ 63-gene panel. Mutations for six major genes within the EGFR pathway (BRAF, KRAS, MAP2K1, NRAS, PIK3CA, and PTEN) were analyzed as categorical and continuous variables, and were evaluated for association with OS using univariate Cox proportional hazards (PH) model. Results: Of the 499 patients randomized to panitumumab, 208 had paired plasma samples at BL and SFU. Of the 113 (54.3%) patients who were WT for all genes at BL, 59 (52%) remained WT at SFU. At BL, 65 patients had single gene mutations in either BRAF (7.7%), KRAS (6.7%), PIK3CA (6.3%), NRAS (5.8%), PTEN (3.8%), or MAP2K1 (1.0%), and 30 patients had mutations in multiple genes (at Citation Format: Michael Boedigheimer, Agnes Lee Ang, Tae Won Kim, Anne Thomas, Peter Gibbs, Paul Ruff, Kristina Hool, Timothy Price. Circulating tumor (ct)DNA mutations in EGFR pathway genes and clinical outcomes for patients with metastatic colorectal cancer (mCRC) treated with panitumumab from the ASPECCT study [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A032.

Objective To evaluate safety, pharmacokinetics and pharmacodynamics of anti-interferon (IFN)-γ monoclonal antibody AMG 811 in subjects with SLE without or with lupus nephritis (LN). Methods In this phase Ib, randomised, multiple-dose escalation study (NCT00818948), subjects without LN were randomised to subcutaneous AMG 811 (6, 20 or 60 mg) or placebo and subjects with LN were randomised to subcutaneous AMG 811 (20, 60 or 120 mg) or placebo every four weeks for three total doses. Outcomes included incidence of adverse events (AEs); pharmacokinetics; levels of serum proteins (CXCL-10, interleukin 18, monocyte chemotactic protein-1); changes in gene transcript profiles and clinical parameters (Safety of Estrogen in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) scores, proteinuria, anti-double-stranded DNA (anti-dsDNA) antibodies, C3 complement, C4 complement). Results Fifty-six subjects enrolled (28 SLE without LN; 28 with LN). Baseline mean SELENA-SLEDAI scores were 2.2 and 12.0 for SLE subjects without and with LN, respectively. Most subjects reported an AE; no meaningful imbalances were observed between AMG 811 and placebo. Pharmacokinetic profiles were similar and mostly dose-proportional in subjects without or with LN. AMG 811 treatment reduced CXCL-10 protein levels and blood-based RNA IFN-γ Blockade Signature compared with placebo. Reductions were less pronounced and not sustained in subjects with LN, even at the highest dose tested, compared with subjects without LN. No effect on SELENA-SLEDAI scores, proteinuria, C3 or C4 complement levels, or anti-dsDNA antibodies was observed. Conclusion AMG 811 demonstrated favourable pharmacokinetics and acceptable safety profile but no evidence of clinical impact. IFN-γ-associated biomarkers decreased with AMG 811; effects were less pronounced and not sustained in LN subjects. Trial registration number NCT00818948; results.

3523 Background: ASPECCT was a clinical trial performed in the chemotherapy-refractory third-line mCRC setting (N = 1010). This biomarker analysis explores the mutational landscape in panitumumab monotherapy subjects. Analysis of plasma ctDNA at baseline and post-treatment (PT) by next-generation sequencing provides a snapshot of the main changes in key genes before and after therapy. Methods: CtDNA collected at baseline and PT was analyzed for mutations using the Plasma Select-R™ 63-gene panel (0.1% limit of detection). Gain or loss of mutation was defined at the amino acid level. Net change is the sum of mutations gained minus the sum of mutations lost. A single individual could have both net gain and/or net loss of mutations within a single gene. Results: Significant tumor clonal diversification was observed during therapy. In 238 subjects with paired plasma samples,29% of subjects had multiple mutations in the same gene at baseline and 41% of subjects had multiple mutations in the same gene PT. At least 10% of subjects demonstrated an on-therapy acquired mutation in at least one of the following genes: APC, EGFR, ALK, HER4, TP53, AR, KRAS, BRAF, PDGFRA, STK11, ESR1, FBXWT, and KIT (ordered by frequency). New mutations were noted both inside and outside the EGFR pathway. Unexpectedly, patients with a large decrease in mutant DNA burden after anti-EGFR treatment were also seen. EGFR pathway genes with significant net gain were: KRAS, EGFR, NRAS, BRAF, MAP2K1, PIK3CA, and AKT1. Non-EGFR pathway mutations gained included: APC, CDK6, SMARCB1, FBXW7, TERT, RB1, CTNNB1, and IDH1. Conclusions: This 63-gene plasma analysis suggests that there are significant dynamic changes in clonal mutational fraction under anti-EGFR selection. Our analysis reveals that increasing global tumor heterogeneity is associated with poorer overall survival. A subset of patients demonstrated an overall decrease in tumor heterogeneity on panitumumab therapy (28%), indicating that under anti-EGFR selective pressure mutational heterogeneity can also decrease. Clinical trial information: NCT01001377.

3584 Background: ASPECCT was a phase III clinical trial performed in the chemotherapy-refractory third-line mCRC setting (N = 1010). This analysis explores the relationship between circulating levels of mutations and clinical outcomes for panitumumab-treated subjects using univariate and multivariate models that treat total mutational load as a continuous measure. Methods: 238 subjects treated with panitumumab had paired plasma samples at baseline and post-treatment (PT). Samples were analyzed for mutations using the Plasma Select-R™ 63-gene panel (0.1% limit of detection). The fraction of mutant RAS reads was evaluated for association with tumor response (by RECIST) and overall survival using univariate and multivariate Cox proportional hazards models. Results: 52% of the subjects who were RAS wild-type by plasma at baseline never developed a RAS mutation. For those with mutant RAS ctDNA ( KRAS+ NRAS) detected at baseline or PT, there was an overall increase in RAS mutant DNA fraction at PT compared to baseline. By non-parametric analysis, there was no difference in the distribution of baseline mutant RAS fraction between those who achieved stable disease (SD) or those with progression ( P = 0.09). There was also no difference in the increase in mutant RAS fraction on therapy between subjects with SD or progressive disease (PD). In addition, RAS mutation was not required for progression: 48% of subjects with PD had no RAS mutant DNA detected. Conclusions: In this exploratory analysis, baseline plasma mutant RAS fraction is an unreliable predictor of subsequent tumor response. Subjects with objective response or SD may have stable or rising levels of mutant RAS DNA. Subjects without any detectable RAS mutation still experience PD. These findings suggest that detectable plasma ctDNA RAS mutations do not necessarily predict response to panitumumab and should be interpreted with caution. Further work is needed to establish clinically relevant and validated thresholds. Clinical trial information: NCT01001377.

Background Improvements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures. Methods The evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance. Results We observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250 ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value

// Anne Y. Saiki 1 , Sean Caenepeel 1 , Elissa Cosgrove 2, 5 , Cheng Su 3 , Michael Boedigheimer 4 , Jonathan D. Oliner 1 1 Oncology Research, Amgen, Inc., Thousand Oaks, California, USA 2 Genome Analysis Unit, Amgen, Inc., South San Francisco, California, USA 3 Biostatistics, Amgen, Inc., Seattle, Washington, USA 4 Molecular Sciences, Amgen, Inc., Thousand Oaks, California, USA 5 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA Correspondence to: Jonathan D. Oliner, e-mail: jonoliner@hotmail.com Keywords: TP53 , MDM2 , amplification Received: October 28, 2014 Accepted: January 08, 2015 Published: February 03, 2015 ABSTRACT Previous reports have provided evidence that p53 mutation is a strong negative predictor of response to MDM2 inhibitors. However, this correlation is not absolute, as many p53 Mutant cell lines have been reported to respond to MDM2 inhibition, while many p53 WT cell lines have been shown not to respond. To better understand the nature of these exceptions, we screened a panel of 260 cell lines and noted similar discrepancies. However, upon extensive curation of this panel, these apparent exceptions could be eliminated, revealing a perfect correlation between p53 mutational status and MDM2 inhibitor responsiveness. It has been suggested that the MDM2 -amplified subset of p53 WT tumors might be particularly sensitive to MDM2 inhibition. To facilitate clinical testing of this hypothesis, we identified a rationally derived copy number cutoff for assignment of functionally relevant MDM2 amplification. Applying this cutoff resulted in a pan-cancer MDM2 amplification rate far lower than previously published.

Thymic stromal lymphopoietin (TSLP) is an epithelial-cell-derived cytokine that may be important in initiating allergic inflammation. AMG 157 is a human anti-TSLP monoclonal immunoglobulin G2λ that binds human TSLP and prevents receptor interaction.In this double-blind, placebo-controlled study, we randomly assigned 31 patients with mild allergic asthma to receive three monthly doses of AMG 157 (700 mg) or placebo intravenously. We conducted allergen challenges on days 42 and 84 to evaluate the effect of AMG 157 in reducing the maximum percentage decrease in the forced expiratory volume in 1 second (FEV1). We also measured the fraction of nitric oxide in exhaled air, blood and sputum eosinophils, and airway hyperresponsiveness. The primary end point was the late asthmatic response, as measured 3 to 7 hours after the allergen challenge.AMG 157 attenuated most measures of allergen-induced early and late asthmatic responses. The maximum percentage decrease in the FEV1 during the late response was 34.0% smaller in the AMG-157 group than in the placebo group on day 42 (P=0.09) and 45.9% smaller on day 84 (P=0.02). In addition, patients receiving AMG 157 had significant decreases in levels of blood and sputum eosinophils before and after the allergen challenge and in the fraction of exhaled nitric oxide. There were 15 adverse events in the AMG-157 group, as compared with 12 in the placebo group; there were no serious adverse events.Treatment with AMG 157 reduced allergen-induced bronchoconstriction and indexes of airway inflammation before and after allergen challenge. These findings are consistent with a key role for TSLP in allergen-induced airway responses and persistent airway inflammation in patients with allergic asthma. Whether anti-TSLP therapeutics will have clinical value cannot be determined from these data. (Funded by Amgen; ClinicalTrials.gov number, NCT01405963.).

The Drosophila expanded (ex) gene encodes a product (Ex) that shares homology with the Protein 4.1 family of proteins, many of which are enriched at specific lateral cell junctions and the apical cellular domain. Ex colocalizes with actin in the apical domain of imaginal disc epithelial cells, where it partially overlaps the distribution of phosphotyrosine (PY)-containing proteins. This suggests that Ex is present in or associated with adherens junctions. Genetic studies show that Ex is necessary for proper regulation of final cell number in adult wings and for the formation of eyes, distal leg, and distal antennal segments. We have generated mitotic clones that lack Ex using the twin spot technique, and demonstrated that the primary function of Ex is to regulate cell proliferation. Overexpressing Ex protein results in a decrease in final cell number in wings, suggesting a direct relationship between Ex function and proliferation rate.

Here we report the discovery and characterization of the Drosophila tartan gene. tartan is transcribed in an unusual embryonic pattern of intersecting stripes which are generated in response to the anterior-posterior and dorsal-ventral regulatory systems. tartan encodes a putative transmembrane protein containing extracellular leucine-rich repeats characteristic of numerous cell surface receptors and adhesion proteins. Its expression is correlated with aspects of segmentation and neurogenesis, including the formation of neuroblasts, sensory mother cells, and peripheral nerves. Mutants homozygous for a recessive lethal tartan loss-function allele exhibit defects in the position and number of cells within peripheral sense organs, the routing of peripheral nerves, and the organization of commissures within the central nervous system. Mutants are also defective in muscle organization. These results suggest that tartan is required for cell surface interactions important for normal organization of epidermal and subepidermal structures.

The expanded gene was first identified by a spontaneous mutation that causes broad wings. We have identified an enhancer-trap insertion within expanded and used it to generate additional mutations, including one null allele. expanded is an essential gene, necessary for proper growth control of imaginal discs and, when mutant, causes either hyperplasia or degeneration depending on the disc. Wing overgrowth in expanded hypermorphs is limited to specific regions along the anterior-posterior and dorsal-ventral axis. expanded encodes a novel 1429 amino acid protein that is localized to the apical surface of disc cells and contains three potential SH3-binding sites. Together, these observations suggest that the Expanded protein engages in protein-protein interactions regulating cell proliferation in discs.

Effective use of microarray technology in clinical and regulatory settings is contingent on the adoption of standard methods for assessing performance. The MicroArray Quality Control project evaluated the repeatability and comparability of microarray data on the major commercial platforms and laid the groundwork for the application of microarray technology to regulatory assessments. However, methods for assessing performance that are commonly applied to diagnostic assays used in laboratory medicine remain to be developed for microarray assays. A reference system for microarray performance evaluation and process improvement was developed that includes reference samples, metrics and reference datasets. The reference material is composed of two mixes of four different rat tissue RNAs that allow defined target ratios to be assayed using a set of tissue-selective analytes that are distributed along the dynamic range of measurement. The diagnostic accuracy of detected changes in expression ratios, measured as the area under the curve from receiver operating characteristic plots, provides a single commutable value for comparing assay specificity and sensitivity. The utility of this system for assessing overall performance was evaluated for relevant applications like multi-laboratory proficiency testing programs and single-laboratory process drift monitoring. The diagnostic accuracy of detection of a 1.5-fold change in signal level was found to be a sensitive metric for comparing overall performance. This test approaches the technical limit for reliable discrimination of differences between two samples using this technology. We describe a reference system that provides a mechanism for internal and external assessment of laboratory proficiency with microarray technology and is translatable to performance assessments on other whole-genome expression arrays used for basic and clinical research.

Background Interferon-gamma (IFN-g) is a pro-inflammatory cytokine that modulates the function of several important immune populations. Evidence from human and animal models suggests that increased levels of Type I and/or Type II IFN are associated with SLE. Objectives This study assessed the safety, tolerability, PK and pharmacodynamic (PD) data from AMG 811, an anti-IFNg mAb, in subjects with active class III or IV LN. Methods Subjects were enrolled if they had new onset or reactivation of biopsy-proven (within 18 months) class III or IV LN, UP/Cr >1 or 24 hr urine protein >1 g after at least 12 wks of treatment with MMF or AZA. Superimposed membranous changes were allowed. Subjects with rapidly progressive GN or significant chronicity were excluded. Subjects could receive oral prednisone at doses up to 20 mg/day. Subjects were randomized to placebo or ascending doses of AMG 811 (20, 60 or 120 mg given SC q4 wks for three doses at 3:1 allocation) in addition to MMF or AZA. The primary endpoints were safety, tolerability, and anti-drug antibodies. Serum AMG 811 concentrations, anti-AMG 811 antibodies and PD biomarkers (blood RNA and serum) were also assessed. Disease related assessments included reduction from baseline in proteinuria (24 hr urine and spot UP/Cr), SLEDAI scores and changes in SLE-related biomarkers. Results 28 subjects were enrolled: 7 in the PBO group and 21 in the AMG 811 treatment groups. The proportion of subjects reporting treatment emergent AEs, including serious AEs, was similar between PBO and AMG 811 groups although a numerically higher proportion of subjects treated with AMG 811 had infectious events. AMG 811 displayed linear PK with half-life of 11-21 days. Exposures after doses of 20 & 60 mg in LN subjects were similar to exposures in non-renal SLE subjects. Antibodies to AMG 811 were not observed at any time. Baseline serum CXCL10 (IP-10) levels and IFNgamma-modulated mRNAs were higher in LN compared to non-renal SLE; both populations had elevated levels compared to healthy controls. AMG 811 (60 & 120 mg cohorts) led to a reduction in these biomarkers, albeit incomplete and transient, suggesting that AMG 811 may have had reduced target coverage in LN relative to non-renal SLE, where levels were reduced into the healthy range. Although numerous subjects demonstrated improvement in proteinuria, no consistent differences between AMG 811 treatment cohorts and PBO were discernable at week 12 in renal outcome or SLE-related serum biomarkers. Conclusions AMG 811 demonstrated a favorable PK and immunogenicity profile in active LN subjects, and the overall safety profile was acceptable in this small study. Inhibition of pharmacodynamic biomarkers with doses up to 120 mg of AMG 811 appeared incomplete in LN subjects. There were no discernible effects of AMG 811 on clinical or SLE-related serologic outcome measures over the treatment period although interpretation is challenging given the small sample size. Disclosure of Interest D. Martin Employee of: Amgen, Z. Amoura: None declared, J. Romero-Diaz: None declared, Y. Chong: None declared, J. Sanchez-Guerrero: None declared, T. Chan: None declared, G. Arnold Employee of: Amgen, M. Damore Employee of: Amgen, W. Sohn Employee of: Amgen, N. Chirmule Employee of: Amgen, K. Chiu Employee of: Amgen, C. Wang Employee of: Amgen, M. Boedigheimer Employee of: Amgen, B. Sullivan Employee of: Amgen, A. Welcher Employee of: Amgen, B. Kotzin Employee of: Amgen, J. Chung Employee of: Amgen