Background Biomarkers predictive of drug efficacy are lacking in rheumatoid arthritis (RA) and would be useful in clinical practice and clinical trials. Single cell network profiling (SCNP) is a multiparametric flow cytometry-based assay that measures induced changes (phosphorylation) in intracellular signaling proteins, providing a functional measure of pathway activity and immune networking in multiple cell subsets without physical separation. Objectives Induced signaling was measured in specific subsets of monocytes, B and T cells from RA patients (pts) initiating new treatment, and analyzed to build models to predict treatment response. Methods PBMCs from RA pts (n=87) starting TNF inhibitors (TNFi) were examined by SCNP of 42 nodes (combinations of modulator and intracellular readout) within 21 immune cell subsets. A subset of ∼200 RA pts from the Treatment Efficacy and Toxicity in Rheumatoid Arthritis Database and Repository (TETRAD) were studied. Blood samples were collected before treatment with TNFi (adalimumab, etanercept, infliximab, golimumab). Clinical data included DAS28 and EULAR response criteria at baseline, 3, 6, and 12 months. For the 53 evaluable patients, ordinal logistic regression and multivariate modeling were performed to identify signaling profiles associated with response to TNFi. Results Immune cell subsets from RA pts before TNFi treatment exhibited heterogeneity in induced intracellular signaling. Of note, T cell receptor (TCR) and IFNα modulation produced cell subset-specific signaling profiles that were associated with response at 3 months. Specifically, in CD4−CD45RA+ (naive and effector) T cells, phosphorylation of CD3ζ after stimulation through the TCR (TCR→p-CD3ζ) was weakest in pts that had a good EULAR response to TNFi (p=0.04). In contrast, phosphorylation of STAT3 after stimulation with IL-6 (IL-6→p-STAT3) in naive CD4+ T cells was weakest in autoantibody-positive pts with no response (p=0.01). Signaling nodes modulated by IFNα, TNFα, and IL-6 were combined to construct models to predict response and compared to models generated with standard clinical variables, including age, sex, and DAS28. Models utilizing cell signaling capacity of samples had greater performance with an internal cross-validated AUROC of 0.75 compared to 0.45 for clinical models. Conclusions This is the first evidence that measurement of peripheral blood immune cell function can: 1) identify patients likely to respond to TNFi, and 2) reveal the biology associated with TNFi response or lack thereof. SCNP has revealed predictive biomarkers that, once replicated in future studies, may enable patient stratification in clinical practice and clinical trials. Disclosure of Interest J. Ptacek Shareholder of: Nodality, Inc., Employee of: Nodality, Inc., R. Hawtin Shareholder of: Nodality, Inc., Employee of: Nodality, Inc., B. Louie Shareholder of: Nodality, Inc., Employee of: Nodality, Inc., E. Evensen Shareholder of: Nodality, Inc., Employee of: Nodality, Inc., J. Cordeiro Shareholder of: Nodality, Inc., Employee of: Nodality, Inc., B. Mittleman Shareholder of: Nodality, Inc., Employee of: Nodality, Inc., M. Atallah Shareholder of: Nodality, Inc., Employee of: Nodality, Inc., A. Cesano Shareholder of: Nodality, Inc., Employee of: Nodality, Inc., G. Cavet Shareholder of: Nodality, Inc., Employee of: Nodality, Inc., C. Bingham III: None declared, S. Cofield: None declared, J. Curtis: None declared, M. Danila: None declared, R. Furie: None declared, M. Genovese: None declared, M. Levesque: None declared, L. Moreland: None declared, P. Nigrovic: None declared, J. O9Dell: None declared, W. Robinson: None declared, N. Shadick: None declared, E. W. St. Clair: None declared, C. Striebich: None declared, G. Thiele: None declared, P. Gregersen: None declared, S. L. Bridges, Jr.: None declared DOI 10.1136/annrheumdis-2014-eular.2057

Abstract Background: Inherited alterations in BRCA1/2 genes increase genomic instability and cancer susceptibility. DNA sequencing detects BRCA1/2 mutations, but has the following limitations; 1) mutations may have unknown functional significance, 2) epigenetic alterations and mutations in other Homologous Recombination (HR) pathway components are not detected, and 3) the combined effects of pathway mutations are not understood. Thus a functional assessment of HR competence at the single cell level remains an unmet need as BRCA1/2 sequencing does not holistically inform on functionality of the HR pathway. Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry-based assay that simultaneously measures, at the single cell level, extracellular surface markers and functional changes in intracellular signaling in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). In this study, we tested the ability of SCNP to detect and quantify functional changes in HR signaling using peripheral blood mononuclear cell (PBMC) samples from BRCA1 mutation carriers (MUT) and wild type (WT) subjects. Methods: HR pathway activity was examined in PBMCs from BRCA1 MUT (n = 21) or WT (n = 20) subjects. Cell lines carrying BRCA1 MUTor WT genes were used as controls. PBMCs were stimulated with anti-CD3 and anti-CD28 for 24 hours to induce T cell proliferation then treated with PARP inhibitor (PARPi) AZD2281 +/− Temozolomide (TMZ) for 48h or 72h to induce DNA damage. DNA damage response (DDR) readouts were measured in both CyclinA2- and CyclinA2+ T cell subsets. Measurements included induced levels of p21, p53 and phosphorylation (p−) of p-H2AX, p-DNA-PKcs, p-RPA2/32, and p-BRCA1. Results: As expected based on the mechanism of action of PARPi, higher levels of induced p-H2AX and p53 were observed in CyclinA2+ cells of BRCA1 MUT versus WT cell line controls. In PBMCs, T cell proliferation (%CyclinA2+) was positively associated with PARPi induced DDR readouts. After controlling for proliferation, statistically significant differences in PARPi induced DDR signaling were observed between BRCA1 MUT and WT samples in many simultaneously assessed readouts including p-H2AX, p53 and p21 (increased in MUT), particularly in CyclinA2+ cells. Additionally, BRCA1 MUT samples displayed lower basal p-BRCA1 but higher induced p-BRCA1 levels compared to BRCA1 WT samples. Conclusions: SCNP was able to detect and quantify functional differences between PBMC samples from BRCA1 MUT (haploinsufficient) and WT donors by quantitatively assessing DDR signaling in CyclinA2+ T cells. Once verified on a larger data set, the assay could form the basis for the development of screening tests to identify subjects at higher risk of developing cancer or stratification tests to inform on cancer patient selection for treatment with PARP inhibitors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-31.

The aim of this study was to explore the clinical and other associated laboratory features of chronic lymphocytic leukemia (CLL) patients with immunoglobulin (Ig) paraproteinemia. Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) were performed to measure serum Ig paraprotein. The correlations between serum Ig paraprotein and other prognostic factors were analyzed. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. In 133 Chinese CLL patients, 27 (20.3%) patients occurred Ig paraproteinemia at diagnosis. According to the correlation analysis, advanced Binet stage (r = 0.314, P < 0.001), direct antiglobulin test (DAT)-positive (r = 0.366, P < 0.001), high level of serum β2-microglobulin (β2-MG) (r = 0.296, P = 0.001) and thymidine kinase (TK) 1 (r = 0.227, P = 0.037), unmutated immunoglobulin heavy chain variable gene (IGHV) status (r = 0.284, P = 0.002), ZAP-70-positive (r = 0.305, P = 0.001), CD38-positive (r = 0.284, P = 0.002), and cytogenetic abnormalities of del(17p13) or del(11q22.3) (r = 0.208, P = 0.032) emerged as factors significantly related to the occurrence of Ig paraproteinemia. Survival analysis showed that the patients with Ig paraproteinemia had significantly shorter survival times than the patients without serum Ig paraprotein (P = 0.013). Binet stage (P = 0.028), high levels of lactate dehydrogenase (LDH) (P = 0.004), IgG paraproteinemia (P = 0.048), IgM paraproteinemia (P = 0.001), ZAP-70-positive (P = 0.003), DAT-positive (P = 0.013), unmutated IGHV status (P = 0.009), and del(17p13) (P = 0.001) were the adverse factors in determining overall survival (OS). Del(17p13) (P = 0.006), ZAP-70 (P = 0.030), and IgM paraproteinemia (P = 0.040) were the variables strongly associated with OS by multivariate Cox regression analysis. It was showed that serum Ig paraprotein might be applied for the assessment of prognosis in patients with CLL.