Multiplex Bead Array Technology Research Articles

Dysregulation of immune checkpoint proteins in patients with newly diagnosed early breast cancer undergoing neoadjuvant chemotherapy: A comparison between patients with triple negative breast cancer (TNBC) and non-TNBC.

e14546 Background: Checkpoint proteins regulate the immune system. Breast cancer is associated with up-regulation or down-regulation of these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (sICMs) can be measured in human plasma. The study aimed to measure the systemic levels of a series of co-stimulatory and co-inhibitory ICMs at diagnosis, post-neo-adjuvant chemotherapy (NAC) and post-surgery in newly- diagnosed BC patients (pts) and compare the biological behaviour of these sICMs between TNBC and non-TNBC patients. Methods: Soluble ICMs were measured using multiplex bead array technology in plasma from 72 BC pts and 45 healthy controls. Data were prospectively obtained and levels were compared between pre-treatment, post-NAC, and post-surgery using non-parametric tests (Mann-Whitney & Kruskal-Wallis). Results: Following NAC, the plasma levels of six soluble co-stimulatory checkpoints (CD28, CD40, ICOS, CD27, CD80, GITR), all involved in the activation of CD8+ cytotoxic T cells, were significantly increased (p < 0.04-p < 0.00001). Four of the soluble co-inhibitory checkpoints (LAG-3, PDL1, TIM-3 and HVEM) increased significantly post-NAC, reaching levels significantly greater than those of the control group. PD-1 remained unchanged, while BTLA and CTLA-4 decreased significantly (p < 0.03 and p < 0.00001, respectively). Normalization of soluble co-stimulatory immune checkpoints seemingly indicates a reversal of systemic immune dysregulation following the administration of NAC in early BC. In contrast, recovery of immune homeostasis may explain the increased levels of several negative checkpoint proteins, albeit with the exceptions of CTLA-4 and PD-1. No significant differences were detected between the changes of 16 sICMs levels at pre-NAC, post-NAC and post-surgery when comparing the TNBC and non-TNBC patients. Conclusions: This study demonstrates low levels of co-stimulatory and co-inhibitory sICMs in newly-diagnosed, non-metastatic BC pts. Following treatment with NAC, the sICMs levels increase substantially, except CTLA-4. In the case of co-stimulatory sICMs, these novel findings are indicative of an immune-restorative mechanism. The pattern of co-inhibitory sICMs (elevated levels of PD-L1, LAG-3, TIM-3 and HVEM), might be indicative of immune-therapeutic resistance, underscoring the augmentative immune-therapeutic promise of targeting these molecules, either individually or in combination, to improve the outcome of pts with early BC. These differences were not significant between TNBC and non-TNBC patients.

Dysregulation of immune checkpoint proteins in patients with newly diagnosed early breast cancer.

3044 Background: Checkpoint proteins regulate the immune system. Breast cancer (BC) cells can up-regulate or down-regulate these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (ICMs) can be measured in human plasma. The study aimed to measure the systemic levels of a series of co-stimulatory and co-inhibitory ICMs at diagnosis, post-neo-adjuvant chemotherapy (NAC) and post-surgery in newly- diagnosed BC patients (pts) relative to those of a healthy control group. Methods: Soluble ICMs were measured using multiplex bead array technology in plasma from 72 BC pts and 45 healthy controls. Data was prospectively obtained and levels compared between pre-treatment, post-NAC, and post-surgery using non-parametric tests (Mann-Whitney & Kruskal-Wallis). Results: Pre-treatment levels of the soluble stimulatory molecules viz. GITR (p<0.0001), GITRL (p< 0.020), CD27 (p< 0.024), CD40 (p< 0.021), ICOS (p< 0.009), as well as the inhibitory molecules PD-L1 (p< 0.0001), CTLA-4 (p< 0.005), TIM-3 (p< 0.0004), HVEM (p< 0.0004) were significantly lower in early BC pts compared to controls. Post-treatment, there were significant increases in most ICM levels (Table), with the exception of CTLA-4, which decreased significantly following treatment. On the other hand, pre-treatment plasma concentrations of CCL5 (RANTES) (84.22 vs. 48.72 pg/mL, p<0.0001), M-CSF (84.41 vs 13.34 pg/mL, p<0.0001), FGF-21 (24.36 vs. 8.64 pg/mL, p<0.001) and GDF-15 (806.82 vs. 430.03 pg/mL, p<0.0001) were significantly increased in the breast cancer pts compared to healthy controls. A pathological complete response (pCR) was documented in 65% of pts (mostly TNBC). There were no correlations between pre-treatment ICM levels, CCL5, M-CSF, FGF-21 and GDF-15 and pCR. Conclusions: We identified low levels of stimulatory and inhibitory ICMs in newly-diagnosed, non-metastatic BC pts compared to healthy controls. Following treatment, with the exception of CTLA-4, most of these pre-treatment abnormalities of systemic ICM levels corrected. NAC is associated with upregulation of sPD-L1 and most other ICMs. These results indicate that early BC is associated with down-regulation of soluble stimulatory and inhibitory ICMs. Newly-diagnosed early BC pts appear to have generalized immune-suppression independent of subtype and stage. To our knowledge, this is the first study to describe the effect of treatment on systemic ICMs in early BC pts. [Table: see text]

Profiling of Cytokine and Chemokine Responses Using Multiplex Bead Array Technology.

Multiplex bead array technology expands upon the principles of the enzyme-linked immunosorbent assay by allowing the simultaneous quantification of a large number of cytokines and chemokines within a single sample. This allows researchers more freedom and opportunities to investigate complex immune responses both in vivo and in vitro. Here we describe and update the detailed assay protocol and technical tips for simultaneous quantification of multiple cytokines and chemokines in mouse biological fluids such as sera, bronchoalveolar lavage fluid, tissue homogenate supernatant, and tissue culture supernatant, using a multiplex bead array assay.

Detection of High Prevalence of TEM/SHV/CTX-M Genes in ESBL Producing and Multidrug Resistant Klebsiella Pneumoniae and Klebsiella Oxytoca

Septic shock remains clinically challenging to manage. The dysregulated immune response seen in septic shock contributes to this complexity. However, biological response modifiers including the cytokine recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) might be important in managing both septicemia and septic shock. We hypothesized that ex vivo stimulation with rHuGM-CSF could restore functional and phenotypic responses of monocytes. The functional responses of complex whole blood and highly-enriched monocyte cultures were assessed from a panel of n=30 human study subjects (n=23, septic shock and n=7 healthy controls). Cultures were initially primed with rHuGM-CSF then stimulated with relevant toll-like receptor (TLR) ligands for short (4 h) or extended (48 h) time-periods. TLR-specific ligands included lipopolysaccharide (LPS, a surrogate of gram-negative bacterial infection) and double-stranded RNA (dsRNA, a surrogate of a viral infection). Endpoints included measurement of cell surface receptors by flow cytometry and cytokine secretion by multiplex bead-array technology. We found depressed expression of immune cell-surface markers on septic shock monocytes as compared to normal controls. However, a trend of partially restored cell surface expression of functionally important phenotypic markers on septic shock monocytes was associated with restored, or even augmented, cytokine secretion following rHuGM-CSF-priming and secondary stimulation with LPS or dsRNA. A dysregulated pattern of cell activation typically characterizes septic shock and may contribute to inadvertent immune suppression of monocytes. We have showed that rHuGM-CSF priming restored several important aspects of monocyte responsiveness to exogenous bacterial and viral stimuli. This approach could offer therapeutic utility that nonetheless requires empirical proof of concept.

Abstract 17206: Osteopontin Levels in Patients at High Cardiovascular Risk

Introduction: Proteins which are involved in the bone metabolism have recently been discovered to have significant implications in the development of vascular disease and complications. Osteopontin (OPN) has been suggested as a biomarker of atherosclerosis and vascular calcification. Hypothesis: OPN levels may be used for risk stratification in patients with peripheral artery disease (PAD). Methods: 361 patients suffering from PAD were studied and followed-up for five years. 60 outcome events, defined as death, non-fatal myocardial infarction or non-fatal stroke, were observed. Multiplex bead array technology was used to measure serum levels of OPN at baseline. For basic analysis, the patients were divided into quartiles according to OPN levels. A Cox regression model was used to identify confounding variables. Results: A significantly different event-free survival could be observed between all groups (log-rank: p=0.049). When comparing the highest quartile to the rest of the cohort, a further increase in significance could be observed (log-rank: p=0.017). A cut-off point accommodating the most favorable sensitivity (50.0%) and specificity (66.4%) was determined for regression analysis. The hazard ratio was 2.13 (1.26-3.60) in the unadjusted model and reached 2.16 (1.21-3.84) after adjustment for age, gender, diabetes, low-density lipoprotein cholesterol, blood pressure, smoking, c-reactive protein and renal function. Conclusions: Our results suggest an association between OPN levels and the rate of cardiovascular events in high risk patients. In our observations this relation was independent of established risk factors.

Abstract 15880: Angiopoietin 2, a Pro-inflammatory Protein, is Associated With Major Adverse Cardiovascular Events in Patients With Peripheral Arterial Disease

Introduction: Angiopoietin 2 (ANG2) is a pro-inflammatory protein which is elevated in several inflammatory conditions and associated with increased morbidity and mortality. Hypothesis: ANG2 is associated with the composite of death, non-fatal myocardial infarction and non-fatal stroke (MACE) in patients with peripheral arterial disease (PAD). Methods: We measured serum ANG2 levels at baseline in 363 consecutive PAD patients with using multiplex bead array technology. Follow-up was five years. In Cox regression, confounding was evaluated by examining the relative change in coefficients (delta-beta) for ANG2 after the omission of the respective potential confounder from the fully adjusted model. Significant Confounding was defined as a delta-beta > 10%. Results: Using receiver operating characteristics, ANG2 exhibited an area under the curve of 0.6 (95%CI: 0.522 - 0.678) for the proper discrimination between MACE and non-events. A cut-off of 3.74 ng/ml resulted in the highest possible combination of specifity (57.5%) and sensitivity (63.3%) leading to an unadjusted hazard ratio (HR) of 2.17 (95%CI: 1.28 - 3.67) in Cox regression (p=0.004). Adjusting for age, sex, diabetes, systolic blood pressure, smoking status and renal function lead to a HR of 1.89 (95%CI: 1.11 - 3.24) for ANG2 >3.74 ng/ml (p=0.020). The additional adjustment for c-reactive protein (CRP) resulted in a no more significant HR of 1.68 (95%CI: 0.97 - 2.92)for high ANG2 (p=0.067). Diabetes and CRP, both significant predictors of MACE in our cohort, proved to be significant confounders with concomitant delta-betas of 19.7% and 22.9%, respectively. Conclusions: We are the first to demonstrate that elevated ANG2 levels are interrelated with MACE in patients with PAD. Inflammation and glucose hemostasis seem to influence this association.

Effect of Adipose Tissue-Derived Inflammatory and Proangiogenic Cytokines on Proliferative Diabetic Retinopathy

Objective: To determine the vitreous and serum concentrations of TNF-α, IL-6, VEGF, IL-1β, IL-8, IL-17, MCP-1, IL-1Ra, IL-10 in patients with proliferative diabetic retinopathy (PDR) and to investigate the effect of adipose tissue on the pathogenesis of PDR. Methods: Twenty-two patients with PDR were prospectively evaluated. Seven cadavers and 11 patients with idiopathic epiretinal membrane or macular hole served as the controls. Multiplex bead array technology was employed to assess the concentrations of the cytokines. Results: The intravitreal levels of VEGF, IL-8, IL-6 were significantly higher in PDR group than control groups. In PDR group, the levels of IL-17, IL-8, IL-6 were significantly elevated in the vitreous, whereas the intravitreal levels of IL-10, IL-1Ra were found to be significantly lower than the serum concentrations. No significant correlation was found between cytokine levels and body mass index (BMI), fasting blood glucose (FBG), or glycated haemoglobin (HbA1c) of diabetic patients. Conclusion: In PDR the balance between the intravitreal pro- and anti- inflammatory adipocytokines is disturbed in favor of proinflammatory and proangiogenic cytokines in the vitreous humour. This study supports the role of adipocytokines in vascular pathology in PDR. It seems that PDR is a local inflammatory disease. However, obesity may not be the root of the inflammatory mediators in PDR.

A Metalloporphyrin Antioxidant Alters Cytokine Responses after Irradiation in a Prostate Tumor Model

The goal of this study was to evaluate cytokine secretion capacity in a mouse model of prostate cancer, both with and without metalloporphyrin antioxidant and radiation treatment. C57BL/6 mice with subcutaneous RM-9 tumors were treated daily for 12 days with MnTE-2-PyP(5+) [Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin], beginning 1 day after injection of RM-9 cells; a 10-Gy tumor-localized dose of (60)Co gamma rays was administered in a single fraction on day 7. Spleen, tumors and plasma were collected on day 12. T cells in the spleen were activated with anti-CD3 antibody and supernatants were collected. Twenty-two cytokines were quantified in spleen supernatants, five in tumor homogenates, and three in plasma using multiplex bead array technology and ELISA. The presence of a tumor had significant effects on many of the cytokines quantified (P < 0.05). Tumor-induced depression was evident for eight spleen cytokines (TNF-alpha, G-CSF, GM-CSF, IFN-gamma, IL10, IP-10, MIP-1alpha and mKC), whereas only three were enhanced (IL1beta, IL6 and MCP-1). Radiotherapy resulted in enhanced splenocyte capacity to produce IL4 and IL13 and increased IL4, MCP-1 and VEGF in tumors (P < 0.05). Addition of MnTE-2-PyP(5+) to radiation decreased the concentrations of IL4, IL13 and TGF-beta1 in spleen supernatants and IL4 and VEGF in tumors (P < 0.05 compared to radiation alone). Some differences were also noted in plasma cytokines. Overall, the findings suggest that administration of MnTE-2-PyP(5+) together with radiotherapy may enhance anti-tumor immune responsiveness and decrease the risk for radiation-induced normal tissue toxicities.