Background: DAPHNe was a prospective trial designed to assess adherence to de-escalated antibody doublet therapy in the adjuvant setting among HER2+ breast cancer patients experiencing a pathologic complete response (pCR) following neoadjuvant taxol (T), trastuzumab (H) and pertuzumab (P). Peripheral blood mononuclear cells (PBMC), were collected from all patients at baseline and after THP completion. The goal of this study was to determine if a patient’s peripheral blood immune profile at baseline, or the longitudinal change with treatment, could predict response to THP. Methods: Blood samples were subjected to high dimensional (28-30 parameter) flow cytometry with comprehensive T- and NK-cell panels. A fully automated computational analysis strategy was undertaken consisting of unsupervised clustering of the high dimensional data into groups of cells with similar immunophenotypic signatures. Clustering was performed using 2 algorithms: Fingerprint-based clustering (Fluster) and High Throughput Mapper (HiTMapper). Clusters were tested using the Wilcoxon rank-sum test for correlation with the clinical response. Responders were those with pCR (=residual cancer burden [RCB] 0) or RCB 1; non-responders were those with RCB 2/3 disease. P values were adjusted with the Benjamini-Hochberg method to control for FDR. In addition to P values, effect size was evaluated using the nonparametric Cliff’s Delta measure. An effect was determined to be large if the magnitude of Delta was >0.4 which corresponded to one cohort coming ahead 70% of the time. In addition, groups of clusters were evaluated using multivariate statistical modeling or dimensionality reduction to determine if there was an association with pCR. Results: Matched baseline and pre-op PBMC were available to perform the NK panel in 66 patients and the T cell panel in 40. In both groups 70% were responders and 30% were non-responders. No cluster produced by Fluster or HiTMapper differed significantly between responders and non-responders however, in the T cell panel, several clusters had a large effect size (table) suggesting the clusters are good at differentiating some, responders from non-responders. Both algorithms agreed that the median responder has more CD4 naïve and CD8 naïve cells than the median non-responder. While no individual cluster differed significantly between responders and non-responders, cross-validated logistic regression analyses showed that 2 clusters, activated CD4 central memory clusters, and activated CD4 naïve clusters, predicted responder status with AUC of 0.70 and 0.68 respectively. Numerous clusters showed robust and significant longitudinal changes between baseline and pre-op samples. Stratifying longitudinal changes by response status revealed no significant differences between responders and non-responders, however evaluation of effect size suggested a naïve CD4 cluster that increased in non-responders and decreased in responders. The latter could be explained as naïve T cells acquiring a memory phenotype in response to treatment in responders. Conclusion: High dimensional flow cytometry suggested a potential role for monitoring several T cell subsets to predict response in HER2+ patients receiving THP. Additional analyses to include cyTOF evaluation of PBMCs are ongoing to further characterize the peripheral immune profile of these HER2+ patients. T cell clusters with high effect sizeMajor PhenotypeOther markersMethodp-valueeffect sizeCD4 CM.actCD38, CD226HiTMapper0.351-0.469CD4 Naive.act.2CD38, CD226HiTMAPPER0.4-0.413CD4 Naive.act.4CD226HiTMapper0.351-0.490CD4 Naive.act.5CD38HiTMapper0.351-0.524CD8 Naive 1CD226HiTMapper0.396-0.427CD3 Neg-Fluster0.3190.476CD3 Neg3CD45RA, CD185, CD197Fluster0.3190.476CD3 Neg4CD45RA, Eomes,tBETFluster0.4240.413CD4 Naive-Fluster0.319-0.517CD8 Naive1-Fluster0.319-0.469Unassigned 20 (CD4)CD45RA, CD27-, CD28-Fluster0.364-0.441 Citation Format: Esther R Ogayo, Adrienne Waks, Wade Rogers, Matei Ionita, Kenechukwu Adigwe, Jillian Alberti, Sapana Kadel, Jonni Moore, Tari King, Ian Krop, Sara Tolaney, Eric Winer, Jennifer Guerriero, Elizabeth Mittendorf. High dimensional flow cytometric analysis or the peripheral immune profile and response to HER2-targeted antibody therapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-15.

BACKGROUND:There is a critical need for development of biomarkers to noninvasively monitor for lung transplant rejection. We investigated the potential of circulating donor lung-specific exosome profiles for time-sensitive diagnosis of acute rejection in a rat orthotopic lung transplant model. METHODS:Left lungs from Wistar transgenic rats expressing human CD63-GFP, an exosome marker, were transplanted into fully MHC-mismatched Lewis recipients or syngeneic controls. Recipient blood was collected between 4 h and 10 d after transplantation, and plasma was processed for exosome isolation by size exclusion column chromatography and ultracentrifugation. Circulating donor exosomes were profiled using antihuman CD63 antibody quantum dot on the nanoparticle detector and via GFP trigger on the nanoparticle flow cytometer. RESULTS:In syngeneic controls, steady-state levels of circulating donor exosomes were detected at all posttransplant time points. Allogeneic grafts lost perfusion by day 8, consistent with acute rejection. Levels of circulating donor exosomes peaked on day 1, decreased significantly by day 2, and then reached baseline levels by day 3. Notably, decrease in peripheral donor exosome levels occurred before grafts had histological evidence of acute rejection. CONCLUSIONS:Circulating donor lung-specific exosome profiles enable an early detection of acute rejection before histologic manifestation of injury to the pulmonary allograft. As acute rejection episodes are a major risk factor for the development of chronic lung allograft dysfunction, this biomarker may provide a novel noninvasive diagnostic platform that can translate into earlier therapeutic intervention for lung transplant patients.

Automated clustering workflows are increasingly used for the analysis of high parameter flow cytometry data. This trend calls for algorithms which are able to quickly process tens of millions of data points, to compare results across subjects or time points, and to provide easily actionable interpretations of the results. To this end, we created Tailor, a model‐based clustering algorithm specialized for flow cytometry data. Our approach leverages a phenotype‐aware binning scheme to provide a coarse model of the data, which is then refined using a multivariate Gaussian mixture model. We benchmark Tailor using a simulation study and two flow cytometry data sets, and show that the results are robust to moderate departures from normality and inter‐sample variation. Moreover, Tailor provides automated, non‐overlapping annotations of its clusters, which facilitates interpretation of results and downstream analysis. Tailor is released as an R package, and the source code is publicly available at www.github.com/matei-ionita/Tailor .

BackgroundWe previously reported the development of a novel high dimensional cytomic assay, the Vascular Health Profile (VHP) based on measurements of angiogenic circulating hematopoietic stem and progenitor cells (CHSPCAng) and extracellular vesicles (EVs), that discovered a unique signature, differentiating the vascular status of diabetics and normal healthy controls. Here, we present data from a 3‐year follow‐up to evaluate the power of the VHP to identify individuals at risk for cardiovascular (CV) events.MethodsThe original data were generated as previously described by measuring a broad panel of progenitor cells and EVs and profiled using cytometric fingerprinting. Subjects were classified into groups according to the occurrence of adjudicated CV events including myocardial infarction, stroke, major adverse cardiovascular events, revascularization, and irregular rhythm. Cross‐validated Linear Discriminate Analysis (LDA) models were constructed and used to predict the occurrence of events, and were evaluated for predictive accuracy (AUC, area under the curve) using receiver operating characteristic (ROC) analysis.ResultsOver the period of this analysis, follow‐up data was obtained on 87 subjects, with 32 events occurring overall, and only in the diabetic group. In all cases, the VHP added significant predictive power, in the form of ROC analysis, for all evaluated outcomes with the exception of irregular rhythm.ConclusionsThe VHP, a relatively simple blood test, can provide sensitive and clinically relevant information on the vascular status of a patient that may be useful for a variety of applications including drug development, clinical risk assessment, and companion diagnostics. © 2015 International Clinical Cytometry Society

Background: Patients with vasculitis (VS) have complex organ- and life-threatening disease manifestations due to autoimmune-mediated inflammatory vascular damage and an increased risk of cardiovascular disease (CVD). Accurate biomarkers do not exist for VS. Cells release extracellular vesicles (EVs) in the setting of activation, injury, or death. In patients with diabetes mellitus and CVD, the effects of the inflammatory cascade, platelets and endothelium, positively correlate with levels of EVs bearing receptors for CD3, CD41, or CD105, and negatively correlate with levels of circulating endothelial progenitor cells (CPCs). Because VS is characterized by systemic inflammation, platelet activation and endothelial damage, this group of diseases may have unique effects on EVs and CPCs. This study evaluated a high dimensional flow cytometry approach to profiling EVs and CPCs in plasma to discern a biomarker pattern in VS. Methods: Levels of CPCs and EVs were measured with a panel of cell surface markers in 10 VS patients and 5 age-similar healthy controls (HC). Data were acquired on a modified Becton Dickinson FACSCanto. Results: Although no significant increase in CPCs in VS compared with HC was detected, the standard deviation of CPC levels in VS was more than double that in HC (SD=574 vs. 165, p=0.081), suggesting heterogeneity in the VS group. EV subsets with lower concentrations in VS, compared with HC, included: Annexin + CD3 + double-positive T Lymphocyte (p=0.026) and CD105 + CD64 + double-positive activated Macrophage (p=0.030). CD144 + CD105 + double-positive endothelial EVs were only detected in a subset of VS and not in HC. CD31 + CD41a + double-positive platelet EVs were increased in VS compared with HC (894 (580) vs. 416 (128), p=0.004). Conclusion: This proof of concept study discovered a distinct EV pattern that correlates with a diagnosis of VS, suggesting that cell- and vesicle-based assays could be useful biomarkers in evaluating VS. This pattern is different from that found in patients with diabetes mellitus and CVD. Larger studies could seek to elucidate correlations between CPCs and disease activity and identify a unique EV signature for VS with increased risk for CVD.

Background Psoriasis, especially when severe, is a risk factor for cardiometabolic disease beyond traditional risk factors. The mechanism of atherogenesis in psoriasis remains unknown. Cell membrane vesicles (ie, microparticles), released upon cell activation or apoptosis, have recently been associated with cardiometabolic disease and may play a pathogenic role. Microparticle levels, particularly from endothelial cells and platelets, are elevated in patients with cardiovascular disorders, metabolic syndrome, other inflammatory diseases, autoimmune conditions, and have been shown to be predictive of cardiovascular outcomes. Methods and Results Concentrations of microparticles with positive expression for any of 7 cell surface markers (Annexin V, CD 3, CD 31, CD 41a, CD 64, CD 105, and CD 144) were measured in blood samples from psoriasis patients (n=53) and control subjects without psoriasis (n=41). Platelet‐free plasma was separated from whole blood by one‐step centrifugation for microparticle analysis. Microparticles were fluorescently labeled and characterized by flow cytometry. Higher concentrations of CD 105 (5.5/μL versus 2.5/μL, P <0.001), CD 31 (31/μL versus 18/μL, P =0.002), CD 41a (50/μL versus 22/μL, P <0.001), and CD 64 (5.0/μL versus 4.1/μL, P =0.02) singly positive microparticles corresponding to endothelial cell‐, platelet‐, and monocyte/macrophage‐derived microparticles, respectively, were found in psoriasis patients compared with controls. These differences persisted after adjustment for traditional cardiometabolic risk factors including body mass index. Conclusions Increased microparticle concentrations, independent of cardiometabolic risk factors, in patients with psoriasis suggest that the presence of increased endothelial cell, platelet, and monocyte/macrophage activation with cell turnover may contribute to the heightened atherogenesis associated with psoriasis.

Objectives: To examine the effect of chemotherapy for ovarian cancer on immunologic function and to define the effect on the serologic response to the influenza vaccine.Methods: Under IRB approved protocols, patients with ovarian cancer were administered seasonal trivalent killed influenza vaccines. Peripheral blood was collected for immunologic assessments. Serum was analyzed for hemagglutination inhibition (HAI) antibody titers. Peripheral blood mononuclear cells were isolated to characterize T and B cell populations and function.Results: Thirty-one patients were recruited: 13 in remission receiving a dendritic cell vaccine with or without a single dose of low-dose cyclophosphamide, 3 in remission not receiving treatment, and 15 undergoing standard therapy. Significant effects on T cell and B cell subset distributions were seen. Functional effects were also seen. Few patients were able to mount a 4-fold HAI antibody response. A 4-fold response was observed for HI NI in 20%, for H3N2 in 26%, and for influenza B in 6%. Pre-existing exposure to influenza was predictive of responders.Conclusions: Despite CDC recommendations that patients undergoing chemotherapy receive influenza vaccine, there is little evidence to support its serologic effectiveness in this population. Patients with ovarian cancer are almost uniformly unable to mount a meaningful antibody response. These findings have serious implications for future resource allocation for both seasonal and novel pandemic influenza outbreak and understanding the immunologic deficits as a result of chemotherapy may improve patient care. (C) 2013 Elsevier Ltd. All rights reserved.

BackgroundAn inexpensive and accurate blood test does not currently exist that can evaluate the cardiovascular health of a patient. This study evaluated a novel high dimensional flow cytometry approach in combination with cytometric fingerprinting (CF), to comprehensively enumerate differentially expressed subsets of pro‐angiogenic circulating progenitor cells (CPCs), involved in the repair of vasculature, and microparticles (MPs), frequently involved in inflammation and thrombosis. CF enabled discovery of a unique pattern, involving both MPs and CPCs and generated a personalized signature of vascular health, the vascular health profile (VHP).MethodsLevels of CPCs and MPs were measured with a broad panel of cell surface markers in a population with atherosclerosis and type 2 diabetes mellitus (DM) and age‐similar Healthy controls (HC) using an unbiased computational approach, termed CF.ResultsCirculating hematopoietic stem and progenitor cell (CHSPCAng) levels were detected at significantly lower concentrations in DM (P < 0.001), whereas levels of seven phenotypically distinct MPs were present at significantly higher concentrations in DM patients and one MP subset was present at significantly lower concentration in DM patients. Collectively, the combination of CHSPCAng and MP levels was more informative than any one measure alone.ConclusionsThis work provides the basis for a personalized cytomic vascular health profile that may be useful for a variety of applications including drug development, clinical risk assessment and companion diagnostics. © 2013 International Clinical Cytometry Society

Objective: Intra-amniotic infection/inflammation are major causes of spontaneous preterm labor and delivery. However, diagnosis of intra-amniotic infection is challenging because most are subclinical and amniotic fluid (AF) cultures take several days before results are available. Several tests have been proposed for the rapid diagnosis of microbial invasion of the amniotic cavity (MIAC) or intra-amniotic inflammation. The aim of this study was to examine the diagnostic performance of the AF Mass Restricted (MR) score in comparison with interleukin-6 (IL-6) and matrix metalloproteinase-8 (MMP-8) for the identification of MIAC or inflammation.Methods: AF samples were collected from patients with singleton gestations and symptoms of preterm labor (n - 100). Intra-amniotic inflammation was defined as 4100 white blood cells/mm(3) (WBCs) in AF; MIAC was defined as a positive AF culture. AF IL-6 and MMP-8 were determined using ELISA. The MR score was obtained using the Surface-Enhanced Laser Desorption Ionization Time of Flight (SELDI-TOF) mass spectrometry. Sensitivity and specificity were calculated and logistic regression models were fit to construct receiver-operating characteristic (ROC) curves for the identification of each outcome. The McNemar's test and paired sample non-parametric statistical techniques were used to test for differences in diagnostic performance metrics.Results: (1) The prevalence of MIAC and intra-amniotic inflammation was 34% (34/100) and 40% (40/100), respectively; (2) there were no significant differences in sensitivity of the three tests under study (MR score, IL-6 or MMP-8) in the identification of either MIAC or intra-amniotic inflammation (using the following cutoffs: MR score >2, IL-6 > 11.4 ng/mL, and MMP-8>23 ng/mL); (3) there was no significant difference in the sensitivity among the three tests for the same outcomes when the false positive rate was fixed at 15%; (4) the specificity for IL-6 was not significantly different from that of the MR score in identifying either MIAC or intra-amniotic inflammation when using previously reported thresholds; and (5) there were no significant differences in the area under the ROC curve when comparing the MR score, IL-6 or MMP-8 in the identification of these outcomes.Conclusions: IL-6 and the MR score have equivalent diagnostic performance in the identification of MIAC or intra-amniotic inflammation. Selection from among these three tests (MR score, IL-6 and MMP-8) for diagnostic purposes should be based on factors such as availability, reproducibility, and cost. The MR score requires a protein chip and a SELDI-TOF instrument which are not widely available or considered "state of the art". In contrast, immunoassays for IL-6 can be performed in the majority of clinical laboratories.

Endothelial microparticles (EMPs) belong to a family of extracellular vesicles that are dynamic, mobile, biological effectors capable of mediating vascular physiology and function. The release of EMPs can impart autocrine and paracrine effects on target cells through surface interaction, cellular fusion, and, possibly, the delivery of intra-vesicular cargo. A greater understanding of the formation, composition, and function of EMPs will broaden our understanding of endothelial communication and may expose new pathways amenable for therapeutic manipulation.

Severe psoriasis is a risk factor for cardiovascular (CV) disease beyond traditional risk factors; however, the mechanism remains unknown. Recent findings suggest that cell membrane vesicles, or microparticles, which are released upon cell activation or apoptosis, are associated with CV disease and

Endothelial progenitor cells (EPCs) are thought to be important for maintaining normal vascular function. We conducted a prospective study evaluating the effect of the erythropoiesis-stimulating agent darbepoetin alfa on EPCs and vascular function in patients with chronic kidney disease (CKD), with or without diabetes. Thirty subjects with CKD (20 subjects with type II diabetes mellitus and 10 without diabetes mellitus) received weekly subcutaneous administration of darbepoetin alfa for 4 weeks. EPCs were measured at baseline and 2 and 4 weeks after drug administration. Vascular function was measured with brachial ultrasound and cell activity was measured with a cell proliferation assay. Cells expressing CD133, CD34, CD146 and CD146/31 were significantly elevated (all p < 0.05), flow-mediated vasodilatation increased 2.1%, 95% CI: (0.4%, 3.8%) and colony-forming units increased twofold, 95% CI: (1.7, 2.3) after 4 weeks of treatment with darbepoetin alfa. Subjects with diabetes exhibited an increase in a subset of EPCs (CD133 + and 34+, p < 0.01 and p = 0.06, respectively), vasodilatation and proliferation. In conclusion, the administration of darbepoetin alfa for 4 weeks increased a subset of EPCs, improved endothelial function and increased cell proliferation, including those with diabetes, which is consistent with a favorable improvement in vascular health.

BACKGROUND:Flow cytometry is used to monitor lymphocyte subsets in both the clinical and research settings. An understanding of the degree of inter- and intrasubject variability of these populations is critical for data interpretation. METHODS:Peripheral blood lymphocytes of 18 healthy adults were analyzed on two separate occasions using a multicolor flow cytometric panel with B, T, and NK cell markers. Variability was calculated using the coefficient of variation and compared between and within individuals using agglomerative clustering. RESULTS:Each subject appears to have B and T cell subset profiles that are stable over the two time points, but differ from the profiles of other subjects. Thus, the range of measurements for a particular B or T cell subset is larger between subjects and narrower for an individual. In addition, the level of variability correlates inversely with the size of the lymphocyte subset. When lymphocyte profiles are analyzed by agglomerative clustering, replicate samples from the same individual tend to cluster. When single samples from different individuals are analyzed, individuals appear to cluster into different subgroups. CONCLUSIONS:Variability of lymphocyte subsets is usually greater between individuals than within a single individual and each person appears to have a characteristic profile of lymphocyte subsets. These results underscore the importance of obtaining a baseline value for each subject when investigating the impact of a treatment on lymphocyte subsets over time. These results also highlight the potential utility of cluster analysis as a tool for immune subset profiling and biomarker discovery. © 2011 International Clinical Cytometry Society.

Manual gating of bivariate plots remains the most frequently used data analysis method in flow cytometry. However, gating is operator-dependent and cumbersome, particularly with the increasing complexity of modern multicolor immunophenotyping data. A method that can remove operator bias, enable systematic and thorough analysis of complex high-dimensional data, correlate temporal changes in different subsets and lead to biomarker discovery is needed. Here we apply such a method, called cytometric fingerprinting (CF), to data obtained on peripheral blood B cells from an adult patient with type-1 diabetes who underwent pancreatic islet transplantation. We establish that CF can be used to analyze longitudinal trends in immunophenotypic data, and show that results from CF are comparable to those obtained with traditional gating methods. Both methods reveal the appearance of transitional B cells and subsequent accumulation of more mature B cells following immunosuppression and transplantation. This pattern is consistent with a temporally ordered process of B cell auto-reconstitution. We also show the comparative efficiency of fingerprinting in recognizing relative changes in B cell subsets with respect to time, its ability to couple the data with statistical methods (agglomerative clustering) and its potential to define novel subsets.

Objective. Examination of the amniotic fluid (AF) proteome has been previously attempted to identify useful biomarkers in predicting the outcome of preterm labor (PTL). Isobaric Tag for Relative and Absolute Quantitation (iTRAQ™) labeling allows direct ratiometric comparison of relative abundance of identified protein species among multiplexed samples. The purpose of this study was to apply, for the first time, the combination of iTRAQ and tandem mass spectrometry to identify proteins differentially regulated in AF samples of women with spontaneous PTL and intact membranes with and without intra-amniotic infection/inflammation (IAI). Methods. A cross-sectional study was designed and included AF samples from patients with spontaneous PTL and intact membranes in the following groups: (1) patients without IAI who delivered at term (n = 26); (2) patients who delivered preterm without IAI (n = 25); and (3) patients with IAI (n = 24). Proteomic profiling of AF samples was performed using a workflow involving tryptic digestion, iTRAQ labeling and multiplexing, strong cation exchange fractionation, and liquid chromatography tandem mass spectrometry. Twenty-five separate 4-plex samples were prepared and analyzed. Results. Collectively, 123,011 MS2 spectra were analyzed, and over 25,000 peptides were analyzed by database search (X!Tandem and Mascot), resulting in the identification of 309 unique high-confidence proteins. Analysis of differentially present iTRAQ reporter peaks revealed many proteins that have been previously reported to be associated with preterm delivery with IAI. Importantly, many novel proteins were found to be up-regulated in the AF of patients with PTL and IAI including leukocyte elastase precursor, Thymosin-like 3, and 14-3-3 protein isoforms. Moreover, we observed differential expression of proteins in AF of patients who delivered preterm in the absence of IAI in comparison with those with PTL who delivered at term including Mimecan precursor, latent-transforming growth factor β-binding protein isoform 1L precursor, and Resistin. These findings have been confirmed for Resistin in an independent cohort of samples using ELISA. Gene ontology enrichment analysis was employed to reveal families of proteins participating in distinct biological processes. We identified enrichment for host defense, anti-apoptosis, metabolism/catabolism and cell and protein mobility, localization and targeting. Conclusions. (1) Proteomics with iTRAQ labeling is a profiling tool capable of revealing differential expression of proteins in AF; (2) We discovered 82 proteins differentially expressed in three clinical subgroups of premature labor, 67 which were heretofore unknown. Of particular importance is the identification of proteins differentially expressed in AF from women who delivered preterm in the absence of IAI. This is the first report of the positive identification of biomarkers in this subgroup of patients.