Identification Of Subset Research Articles

Identification of Lymphocyte Subsets Associated with Outcomes in Patients with Hematological Malignancy Following Allogeneic Stem Cell Transplantation: A Single Institute Study

Background: Immune reconstitution after allogeneic stem cell transplantation (SCT) is a complicated process influenced by factors such as preconditioning regimens, graft-versus-host disease (GVHD) prophylaxis, and grafts. We studied the association between the kinetics of lymphocyte subsets and transplant outcome to clarify the differences in immune reconstitution after hematopoietic cell transplantation according to stem cell sources and its clinical significance.Patients and Methods: Clinical data were collected from patients' medical charts at Kanagawa Cancer Center, Yokohama, Japan. Patients with hematological malignancies aged ≥18 years old who underwent SCT between April 2009 and December 2017 were initially selected. Those who died or experienced disease relapse before day 100 post SCT were excluded. We measured absolute lymphocyte count (ALC) and lymphocyte subsets by flow cytometry with antibodies against CD2, CD3, CD4, CD8, CD11b, CD11c, CD16, CD25, CD29, CD56, CD57, CD45RA, and CD45RO on days 100, 180, 365, and 730 post SCT.Results: The final cohort included 314 patients (acute leukemia, n = 249; myelodysplastic syndrome, n = 44; chronic myelogenous leukemia, n = 9; malignant lymphoma, n = 6; and others, n = 6). The median age was 51 (range: 18- 69) years, with 184 males and 130 females. The disease risk at transplantation was standard in 209 and high in 105 patients. Myeloablative preconditioning was administered to 114 and reduced-intensity preconditioning to 200 patients. Bone marrow transplantation (BMT), peripheral blood SCT (PBSCT), and cord blood transplantation (CBT) were performed in 121, 57, and 136 patients, respectively. A calcineurin inhibitor with short-term methotrexate was mainly used for GVHD prevention. The median follow-up of surviving patients was 869 (range: 103-3074) days. The 2-year overall survival (OS), cumulative incidence of relapse (CIR), and non-relapse mortality (NRM) in BMT, PBSCT, and CBT were 62%, 68%, and 76% (P = 0.023); 33%, 38%, and 27% (P = 0.068); and 17%, 16%, and 13% (P = 0.82); respectively. The 2-year cumulative incidence of chronic GVHD was 43% in BMT, 45% in PBSCT, and 28% in CBT (P = 0.027).There were significant differences between lymphocyte subset recovery and stem cell sources (Table). ALC; CD20+ B cell; CD4+, CD4+CD29+, CD4+CD45RO+, CD4+CD45RO− , and CD4+CD45RA+ T cell subsets; and CD3−CD56+ and CD16+CD57− natural killer (NK) cell subsets were significantly elevated in CBT compared with BMT and PBSCT at day 100 post SCT. Conversely, CD8+CD11b+ and CD8+CD11b− T cell subsets and CD3+CD56+ NKT cells were significantly lower in CBT than in BMT and PBSCT on day 100. Univariate analysis revealed that lymphocyte subsets exhibiting higher levels of CD20+ B cell; CD16+CD57− and CD3−CD56+ NK cells; and CD4+CD25+, CD4+CD29+, CD4+CD45RA+, CD8+CD11b+, and CD8+CD11b− T cell subsets at day 100 were associated with a better 2-year OS. There were strong correlations with a lower CIR and higher CD16+CD57+ and CD16+CD57− NK cell levels . A higher incidence of chronic GVHD was associated with lower levels of CD16+CD57− NK cells and CD4+CD25+ T cells and with higher levels of CD8+CD11b+ T cells and CD8+CD11b− and CD3+CD56+ NKT cells. Further, a lower NRM correlated with higher levels of CD20+ B cells and CD8+CD11b− T cells.The lymphocyte subsets were used for multivariate analysis. Favorable factors for better OS were higher levels of CD16+CD57− NK cells [hazard ratio (HR), 0.62; 95% confidence index (CI), 0.38-0.81; P = 0.024] and CD20+ B cells (HR, 0.56; 95% CI, 0.31-0.0.98; P = 0.048). Prognostic factors for lower CIR were higher levels of CD16+CD57+ NK cells (HR, 0.51; 95% CI, 0.27-0.95; P = 0.034) and CD16+CD57− NK cells (HR, 0.52; 95% CI, 0.28-0.99; P = 0.048). A lower incidence of NRM was associated with higher levels of CD8+CD11b− T cells (HR, 0.18; 95% CI, 0.08-0.39; P < 0.001) and CD20+ B cells (HR, 0.24; 95% CI, 0.08-0.70; P = 0.0088). High levels of CD8+CD11b+ T cells were an independent predictor for a higher incidence of chronic GVHD (HR, 2.38; 95% CI, 1.22-4.95; P = 0.012).Conclusions: The distinct differences in immune reconstitution according to stem cell sources and lymphocyte subset analysis at day 100 post SCT are useful for predicting transplant outcome. Furthermore, the results suggest that characteristic immune recovery in CBT positively affects transplant outcome. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hypodiploidy in Childhood Acute Myeloid Leukemia: A Retrospective Cohort Study within the International Berlin-Frankfurt-Münster Study Group

Background: Despite major improvements in survival, relapse is still a frequent and severe event in pediatric acute myeloid leukemia (AML). Cytogenetic abnormalities represent important predictors of outcome. Improved risk stratification encompassing the identification of novel cytogenetic subsets may allow refinements in treatment strategies and increase survival rates.Hypodiploidy, defined as a modal number (MN) below 46 chromosomes, is associated with an adverse outcome in pediatric acute lymphoblastic leukemia. In childhood AML, the predominant cause of hypodiploidy is loss of a sex chromosome accompanying t(8;21). The most commonly lost autosome is chromosome 7, associated with a poor prognosis. However, other chromosome losses in pediatric AML have not been independently explored.We aimed to investigate the occurrence, genetic characteristics, and prognostic impact of hypodiploidy in childhood AML.Methods: This study was conducted as a retrospective cohort study within the I-BFM-AML framework. Children 0-18 years of age diagnosed with de novo AML between January 2000 to December 2015 and a hypodiploid karyotype were eligible for inclusion. Patients with constitutional hypodiploidy, monosomy 7, composite karyotypes, and t(8;21) with loss of a sex chromosome were excluded.Cytogenetic review was performed according to International System for Human Cytogenetic Nomenclature. Hypodiploidy was considered clonal when occurring in at least three metaphases. The clone with the lowest MN defined the ploidy level. The log-rank test was applied for estimating difference in survival. Considering allografting in first complete remission (CR1) as a time-dependent event, the effect of stem cell transplantation (SCT) on overall survival (OS) was estimated by the Mantel-Byar method. Multivariate analyses included sex, complex karyotype (≥3 non-recurrent aberrations), FLT3-ITD mutation status, and white blood cell count as covariates. As all patients with FLT3-ITD mutations had same MN (MN=45) leading to a skewed imputation model, multiple imputation was not performed, and multivariate analyses included complete cases only (n=54).Results: Hypodiploidy was detected in 81/6,409 patients with full karyotyping from 14 collaborative study groups, yielding a frequency of 1.2% in childhood AML. The cohort displayed a balanced sex distribution (male/female: 42/39) and a median age of 6 (range: 0-17). MNs 45 (n=66), 44 (n=10) and 43 (n=5) were observed. No patient had MN below 43. Eight patients (9.8%) had chromosome loss as the only aberration. Forty-eight patients (59%) had a complex karyotype. Core-binding factor abnormalities were observed in three patients (3.7%). Four patients harbored FLT3-ITD mutations (9.3%, 54 tested).Median follow-time for patients alive was 4.5 years (range: 0.2-15.3). Five-year event-free survival (EFS) and OS were 34% (CI 95%: 23% - 45%) and 52% (CI 95%: 40% - 63%), respectively. Cumulative incidence of relapse was 50% with a median time-to-relapse of 0.8 years. Twelve out of 38 relapsed children (32%) were alive at the end of follow-up. Children with MN≤44 (n=15) had lower EFS (21%, CI 95%: 4% - 46%) and OS (33%, CI 95%: 10% - 59%) than children with MN=45 (n=66, EFS: 37%, CI 95%: 24% - 49%, OS: 56%, CI 95%: 42% - 68%). Crude hazard ratios (HR) for MN≤44 vs MN=45 were 1.8 (p=0.08) for EFS and 1.9 (p=0.1) for OS. After adjustment HRs increased to 4.9 (p=0.001) and 6.1 (p=0.003), respectively. Loss of chromosome Y (n=12) displayed a non-significant superior survival (EFS: 58%, OS: 75%). Poor survival rates were especially observed in monosomy 9 (n=11, EFS: 14%, OS 15%), 10 (n=5, EFS: 20%, OS: 20%), and 16 (n=5, EFS: 25%, OS: 25%), although not reaching statistical significance.Allogenic SCT did not offer any advantage in survival. For children receiving SCT in CR1 (n=18), crude HR for OS was 1.4, p=0.39. Multivariate analysis showed HR for OS at 1.5, p=0.42. Exclusion of children not reaching CR1 (n=7) did not significantly change HRs.This large international study reveals that hypodiploidy is rare in pediatric AML. Children with loss of chromosome 9, 10, or 16 all displayed unfavorable survival rates, whereas loss of chromosome Y did not have influence on prognosis. SCT in CR1 did not improve survival. Our results suggest that modal number has an impact on outcome and may be important for risk stratification. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical and Genetic Features of Epstein-Barr Virus-Triggered Late-Onset Primary Hemophagocytic Lymphohistiocytosis: 10 Pedigrees Study

Introduction: It is widely accepted that primary Hemophagocytic Lymphohistiocytosis (PHLH) in infants and children is a genetic disease inherited in an autosomal recessive manner. However, it is proposed recently that the late-onset PHLH could result from the synergetic effects of the atypical heterozygous HLH-associated mutations and trigger of environmental factors such as viral infection. EBV is the most common viral pathogen linked to the development of HLH. EBV is known as a trigger to induce secondary HLH in some patients; it also can be a trigger of PHLH. However, the subpopulation of EBV infection in PHLH is unknown. Identification of EBV-infected lymphocyte subsets will help to clarify the role of EBV in the development of late-onset PHLH. This study identified the EBV infected cells subpopulation and summarized the clinical and genetic characteristics of 10 late-onset PHLH patients.Methods: Between June 2013 and June 2018, 10 patients with Epstein-Barr virus-triggered Late-onset PHLH and their family members were investigated, with a median age of 22.3 years (range, 12-45 years). We quantified EBV load by PCR, examined Natural killer (NK) cell activity by Lactate dehydrogenase (LDH) assay, and performed flow cytometry to analyze perforin expression and degranulation in addition to collecting clinical characteristics of 10 EBV-triggered PHLH patients. Next-generation sequencing was used to detect HLH-associated genes mutations. Immunobead sorting followed by quantitative PCR and fluorescence in situ hybridization were conducted to identify EBV-infected cells. We also performed pedigrees investigation and detected EBV loads and HLH-associated genes mutations in these patients' family members.Results: All patients had the acute onset and rapid progression of a disease and met the Diagnostic Guidelines for HLH (2004) proposed by the Histiocyte Society. Reduced NK-cell activity was observed in 6 late-onset PHLH patients (normal range: 30%-50%), while NK-cell activity defect (<5%) was found in 4 patients. Substantially reduced perforin expression (normal range: > 50%) on CD3-CD56+ NK cells were detected in 7 of 10 patients (70%). One of 10 cases (10%) had abnormal resting NK-cell degranulation, and 4 of 10 (40%) had defective resting NK-cell degranulation. Abnormal activated NK-cell degranulation (normal range: > 20%) was recorded in 3 of 10 cases (30%). Nine of 10 cases (90%) had either reduced perforin expression or decreased activated NK-cell degranulation. The EBV-DNA load and genetic characteristics of the late-onset PHLH cases and their families were summarized in Figure A. All the details of 10 pedigrees are shown in Figure B. The family members had the same type of mutations as the patients but did not develop the disease. High load of EBV was detected in the peripheral blood mononuclear cells of the probands, whereas a much lower load of EBV were found in the probands' family members by quantitative PCR. The results of sorting-PCR demonstrated that three group of cells (T cells, B cells, and NK cells) in all the patients were infected with EBV, in 9 of them EBV significantly infected NK cells, and only in 1 patient EBV infected both NK cells and T lymphocyte cells primarily. More than half of the patients (7 of 10) died of disease progression or complications, and median survival time is only 95 days. Only three patients who received allogeneic hematopoietic stem cell transplantation from unrelated donor acquired long-term survival, currently alive.Conclusions: The late-onset PHLH is a devastating disorder, and the prognosis is poor. Unlike sporadic HLH, EBV-associated late-onset PHLH patients usually have 1 or 2 pathologic mutations of PHLH-associated genes, reduced perforin expression or degranulation. Some of them have abnormal T or NK cells in bone marrow or lymph node; most of them have high load EBV infection, and the target cells of EBV infectious are usually NK cells, sometimes combined with T cells. HLH gene sequencing and pedigree investigation can help clinicians classify patients as having late-onset PHLH or sporadic HLH. Based on genetic mutations, EBV-Infected NK/T lymphocyte subtypes may be involved in the development of Late-onset PHLH. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase 1b Dose Escalation Study of BI 836858 and Azacitidine in Previously Untreated AML: Results from Beat AML S2

Phase 1b Dose Escalation Study of BI 836858 and Azacitidine in Previously Untreated AML: Results from Beat AML S2

A 36-Dimensional Cytometry by Time of Flight (CyTOF) Analysis of De Novo Acute Myeloid Leukemia (AML) Patients Eligible for Intensive Chemotherapy

IntroductionChemotherapy induced clonal selection and development are major contributors to disease relapse and resistance to therapy in AML. An immediate response to chemotherapy in intracellular signalling networks can be detected within minutes in vivo. (Irish J. et al 2004) We hypothesize that by analyzing the immediate intracellular chemotherapy response we might reveal which cancer clones are resistant to therapy and how they might be targeted. (Anensen N. et al Clin Cancer Res 2006), (Øyan M. et al BMC Cancer 2009)CyTOF combines the principles of conventional flow cytometry with time-of-flight mass spectrometry to allow analyses of multiple parameters simultaneously in single cells. This dramatically increases the numbers of antibodies that can be applied and permits a simultaneous analysis of both intracellular signaling networks and phenotypic characterization of cells.Material and methodsWe have performed a 36- dimensional mass cytometry analysis (CyTOF), containing 21 surface markers (CD66b, CD16, CD45, CD11b, CD25, CD3, CD4, CD8, CD56, CD20, CD38, CD34, CD117, CD33, CD64, CD14, HLA-DR, Axl-1H12, CD90, CD7, CD123) and 15 intracellular markers (cCaspase-3, p-4EB-P1, p-Stat 5, p-Stat 3, p-Stat 1, p-p38, p- ERK ½, p-Akt, p-NFkB, p-CREB, p-S6, p-Axl, p-Rb, Cyclin B1, p-Histone H3) of over 40 de novo AML patients eligible for intensive chemotherapy. Peripheral blood was sampled at 4 hours and at 18- 24 hours after start of standard “3+7” induction therapy (Lowenberg B. et al Blood 2017). Bone marrow and peripheral blood from healthy donors were also included in the analysis.ResultsWell-characterized surface markers were used to perform an unsupervised clustering of the high dimensional single cell data from each AML patient before start of induction therapy (Figure 1A). This clustering resulted in 15 unique cell clusters throughout the patient cohort, illustrated by viSNE in Figure 2A. The heatmap in Figure 1A show the expression of surface markers in each cluster. Healthy cells in AML patients clustered together and were recognized by their characteristic phenotype, for example granulocytes in cluster 13 (CD66b+ and CD11b+), CD8 T-cells in cluster 1 (CD3+, CD7+ and CD8+) and monocytes in cluster 11 (CD45+, CD64+, CD38+ and HLA-DR+). Cluster 3 and 4 had high expression of the immature marker CD34 and thereby likely to represent blast populations.Intracellular signaling at time of diagnosis was then compared to 24 hours after start of induction therapy in the same populations. A lasso regression analysis identified intracellular markers that significantly correlated to patient survival, based on their change in expression between pre-treatment and at 24 hours. Figure 1C show a Kaplan-Meier curve of patient survival (24 months) between patients that had high or low expression ratio of an intracellular phosphor-specific antibody in cluster 3 and 15. Both clusters have expression of CD34 and CD117 that are frequently expressed on leukemic cells. The expression ratio of this intracellular marker was significantly correlated to survival (p<0,0001). Furthermore, these CyTOF data will be correlated to RNA-seq data, mutation status and proteomic data from the same patients.ConclusionsBy applying unsupervised clustering analysis of over 40 de novo AML patients we identified leukemic cell subpopulations that had unique patterns of intracellular phospho-specific antibodies during start of induction therapy. Changes in intracellular markers during the first 24 hours of induction therapy could significantly be related to patient survival. By analyzing molecular alterations short time after chemotherapy, we aim to reveal the sub-clonal diversity and the heterogeneous responses to treatment. Identification of cancer cell subsets that are resistant to conventional chemotherapy may in the future be used to guide therapy in AML. [Display omitted] DisclosuresGjertsen:Alden Cancer Therapy 2: Patents & Royalties: Alden Cancer Therapy II patent application in relation to CryoIT trial.; BerGenBio: Consultancy; Novartis: Consultancy; Alden Cancer Therapy 2: Membership on an entity's Board of Directors or advisory committees; Alden Cancer Therapy 2: Equity Ownership; Kinn Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Kinn Therapeutics: Equity Ownership; Boehringer Ingelheim: Research Funding.

Pre-Transplant Monocytic Myeloid-Derived Suppressor Cell Frequency Has No Prognostic Role for Outcome after Allogeneic Hematopoietic Cell Transplantation for Acute Myeloid Leukemia in Remission

INTRODUCTION: Emerging data indicate myeloid-derived suppressor cells (MDSCs) adversely impact outcomes of patients with a variety of malignancies. However, it is less clear how MDSCs affect acute myeloid leukemia (AML) disease biology or the response of AML patients to treatment. Here, we studied a cohort of people with AML undergoing hematopoietic cell transplantation (HCT) in first complete remission (CR1) to examine the relationship between the monocytic subtype of MDSCs (M-MDSCs), pre-transplant measurable residual disease (MRD) status, and outcome after HCT.PATIENTS AND METHODS: Adults ≥18 years of age were included if they underwent first allogeneic HCT with myeloablative or nonmyeloablative conditioning for AML in CR1 from April 2006 until October 2014. Prospective MRD testing in bone marrow aspirates was performed routinely via 10-color multiparameter flow cytometry (MFC) as part of the pre-transplant work-up. Pre-transplant MFC data were used retrospectively to quantify M-MDSCs as the proportion of monocytes (identified by side scatter properties and CD14 positivity) with low or negative expression of HLA-DR. M-MDSC frequency was then expressed as a percentage of the total number of viable cells in the sample. Available combinations of antigens measured by MFC did not allow for the identification of other MDSC subsets (e.g. granulocytic MDSCs).RESULTS: We identified 349 adults undergoing allogeneic HCT for AML in CR1 for whom pre-transplant MRD testing was performed and for whom follow-up data were available. Of these, 8 had to be excluded because of insufficient MFC events available for identification of M-MDSCs. In the remaining 341 patients, M-MDSC frequency ranged from <0.001% to 6.53%, with a median of 0.18% (25th percentile: 0.06%; 75th percentile: 0.40%). Patients with lower (i.e. <median) frequency of M-MDSCs were less likely male (p=0.01) and more likely had recovered neutrophil and platelet counts (p=0.001) than patients with higher (i.e. ≥median) frequency of M-MDSCs. There was no difference with regard to all other examined characteristics (age, WBC and cytogenetic risk at diagnosis, type of AML, median CR duration before HCT, donor type, and conditioning intensity). The proportion of MRD positive (“MRDpos”) patients was similar between those with lower vs. higher M-MDSC frequency (18.8 vs. 24.6%, p=0.20). Moreover, while MRD negative (“MRDneg”) patients (n=267) differed statistically significantly from MRDpos patients (n=74) with regard to several characteristics such as more favorable disease risk and lower proportion of secondary AML, there was only a statistically non-significant trend toward lower M-MDSC frequency in MRDneg patients (0.16% [range <0.001-5.61%] vs. 0.22% [range <0.001-6.53%], p=0.09).The median follow-up time after HCT among all survivors was 5.8 (range, 2.0-11.4) years. The time was slightly longer for survivors with lower compared to those with higher M-MDSC frequency (6.1 [range 2.6-11.4] vs. 5.1 [range 2.0-11.3] years; p=0.0076). Stratified by the median M-MDSC frequency, the 3-year estimates of overall survival were similar for patients with lower and higher pre-HCT M-MDSCs (58.2% [95% confidence interval 50.4-65.2%] vs. 57.6% [49.8-64.7%]), as were 3-year estimates for relapse free survival (53.5% [45.7-60.7%] vs. 48.3% [40.6-55.6%]), 3-year estimates of the cumulative incidence of relapse (31.8% [24.9-38.8%] vs. 34.6% [27.6-41.8%]), and 3-year estimates of non-relapse mortality (14.7% [9.9-20.5%] vs. 17.0% [11.8-23.0%]). Analyses restricted to the subset of patients undergoing myeloablative (MA) conditioning or those undergoing MA conditioning in MRDneg remission showed qualitatively similar results.CONCLUSIONS: In the largest study to date examining the role of M-MDSCs in AML, we found no convincing evidence for a prognostic role of these cells for adults undergoing allografting in CR1. DisclosuresWalter:Actinium Pharmaceuticals, Inc.: Other: Clinical trial support, Research Funding; Amgen Inc.: Other: Clinical trial support, Research Funding; Amphivena Therapeutics: Consultancy, Equity Ownership, Other: Clinical trial support, Research Funding; Aptevo Therapeutic: Consultancy, Other: Clinical trial support, Research Funding; Covagen AG: Consultancy, Other: Clinical trial support, Research Funding; Seattle Genetics, Inc.: Consultancy, Other: Clinical trial support, Research Funding; Boehringer Ingelheim Pharma GmbH & Co. KG: Consultancy; Pfizer: Consultancy.

Neoadjuvant therapy for esophageal cancer: Who, when, and what?

The perioperative combination of 5‐fluorouracil, oxaliplatin, and docetaxel is a new standard treatment in patients with esophageal carcinoma. However, chemoradiation results in higher R0 surgical resection rates, pathologic complete response, and negative lymph node status. The identification of molecular subsets may influence the design of future studies and guide drug development.

In silico identification and characterization of a diverse subset of conserved microRNAs in bioenergy crop Arundo donax L.

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the post-transcriptional regulation of gene expression in plants. Arundo donax L. is a perennial C3 grass considered one of the most promising bioenergy crops. Despite its relevance, many fundamental aspects of its biology still remain to be elucidated. In the present study we carried out the first in silico mining and tissue-specific characterization of microRNAs and their putative targets in A. donax. We identified a total of 141 miRNAs belonging to 14 families along with the corresponding primary miRNAs, precursor miRNAs and a total of 462 high-confidence predicted targets and novel target sites were validated by 5′-race. Gene Ontology functional annotation showed that miRNA targets are constituted mainly by transcription factors, but three of the newly validated targets are enzymes involved in novel functions like RNA editing, acyl lipid metabolism and post-Golgi trafficking. Folding variability of pre-miRNA loops and phylogenetic analyses indicate variable selective pressure acting on the different miRNA families. The set of miRNAs identified in this study will pave the road to further miRNA research in Arundo donax and contribute towards a better understanding of miRNA-mediated gene regulatory processes in other bioenergy crops.

Elliptic string solutions on mathbb {R}times hbox {S}^2 and their pohlmeyer reduction

We study classical string solutions on mathbb {R}times hbox {S}^2 that correspond to elliptic solutions of the sine-Gordon equation. In this work, these solutions are systematically derived by inverting the Pohlmeyer reduction. A mapping of the physical properties of the string solutions to those of their Pohlmeyer counterparts is established. An interesting element of this mapping is the association of the number of spikes of the string to the topological charge in the sine-Gordon theory. Finally, the adopted parametrization of the solutions facilitates the identification of a dense subset of the moduli space of solutions, where the dispersion relation can be expressed in a closed form, arbitrarily far from the infinite size limit.

Integrating Immunology and Microfluidics for Single Immune Cell Analysis.

The field of immunoengineering aims to develop novel therapies and modern vaccines to manipulate and modulate the immune system and applies innovative technologies toward improved understanding of the immune system in health and disease. Microfluidics has proven to be an excellent technology for analytics in biology and chemistry. From simple microsystem chips to complex microfluidic designs, these platforms have witnessed an immense growth over the last decades with frequent emergence of new designs. Microfluidics provides a highly robust and precise tool which led to its widespread application in single-cell analysis of immune cells. Single-cell analysis allows scientists to account for the heterogeneous behavior of immune cells which often gets overshadowed when conventional bulk study methods are used. Application of single-cell analysis using microfluidics has facilitated the identification of several novel functional immune cell subsets, quantification of signaling molecules, and understanding of cellular communication and signaling pathways. Single-cell analysis research in combination with microfluidics has paved the way for the development of novel therapies, point-of-care diagnostics, and even more complex microfluidic platforms that aid in creating in vitro cellular microenvironments for applications in drug and toxicity screening. In this review, we provide a comprehensive overview on the integration of microsystems and microfluidics with immunology and focus on different designs developed to decode single immune cell behavior and cellular communication. We have categorized the microfluidic designs in three specific categories: microfluidic chips with cell traps, valve-based microfluidics, and droplet microfluidics that have facilitated the ongoing research in the field of immunology at single-cell level.

Natural regulatory plasma cells

B cells can generate several types of antibody-secreting cells, including plasmablasts that divide and are short lived, as well as plasma cells that do not proliferate and can persist for extended time periods. Here, we discuss the identification of a novel subset of non-dividing plasma cells specialized in the production of interleukin(IL)-10. These cells develop at steady state, including in germ-free mice, via a mechanism dependent on the B cell receptor for antigen and possibly involving the recognition of damaged cells. They are characterized by the expression of the inhibitory receptor LAG-3, and also express CD200, PD-L1, as well as PD-L2. Their specialized epigenome allows them to produce IL-10 within hours after stimulation, which altogether qualify these cells as natural regulatory plasma cells.

The Fast Real-time Assessment of Combination Therapies in Immuno-ONcology (FRACTION) program: innovative, high-throughput clinical screening of immunotherapies

BackgroundThe unprecedented success of immuno-oncology (I-O) agents targeting the cytotoxic T lymphocyte–associated antigen 4 and programmed death-1/programmed death-ligand 1 pathways has stimulated the rapid development of other I-O agents against novel immune targets. Bristol-Myers Squibb has designed a novel phase II platform trial, the Fast Real-time Assessment of Combination Therapies in Immuno-ONcology (FRACTION) Program, to efficiently identify promising combinations for patients with specific malignancies. The concept and study design of the FRACTION Program—currently ongoing in patients with advanced non-small-cell lung cancer (FRACTION-Lung), gastric cancer (FRACTION-Gastric Cancer) and renal cell carcinoma (FRACTION-RCC)—are described. MethodsThe FRACTION Program comprises open-label, phase II studies that use adaptive randomisation designs with rolling combination regimens. Master Protocols provide the overall study design framework, whereas Sub-Protocols introduced over time provide details on specific I-O combination therapies to which patients may be randomised. In a Master Protocol, patients are enrolled into different Study Tracks based on characteristics such as prior I-O therapy experience. Patients who progress may be rerandomised to other combination regimens from any ongoing Sub-Protocol. Primary objectives are to assess objective response rate, median duration of response and progression-free survival rate at 24 weeks; the secondary objective is to investigate safety and tolerability. Biomarker collection before and on treatment will facilitate identification of patient subsets who benefit most from each therapy. ConclusionsThe FRACTION Program allows for the evaluation of multiple I-O combinations through individual studies for specific tumours using an adaptive trial design and continuous enrolment.

Clinical significance of rare serum autoantibodies in rheumatic diseases: a systematic literature review

The identification of serum autoantibodies is central in the diagnosis of systemic autoimmune rheumatic disease (SARD), and an increasing number of specificities have been detected in the past years. This allows an early diagnosis in the active phases of diseases, with the identification of specific disease subsets that may ultimately improve the disease outcomes. Thanks to the use of old and new laboratory techniques that are becoming increasingly available worldwide, the number of rheumatic patients with a specific autoantibody is increasing and this is improving also our knowledge of disease trigger mechanisms. The paradigmatic example is the plethora of serum autoantibodies described in polymyositis and dermatomyositis, coined myositis-specific antibodies (MSA) which include antibodies directed against tRNA synthetases, anti-SRP, anti-Mi-2, and anti-TIF-1γ and can discriminate disease subtypes, particularly when associated with the risk of cancer. As a further example, anti-HMGCR antibodies have been reported in several studies in association with necrotizing autoimmune myositis that may follow statin use. To clarify the current knowledge on these rare specificities, we performed a systematic literature review. We focused on the main features associated to specific autoantibodies that are rarely identified in rheumatic disease, to increase the awareness and scientific knowledge on these autoantibodies in different ethnic groups worldwide.

Modeling of cigarette smoke constituents - From intense to less intense smoking regime

Since it was first required to measure and to report NFDPM and nicotine yields in a limited number of countries, there has been an increasing trend for more testing and reporting requirements. Historically, the ISO 3308 smoking regime has been used to determine NFDPM and nicotine yields. However recommendations from the World Health Organization, now include the use of two smoking regimes such as the ISO 3308 and the WHO TobLabNet Official Method SOP01, the latter being considered as an intense smoking regime. Considering the increase in data produced and similarities between some smoke constituents formed during combustion, we explored possible correlations between emissions under intense and less intense smoking conditions. A set of 22 commercial cigarettes was tested. Eighty five smoke constituents were determined under both intense and less intense regimes. In addition 36 tobacco constituents, 14 cigarette design parameters and eight cigarette burning features were determined. A computational process was designed to implement multiple linear regression analyses enabling the identification of the best subsets of explanatory variables among emissions under intense conditions, cigarette design parameters, tobacco constituents and burning parameters. We succeeded in building simple linear models, involving four to six variables, while reaching satisfactory goodness of fit and R-squared values ranging from 0.87 to 1.00. Our findings suggest, in the range of products tested, that the additional data gained by using a second smoking regime does not necessarily increase the volume of information and consequently does not necessarily improve knowledge. This study supports the premise that the application of two smoking regimes does not produce a more comprehensive product characterisation compared to using one.

Identification of Distinct Unmutated Chronic Lymphocytic Leukemia Subsets in Mice Based on Their T Cell Dependency

Chronic lymphocytic leukemia (CLL) can be divided into prognostically distinct subsets with stereotyped or non-stereotyped, mutated or unmutated B cell receptors (BCRs). Individual subsets vary in antigen specificity and origin, but the impact of antigenic pressure on the CLL BCR repertoire remains unknown. Here, we employed IgH.TEμ mice that spontaneously develop CLL, expressing mostly unmutated BCRs of which ~35% harbor VH11-2/Vκ14-126 and recognize phosphatidylcholine. Proportions of VH11/Vκ14-expressing CLL were increased in the absence of functional germinal centers in IgH.TEμ mice deficient for CD40L or activation-induced cytidine deaminase. Conversely, in vivo T cell-dependent immunization decreased the proportions of VH11/Vκ14-expressing CLL. Furthermore, CLL onset was accelerated by enhanced BCR signaling in Siglec-G−/− mice or in mice expressing constitutively active Bruton's tyrosine kinase. Transcriptional profiling revealed that VH11 and non-VH11 CLL differed in the upregulation of specific pathways implicated in cell signaling and metabolism. Interestingly, principal component analyses using the 148 differentially expressed genes revealed that VH11 and non-VH11 CLL clustered with BCR-stimulated and anti-CD40-stimulated B cells, respectively. We identified an expression signature consisting of 13 genes that were differentially expressed in a larger panel of T cell-dependent non-VH11 CLL compared with T cell-independent VH11/Vκ14 or mutated IgH.TEμ CLL. Parallel differences in the expression of these 13 signature genes were observed between heterogeneous and stereotypic human unmutated CLL. Our findings provide evidence for two distinct unmutated CLL subsets with a specific transcriptional signature: one is T cell-independent and B-1 cell-derived while the other arises upon antigen stimulation in the context of T-cell help.

Spectral clustering for non-reversible Markov chains

Spectral clustering methods are based on solving eigenvalue problems for the identification of clusters, e.g., the identification of metastable subsets of a Markov chain. Usually, real-valued eigenvectors are mandatory for this type of algorithms. The Perron Cluster Analysis (PCCA+) is a well-known spectral clustering method of Markov chains. It is applicable for reversible Markov chains, because reversibility implies a real-valued spectrum. We also extend this spectral clustering method to non-reversible Markov chains and give some illustrative examples. The main idea is to replace the eigenvalue problem by a real-valued Schur decomposition. By this extension non-reversible Markov chains can be analyzed. Furthermore, the chains do not need to have a positive stationary distribution. In addition to metastabilities, dominant cycles and sinks can also be identified. This novel method is called GenPCCA (i.e., generalized PCCA), since it includes the case of non-reversible processes. We also apply the method to real-world eye-tracking data.

Bayesian negative binomial mixture regression models for the analysis of sequence count and methylation data.

In this article, we develop a Bayesian hierarchical mixture regression model for studying the association between a multivariate response, measured as counts on a set of features, and a set of covariates. We have available RNA-Seq and DNA methylation data measured on breast cancer patients at different stages of the disease. We account for the heterogeneity and over-dispersion of count data (here, RNA-Seq data) by considering a mixture of negative binomial distributions and incorporate the covariates (here, methylation data) into the model via a linear modeling construction on the mean components. Our modeling construction includes several innovative characteristics. First, it employs selection techniques that allow the identification of a small subset of features that best discriminate the samples while simultaneously selecting a set of covariates associated to each feature. Second, it incorporates known dependencies into the feature selection process via the use of Markov random field (MRF) priors. On simulated data, we show how incorporating existing information via the prior model can improve the accuracy of feature selection. In the analysis of RNA-Seq and DNA methylation data on breast cancer, we incorporate knowledge on relationships among genes via a gene-gene network, which we extract from the KEGG database. Our data analysis identifies genes which are discriminatory of cancer stages and simultaneously selects significant associations between those genes and DNA methylation sites. A biological interpretation of our findings reveals several biomarkers that can help understanding the effect of DNA methylation on gene expression transcription across cancer stages.

Study of Serum YKL-40 in Patients with Postmenopausal Bleeding and Thickened Endometrium

YKL-40 is a secreted glycoprotein that has been described as a diagnostic and prognostic marker for a number of cancers including epithelial ovarian cancer. In this study, we examined the frequency of its serum elevation in patients with postmenopausal bleeding and thickened endometrium for its possible use in assessment of endometrial pathologies as atypical hyperplasia and malignancy. Preoperative serum levels of YKL-40 were evaluated by ELISA for all endometrial cancer, atypical hyperplasia patients recruited from El Shatby Maternity University Hospital, Gynecological Oncology specialized in between the years 2015 and 2017 and compared to a cohort of normal individuals. YKL-40 value of 39 ng/mL has previously been determined to represent the upper limit of normal. YKL-40 values were correlated with clinical characteristics, including tumor grade, histology, clinical stage, and risk groups. YKL-40 was elevated (> 39 ng/mL) in 31 endometrial cancer patients compared to 31 atypical hyperplasia patients and 31 normal control cases. Mean preoperative YKL-40 value was 105.89 ± 96.99 ng/mL for endometrial cancer patients compared with 51.56 ± 43.47 ng/mL for atypical hyperplasia patients (P < 0.001*) and compared with 29.09 ± 10.54 ng/mL for normal healthy subjects (P < 0.001*). There was statistically significant association of YKL-40 with tumor grade, histological result, stage, and risk group. Preoperative serum YKL-40 is frequently elevated and may represent a novel marker for the detection of endometrial cancer and the identification of high-risk subsets of patients with worse clinical outcome.

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of "contaminating" progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

Identification of a new subset of lymph node stromal cells involved in regulating plasma cell homeostasis

Antibody-secreting plasma cells (PCs) arise rapidly during adaptive immunity to control infections. The early PCs are retained within the reactive lymphoid organ where their localization and homeostasis rely on extrinsic factors, presumably produced by local niche cells. While myeloid cells have been proposed to form those niches, the contribution by colocalizing stromal cells has remained unclear. Here, we characterized a subset of fibroblastic reticular cells (FRCs) that forms a dense meshwork throughout medullary cords of lymph nodes (LNs) where PCs reside. This medullary FRC type is shown to be anatomically, phenotypically, and functionally distinct from T zone FRCs, both in mice and humans. By using static and dynamic imaging approaches, we provide evidence that medullary FRCs are the main cell type in contact with PCs guiding them in their migration. Medullary FRCs also represent a major local source of the PC survival factors IL-6, BAFF, and CXCL12, besides also producing APRIL. In vitro, medullary FRCs alone or in combination with macrophages promote PC survival while other LN cell types do not have this property. Thus, we propose that this FRC subset, together with medullary macrophages, forms PC survival niches within the LN medulla, and thereby helps in promoting the rapid development of humoral immunity, which is critical in limiting early pathogen spread.