Standard Flow Cytometry Research Articles

(696) - Old Dogs with New Tricks: A Standardized Flow Cytometry Panel Measuring Donor-Recipient Alloreactivity

(696) - Old Dogs with New Tricks: A Standardized Flow Cytometry Panel Measuring Donor-Recipient Alloreactivity

Biophysics: A toehold in cell surface dynamics.

A combination of standard fluorescence microscopy and flow cytometry offers a practical new approach to study encounter rates and preferences during various live cell membrane signalling events.

Changes in T-cell subpopulations and cytokine network during early period of ibrutinib therapy in chronic lymphocytic leukemia patients: the significant decrease in T regulatory cells number.

B cell receptor (BCR) stimulation signal plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), and kinase inhibitors directed toward the BCR pathway are now the promising anti-leukemic drugs. Ibrutinib, a Bruton tyrosine kinase inhibitor, demonstrates promising clinical activity in CLL. It is reported that ibrutinib, additionally to directly targeting leukemic cells, also inhibits the interactions of these cells with T cells, macrophages and accessory cells. Assessment of these mechanisms is important because of their non -direct anti-leukemic effects and to identify possible side effects connected with long-term drug administration.The aim of this study was to assess the in vivo effects of ibrutinib on T-cell subpopulations and cytokine network in CLL. The analysis was performed on a group of 19 patients during first month of ibrutinib therapy. The standard multicolor flow cytometry and cytometric bead array methods were used for assessment of T-cell subsets and cytokines/chemokines, respectively.The data obtained indicates that Ibrutinib treatment results in changes in T-cell subpopulations and cytokine network in CLL patients. Particularly, a significant reduction of T regulatory cells in peripheral blood was observed. By targeting these populations of T cells Ibrutinib can stimulate rejection of tumor cells by the immune system.

Microparticles in nasal lavage fluids in chronic rhinosinusitis: Potential biomarkers for diagnosis of aspirin-exacerbated respiratory disease

Microparticles (MPs) are submicron-sized shed membrane vesicles released from activated or injured cells and are detectable by flow cytometry. MP levels have been used as biomarkers to evaluate cell injury or activation in patients with pathological conditions. We sought to compare MP types and levels in nasal lavage fluids (NLFs) from controls and patients with chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), and aspirin-exacerbated respiratory disease (AERD). We collected NLFs from patients with CRSsNP (n=33), CRSwNP (n=45), and AERD (n=31) and control (n=24) subjects. Standardized flow cytometry methods were used to characterize the following MP types: endothelial MPs, epithelial MPs (epithelial cell adhesion molecule [EpCAM](+)MPs, E-cadherin(+)MPs), platelet MPs (CD31(+)CD41(+)MPs), eosinophil MPs (EGF-like module-containing mucin-like hormone receptor-like 1[EMR1](+)MPs), mast cell MPs (high-affinity IgE receptor [FcεRI](+)c-kit(+)MPs), and basophil MPs (CD203c(+)c-kit(-)MPs). Basophil activation was evaluated by the mean fluorescence intensity of CD203c on basophil MPs. Activated mast cell MPs (CD137(+) FcεRI(+)c-kit(+)MPs) were significantly increased in NLFs of controls compared with NLFs of patients with CRSsNP (2.3-fold; P<.02), CRSwNP (2.3-fold; P<.03), and AERD (7.4-fold; P<.0001). Platelet MPs (3.5-fold; P<.01) and basophil MPs (2.5-fold; P<.05) were increased only in patients with AERD. Mean fluorescence intensity of CD203c on MPs was increased in patients with CRSwNP (P<.002) and AERD (P<.0001), but not in patients with CRSsNP. EpCAM(+)MPs in patients with CRSwNP were no different from control (P=.91) and lower than those in patients with CRSsNP (P<.02) and AERD (P<.002). Based on released MPs, mast cells, platelets, and basophils were more highly activated in patients with AERD than in patients with CRS. Epithelial injury was lower in patients with CRSwNP than in patients with CRSsNP and AERD. MP analysis may help identify phenotypes of CRS, and in distinguishing AERD from CRSwNP.

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients

BackgroundGlycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets.ResultsWe found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells.ConclusionsOur data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia

AbstractA fully-standardized EuroFlow 8–color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10−5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)–based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR–based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10−5), if sufficient cells (>4 × 106, preferably more) are evaluated.

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

Clinical Manifestation and Errors in the Diagnosis of Classical Paroxysmal Nocturnal Hemoglobinuria:

Background. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal hematopoietic stem cell disorder, characterized by intravascular hemolysis, cytopenia and thrombosis. Diagnostic errors with delayed diagnosis of PNH are often due to the variety of the clinical presentation and the lack of awareness of the doctors of this rare disease. Aim. The aim of the study was to characterize the spectrum of clinical manifestations and the complexity of diagnosis of classical PNH. Materials &amp; Methods. The study included 150 patients with classical PNH. The inclusion criteria were: 1) clinical and laboratory signs of intravascular hemolysis; 2) verification of the diagnosis using standard flow cytometry; 3) absence of aplastic anemia, myelodysplastic syndrome and primary myelofibrosis. Results. The study population consisted of 89 (59 %) women and 61 (41 %) men. Median age was 34 years (13-72 years). The time before the diagnosis ranged from 0 to 455 months (median 33 months). The median size of the PNH clone among granulocytes and erythrocytes was 95 % and 41 %, respectively. The median of the lactate dehydrogenase was 7.2 times the upper limit of normal (ULN). Cytopenia occurred in 65 % of patients, including a combination of thrombocytopenia and neutropenia in 29 % of cases. Weakness and fatigue (99 %), hemoglobinuria (57 %), pain (52 %), icterus (46 %), dysphagia (37 %) and infection/fever (23 %) were the most common symptoms on the onset of the disease. Before the diagnosis of PNH, thrombosis or acute kidney injury was found in 22 % and 12 % of patients, respectively. Only 22 % of patients were initially diagnosed with PNH. In the remaining patients, the primary diagnosis was incorrect. Conclusion. The clinical manifestation of PNH is characterised by the presence of hemoglobinuria, cytopenia and early thrombosis in 57 %, 65 % and 22 % of patients, respectively. Errors of the primary diagnosis reach 78 % and lead to inadequate treatment. The results of the study showed the need for multidisciplinary approach and strict adherence to diagnostic algorithms of PNH in the risk groups, according to current recommendations.

Ferroptosis and Cell Death Analysis by Flow Cytometry.

Cell death and its recently discovered regulated form ferroptosis are characterized by distinct morphological, electrophysiological, and pharmacological features. In particular ferroptosis can be induced by experimental compounds and clinical drugs (i.e., erastin, sulfasalazine, sorafenib, and artesunate) in various cell types and cancer cells. Pharmacologically, this cell death process can be inhibited by iron chelators and lipid peroxidation inhibitors. Relevance of this specific cell death form has been found in different pathological conditions such as cancer, neurotoxicity, neurodegeneration, and ischemia. Distinguishing cell viability and cell death is essential for experimental and clinical applications and a key component in flow cytometry experiments. Dead cells can compromise the integrity of the data by nonspecific binding of antibodies and dyes. Therefore it is essential that dead cells are robustly and reproducibly identified and characterized by means of cytometry application. Here we describe a procedure to detect and quantify cell death and its specific form ferroptosis based on standard flow cytometry techniques.

Evaluation of a Methodology for Automated Cell Counting for Streak Mode Imaging Flow Cytometry

Identification of Circulating Tumor Cells (CTCs) has shown promising clinical applications, but since CTCs are found in very low concentration in blood large sample volumes are needed for meaningful enumeration. This issue impedes the analysis of CTCs using standard flow cytometry due to its low throughput. To address this issue, a high throughput microfluidic cytometer was recently developed using a wide field flow- flow cell instead of the conventional narrow hydrodynamic focusing cells (used in traditional flow cytometry) enabling analysis of large volumes at lower flow rate. This wide-field flow cytometer adopts a technique known as “streak photography” where exposure times and flow velocities are set such that the particles are imaged as short “streaks”. Since streaks are imaged with large number of pixels, they are easily distinguished from the noise which appears as “speckles” increasing the detection capabilities of the device, making it more suitable for analysis using current low sensitivity, high noise webcams or mobile phone cameras. The non-stationary nature of the high noisy background found in streak cytometry introduces additional challenges for automated cell counting methods using traditional cell detection techniques such TLC, CellProfiler, CellTracker and other tools based in traditional edge detection (e.g., Canny based filters) or manual thresholding. In order to address this issue, we developed a new automated enumeration approach that does not rely on edge detection or manual thresholding of individual cells, rather is based in image quantizing, morphological operations, 2D order-statistic filtering and decisions rules that take into account knowledge of the structure and expected location of the streaks in consecutive frames. We evaluated our approach comparing it with two current methods representing the major computational modalities for cell detection: CellTrack (based in edge detection) and MTrack2 (based in manual thresholding). Samples of 1 cell/mL nominal concentration were analyzed in batch size of 30 mL at flow rate of 10 mL/min and imaged at 4 frames per second (fps), the files were saved in uncompressed AVI format files. The cells were annotated and the signal to noise ratio (SNR) was calculated. For samples with average SNR greater than 4.4 dB, our method achieved a sensitivity of 91% compared to CellTrack (60%) and MTrack2 (71%). The True Positive Rate (TPR) of cells detected was 0.93 for our method compared with 0.80 for Mtrack2 and 0.29 for CellTrack. This demonstrated the ability of the algorithm to count rare cells with high accuracy for concentrations of 1 cell/mL with SNR greater than 4.4 dB. This cell counting capability will enable to automate low cost imaging flow cytometry based on CCD detector and the expansion of cell-based clinical diagnostics in resource-poor settings.

Flow Cytometric Response Monitoring in MDS with Del(5q) Using a Simple 5-Parameter-Score - an Elnet Imds-Flow Experience

Recently, we have established a 5-parameter flow cytometry (FCM)-based score allowing for a precise prediction a deletion (5q) in therapy naïve MDS patients. The aim of this prospective study was to test, whether this FCM-based profiling is at least equal to the cytogenetics/FISH-based del(5q) detection for monitoring response to treatment.Overall, 229 FCM investigations were performed in 57 patients with MDS and del(5q) (IPSS-R very low/low: n=24, int: n119, high/very high n=22) including 39 patients with isolated del(5q) or one additional cytogenetic abnormality. The majority of analyses were performed in patients receiving lenalidomide or azacitidine (n=28 and n=21 patients), or in patients receiving chemotherapy and/or allogeneic transplantation (n=3), or growth factors (n=5). The del(5q)-FCM-score includes the following 5 parameters: myeloid progenitors (myPC > 2%) = 3 points, CD45 MFI ratio (lymphocytes: myPC ≤ 7.0) = 10, SSC ratio (granulocytes : lymphocytes < 6.0) = 2, CD71 (≤ 20%) on granulocytes = 1.5, and female gender = 1.5; a score ≥ 15.0 indicates the presence of del(5q). A standardized FCM (lyse-stain-wash) and cytogenetics/FISH were performed according to ELN guidelines at the TU of Dresden, VUMC of Amsterdam, UH of Bristol, and UH of Guadalajara.Before therapy, in 61 cytogenetic/FISH analyses in 40 MDS patients a del(5q) was detectable. In 53/561 (87%) FCM measurements, performed in parallel, a del(5q) could be predicted using the del(5q)-FCM-score. Monitoring response to treatment, a cytogenetic complete response (CCR) with the disappearance of del(5q) was accompanied by the disappearance of the typical del(5q)-FCM-profile in all of the 50 (19 patients) FCM measurements (sensitivity=100%). Otherwise, in patients without CCR the presence of del(5q) could be predicted by FCM in 96/118 measurements (specificity=81%).Aiming at a still higher specificity, in the following analyses we included only patients who presented with a typical del(5q)-FCM-score before therapy (33 patients; 54 measurements; median score=16.5) resulting in the above described sensitivity of 100% (32 measurements; median score=13.0) as well as a remarkably high specificity of 97% (59/61 measurements; median score=16.5) for response prediction.Next, we compared cytogenetics/FISH and del(5q)-FCM-score with further methods for response monitoring as cytomorphology, two well established diagnostic FCM scores: FCSS (flow cytometry scoring system; Wells et al. 2003) and the diagnostic score (Ogata et al. 2009), as well as the assessment of hematological improvement (HI). Thus, cytomorphology and FCSS, analyzing dyspoiesis of multiple cell lineages, showed a CR or disappearance of a MDS typical FCSS in less than the half of all investigations being in cytogenetic CR (sensitivity: 41% and 38%), but those two methods revealed a high specificity (98% and 97%). On the other side, the analysis of HI was high sensitive (91%) but not specific (49%). The use of the diagnostic Ogata score, considering almost only abnormalities of the myeloid progenitors, ended up with a slightly lower sensitivity (91%) and specificity (89%) as cytogenetics/FISH and the del(5q)-FCM-score.Finally, response monitoring using the del(5q) FCM-score allowed for a better separation of overall survival compared to cytogenetics/FISH (p=0.039 vs. p=0.1725).Flow cytometry-based detection of a del(5q)-specific immunophenotype is feasible and can serve as a rapid tool for response monitoring during treatment with disease-modifying drugs. At the moment, we test whether the better separation of the patients' survival curves applying the FCM-score holds true in a larger patient cohort to be analyzed within the ELNet iMDS-Flow working group. DisclosuresPlatzbecker:TEVA: Honoraria, Research Funding.

The Detection and Determination of the Genomic Profile of Low-Frequency 17p Deletion Clones in Chronic Lymphocytic Leukaemia (CLL)

Novel gene mutation discovery has resulted in the increasing utility of targeted therapies. This is of particular relevance where traditional therapies have failed, resulting in increasing drug resistance and genetic instability. The incremental rise of subclonal populations of drug resistant cells is well recognized in CLL, however exactly how these subclones contribute to the overall disease course of the patient is unknown. Critical to further understanding the relevance of early minor subclones is the determination of the genetic profiles of these subclones and the identification of potential driver mutations.While a high level of resolution of genetic mutations can be revealed using ultra-deep next generation sequencing of CLL cells, this method does not determine which actual subclone contain the mutations, and requires approximately 2000 fold coverage.One of the most important prognostic markers in CLL is a deletion of the short arm of chromosome 17 (del17p), which includes deletion of TP53 gene. Whilst del17p is uncommon at diagnosis (only 5%-10% of all CLL patients), this proportion significantly increases to rougly 40-50% of chemo-refractory CLL. Therefore, we hypothesise that there are specific mutations in the del17p cells, including but not limited to TP53, which drive these subclones through clonal evolution, creating genetically unstable cells which are then refractory to treatment. We are particularly interested in those cases of CLL that carry a low frequency del17p subclone (<20% CLL cells), as these patients represent the greatest challenge to clinicians to decide the most appropriate course of treatment.Current methods to detect these 17p-deleted cells, such as microscopy-based fluorescence in situ hybridization (FISH) and karyotyping, have restrictions on their lower limit of detection due to the low number of cells targeted. We have developed a sensitive method of detecting and flow sorting del17p cells to facilitate specific subclone analysis. FISH in suspension (FISH-IS) incorporates a flow cytometry-based imaging approach with automated analysis of thousands of cells, and is highly applicable to detecting del17p in CLL samples.Methods:The FISH-IS workflow was used with 17p locus-specific identifier (LSI) probes in CLL samples. A fluorescently labelled contig of multiple BAC clones covering the TP53 region was hybridised to CLL cells in suspension. Data was collected through the Image Stream X flow cytometer (Amnis) and IDEAS software was used to carry out the analysis.Results:In preliminary experiments CLL cells were mixed in fixed ratios with wild type 17p cells (wt 17p). We have shown that FISH-IS is able to accurately enumerate the 17p allele status (monoallelic vs biallelic) based on fluorescence intensity. Furthermore, the sensitivity of detection of del17p cells amongst 20,000 analysed cells was precisely identified to a 5% limit (Figure 1).The second phase involved developing a methodology capable of enriching del17p low-frequency subclones in CLL samples by standard flow cytometry. Flow cytometry was used to sort cells based on their mean fluorescence intensity. Analysis of common polymorphisms within TP53 were used to demonstrate enrichment by collecting predefined fractions from the flow cytometer, based on fluorescence intensity and predicted 17p deletion status.We confirmed this method on CLL samples carrying high-frequency del17p clones due to sample availability. Our data clearly shows that this method is able to enrich for the low frequency clone as evidenced by analysis of targeted heterozygous SNPs located in the deleted region of 17p (Figure 2).Further sample analysis and exome sequencing is underway to determine sub-clonal mutation architecture. Original findings in this specific and novel approach to sub-clone analysis will be presented.Conclusion:This is the first time the genomic landscape of these low-frequency subclones has been interrogated in an unbiased manner. This data will enable a specific and in-depth genetic analysis of the untreated low-frequency del17p subclone, with a view to being able to identify the mechanisms of development of a chemorefractory and aggressive CLL phenotype. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tocilizumab-Refractory Cytokine Release Syndrome (CRS) Triggered By Chimeric Antigen Receptor (CAR)-Transduced T Cells May Have Distinct Cytokine Profiles Compared to Typical CRS

Background: Cytokine release syndrome (CRS) in the setting of CAR T cell therapy manifests as a wide constellation of symptoms with multi-organ involvement. CRS can vary from a mild, self-limited course to a life-threatening systemic inflammatory response which, in severe cases, may be associated with manifestations similar to those seen in hemophagocytic lymphohistiocytosis (HLH). Tocilizumab, an anti-human IL-6 receptor antibody, has become a widely accepted pharmacologic intervention of first choice in severe CRS based on the observation of elevated levels of inflammatory cytokines, most notably interleukin (IL)-6 and interferon (IFN)-γ. Indeed, following administration of tocilizumab, most patients show rapid signs of improvement. However, in rare circumstances, CRS may be refractory to tocilizumab, and repeat administration or institution of additional immunosuppression is needed. Here we summarize cytokine profiles and CRS seen in patients treated with anti-CD22-CAR T cells and propose tocilizumab-refractory CRS as a potentially distinct pathophysiological entity from typical CRS that may merit alternative immunosuppressive interventions other than tocilizumab.Methods: Children and young adults with relapsed/refractory CD22+ ALL were treated with anti-CD22 CAR T cells. Serial samples for serum cytokine levels (IFN-γ, IL-6, IL-2, IL-10, IL-12p70, IL-1β, IL-15, IL-13, IL-4, IL-8, TNF-α, GM-CSF, MIP1-α) were obtained at pre-specified time points (0, 12, 24, 48, 72 hours, then daily on days 4 - 14 and 28 following CAR T cell infusion). Transduced CAR T cell dosage ranged from 3x10e5 cells/kg (dose level [DL] 1), 1x10e6 cells/kg (DL2), and 3x10e6 cells/kg (DL3). CRS severity was determined according to recently proposed grading system (Lee DW et al., Blood. 2014). Disease burden was assessed using standard morphology and flow cytometry analysis of bone marrow and peripheral blood samples.Results: Cytokine profiles are available on 10 patients treated: first 9 patients enrolled in our phase I trial (NCT02315612), and the tenth patient was treated off-protocol on an emergency investigational new drug protocol given lack of alternative treatment option for rapidly progressing disease. All subjects, median age 20 years (range, 6-22 years), had a diagnosis of multiply relapsed ALL. Seven of 10 subjects developed CRS. Five subjects with CRS were complete responders to CAR therapy (Table). The median time to the onset of CRS was 9 days (range, 7-12 days) post-infusion and resolved within 1 week with supportive care alone except in one patient who received pharmacologic intervention for grade 4 CRS. Rise in C-reactive protein (CRP) tended to correlate with clinical severity of CRS. Chronological changes in the level of IFN-γ, IL-6, IL-1β, IL-8, TNF-α, and MIP1-α generally mirrored the CRP trend, typically preceding CRP change by 1-2 days.In contrast to the CRS (maximum grade 2) seen in the first 9 patients, the 10th patient treated at DL3 developed grade 4 CRS with manifestations characteristic of HLH unresponsive to tocilizumab. Cytokine profile for this patient, compared to those of other CRS patients, was notable for a substantially higher serum IL-2 (35 pg/mL vs median 6.1 (range, 1.2-13.5)) and GM-CSF level (28 pg/mL vs median 1.0 (range, 0-6.1)) at 12 hours post infusion. Subsequent CRP elevation was not initially accompanied by a rise in IL-6 as in other patients, which may have explained the lack of response to tocilizumab (Figure). Evaluation for a genetic cause of HLH did not reveal any mutations (PRF1, MUNC13-4, RAB27A, STX11, STXBP2). Although this patient was a complete responder to therapy, the clinical course was complicated by pre-existing respiratory compromise and bacteremia, which may have contributed to increased CAR toxicity with variability in the cytokine profile.Conclusion: Based on our early experience, we postulate that patients with an early increase in GM-CSF and IL-2 may potentially experience more atypical and severe CRS, which without a concomitant rise in IL-6, may not respond to tocilizumab and thus early intervention with other immunosuppression may be indicated. Analysis of larger numbers of patients is required to better delineate clinical confounders and to develop rational pharmacological approach to CAR-mediated inflammatory responses. Ongoing efforts are underway to further analyze clinical samples for biomarkers. [Display omitted] [Display omitted] DisclosuresMackall:NCI: Patents & Royalties: B7H3 CAR.

Response Adapted Induction Treatment Improves Outcomes for Myeloma Patients; Results of the Phase III Myeloma XI Study

Response Adapted Induction Treatment Improves Outcomes for Myeloma Patients; Results of the Phase III Myeloma XI Study

Early Achievement of MRD-Negativity in IGHV-Mutated (IGHV-M) Patients Portends Highly Favorable Outcomes after First-Line Treatment of CLL with Fludarabine, Cyclophosphamide and Rituximab (FCR). Serial Monitoring for Minimal Residual Disease (MRD) in Blood after Achieving MRD-Negativity Predicts Subsequent Clinical Relapse

IntroductionAchieving MRD-negative remission after FCR therapy is an important predictor of longer progression-free (PFS) and overall survival (OS); however, with long-term follow-up, many MRD-negative patients subsequently relapse. The time course and predictive factors for relapse in initially MRD-negative patients have not been extensively characterized.MethodsWe prospectively analyzed 289 patients treated first-line with FCR for CLL at M.D. Anderson Cancer Center from 2008-15, to determine pre-treatment characteristics associated with post-treatment MRD negativity and the time course of relapse in MRD-negative patients. MRD analysis was performed in BM after course 3 (n=239) and at end of therapy (EOT) (n=231) using standardized 4-color flow cytometry (FLC). Ninety-five MRD-negative patients had 12-monthly serial MRD monitoring on blood.ResultsPretreatment characteristics are shown below (N=289).For the total cohort, the only pre-treatment characteristic significantly associated with PFS was IGHV-unmutated (IGHV-UM) (HR 3.0 [1.6-5.5], p<0.001). Analyzed separately, among IGHV-mutated (IGHV-M) patients, only ZAP70 positivity (38/92 [41%]) by immunohistochemistry was significantly associated with shorter PFS (HR 3.2 [1.3-8.2], p=0.01); among IGHV-UM patients only B2M ≥4.0mg/L (64/146 [44%]) was significantly associated with shorter PFS (HR 1.9 [1.2-3.0], p=0.005).Overall response rate was 96%. 46/239 patients (19%) after 3 courses and 120/231 (51%) at EOT achieved MRD negativity in BM. The following pre-treatment characteristics were significantly associated in univariableanalysis with achieving MRD-negativity at EOT in all patients: negative FISH (OR 3.1 [1.3-7.0], p=0.01), trisomy 12 (OR 2.7 [1.1-6.3], p=0.03, IGHV-M [OR 3.2 (1.8-5.9), p<0.001], β2-microglobulin <4.0mg/L [OR 1.85 (1.1-3.1), p=0.02] and ZAP70 negativity [OR 1.8 (1.03-3.2), p=0.04]. On multivariable analysis, only IGHV-M was significantly associated with MRD-negativity [OR 3.7 (1.7-8.2), p=0.001]. There were trends toward association between negative FISH [OR 5.4 (0.9-31.8), p=0.07] and trisomy 12 [OR 4.9 (0.8-29.8), p=0.08] with MRD-negativity. MRD negativity at EOT correlated with PFS, independent of IWCLL response category, and correlated with PFS in both IGHV-M and IGHV-UM patients (Figure A). In IGHV-M patients who were MRD-negative at EOT, there was a trend toward superior PFS if MRD-negativity was attained after 3 courses of FCR, even though 11/22 patients who were MRD-negative after 3 courses received no further therapy and only 3/22 received 6 courses (Figure B).Re-emergence of MRD in blood was detected in 45/95 MRD negative patients, at a median of 49 months post- treatment; this appeared less frequent in IGHV-M patients (Figure C). Re-emergence of MRD preceded clinical relapse by a median of 25 months. There were no significant differences in time-to-subsequent clinical relapse after re-emergence of MRD according to mutation status (Figure D). Among IGHV-M patients who were MRD-negative after 3 cycles, 4/22 had subsequent re-emergence of MRD.ConclusionsAmong patients with CLL receiving first-line FCR, the best pre-treatment predictor of achieving MRD-negativity and subsequent longer PFS was IGHV-M. Post-treatment MRD-negativity correlated with subsequent PFS; patients with IGHV-M who achieved MRD-negativity after 3 courses had a particularly favorable outcome, even after abbreviation of therapy. However, despite achieving MRD-negativity, many patients subsequently relapse; serial MRD monitoring in peripheral blood can herald relapse with a lead-time of approximately 2 years and potentially direct monitoring and/or early intervention strategies. [Display omitted] [Display omitted] DisclosuresThompson:Pharmacyclics: Consultancy, Honoraria. O'Brien:Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Jain:Incyte: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding; Servier: Consultancy, Honoraria; Abbvie: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Novimmune: Consultancy, Honoraria; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Genentech: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding. Kadia:Novartis: Honoraria; BMS: Research Funding. DiNardo:Novartis: Research Funding; Daiichi Sankyo: Research Funding; Agios: Research Funding; Abbvie: Research Funding; Celgene: Research Funding. Wierda:GSK/Novartis: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Genentech/Roche: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Emergent: Consultancy, Research Funding; Sanofi: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; Merck: Consultancy; Karyopharm: Research Funding; Acerta: Research Funding; Janssen: Research Funding; Juno Therapeutics: Research Funding; KITE Pharma: Research Funding.

Characterization of Myelodysplastic Syndromes (MDS) with T-Cell Large Granular Lymphocyte Proliferations (LGL)

▪Introduction:MDS include a spectrum of hematopoietic stem cell malignancies characterized by bone marrow failure and dysplastic morphology. LGL is a clonal proliferation of cytotoxic T cells, which manifest as neutropenia, anemia, and thrombocytopenia and is associated with autoimmune disorders. LGL in association with MDS has been previously reported. However, clinicopathological features, prognostic, and predictive factors in those patients diagnosed with both LGL and MDS is not well studied.Methods:We identified patients at Moffitt Cancer Center (MCC) diagnosed with MDS who were previously tested for the presence of LGL clonal populations by peripheral blood flow cytometry at time of first visit. An LGL population was defined by the standard flow cytometry immunophenotype and clonality confirmed by T-cell receptor gamma and beta gene rearrangement.. Next Generation sequencing data was available for 151 patients. Recurrent somatic gene mutations were compared between patients with an LGL clone and those without.Results:Of the 675 patients with MDS tested for LGL in the database, 206 (30.5%) had an LGL clonal population. The mean LGL absolute cell count in the peripheral blood was 335/µL. Table-1 summarizes the baseline characteristics of the two groups.There was no difference in response to azacitidine therapy. Among 50 patients with LGL clone who received azacitidine with available data on response, the rate of hematological improvement or better (HI+) was 38%. The (HI+) was 28% among 105 patients evaluable for response without LGL clone. P .14The median overall survival (OS) was for patients with no LGL clone was 65 months (mo) compared to 46 mo (p .024). The median OS for lower risk MDS patients (low/int-1 by International Prognostic Scoring System [IPSS]) was 68 mo versus 97 mo for those with or without LGL proliferation, respectively (P .005). In higher risk MDS, there was no difference in median OS between those with or without LGL expansion, respectively (20 mo versus 16 mo, p .7). The median OS for patients with very low/ low Revised-Internatinal Prognostic Scoring System (R-IPSS) was 96 mo if LGL proliferation was detected compared to 128 mo if it was not, (p value .016). For intermediate R-IPSS the median OS was 65 mo and 41 mo with or without LGL proliferation (p .16). Finally, for high/very high R-IPSS the median OS was 18 and 16 mo with or without LGL proliferation, (p .84) In cox regression analysis the presence of an LGL clone was independently prognostic for OS after adjusting for age and R-IPSS, Hazard ratio 1.3, p = .05. Somatic gene mutation data were available for 151 patients; there was no statistically significant difference in the distribution of any mutation except IDH-2 (Table-2). The most common somatic mutations observed among patients with LGL clone were SF3B1 19%, TET-2 16%, U2AF1 13%, IDH-2 13%, RUNX-1 13%, and ASXL-1 10%. In patients without an LGL clone the most common somatic mutations were TET-2 26%, ASXL-1 20%, DNMT3A 16%, TP53 13%, SF3B1 12%.Conclusion:An LGL clone is demonstrable in approximately 30% of patients with MDS in association with advancing age. The presence of LGL proliferation was associated with worse OS in lower risk MDS pts. Although the spectrum of somatic gene mutations were similar, the presence of IDH-2 mutation and absence of DNMT3A or TP53 gene mutationscharacterized LGL+ cases. [Display omitted] [Display omitted] DisclosuresRoe:Celgene: Speakers Bureau; Alexion: Speakers Bureau; Seattle Genetics: Speakers Bureau. Sweet:Pfizer: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Incyte Corporation: Research Funding; Karyopharm: Honoraria, Research Funding. Sokol:Seattle Genetics: Consultancy; Spectrum: Consultancy. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy.

Genetic tools for advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis.

BackgroundSuccessful implementation of modified cyanobacteria as hosts for industrial applications requires the development of a cyanobacterial chassis. The cyanobacterium Synechococcus sp. PCC 7002 embodies key attributes for an industrial host, including a fast growth rate and high salt, light, and temperature tolerances. This study addresses key limitations in the advancement of Synechococcus sp. PCC 7002 as an industrial chassis.ResultsTools for genome integration were developed and characterized, including several putative neutral sites for genome integration. The minimum homology arm length for genome integration in Synechococcus sp. PCC 7002 was determined to be approximately 250 bp. Three fluorescent protein reporters (hGFP, Ypet, and mOrange) were characterized for gene expression, microscopy, and flow cytometry applications in Synechococcus sp. PCC 7002. Of these three proteins, the yellow fluorescent protein (Ypet) had the best optical properties for minimal interference with the native photosynthetic pigments and for detection using standard microscopy and flow cytometry optics. Twenty-five native promoters were characterized as tools for recombinant gene expression in Synechococcus sp. PCC 7002 based on previous RNA-seq results. This characterization included comparisons of protein and mRNA levels as well as expression under both continuous and diurnal light conditions. Promoters A2520 and A2579 were found to have strong expression in Synechococcus sp. PCC 7002 while promoters A1930, A1961, A2531, and A2813 had moderate expression. Promoters A2520 and A2813 showed more than twofold increases in gene expression under light conditions compared to dark, suggesting these promoters may be useful tools for engineering diurnal regulation.ConclusionsThe genome integration, fluorescent protein, and promoter tools developed in this study will help to advance Synechococcus sp. PCC 7002 as a cyanobacterial chassis. The long minimum homology arm length for Synechococcus sp. PCC 7002 genome integration indicates native exonuclease activity or a low efficiency of homologous recombination. Low correlation between transcript and protein levels in Synechococcus sp. PCC 7002 suggests that transcriptomic data are poor selection criteria for promoter tool development. Lastly, the conventional strategy of using promoters from photosynthetic operons as strong promoter tools is debunked, as promoters from hypothetical proteins (A2520 and A2579) were found to have much higher expression levels.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0584-6) contains supplementary material, which is available to authorized users.

Intracellular Flow Cytometry Improvements in Clinical Studies.

Flow cytometry has become a basic of biological research and clinical diagnostics, and its application has been crucial to numerous advances in immunology and cell biology. However, several issues remain when considering intracellular stainings, especially in the context of a daily routine use and in multicenter clinical research protocols including large cohorts of patients. The requirements for multiple protocol steps are not only time-consuming but also frequently associated with high cell loss and nonspecific binding or reduced fluorescence. These drawbacks make standardized intracellular flow cytometry use in multicenter studies struggling. As a consequence, intracellular flow cytometry has mostly remained a tool for experimental and clinical research. In the current chapter, we will complete flow cytometry protocols described in the previous edition by presenting novel intracellular protocols usable in clinic. These present with many advantages including shorter time-to-results, one-step whole blood procedures, lyse-no-wash-no-centrifuge protocols, improved staining quality, and lyophilized coated reagents in ready-to-use tubes. This opens novel perspectives for standardization and feasibility in clinical studies, for drug efficacy monitoring and for patients' stratification within a context of personalized medicine. Here, we present illustrative examples taken from septic patients' immunomonitoring. We consider the evaluation of myeloperoxidase and lactoferrin expressions in neutrophils, FOXP3 lymphocyte expression, and STAT5 phosphorylation in lymphocyte subsets.

189 - Nanozyme and Antioxidant Mimetic Cerium Oxide Nanoparticles Confer Excellent Protection against UV Induced Oxidative Damage in Skin

Generation of reactive oxygen species (ROS) is well accepted phenomenon in the skin exposed to Ultraviolet radiations. Ultraviolet radiations initiate an acute cascade of photochemical reactions culminating in the generation of reactive oxygen species such as superoxides. The downstream effects of these acute changes are directly involved in various cutaneous changes such as sunburn erythema, phytocarcionoma and photoaging. The risk and exposure of UV radiations varies dramatically over time, space and topology across the globe suggesting the clinical relevance of identifying efficient UV protective agents. Based on the prior understanding from our lab and other studies cerium oxide has emerged as one of the promising antioxidants due to its superoxide dismutase and catalase mimetic activity. In this study we evaluated the UV protective efficiency of custom synthesized cerium oxide nanoparticles tagged with polyethyleneglycol (PEG-CNPs). The internalization of nanoparticles was successfully evaluated using DiD florescent tags. Preliminary studies were conducted on primary keratinocyte culture (HAEK cell line) using standard flow cytometry and microscopy. Significant reduction in ROS was observed in UV irradiated cells pretreated with 25 μM PEG-CNPs for 6 hours with concomitant reduction in protein modification such as carbonylation and nitrosylation and eventually the cell death. Moreover, on gaining confidence in cell culture model the hypothesis was tested in rat skin exposed to known doses of UV the PEG-CNPs showed significant reduction in clinical cutaneous parameters such as erythema. Histopatholical examination and basic biochemical indicators of oxidative stress including 8-OHdG, protein carbonyl, nitrosylation was significantly reduced on topical application prior to UV exposure. Apart from evaluating the minimum effective dose we also evaluated basic toxicological assessment and developed a topical formulation with hydrophobic solvents. In conclusion, the PEG-CNPs were highly efficient in protein skin from UV damage therefore providing a promising cosmaceutical application of this novel class of antioxidants.

The DAU cluster: a comparative analysis of 18 RHD alleles, some forming partial D antigens.

The Rh system is the most complex and polymorphic blood group system in humans with more than 460 alleles known for the RHD gene. The DAU cluster of RHD alleles is characterized by the single-nucleotide change producing the p.Thr379Met amino acid substitution. It is called the DAU-0 allele and has been postulated to be the primordial allele, from which all other alleles of the DAU cluster have eventually evolved. For two novel DAU alleles, the nucleotide sequences of all 10 exons as well as adjacent intronic regions, including the 5' and 3' untranslated regions (UTR), were determined for the RHD and RHCE genes. A phylogenetic tree for all DAU alleles was established using the neighbor-joining method with Pan troglodytes as root. Standard hemagglutination and flow cytometry tests were performed. We characterized two DAU alleles, DAU-11 and DAU-5.1, closely related to DAU-3 and DAU-5, respectively. A phylogenetic analysis of the 18 known DAU alleles indicated point mutations and interallelic recombination contributing to diversification of the DAU cluster. The DAU alleles encode a group of RhD protein variants, some forming partial D antigens known to permit anti-D in carriers; all are expected to cause anti-D alloimmunization in recipients of red blood cell transfusions. The DAU alleles evolved through genomic point mutations and recombination. These results suggest that the cluster of DAU alleles represent a clade, which is concordant with our previous postulate that they derived from the primordial DAU-0 allele.