Multidimensional Flow Research Articles

Efficient two-dimension particle element method of interior ballistic two-phase flow

Reducing the calculation cost is of great importance as the demand for interior ballistic calculation is increasing. The key factor of interior ballistic simulation is the quest to develop a more efficient method. In this paper, an efficient two-dimension particle element method (PEM) is proposed to simulate the detailed multidimensional flow of the gas and propellant particle in the chamber. Firstly, several real particles are packed as the particle element to reduce the calculation of the solid phase. In particular, the particle element matrix is established to describe the distribution of particle parameters. Secondly, the particle element boundary is adjusted according to the particle’s movement to reduce the computational cost of grid generation. Besides, the dynamic self-adapting mesh map method is adopted to realise the coupling computation between gas phase and the particle element. The application of a standard virtual gun as a standard benchmark for interior ballistic codes is used to validate the accuracy and reliability with 1.68% error. The particle element model accurately describes the distribution of flow field in the chamber. Compared with the two-fluid method, the PEM significantly improves the computational efficiency by 21.7%. The PEM may be promising for the rapid simulation of two-phase flow in interior ballistic.

Gut virome-colonising Orthohepadnavirus genus is associated with ulcerative colitis pathogenesis and induces intestinal inflammation in vivo

ObjectivesUlcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut virome dysbiosis is fundamental in UC progression, although its role in the early phases of the disease is...

Boosted Mechanical Properties of Polyamide 11 Tube Originated from the Hierarchical Architecture Manipulation by Helical Flow Field

Polyamide 11 (PA11) is a biobased polymer originated from the renewable plant-based resources and thus holds promising potential in reducing the consumption of limited fossil resources and protecting the environment. Compared to the commonly investigated polyolefin, the flow manipulation strategy for improving the mechanical properties of PA11 still remained a huge challenge because the strong intermolecular interactions elevated the difficulty of stretching the polar polymer chains. Herein, a helical flow was introduced in the PA11 tube extrusion by superposing a hoop flow from mandrel rotation onto an axial flow. Then, the helical flow-induced hierarchical architecture was investigated to reveal the effect of intermolecular interactions on the orientation and crystalline structure. Limited by strong hydrogen bonds between the amide groups, the PA11 chains were not stretched sufficiently to form anisotropic shish-kebab crystals but assembled into a helically aligned configuration in amorphous regions, similar to that in natural materials, endowing the as-prepared tube with enhanced hoop kink resistance and axial compression properties which were favorable to the practical application of the thin-wall tube. This study not only can provide a feasible way to tailor the mechanical properties of the PA11 tube by constructing a special multidimensional flow but also is beneficial to the fundamental understanding on the flow-induced crystallization mechanism of polar polymers with strong intermolecular interactions.

OP32 The gut virome-colonizing Orthohepadnavirus genus is associated with ulcerative colitis pathogenesis and induces intestinal inflammation in vivo

Abstract Background Ulcerative colitis (UC) is a chronic inflammatory disorder with an unknown etiology1. Over recent years, a growing body of evidence has been pinpointing gut virome dysbiosis as a fundamental component in its progression2, although its role during the early phases of chronic inflammation is far from being fully defined. Therefore, we sought to investigate the role of a virome-associated protein encoded by the Orthohepadnavirus genus, the Hepatitis B virus X protein (HBx)3, during UC aetiopathogenesis. Methods HBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors. C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and control animals. Transepithelial electrical resistance measurement, proliferation assay, ChIP-Seq, and RNA-Seq were performed on in vitro models of the gut barrier. Results HBx was detected in about 45% of patients with UC (Figure 1) and found to induce colonic inflammation in mice (Figure 2A-2D), while its silencing reverted the colitis phenotype in vivo (Figure 2E-2H). HBx acts as a transcriptional regulator in epithelial cells (Figure 3), provoking barrier leakage and altering inflammatory response (Figures 3 and 4). Conclusion This study paves the way for the understanding of the aetiopathogenesis of UC and provides a brand-new standpoint that looks at the virome as a target for tailored treatments, possibly leading to an entirely new approach to therapeutic intervention.

An integrated coupling model for solving multiscale fluid-fluid coupling problems in SAM code

In this study, an integrated coupling method has been developed for solving multiscale fluid-fluid coupling problems in plant-scale safety analysis models in SAM (System Analysis Module) code. In this method, a higher-fidelity multi-dimensional (3D) flow module is used for reactor components of complex flow features (e.g., reactor core) and a lumped parameter one-dimensional (1D) flow module for plant-scale flow loops (e.g., primary loop pipe network), respectively. In this method, the 3D fluid equation/domain and 1D fluid equation/domain are tightly coupled at the residual level and solved simultaneously using the Newton’s method to overcome the convergence issues typically seen in existing approaches like separate domain approach, where the 3D fluid equation and 1D fluid equation are solved separately. Extensive and successful code verifications and demonstrations have been performed for this newly developed method. This new modeling approach significantly simplify the work flow in developing high-fidelity plant-scale safety analysis model, e.g. for pool-type reactors and pebble-bed reactors.

Analysis of Immune-Cell Distribution of Bone Marrow in Patients with Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) immunity plays an important role in the proliferation and apoptosis of aberrant cells. Immune dysregulation has been studied in various prognostic subgroups. This study analyzed 60 patients with MDS via multidimensional flow cytometry to evaluate the expression of aberrant markers, such as CD7 and cytoplasmic CD3 on lymphocytes. The Revised International Prognostic Scoring System (IPSS-R) scores were used to classify the patients into risk groups. The results showed a significant downregulation of CyCD3- T cells in low-intermediate versus high-risk patients (p = 0.013). This study is the first to show that a significant decrease in cyCD3- T cells in patients with a lower IPSS-R score may indicate microenvironmental changes conducive to transformation in MDS.

Asymptotic stability of planar rarefaction wave to a multi-dimensional two-phase flow

<p style='text-indent:20px;'>We are concerned with the time-asymptotic stability of planar rarefaction wave to a non-conservative two-phase flow system described by two-dimentional compressible Euler and Navier-Stokes equations through drag force. In this paper, we show the planar rarefaction wave to a non-conservative compressible two-phase model is asymptotically stable under small initial perturbation in <inline-formula><tex-math id="M1">\begin{document}$ H^3 $\end{document}</tex-math></inline-formula>. The main difficulties overcome in this paper come from the non-viscosity of one fluid and the interaction between two fluids caused by drag force. The stability result is proved by the energy method.</p>

Multidimensional Governing Equations of Matrix Flow Component of Subsurface Stormflow as Function of Bedrock Surface Geometry

AbstractSubsurface flow is a critical component in the hydrological cycle, since it controls the quantity and timing of surface runoff and groundwater flow. Field studies have shown the fundamental influence of the bedrock surface geometry on subsurface stormflow (SSSF) and that the SSSF process consists of at least two major components: the matrix flow component and the macropore flow component that are in dynamical interaction toward forming the SSSF. Furthermore, field studies have shown that the bedrock surface that underlies the SSSF has essentially a two‐dimensional (2D) geometry, where not only the longitudinal profile along the hillslope but also the profile in the transverse direction influence the subsurface stormflow over the bedrock. The macropore flow itself being a multidimensional flow process, requires a multidimensional matrix flow component in order to quantify realistic dynamical interactions between these components within the subsurface stormflow module of a watershed hydrology model. Within this framework, the critical research question is how to extend the existing 2D Boussinesq model for the matrix flow over an inclined flat 2D surface in the longitudinal hillslope direction by developing the governing equations of matrix flow at hillslope scale over various 2D bedrock surface geometries that are reported in the field studies of SSSF. Then, numerical experiments are performed by applying the developed governing equations to various bedrock surface geometries. The results of the numerical simulations show (a) the proposed governing equations can simulate various two‐dimensional bedrock surface geometries, and are capable of incorporating the upward or downward concavity of bedrock surface in longitudinal and transverse directions into SSSF by modulating the timing and magnitude of the flows and (b) the proposed governing equations for matrix flow provide an appropriate setting for the dynamical interaction of the matrix flows with the macropore flows, creating a promising modeling framework for SSSF.

SAM-ML: Integrating data-driven closure with nuclear system code SAM for improved modeling capability

Advanced reactors often involve complicated thermal-fluid (T-F) phenomena. Modeling such phenomena withthe traditional one-dimensional (1-D) system code is a challenging task.The System Analysis Module (SAM), a modern nuclear system code, has developed a coarse mesh multi-dimensional (multi-D) flow modelto capture the spatial effect of T-F phenomena in advanced reactors. As a coarse mesh solver, constitutive relations are required for SAM’s multi-D model for unresolved fine-scale physics, such as turbulence.This work presents a novel approach that integrates neural networks as data-driven closurefor SAM’s multi-D flow model. The data-driven closure is trained with fine-resolution data to ensure its accuracy while maintaining a coarse mesh setup to ensure its efficiency and consistency with SAM. We demonstrate the applicability of this SAM-ML capability in an open volume thermal stratification problem, where a neural network model serves as the eddy viscosity closure. A customized interface between the neural network and SAM is developed to ensure flexible and efficient data exchange. The SAM-ML results demonstrate superior performance compared to SAM’s built-in zero-equation eddy viscosity closure.The case study shows that although the generalization capability of the data-driven closure still needs to be improved for different transient case or different geometric setup, SAM-ML demonstrates good potential for challenging simulation problems with improved accuracy and computational efficiency.

Immune Biomarkers of Survival and Severe Infection in Newly Diagnosed Multiple Myeloma (NDMM) Patients (pts) Treated with the Backbone Regimen Lenalidomide and Dexamethasone (Rd)

Introduction: Rd is a backbone of treatment in MM. The strong immunomodulatory effects of this regimen would suggest that immune profiling might complement conventional prognostic factors. However, immune signatures predictive of survival were not well defined in the past, and nowadays it is challenging to detach the effect of Rd from other backbone drugs in triplets or quadruplets, as well from high dose therapy in transplant eligible patients. Aim: Identify immune biomarkers in elderly NDMM pts treated with Rd. Methods: The GEM-CLARIDEX trial randomized transplant ineligible NDMM pts to Rd +/- clarithromycin until disease progression. Because clarithromycin had no effect in survival, the trial design yielded a unique opportunity to identify immune biomarkers in elderly MM pts treated with Rd. Immune profiling was performed in bone marrow (BM) at baseline in 186 pts, using multidimensional and computational flow cytometry together with lasso penalized Cox regression and 10-fold cross validation, for a holistic and unbiased definition of prognostic immune signatures. 38 cell populations were identified, including seven myeloid/erythroid and 29 lymphoid subsets plus normal and clonal plasma cells (PC). Single cell RNA sequencing (scRNAseq, 10x Genomics) was performed on BM immune cells sorted from healthy adults, MGUS, smoldering and active MM pts (n = 17). Results: An immune signature associated with inferior survival was defined based on increased frequency of cytotoxic CD314+ NK and CD4+ central memory (CM) CD127+ PD1+ T cells, and decreased percentages of CD4+ CM CD127+ PD1- and CD8+ CD127low PD1+ terminally differentiated T cells, as well as of B cell precursors. Median progression-free survival (PFS) of NDMM pts with high risk immune score (106/186 [57%]) was 18 months vs not reached in those with low risk (80/186 [43%]) (p=.0008, Fig 1A). The respective median overall survival (OS) rates at three years were 50% and 80% (p = .0021, Fig 1B). In multivariate analyses including age, ISS, LDH and cytogenetic risk, immune profiling showed independent prognostic value for PFS (HR: 2.6, p = .001) and OS (HR: 2.9, p = .003). Increased frequency of cytotoxic CD314+ NK cells was the immune biomarker with the highest negative impact in the survival predictive model. In an attempt to better understand this finding, we analyzed scRNAseq data of 61,637 NK cells from normal and tumor BM. Up to 47 NK-cell clusters were identified, and the one showing the highest expression of CD314 also displayed upregulation of granzymes K, H and B, perforin, CD16, CD38, CD69 and IFNγ, and therefore appeared to be associated with cytolytic activity and inflammation. Interestingly, there was a progressive expansion of this cluster within the NK cell compartment of normal vs pre-malignant vs neoplastic BM samples. Similarly, in genetically engineered BIcγ1 mice that develop active MM preceded by precursor states, there was a progressive expansion of CD314+ NK cells in normal vs benign vs malignant BM (p = .001). Being infection an important cause of morbidity and mortality in elderly MM pts as those included in this study, we next investigated if immune profiling at diagnosis could help predicting severe infection (≥ grade 3 CTCAE v4.0, n=43/186). The combination of five clinical features - age ≥ 75y, low hemoglobin and albumin, high β2m and LDH - plus seven immune biomarkers - increased percentages of eosinophils, together with decreased frequencies of CD4+ CM CD127+ PD1-, CD8+ naïve and CD127low PD1+ and PD1- terminally differentiated T cells, B cell precursors and normal PC - resulted in the most effective model to predict severe infection. B cell precursors and eosinophils, which are easily identified using flow cytometry at diagnosis, showed the strongest association. The area under the curve (AUC) of the model was 0.90 with a 4-fold cross validation showing a mean AUC = 0.86 ± 0.06. Conclusions: This study shows the clinical value of immune profiling at diagnosis and defined immune cell types, including a previously unidentified subset of CD314+ cytotoxic NK cells, which were predictive of survival in transplant ineligible NDMM pts treated with the backbone regimen Rd. Furthermore, this study uncovered immune biomarkers complementary to clinical features for predicting severe infection in elderly MM pts, which could be used to intensify prophylactic measures in specific subgroups that are more susceptible to severe infection. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transcriptional and Genomic Characterization of Measurable Residual Disease (MRD) Cells in Acute Myeloid Leukemia (AML)

Background: Failure to achieve complete response (CR) and undetectable MRD is associated with inferior survival in AML. However, there is virtually no data on the molecular traits of treatment resistant cells to understand the mechanisms of MRD resistance. This would be particularly relevant in elderly AML, for which outcomes remain dismal despite increasing CR rates with novel combinations. Aim: Uncover mechanisms of MRD resistance in elderly AML by comparing the transcriptional and genomic profile of patient-matched leukemic cells at diagnosis and after treatment with semi-intensive chemotherapy or hypomethylating agents. Methods: 285 AML patients with a median age of 75 were included in the phase 3 PETHEMA-FLUGAZA trial and were randomized to receive induction and consolidation with fludarabine and cytarabine (FLUGA) vs 5-azacitidine (AZA). After consolidation, patients continued with the same treatment if MRD ≥ 0.01% or stopped if MRD < 0.01%. MRD was assessed using multidimensional flow cytometry, and highly-purified MRD cells were isolated according to patient-specific aberrant phenotypes using FACS. Leukemic blasts and T cells (germline control) were also sorted at diagnosis. A 3' end RNAseq method optimized for generating libraries from low-input starting material (MARSeq) was used. RNAseq was performed at diagnosis in 220 patients, and in paired diagnostic-MRD leukemic cells from 22 patients. Whole-exome sequencing (WES) was performed using molecular barcoding in paired diagnostic-MRD leukemic cells from 14 patients. Results: After induction, 35 patients were in partial remission (PR), 51 attained CR/CRi but persistent MRD (CR/MRD+), and 13 achieved CR/CRi and undetectable MRD (CR/MRD-). There were no differences in median overall survival (OS) between patients in PR vs CR/MRD+ (median of 21 vs 20 months, p = .603), and both were numerically inferior to that observed in CR/MRD- patients (median of 27 months, p = .097). There was only one gene (PIEZO2) differentially expressed between leukemic cells at diagnosis vs after treatment in patients achieving PR (n = 10). By contrast, there were 117 differentially expressed genes between leukemic cells at diagnosis vs after treatment in CR/MRD+ patients (n = 12). To confirm that gene deregulation was intrinsically associated with treatment resistance, the prognostic value of gene (over or under) expression in accordance with the differential observed between diagnostic and MRD leukemic cells, was subsequently analyzed in the 220 patients with RNAseq data at diagnosis. Over expression of 3 genes (IGFBP2, LINC00475 and PLEKHG3) and under expression of 12 genes (eg, CASC15, KDM7A and MEX3B) among the 117 identified above, was significantly associated with inferior OS. Interestingly, none of the genes was prognostic in the BeatAML cohort, suggesting that the transcriptome of MRD resistance may be specific of treatment. A total of 6,054 mutations were detected after WES of matched leukemic cells at diagnosis and after treatment, in 14 patients achieving CR/MRD+. Amongst these, 4,708 (78%) were detected at both time points, 354 (6%) were present at diagnosis while absent in MRD blasts, and 992 (16%) emerged during MRD resistance (p ≤ 0.008). There were no differences between treatment arms in the number of shared and private mutations in diagnostic vs MRD cells. Amongst the 1,346 mutations that either became undetectable or present at MRD, recurrence (ie, ≥ 3 patients) was observed in 47 genes. Acquired mutations in AVP and UBXN11 were observed in MRD cells from 6 of the 14 patients. Conclusions: This study showed that elderly AML patients treated with semi-intensive chemotherapy or hypomethylating agents, have dire survival regardless of achieving PR vs CR/MRD+. However, RNAseq analyses of resistant cells uncovered that whereas PR is characterized by primary resistance and transcriptionally stability from diagnosis, CR/MRD+ is associated with the emergence of molecular traits of acquired resistance. These findings could help explaining why additional treatment with the same schema is unable to overcome the poor prognosis of persistent MRD in elderly AML patients achieving CR/CRi. First-ever simultaneous RNAseq and WES of patient-matched diagnostic and MRD blasts, suggests that a few deregulated genes and recurrent mutations with limited or previously unknown role in AML pathobiology, are associated with MRD resistance and inferior OS.

Trial in Progress: Tagraxofusp-Based Therapy to Eradicate Measurable Residual Disease of Acute Myelogenous Leukemia Prior to Allogeneic Hematopoietic Cell Transplantation

Background Numerous studies convincingly demonstrate that measurable residual disease (MRD) in AML at time of allogeneic cell transplantation (alloHCT) is a powerful, independent predictor of subsequent relapse. Attempts to eradicate AML MRD pre-transplant with consolidation chemotherapy have been unsuccessful, with reported MRD conversion rates as low as 20% (Appelbaum et al., Blood 2019). Tagraxofusp is an IL-3 truncated diphtheria toxin recombinant protein that binds CD123 with high affinity. Preliminary data using tagraxofusp as consolidation therapy in AML patients has demonstrated good tolerability (Lane et al., Blood 2017). Pathways of resistance to tagraxofusp have been identified preclinically as loss of the intracellular target for diphtheria toxin, diphthamide, through methylation of an enzyme involved in diphthamide synthesis. Treatment with a hypomethylating agent has the potential to restore expression of diphthamide and increase tagraxofusp sensitivity in AML cells (Togami et al., J Clin Invest 2019). Design This is a single-arm, open-label, multi-center phase Ib/II clinical trial to evaluate the safety and effectiveness of tagraxofusp +/- azacitidine in AML patients who are in first or second remission with detectable MRD and are planned to undergo alloHCT. MRD will be measured by multi-dimensional flow cytometry with sensitivity of 0.02% (Hematologics). Patients will be enrolled in a modified 3+3 dose escalation design with two cohorts: cohort A) tagraxofusp 12 mcg/kg IV qday on days 1-5; and cohort B) azacitidine 75 mg/m2 subq qday on days 1-5 plus tagraxofusp 12 mcg/kg qday on days 8-12. Patients achieving MRD-negativity after cycle 1 will proceed to alloHCT. If a patient remains MRD-positive, an additional cycle of investigational therapy will be administered. All patients will proceed to alloHCT after no more than 2 cycles of investigational therapy. Both cohorts contain a dose escalation and a dose expansion phase. Cohort B will open if cohort A is not efficacious. Up to 44 participants will be enrolled in this trial at University of California Los Angeles and University of California San Francisco (two constituent sites of the University of California Hematologic Malignancies Consortium). Objectives The primary objective is to estimate the rate of conversion from MRD-positive pre-investigational therapy to MRD-negative after 1 or 2 cycles of investigational therapy. Key secondary objectives include to determine the safety of tagraxofusp +/- azacitidine in this patient population and to evaluate if adverse effects of this investigational therapy lead to delays in alloHCT. Exploratory objectives will involve use of single cell RNA sequencing to investigate if response to investigational therapy is predicted by leukemia-specific biomarkers and to evaluate if putative cell surface markers (inferred from RNA detection) are associated with MRD persistence. Main Outcomes The primary endpoint is MRD status after 1 or 2 cycles of investigational therapy. Key secondary endpoints include dose-limiting toxicities (any grade 4 or 5 non-hematologic toxicity or any grade 3 or greater hematologic toxicity at day 42) and number of days between investigational therapy and alloHCT. Conclusions There is an unmet need to improve outcomes in AML with detectable MRD pre-transplant. The strategy in this study is to employ tagraxofusp as an alternative mechanism of action to eradicate chemo-resistant AML cells responsible for subsequent relapse. In order to mitigate any potential resistance the residual leukemia population may have against tagraxofusp, azacitidine will be added to optimize the likelihood for MRD negativity and the possibility for improved outcomes after alloHCT. This study was sponsored by the ASH CRTI. References Appelbaum J, Loeb A, Gardner K, et al. Additional Cytotoxic Chemotherapy Is Unlikely to Eliminate Measurable Residual Acute Myeloid Leukemia (AML). Blood. 2019;134(Supplement_1):260-260. Lane AA, Sweet KL, Wang ES, et al. Results from Ongoing Phase 1/2 Trial of SL-401 As Consolidation Therapy in Patients with Acute Myeloid Leukemia (AML) in Remission with Minimal Residual Disease (MRD). Blood. 2017;130(Supplement 1):2583-2583. Togami K, Pastika T, Stephansky J, et al. DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance. J Clin Invest. 2019;129(11):5005-5019. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunoparesis Recovery in Transplant-Ineligible Newly Diagnosis Multiple Myeloma Patients: An Independent Prognosis Factor That Could Complement Prognostic Value of Minimal Residual Disease

Introduction: Immunoparesis (IP) or the suppression of polyclonal uninvolved immunoglobulins (Ig) is one of the immune system alterations present in patients with multiple myeloma (MM) at diagnosis. Several studies have shown that the lack of IP recovery after treatment is associated with worse prognosis. However, these are observational retrospective studies and patients were not homogeneously treated. On the other hand, the depth of response, specially reaching minimal residual disease (MRD) negative, is one of the most important prognostic factors in patients with MM. We decided to study, in a prospective way, the prognostic value of IP recovery in a group of elderly patients not suitable for transplantation and treated in a similar way and compare it with the depth of response to treatment, especially with MRD status. Methods: We analyze patients included and treated in the PETHEMA/GEM2010MAS65 study (registered at www.clinicaltrial.gov as #NCT01237249) who reached CR or VGPR. All patients included in this trial were older than 65 years old and transplant-ineligible and were planned to receive 9 cycles of bortezomib plus melphalan and prednisone (VMP) and 9 cycles of lenalidomide plus dexamethasone (LD). Polyclonal Ig were analyzed by nephelometry or turbidimetry at diagnosis, at the end of the treatment (after 18 cycles) and during subsequent follow up. Minimal residual disease (MRD) was analyzed at the end of the treatment by multidimensional flow cytometry (MFC) with a sensitivity level of 10-5. Results: Of the 233 patients included in the study, 161 reached CR or VGPR, but the protocol design did not included determination of uninvolved Igs at diagnosis and for this reason they were only available in 120 patients. Only 9 cases (7,5%) had normal Ig at diagnosis while 111 patients (92,5%) had IP. There were no differences in progression free survival (PFS) and overall survival (OS) between patients with or without IP. We had Ig determination of 117 cases at the end of the treatment and during follow up. Those patients who had IP at diagnosis and recover it after treatment had better PFS (p<0,001; HR 0,33) and better OS (p=0,001; HR 0,35) than those who do not recover it (Figure 1A). We found no differences between those patients who had recovery of IP at cycle 18 than those who recover IP during follow up. When we focused the analysis in CR patients and VGPR patients separately, in both groups recovery of IP was associated with a better PFS and OS. In fact, when we compared both groups, we found that those patients with VGPR and complete recovery of IP had better PFS (p=0,001; HR 0,12) and OS (p=0,015; HR 0,22) than those with CR and no recovery of IP. These data suggest that IP recovery could be a prognostic factor equal or more important than conventional response. MRD information by MFC was available in 112 cases. As previously reported, in this trial those patients with MRD negative (-ve) had longer PFS and OS than MRD positive (+ve). When we analyzed IP recovery in MRD -ve patients we found that patients with MRD -ve and IP recovery (33 cases) had better PFS (p=0,004; HR 0,30) and better OS (p=0,010; HR 0,21) than patients with MRD -ve without IP recovery (11 cases), although it should be recognized that the number of patients within this latter subgroup was rather small (Figure 1B). We performed a multivariate analysis including other prognostic factors, and we found that both MRD and IP recovery have independent prognostic value for PFS, but only MRD and no IP recovery, is an independent prognosis factor for OS. Conclusions: Recovery of IP after non-continuous treatment in elderly patients with CR or VGPR confers better prognosis (PFS and OS). Prognostic value of IP recovery could be equal or more important than conventional response to treatment. Prognostic value of MRD could be complemented when combined with IP recovery, but these data must be confirmed in other series. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Molecular Mechanisms Governing CAR-T Cell Response in MM Patients Using Single Cell Transcriptomics

Background: CAR-T therapies have revolutionized cancer immunotherapy, especially for hematological malignancies, representing a promising advanced therapy for relapsed/refractory (R/R) multiple myeloma (MM) patients. Nevertheless, despite the high remission rates observed with BCMA CAR-T cells, not every patient achieves a complete response, and a significant number of patients still relapse. Application of single-cell technologies to study CAR-T cells has helped to shed light on important aspects of their expansion and evolution after administration. Methodology: CAR-T cells were FACS-isolated from infusion products (IP) as well as from bone marrow (BM) and peripheral blood (PB) from three MM patients (HCB08, IBS08 and HVA02), enrolled in the academic clinical trial CARTBCMA-HCB-01 (NCT04309981) assessing ARI0002h, an autologous CAR T-cell product targeting BCMA1, at different times after cell infusion (1 and 3 months). Single-cell analysis was performed using the Chromium Single-Cell Immune Profiling solution from 10X Genomics that allows simultaneous analysis of gene expression and paired T-cell receptors. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel computational method developed by our group, that infers regulatory dissimilarities2. Phenotypic analyses were performed by multidimensional flow cytometry. Results: scRNAseq analysis revealed that although CAR-T cells from IP presented similar phenotype, with CD4+ and CD8+ memory cells, and a high number of proliferative cells, CAR-T cells remaining after cell infusion were mainly resting CD8+ cells, with an effector/effector-memory phenotype. In addition, cell type distribution varied among patients, with a population of terminally differentiated effector cells in HCB08 patient, that presented a partial response and an early relapse, that increased from month 1 to 3 and presented a more exhausted phenotype. In contrast, IBS08 and HVA02 (complete responders) presented CD8+ cells in transition to central memory phenotype and a relatively constant pool of effector memory CD8+ CAR-T cells, respectively. Interestingly, CAR-T cells infiltrating BM presented increased expression of cytotoxic (GZMA, PRF1) and exhaustion (LAG3, HAVCR2, TIGIT) markers, compared to their PB counterparts. Functional characterization showed enrichment of T cell activation and cell-cell adhesion pathways in CAR-T cells from BM. GRN analysis at single-cell level with SimiC showed several regulons, such as IRF3 and PRDM1, presenting increased activity in BM. IRF3, a key transcriptional regulator of type I interferon response, was associated to DUSP2, IL32 and CCL5, genes associated to lymphocyte activation and inflammation. PRDM1, with crucial role in maintaining effector T cells homeostasis, was correlated with CST7, PRF1, MAP4K1 and CYTOR, genes associated with effector function and proliferation. All these data suggest that these regulons would be modulating the increased activation and inflammatory response of CAR-T cells observed in the BM. Further analysis using single-cell TCRseq allowed the identification of a hyperexpanded CAR-T cell clone in the BM of HCB08 patient (with an early relapse). Deeper characterization showed that this clone had higher expression of cytotoxicity (PRF1, GZMB) and activation markers (HLA-DRA, TNFRSF9). Strikingly, we also found increased expression of IL10, an immunosuppressor cytokine. Further analysis of this clone with SimiC showed increased activity of regulons from TFs related to exhausted CD8+ T cells (CREM, BHLHE40), meanwhile regulons from TFs associated to naïve and memory T cells (FOXP1, KLF2) had reduced activity. Interestingly, PRDM1, that is implicated in IL10 production in Treg, also presented increased activity in this clone. These data would suggest mechanistic insights regulating the phenotype of this hyperexpanded clone, that could represent a mechanism of therapeutic failure and/or early relapse. Conclusions: Overall, our data show that multiomic analysis are useful tools to characterize CAR-T cells and their dynamics after infusion, that presented different characteristics regarding their location (PB vs BM) and a heterogeneous behavior within the patients. Moreover, GRN analysis at single cell level offers an important tool to understand mechanisms modulating CAR-T cell response and to identify possible mechanisms of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 13216: Evaluation of Left Ventricular Flow Propagation Velocity From Multi-Dimensional Cardiac Imaging

Introduction: The propagation velocity (Vp) during the left ventricular (LV) early diastole has been proposed as a marker of the diastolic function. However, the measurement of Vp is limited to one-dimensional data from Color M-mode (CMM) echocardiography and does not consider the spatiotemporal variation of Vp. Methods: We introduce a new method to evaluate the LV diastolic flow propagation from multi-dimensional cardiac flow imaging. The proposed method estimates the local and instantaneous flow propagation velocity (V prop ) by fitting the first order wave equation to the velocity gradients with weighted least-squares. The proposed method was validated using the synthetic vortex ring flow data. The method was applied to the LV flow data acquired with two-dimensional phase-contrast magnetic resonance imaging (pc-MRI) and 4D flow MRI. Results: The V prop estimated from multi-dimensional data has about 50% less error than the V prop from one-dimensional data. Fig. 1(a) shows the waveforms of the mitral inflow velocity, the IVPD, and the propagation intensity (I prop ) during the LV diastole of a normal filling patient. During early diastole, the timing of peak flow propagation coincides with the peak intraventricular pressure difference. Fig. 1(b) presents the fields of the blood flow velocity, the V prop , and the relative pressure from the timeframes indicated using the dotted lines in the waveform plots. The flow propagation towards the apex is mainly located at the front of the inflow jet and downstream of the vortex ring formed near mitral valve tips. Conclusions: This study introduces a novel propagation velocity measurement method for multi-dimensional cardiac flow imaging and demonstrated the V prop ’s spatial distribution temporal evolution.

Dynamics of T cell repertoire renewal following autologous hematopoietic stem cell transplantation in multiple sclerosis.

Autologous hematopoietic stem cell transplantation (aHSCT) is a highly effective treatment of multiple sclerosis (MS). It depletes autoreactive cells and subsequently renews adaptive immune cells. The possible proinflammatory potential of surviving T cells early after aHSCT has not been studied. Here, we examined the dynamics of new and surviving T cells in 27 patients after aHSCT by multidimensional flow cytometry, T cell receptor (TCR) sequencing, specificity testing, telomere length profiling, and HLA genotyping. Early after aHSCT, naïve T cells are barely detectable, whereas effector memory (EM) T cells quickly reconstitute to pre-aHSCT values. EM CD4+ T cells early after aHSCT have shorter telomeres, have higher expression of senescence and exhaustion markers, and proliferate less than those before aHSCT. We find a median TCR repertoire overlap of 26% between the early post-aHSCT EM CD4+ T cells and pre-aHSCT, indicating persistence of EM CD4+ T cells early after transplantation. The EM CD4+ TCR repertoire overlap declines to 15% at 12 months after aHSCT, whereas the naïve TCR repertoire entirely renews. HLA-DR-associated EM CD4+ T cell reactivity toward MS-related antigens decreased after aHSCT, whereas reactivity toward EBV increased. Our data show substantial survival of pre-aHSCT EM CD4+ T cells early after transplantation but complete renewal of the T cell repertoire by nascent T cells later.

Sharp Interface Methods for Simulation and Analysis of Free Surface Flows with Singularities: Breakup and Coalescence

A common feature of many free surface flows—drop/bubble breakup or coalescence and film/sheet rupture—is the occurrence of hydrodynamic singularities. Accurately computing such flows with continuum mechanical, multidimensional free surface flow algorithms is a challenging task given these problems’ multiscale nature, which necessitates capturing dynamics occurring over disparate length scales across 5–6 orders of magnitude. In drop breakup, the thinning of fluid threads that form and eventually pinch-off must be simulated until the thread's radius is about 10 nm. When two drops approach one another, the thickness of the fluid film separating them must fall below 10 nm before coalescence is said to have occurred. If the initial drop radii are 1 mm, simulations must remain faithful to the physics as thread radius or film thickness falls from 10−3 m to below 10−8 m. Here we review significant findings in interfacial flows with hydrodynamic singularities spearheaded by sharp interface algorithms. These multidimensional algorithms can achieve resolution that to date has only been possible with the use of simple 1D evolution equations.

Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia

AbstractClonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workups. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and fluorescence-activated cell sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly diagnosed patients with AML. Presence of dysplasia according to MFC and World Health Organization criteria had no prognostic value in older adults. NGS of dysplastic cells and blasts isolated at diagnosis identified 3 evolutionary patterns: stable (n = 12 of 21), branching (n = 4 of 21), and clonal evolution (n = 5 of 21). In patients achieving complete response (CR), integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation, and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in ∼80% of patients with newly diagnosed AML, using techniques other than single-cell multiomics.

B-Cell Reconstitution After Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis.

Background and ObjectivesAutologous hematopoietic stem cell transplantation (aHSCT) is increasingly used to treat aggressive forms of multiple sclerosis (MS). This procedure is believed to result in an immune reset and restoration of a self-tolerant immune system. Immune reconstitution has been extensively studied for T cells, but only to a limited extent for B cells. As increasing evidence suggests an important role of B cells in MS pathogenesis, we sought here to better understand reconstitution and the extent of renewal of the B-cell system after aHSCT in MS.MethodsUsing longitudinal multidimensional flow cytometry and immunoglobulin heavy chain (IgH) repertoire sequencing following aHSCT with BCNU + Etoposide + Ara-C + Melphalan anti-thymocyte globulin, we analyzed the B-cell compartment in a cohort of 20 patients with MS in defined intervals before and up to 1 year after aHSCT and compared these findings with data from healthy controls.ResultsTotal B-cell numbers recovered within 3 months and increased above normal levels 1 year after transplantation, successively shifting from a predominantly transitional to a naive immune phenotype. Memory subpopulations recovered slowly and remained below normal levels with reduced repertoire diversity 1 year after transplantation. Isotype subclass analysis revealed a proportional shift toward IgG1-expressing cells and a reduction in IgG2 cells. Mutation analysis of IgH sequences showed that highly mutated memory B cells and plasma cells may transiently survive conditioning while the analysis of sequence cluster overlap, variable (IGHV) and joining (IGHJ) gene usage and repertoire diversity suggested a renewal of the late posttransplant repertoire. In patients with early cytomegalovirus reactivation, reconstitution of naive and memory B cells was delayed.DiscussionOur detailed characterization of B-cell reconstitution after aHSCT in MS indicates a reduced reactivation potential of memory B cells up to 1 year after transplantation, which may leave patients susceptible to infection, but may also be an important aspect of its mechanism of action.

Velocity vector comparison between vector flow imaging and computational fluid dynamics in the carotid bifurcation

It has been largely documented that local hemodynamic conditions, characterized by low and oscillating wall shear stresses, play a key role in the initiation and progression of vascular atherosclerotic lesions. Thus, investigation of the flow field in the carotid bifurcation can lead to early identification of vulnerable plaques. In this scenario, the development of novel non-invasive imaging tools that can be used in routine clinical practice to identify disturbed and recirculating blood flow becomes crucial. In this context, Vector Flow Imaging is becoming a relevant tool as it provides an angle independent assessment of blood flow velocity and multidimensional flow vector visualization. The purpose of the present study was to validate, in several locations of the carotid bifurcation, the high-frame rate vector flow imaging (HiFR-VFI) technique by comparing with computational fluid dynamic simulations (CFD).In all eight carotid bifurcations, HiFR-VFI accurately detected regions of laminar flow as well as recirculation and unsteady flow areas. An accurate and statistically significant agreement was observed between velocity vectors obtained by HiFR-VFI and those computed by CFD, both for vector magnitude (R = 0.85) and direction (R = 0.74).Our study demonstrated that HiFR-VFI is a valid technique for rapid and advanced visual representation of velocity field in large arteries. Thus, it has a great potential in research-based clinical practice for the identification of flow recirculation, low and oscillating velocity gradients near vessel wall. The use of HiFR-VFI may provide a great improvement in the investigation of the role of local hemodynamics in vascular pathologies, as well in the assessment of the effect of pharmacological treatments.