Cryopreserved Bone Marrow Research Articles

Abstract 3604: NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts

Abstract Introduction: Acute myeloid leukemia (AML) is a malignancy of immature myeloid cells characterized by rapid proliferation of abnormal myeloblasts. Treatment options for patients with AML are limited, especially for relapsed/refractory (r/r) disease. Leukemic cells with stem cell features, known as leukemic stem cells (LSCs), have been implicated as the origin of relapse in minimal residual disease positive (MRD+) AML (Khaldoyanidi et al. 2022, Kamel et al. 2022). NKX101 is an allogeneic, off-the-shelf, healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D CAR and membrane bound IL-15. In a clinical trial, NKX101 has shown promise for the treatment of r/r AML, including generating complete responses (CR) with MRD negativity (MRD−). In this study, we utilize a flow cytometry-based approach to (i) evaluate the cell surface expression of NKG2D-Ls on normal cells vs. primary AML blasts, (ii) assess the expression pattern of NKG2D-Ls on AML blast subsets, and (iii) determine NKX101 cytotoxic killing of LSC and non-LSC AML blasts. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. Cryopreserved bone marrow mononuclear cells (BMMCs) were obtained from AML patients (n=20) and healthy donors (n=10) in accordance with approved IRB protocols. AML somatic mutation status was determined using an Illumina TruSight® Myeloid Sequencing Panel. NKX101 cytotoxicity against patient-derived AML blasts was assessed using a flow cytometry-based assay that measured specific cytotoxicity and AML blast/LSC markers. NKG2D-L expression was determined using flow cytometry with antibodies specific to individual NKG2D-Ls. Results: Utilizing patient-derived primary samples, we demonstrate that NKG2D-Ls are expressed on AML blast cells and expressed at higher levels on AML blast cells compared to healthy normal hematopoietic cells (p = 0.002). Furthermore, we show that NKG2D-Ls are expressed at similar levels on LSCs compared to non-LSC AML blast populations. Target cell specific flow cytometry-based cytotoxicity assays revealed that NKX101 potently kills patient-derived AML blasts in a dose-dependent manner. Moreover, NKX101 had similar cytotoxic potency against both LSC and non-LSC AML blasts. Conclusions: In summary, we show that NKG2D-Ls are expressed on the surface of both LSC and non-LSC AML blasts and at levels above those observed in normal hematopoietic cells. Additionally, the ability of NKX101 to potently kill AML LSC blasts suggests that NKG2D-L targeting may be a viable mechanism for eliminating LSCs in the blood and bone marrow of patients with r/r AML. Taken together, these data support NKG2D-Ls as promising therapeutic targets for AML and support further investigation of NKX101 CAR-NK therapy for the treatment of r/r AML. Citation Format: Kyle Hansen, Cynthia Cho, Nishi Kothari, David Shook, James Trager. NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3604.

Prospects for the Use of Cryopreserved Bone Marrow Mesenchymal Stem Cells in the Treatment of Neurological Diseases and Injuries

Prospects for the Use of Cryopreserved Bone Marrow Mesenchymal Stem Cells in the Treatment of Neurological Diseases and Injuries

Minimal Residual Disease Detection By Single Cell DNA Sequencing Technology: A Feasible Approach for Clinical Application and Identification of the Landscape of MRD Clones

Introduction:Despite recent advances in AML treatment, patients continue to relapse and become chemotherapy resistant, leading to poor long-term overall survival. The identification of this measurable residual disease (MRD), defined as the post-therapy presence of leukemic cells, currently stands as one of the most well-established risk factors. The abnormal phenotypic and molecular characteristics of AML cells offer an opportunity for disease monitoring using techniques like multiparameter flow cytometry (MFC) and qPCR, which are currently considered the gold standard for MRD detection. However, these techniques have limitations. The aim of this study was to validate a different approach using single-cell DNA sequencing (scDNAseq) technology to detect MRD in patients achieving complete response (CR) and to characterize the genomic landscape of MRD clones and the potential identification of clonal evolution. Methods:We selected 24 cryopreserved bone marrow samples from 15 AML patients who participated in the QUIWI-PETHEMA clinical trial (NCT04107727) and achieved CR after induction and consolidation (n=20). 4 diagnosis samples were analyzed to determine genomic differences between the MRD clone and the initial leukemic population. All patients had bulk targeted NGS data available at diagnosis. Selection of CD34+ and/or CD117+ cells was performed using magnetic beads for enrichment of blasts and Mission Bio multiome single cell DNA+protein was performed using an AML-related 469 amplicon panel and 19 surface antibody mix. Multiplex of 3 independent samples was performed in each library preparation. Analysis was done according to manufacturer's instructions. MRD was also analyzed using MFC (n=14) EuroFlow panel with a limit of detection of 0.1% aberrant cells. RNA qRT-PCR was used in cases with NPM1 mutations (n=6). Quantification of MRD by scDNAseq was calculated according to the enrichment performance obtained from the manufacturer and the percentage of mutant cells. Results: The concordance between gold standard techniques for MRD and scDNAseq was 75% (15/20) (Table 1). Concordance between MFC and scDNAseq was 78% (11/14). The 3 discordant cases, positives by scDNAseq, MRD levels ranged between 0.04-0.09%, below the consensus cutoff of 0.1% to define MRD+. These results suggest that scDNAseq may complement MFC in the detection of very low levels of MRD. Concordance with qRT-PCR was 66% (4/6), but we only detected one patient with a persistent NPM1 clone. Taking advantage of the single cell approach, we were able to assess the genomic landscape of MRD clones and the clonal evolution in sequential samples. The number of clones/subclones and the number of variants per clone varied between patients with positive MRD as shown in Table 1. Interestingly, we analyzed 4 samples at diagnosis by scDNAseq and observed 2 cases in which MRD mutation was already present at diagnosis but was not informed as VAF was < 1%. In 4 patients, we detected small clones (around 1%) that remained unchanged in size despite treatment, suggesting that they likely represent clonal hematopoiesis. In 6 cases, consecutive samples were obtained showing clearance of some clones with other ones remaining stable (UPN2), progressive clearance of clones (UPN3, UPN4), acquisition of new clones (UPN9) and clearance of some clones and acquisition of new ones (UPN15) (Figure 1). Integration of scDNA and cell surface protein expression in the same cell allowed us to perform mutation-clone specific immunophenotypic analysis. Some cases showed a clear pattern in which one of the mutant clones had a significantly higher expression of some markers that was correlated with previous flow cytometry data (e.g. UPN1). Conclusions: Our study suggests that the use of scDNA technology is a feasible approach for detection of MRD in AML patients. Moreover, the use of this approach in sequential samples may allow deciphering clonal evolution, the co-occurrence of different mutations including potential clonal hematopoiesis mutations and identifying winner clones potentially responsible for disease persistence and relapse. Finally, the integration of mutations and surface antibody markers in the same cell provides a means for identifying the presence of mutations in different cell populations. Validation of these results in larger series of patients and correlation with clinical outcome are the next steps for validation of this technology.

Immunological Footprints of Pediatric Acute Myeloid Leukemia in Local Bone Marrow T Cells

Introduction: Despite notable advancements, childhood acute myeloid leukemia (AML) remains associated with one of the poorest prognoses among pediatric malignancies. Conventional chemotherapies carry significant toxicity, underscoring the urgent need for novel e.g. immunotherapy-based approaches. Despite AML being previously considered non-immunogenic due to its low mutational burden, the complex interplay between leukemic blasts and T cells within the bone marrow in pediatric patients remains largely unexplored. This study investigates the AML-induced footprint on local bone marrow T cells (bmT cells) representing tumor-infiltrating lymphocytes, with the aim to reveal functional points of action for future immunotherapy. Methods: Cryopreserved bone marrow samples from both pediatric AML patients (n=29) and age-matched healthy bone marrow donors (HD, n=9) were analyzed. Multicolor flow cytometry quantified surface expression of inhibitory signaling and activation receptors, as well as T cell differentiation stages. RNA-Seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) were performed of sorted CD8 + bmT cells. Bulk RNA and ATAC-Seq data was visualized by principal component analysis (PCA). Another PCA was performed on the combined dataset of RNA- and ATAC-Seq. Differential analysis using DESeq2 (fitType “parametric”, testType “Wald”) identified 135 differentially expressed genes in CD8 + bmT cells between HD and AML patients based on fold change (≥1/≤-1) and statistical criteria (s-value<0.05). Results: Based on CD45RO, CD62L and CD95 surface expression, late differentiation stages of bmT cells were enriched in AML. Namely, the frequency of naïve bmT cells was reduced (33.9% vs. 57.5%, mean values, p=0.0018), while the frequency of effector memory bmT cells was increased in AML patients (21.5% vs. 9.4%, p=0.0010). This could be validated by RNA-Seq of sorted CD8 + bmT cells which documented higher expression of cytotoxic effector T cell genes such as Perforin, Granulysin and Granzyme B. In addition, our analysis also revealed higher expression of functional relevant genes associated with T cell cytotoxity (ADGRG1), effector function (BATF/TPX2) or exhaustion (SLAMF7) in AML patients. Next, surface expression of receptors for inhibitory signaling and activation was analyzed. Several of these receptors have also been described as immune checkpoints (ICP). Analyzing differential expression between HD and AML patients showed pronounced upregulation of inhibitory markers (TIM-3: 7.1% vs. 1.7%, p<0.0001, CD39: 7.7% vs. 2.0%, p=0.0103, LAG-3: 8.0% vs. 2.9%; CTLA-4: 3.9% vs. 1.6%, p=0.0024; PD-1: 34.8% vs. 21.3%, p=0.0016). Since CD8 + bmT cells showed the most promising effects in the flow cytometry analysis, this subpopulation of bmT cells was further characterized by RNA- and ATAC-Seq. Here, clustering of HD and AML patients' CD8 + bmT cells was observed in PCA. Interestingly, PCA also revealed differential clustering of CD8 + bmT cells from AML primary and relapse samples. Confirming the observed differences in phenotype of bmT cells in AML patients, we identified distinct bmT cell populations specifically induced in AML patients compared to HD, based on phenotype and differential ICP expression patterns. Through tSNE analysis, we discovered a CD8 + effector memory T cell population with a TIM-3 +PD-1 +2B4 + phenotype, comprising 2.3% of all bmT cells in AML samples compared to only 0.03% in HD. Conclusion: This study reveals significant alterations in the activation and inhibition potential of bmT cells in pediatric AML compared to HD, as confirmed by comprehensive analysis of the epigenome, transcriptome and surfaceome. We confirm the observed differences through the identification of a T cell population with a typically exhausted phenotype specific for AML patients when compared to HD, implying persistent antigen exposure and potential direct or indirect interactions with leukemic blasts. Although prospective clinical trials will have to confirm functional relevance in patient cohorts, this study provides crucial insights into the immune landscape of pediatric AML, underscoring the potential for targeted immunotherapeutic interventions.

Differences within the Immune Microenvironment May Account for Poorer Outcomes in African American AML Patients Compared to European American Patients with AML

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy arising from an immature myeloid progenitor with notoriously poor outcomes. A critical factor responsible for the difficulty in treating this disease evasion of the host immune system by AML. Both the innate and adaptive immune systems have been shown to be dysfunctional in AML with poor type I interferon responses, decreased STING signaling and increased populations of granulocytic and monocytic myeloid derived suppressor cells ultimately leading to poor adaptive response. Studies in solid tumors have demonstrated that variation in immunity contributes to racial disparities in cancer and outcomes. This is felt to be due to variation in geographic ancestry leading to differences in the innate immune genome required due to the need for protection against environmental pathogens. However, no data exists comparing innate and adaptive immunity by race in patients (pts) with AML. Here we sought to characterize the immune microenvironment with treatment outcomes in racially diverse AML pts. Methods: Adults with newly diagnosed AML were identified who had corresponding cryopreserved bone marrow mononuclear cells available within Roswell Park Hematologic Procurement bank. Pts were matched as closely as possible for age, gender, cytogenetic risk per ELN 2017 and molecular profile. Transcriptional differences between African American (AA, n=26) and European American (EA, n=26) AML pts and healthy controls (n=10) were analyzed using transcriptomics data from NanoString Pan Cancer Immune Profile panels. AA and EA AML samples were compared to each other, as well as their race-matched healthy controls. The ‘Limma’ package in R was utilized for differential gene expression analysis. Research was conducted according to the Declaration of Helsinki in accordance with the Institutional Review Board regulated protocol. Results: The median age of the entire cohort (n=52) was 65 years (range: 28-92), with 28 female patients (53.8%) ( Table 1). Within the AA cohort, the majority of pts had intermediate risk cytogenetics per ELN 2017 (n=19; 73.1%, and 6 pts had FLT3 mut (26.1%). Three pts (11.5%) underwent allogeneic stem cell transplant, with a median overall survival (OS) censoring for transplant of 5 months (range; 0.5-68 months) ( Figure 1). Within the EA cohort, most pts had poor risk cytogenetics (n=15; 57.7%). Six pts had FLT3 mut (26.1%), and three pts had TP53 mut (12%). Five pts underwent allogeneic stem cell transplant (19.2%), with a median OS of 16.4 months (range; 0.8-67 months). Sixty-nine significantly (p<0.05, |logFC|>1.5) differentially expressed genes in AA vs. EA pts were identified. Gene set enrichment analysis (GSEA) revealed alterations to Interferon, Jun kinase and STAT signaling pathways in AA pts. In contrast, EA pt samples were enriched for pathways related to amyloid metabolism and other T and B cell immune pathways. Master regulator analysis using LISA highlighted transcriptional dysregulation attributed to PDGB5, CTCF, AR, and NOTCH in AA pts and to GLIS2, SMAD1 and NFE2 in EA pts. Conclusions: As previously reported, we found that a clinically matched cohort of AA AML patients experienced significantly worse OS as compared to EA counterparts. We have identified several significant alterations within the immune microenvironment between cohorts, which may potentially explain this discrepancy in survival in AA patients. These differences could potentially serve as targetable alterations for therapeutic purposes. This work represents the first of its kind evaluating the immune microenvironment by race in AML patients and are awaiting confirmation in a larger cohort.

NKX101, an Allogeneic Off-the-Shelf CAR NK Therapy Targeting NKG2D-Ls, Has Potent In Vitro Cytotoxicity Against Patient-Derived AML Leukemic Stem Cell (LSC) and Non-Leukemic Stem Cell (Non-LSC) Blasts

Introduction Acute myeloid leukemia (AML) is a malignancy of immature myeloid cells characterized by rapid proliferation of abnormal myeloblasts. Treatment options for patients with AML are limited, especially for relapsed/refractory (r/r) disease. Leukemic cells with stem cell features, known as leukemic stem cells (LSCs), have been implicated as the origin of relapse in minimal residual disease positive (MRD +) AML (Khaldoyanidi et al. 2022, Kamel et al. 2022). Additionally, absence of NKG2D-ligand (NKG2D-L) expression has been reported to be associated with stemness and the LSC population in AML (Paczulla et. al, 2019). NKX101 is an allogeneic, off-the-shelf, healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR as well membrane bound IL-15. In a clinical trial, NKX101 has shown promise for the treatment of r/r AML, including generating complete responses (CR) with MRD negativity (MRD -). In this study, we utilize a flow cytometry-based approach to (i) evaluate NKG2D-L cell surface expression on normal hematopoietic cells vs. primary AML blasts, (ii) assess the expression pattern of NKG2D-Ls on AML blast subsets, and (iii) determine the potency of NKX101 cytotoxic killing of both LSC and non-LSC AML blasts. Methods NKX101 cells were generated from peripheral blood leukopaks from healthy donors. Cryopreserved bone marrow mononuclear cells (BMMCs) were obtained from AML patients in accordance with approved IRB protocols (n=20). BMMCs from healthy donors were used as controls (n=10). AML somatic mutation status was determined using an Illumina TruSight® Myeloid targeted sequencing panel. NKX101 cytotoxicity against patient derived AML blasts was assessed using a flow cytometry-based assay that simultaneously measured specific cytotoxicity AML blast/LSC markers. NKG2D-L protein expression on LSC and non-LSC subsets was determined using flow cytometry with fluorescently labeled monoclonal antibodies specific to MICA, MICB, and ULBP1-6. Results Utilizing patient-derived primary samples, we demonstrate that NKG2D-Ls are expressed on AML blast cells and expressed at higher levels on AML blast cells compared to healthy normal hematopoietic cells (p = 0.002). Furthermore, we show that NKG2D-Ls are expressed at similar levels on both LSC and non-LSC AML blast populations. Target cell specific flow cytometry-based cytotoxicity assays revealed that NKX101 cells potently kill patient-derived AML blasts in a dose-dependent manner. Moreover, NKX101 cells had similar cytotoxic potency on both LSC and non-LSC AML blasts subsets. Conclusions In summary, we show that NKG2D-Ls are expressed on the surface of AML blasts at levels above those observed in normal hematopoietic cells. NKG2D-Ls are expressed on both non-LSC AML blasts and on the LSC population implicated as the origin of relapse in AML. Additionally, the ability of NKX101 to potently kill AML LSC blasts suggests that NKG2D-L targeting may be a viable mechanism for eliminating LSC in the blood and bone marrow of patients with r/r AML. Taken together, these data support NKG2D-Ls as promising therapeutic targets for AML and support further investigation of NKX101 CAR NK cell therapy for the treatment of r/r AML.

Comparative Analysis of Biological Signatures between Freshly Preserved and Cryo-Preserved Bone Marrow Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) can differentiate into multiple connective tissue lineages, including osteoblasts, chondrocytes, and adipocytes. MSCs secrete paracrine molecules that are associated with immunomodulation, anti-fibrotic effects, and angiogenesis. Due to their orchestrative potential, MSCs have been therapeutically applied for several diseases. An important aspect of this process is the delivery of high-quality MSCs to patients at the right time, and cryo-biology and cryo-preservation facilitate the advancement of the logistics thereof. This study aimed to compare the biological signatures between freshly preserved and cryo-preserved MSCs by using big data sourced from the Pharmicell database. From 2011 to 2022, data on approximately 2300 stem cell manufacturing cases were collected. The dataset included approximately 60 variables, including viability, population doubling time (PDT), immunophenotype, and soluble paracrine molecules. In the dataset, 671 cases with no missing data were able to receive approval from an Institutional Review Board and were analyzed. Among the 60 features included in the final dataset, 20 were selected by experts and abstracted into two features by using a principal component analysis. Circular clustering did not introduce any differences between the two MSC preservation methods. This pattern was also observed when using viability, cluster of differentiation (CD) markers, and paracrine molecular indices as inputs for unsupervised analysis. The individual average PDT and cell viability at most passages did not differ according to the preservation method. Most immunophenotypes (except for the CD14 marker) and paracrine molecules did not exhibit different mean levels or concentrations between the frozen and unfrozen MSC groups. Collectively, the biochemical signatures of the cryo-preserved and unfrozen bone marrow MSCs were comparable.

Disturbed Cytokine Profile in Adjuvant Arthritis – A Target of the Therapeutic Potential of Dendritic Cells Derived From Cryopreserved Precursors

Background. One of the primary causes of rheumatoid arthritis development is the disruption of the immune system's natural tolerance to its own antigens, leading to an imbalance in the body's cytokine profile. A promising method of correcting such a condition is restoring antigen-specific tolerance, in the formation of which tolerogenic dendritic cells (tolDCs) take part. Objective. Experimental substantiation of the possibility of correcting the cytokine profile of animals with adjuvant arthritis (AA) by using tolDCs from cryopreserved bone marrow precursors. Methods. The study was carried out on the CBA/H mice. The development of AA was assessed using a clinical indicator – the arthritis index. The levels of pro- (TNF-a, IL-6, IFN-g) and anti-inflammatory (IL-10, IL-4) cytokines in the blood serum of AA-afflicted animals were measured before and after administration of tolDCs. These tolDCs were obtained from native (NatDCs) or cryopreserved (CryoDCs) using different methods bone marrow mononuclear cells (MNCs). On the 14th day after inducing AA, the animals received intravenous injections of tolDCs (5´105/mouse). One week later, the cytokine levels in the animals' blood serum and the arthritis index were assessed. Results. Throughout the development of AA, a unidirectional increase in TNF-α and IL-6 levels and a reduction in the content of anti-inflammatory cytokines were observed, which was accompanied by joint swelling in the animals. CryoDCs exhibited a more pronounced corrective effect on both pro- and anti-inflammatory cytokines compared to NatDCs, as evidenced by a decrease in the arthritis index, a clinical manifestation of the pathology. Conclusions. The possibility of correcting the disturbed cytokine profile and the clinical state of animals during the development of AA through the use of tolDCs derived from cryopreserved MNCs has been proven. Specific cryopreservation conditions for MNCs have been developed, which facilitate the generation of tolDCs from them with a greater capacity, compared to derivatives of native MNCs, to correct the cytokine profile and clinical status of animals with AA.

Characterization and Function of Cryopreserved Bone Marrow from Deceased Organ Donors: A Potential Viable Alternative Graft Source

Despite the readily available graft sources for allogeneic hematopoietic cell transplantation (alloHCT), a significant unmet need remains in the timely provision of suitable unrelated donor grafts. This shortage is related to the rarity of certain HLA alleles in the donor pool, nonclearance of donors owing to infectious disease or general health status, and prolonged graft procurement and processing times. An alternative hematopoietic progenitor cell (HPC) graft source obtained from the vertebral bodies (VBs) of deceased organ donors could alleviate many of the obstacles associated with using grafts from healthy living donors or umbilical cord blood (UCB). Deceased organ donor-derived bone marrow (BM) can be preemptively screened, cryogenically banked for on-demand use, and made available in adequate cell doses for HCT. We have developed a good manufacturing practice (GMP)-compliant process to recover and cryogenically bank VB-derived HPCs from deceased organ donor (OD) BM. Here we present results from an analysis of HPCs from BM obtained from 250 deceased donors to identify any substantial difference in composition or quality compared with HPCs from BM aspirated from the iliac crests of healthy living donors. BM from deceased donor VBs was processed in a central GMP facility and packaged for cryopreservation in 5% DMSO/2.5% human serum albumin. BM aspirated from living donor iliac crests was obtained and used for comparison. A portion of each specimen was analyzed before and after cryopreservation by flow cytometry and colony-forming unit potential. Bone marrow chimerism potential was assessed in irradiated immunocompromised NSG mice. Analysis of variance with Bonferroni correction for multiple comparisons was used to determine how cryopreservation affects BM cells and to evaluate indicators of successful engraftment of BM cells into irradiated murine models. The t test (with 95% confidence intervals [CIs]) was used to compare cells from deceased donors and living donors. A final dataset of complete clinical and matched laboratory data from 226 cryopreserved samples was used in linear regressions to predict outcomes of BM HPC processing. When compared before and after cryopreservation, OD-derived BM HPCs were found to be stable, with CD34+ cells maintaining high viability and function after thawing. The yield from a single donor is sufficient for transplantation of an average of 1.6 patients (range, 1.2 to 7.5). CD34+ cells from OD-derived HPCs from BM productively engrafted sublethally irradiated immunocompromised mouse BM (>44% and >67% chimerism at 8 and 16 weeks, respectively). Flow cytometry and secondary transplantation confirmed that OD HPCs from BM is composed of long-term engrafting CD34+CD38-CD45RA-CD90+CD49f+ HSCs. Linear regression identified no meaningful predictive associations between selected donor-related characteristics and OD BM HPC quality or yield. Collectively, these data demonstrate that cryopreserved BM HPCs from deceased organ donors is potent and functionally equivalent to living donor BM HPCs and is a viable on-demand graft source for clinical HCT. Prospective clinical trials will soon commence in collaboration with the Center for International Blood and Marrow Research to assess the feasibility, safety, and efficacy of Ossium HPCs from BM (ClinicalTrials.gov identifier NCT05068401).

Prognostic Predictability of 2022 European Leukemianet (ELN) Risk Stratification in the Real World

Introduction The European LeukemiaNet (ELN) reported 2022 guideline of the recommendation for diagnosis and management of acute myeloid leukemia (AML). Compared to 2017, ELN 2022 recommendation has some significant changes in risk classification. First, an exclusion of the FLT3-ITD allelic ratio in the risk classification, and FLT-ITD without NPM1 mutation is no longer classified as adverse risk because of the incorporation of FLT3 inhibitor. Next, in-frame mutations affecting the basic leucine zipper region (bZIP) of CEBPA regardless of monoallelic or biallelic is classified as favorable risk group. Another important change is the inclusion of myelodysplasia-related gene mutations, and patients with these gene mutations are now categorized as adverse risk group. The purpose of this study was to verify and compare the prognostic predictability between previous and new recommendation in the real world. Patients and methods A total of 624 patients who were newly diagnosed with AML from October 1998 to October 2014 in seven institutions were included in this study. Patients who achieved complete remission (CR) received consolidation chemotherapy with or without allogeneic hematopoietic stem cell transplantation (HCT), depending on the availability of a matched donor. Genetic factors were not considered while considering allogeneic HCT. Cryopreserved bone marrow or peripheral blood samples obtained at diagnosis were archived. Genomic DNA was extracted using QIAamp DNA blood mini-kits (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Genetic profiling included the targeted deep sequencing of 45 genes, which had been selected based on recurrent driver mutations from previous studies. Agilent custom probes were designed to cover the entire exon regions of targeted genes and sequenced using an Illumina HiSeq 2000 sequencer. The threshold of mutation positivity was defined as a variant allele frequency≥2%. Results All of the 624 patients were older than 18 years and age with median age was 51 years (range 18-84). All patients were treated with induction chemotherapy using a standard 3+7 protocol, and 523 patients (83.8%) achieved CR. Among them, 235 patients (44.9%) proceeded to allogeneic HCT. After median follow-up of 84.4 months, median overall survival (OS) was 22.0 months and median event-free survival (EFS) was 16.2 months in all patients. By applying the 2022 ELN risk classification, each risk groups showed significant prognostic difference with Kaplan-Meier analysis of OS (P<0.001) and EFS (P<0.001) in all patients. According to ELN 2017 risk stratification, 218 patients (34.9%) were in favorable risk group, 298 patients (47.8%) were in intermediate risk group, and 108 patients (17.3%) were in adverse risk group. Among the previously favorable group, 31 patients were reclassified as intermediate risk group and 3 patients as adverse risk group, and there was no significant prognostic difference in OS (P=0.289) and EFS (P=0.279) between patients newly classified as favorable risk group and those reclassified as intermediate or adverse risk groups. But in patients without allogeneic HCT, favorable group by 2022 edition showed significantly longer OS (P=0.003) and EFS (P<0.000) than intermediate or adverse groups. Among the previously intermediate risk group patients, 21 patients were reclassified as favorable group and 46 patients as adverse risk group, and each group showed significant difference of OS (P<0.001) and EFS (P<0.001). Also, in adverse risk group patients by the 2017 edition, 33 patients were reclassified as intermediate risk group. Because the anti-FLT3 inhibitor therapy was not available at that times, patients who reclassified as intermediate group showed no survival difference compared to the patients remaining in the adverse group. The survival analyses comparing ELN 2017 and ELN 2022 are described in figure 1. Conclusion ELN 2022 guideline is superior to the ELN 2017 for risk stratification and better predicts the prognosis of patients with AML in the real world. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Activation of Distinct Inflammatory Pathways in LR-MDS Is Determined By Genetics

Background Dysregulated innate immune signaling has been identified as a driver of the complex pathophysiology of myelodysplastic syndromes (MDS), predominantly in low risk (LR)-MDS (Basiorka et al., Blood 2016). The pharmacological targeting of inflammation may therefore help to retard disease development. However, since MDS is a heterogeneous disease, a personalized approach is likely to be necessary, requiring a more detailed knowledge of distinct inflammatory patterns and their variation between genetically defined subgroups of LR-MDS patients (Winter et al., J Clin Oncol 2020). Aims The aim of this work was to describe inflammation states in the bone marrow of LR-MDS patients (IPSS-R ≤ 3.5) compared to controls with and without clonal hematopoiesis of indeterminate potential (CHIP) and to high-risk (HR)-MDS patients (IPSS-R ≥ 4). Methods Cryopreserved bone marrow mononuclear cells (BM-MNCs) and corresponding bone marrow plasma samples from non-CHIP controls (n = 15), CHIP individuals (n = 12) and patients with LR-MDS (n = 47) or HR-MDS (n = 14) were obtained from the MDS registry and the BoHemE study (NCT02867085) at the University Hospitals in Dresden and Leipzig. Targeted expression analysis of 11 inflammasome-related genes (S100A9, NLRP3, PYCARD, CASP1, IL1B, IL18, NLRP1, NLRC4, AIM2, CASP3, CASP4) and multiplex cytokine measurements were performed. To investigate heterogeneity in the LR-MDS cohort, a principle component analysis (PCA) was performed and a cumulative gene expression score was calculated as summed log2 fold changes (mean non-CHIP = 0) of all 11 mRNA expression values for each sample. Furthermore, BM-MNCs of selected non-CHIP (n = 3) and CHIP (n = 3) individuals as well as LR-MDS (n = 14) and HR-MDS (n = 5) patients were FACS-sorted for hematopoietic stem and progenitor cells (HSPCs), monocytes, monocytic and polymorphonuclear myeloid-derived suppressor cells (MDSCs) and lymphocytes. The expression of IL1B, IL18, S100A9, NLRP3, PYCARD, CASP1 and NLRC4 was assessed in each population by qRT-PCR. Finally, colony forming assays were used to assess the effect of the IL-1β-neutralizing antibody canakinumab (100 µg/ml) on the hematopoietic potential of healthy CD34+ HSPCs pre-cultured with CD14+ monocytes from patients with SF3B1-mutated (n = 3) or del(5q) (n = 3) LR-MDS. Results The expression of inflammasome-related genes in the bone marrow increased from non-CHIP through CHIP to LR-MDS individuals, with overall levels of inflammasome-related gene expression being 40% higher in LR-MDS than in non-CHIP controls (p = 0.002). The precise signature of inflammation gene expression varied widely within LR-MDS patients. A PCA identified two separable inflammation states with low (cluster 1, n = 24) and high (cluster 2, n = 23) inflammation patterns (Figure 1), with CASP1, PYCARD and NLRC4 contributing most to the PCA dimensions. The clusters were associated with distinct clinical features and disease genotypes: more than 80% of the SF3B1 mutations were found in cluster 1, while all del(5q) cases were in cluster 2 (Figure 1). Compared to del(5q) samples, those with SF3B1-mutation showed lower expression of IL-1β at both mRNA (3.9-fold decrease, p = 0.0005) and protein level (8.3-fold decrease, p = 0.03). Targeted gene expression analysis of sorted cell populations revealed distinct contributions to the differential patterns of inflammation. IL1B, NLRP3, NLRC4 and S100A9 were expressed in all disease states predominantly by monocytes and monocytic MDSCs, consistent with a dominant role for monocytic cells in determining the inflammatory bone marrow environment. However, IL18 mRNA levels were highest in HSPCs. Compared to untreated controls, canakinumab augmented the colony forming activity of healthy HSPCs pre-cultured with monocytes from SF3B1-mutated MDS (+70%, p = 0.002). The effect in co-cultures with del(5q) MDS-derived monocytes was weaker (+56%, p = 0.188), showing that canakinumab efficacy in this assay does not depend on IL-1β expression level alone. Conclusion We conclude that inflammation patterns in LR-MDS are heterogeneous, highlighting the need for personalization of targeted anti-inflammatory therapies currently entering clinical trials. Our analysis suggests that high resolution studies of gene expression in specific populations or single cells will further resolve a spectrum of inflammation states relevant to prognosis and personalized therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Development of a Pediatric AML Patient Derived Xenograft Program

Background: Survival rates for pediatric acute myeloid leukemia (AML) still lag far behind those for most other pediatric cancers. New agents are desperately needed, but new drug development has been hampered by a lack of clinically relevant pediatric AML patient-derived xenograft (PDX) models for research. Texas Children's Hospital (TCH) has the patient volume, a close collaboration between clinical groups and research laboratories, and the infrastructure to address this gap. Methods: TCH patients diagnosed with AML are offered participation in local research. Most samples from local patients are enriched for mononuclear cells (MNCs) and injected fresh on the day of collection. Additionally, cryopreserved bone marrow samples from patients who enrolled on Children's Oncology Group clinical trials and consented to bank tissue for research have been used for PDX generation. Leukemia cells are tail vein injected (2x10e5 cells per mouse) into immunodeficient mice without conditioning. Most models are generated in the NOD.scid.Il2Rγc-/-/SCFh/h/GM-CSFh/h/IL-3h/h (NSGS) strain. Mice are closely monitored for human CD45+/CD33+/CD3- (hAML) cells in peripheral blood by flow cytometry. Once moribund, the mice are humanely euthanized, and bone marrow, peripheral blood, and spleen are harvested and evaluated by flow cytometry for disease burden. Bone marrow cells are injected by tail vein into secondary recipients. Serially transplanting PDX models are validated and characterized by short tandem repeat (STR) fingerprinting and DNA/RNA sequencing. Results: Thus far, we have developed 24 serially transplanting pediatric AML PDX models representing a variety of clinical and cytomolecular categories. The patients from whom these models were derived ranged in age from 0.5-20 years of age at diagnosis (mean 9.4 years). Most are derived from samples collected at diagnosis. For AML005, we have a second serially transplanting model derived from relapse. Our collection includes 8 models with KMT2A fusions, 3 with NPM1 mutations, 4 with WT1 mutations, 3 with FLT3 internal tandem duplications or point mutations, 1 with a NUP98 rearrangement, and 1 CBFA2T3-GLIS2 model. Model identities are validated by comparing the PDX STRs, flow cytometry, and fluorescence in situ hybridization results with matched samples from the patient of origin. For 3 models we also have targeted DNA sequencing for 174 recurrent leukemia-associated mutations for both the patient and the corresponding PDX model. All mutations detected in the patient samples were also detected in the PDX models. The Pearson correlation coefficients comparing the variant allele frequencies for each patient-PDX pair range from 0.82 to 0.99, indicating that the models are faithful representations of the original disease even at the subclone level. A summary of the current collection of serially transplanting models is provided in Table 1. Over 15 additional samples have engrafted once and are currently in secondary recipients. Data on established and validated models are being made publicly available through the Baylor PDX Portal (https://pdxportal.research.bcm.edu), where investigators from Baylor and labs worldwide can access information about our collection and request viably frozen samples for their own research purposes. Our models have been distributed to labs around the world for preclinical studies of novel therapeutics. Conclusion: Through development and distribution of unique pediatric AML PDX models, we hope to create a renewable resource to speed preclinical research and expedite development of drugs for clinical testing for children with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Dimensional Mass Cytometric Analysis of the Bone Marrow Compartment Identifies a Disease-Specific Immune Cell Network in Aplastic Anemia

Idiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST). However, the immune response that underlies the pathogenesis of AA remains incompletely understood. Previous studies have extensively studied the immune cell composition in peripheral blood in AA, but a comprehensive overview of the immune landscape at the site of hematopoietic stem and progenitor cell (HSPC) destruction, the bone marrow, is lacking. We investigated the immune composition of the bone marrow in AA to interrogate which immune populations may be implicated in the pathogenesis of AA. A mass cytometric analysis of bone marrow aspirates was performed using a 39-marker panel for broad immunophenotyping of all major immune lineages and HSPCs. Cryopreserved bone marrow nuclear cells from adult AA patients (n=7) before start of first-line IST with horse-derived anti-thymocyte globulin (hATG) in combination with ciclosporin and age- and sex-matched healthy donors (HDs, n=7) were studied. 6 of 7 AA patients responded to IST. For 3 IST responders paired bone marrow samples taken at six months after start of IST were also included. A Hierarchical Stochastic Neighborhood Embedding (HSNE) analysis in Cytosplore revealed that the bone marrow in AA pre-IST features a distinct immune cell composition compared to HDs. At the major immune lineage level, HSPC and myeloid cell depletion (0.6% vs 33% of all immune cells, p<0.001), decreased B-cell frequencies (9% vs 16%, p=0.007), increased CD4+ T-cell frequencies (37% vs 28%, p=0.04) and increased CD8+ T-cell frequencies (37% vs 20%, p=0.007) were observed. Detailed analysis of these 4 lineages identified 121 phenotypically distinct cell clusters, 45 of which were differentially present (p<0.05) in AA patients pre-IST compared to HDs. To capture the relationships between these 45 immune clusters a Spearman's rank correlation analysis was performed using the cluster frequencies. Two cell networks were identified (Figure 1A), one of which (network AA) was significantly increased in frequency in AA pre-IST (22% vs 5% of all immune cells, p=0.001; Figure 1B). This network consisted of CD16+ myeloid cells, CCR6++ B-cells, Th17-like CCR6+ memory CD4+ T-cells and KLRG1+ terminally differentiated effector memory (EMRA) CD8+ T-cells, compatible with a state of chronic inflammation. Successful treatment with IST largely normalized the immune cell composition at the immune lineage level and strongly reduced the levels of CD16+ myeloid cells. However, at six months after start of IST the frequencies of CCR6++ B-cells, CCR6+ memory CD4+ T-cells and KLRG1+ EMRA CD8+ T-cells were not normalized, which could indicate that ciclosporin is still needed. This study provides a unique overview of the immune landscape in bone marrow in AA pre- and post-IST at a single-cell level. Previously reported cell subsets could be further defined and possible new players that contribute to the pathogenesis of AA were identified. Our findings show that AA is characterized by high frequencies of immune cells compatible with a state of chronic inflammation and provide a basis for future studies to characterize the function of the identified subsets in the pathogenesis of AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cryopreservation Impairs Engraftment in Bone Marrow Transplantation

Introduction: Due to the covid-19 pandemic, a recommendation was made, especially for non-related allogeneic hematopoietic stem cell transplantation (allo-HSCT), to cryopreserve hematopoietic stem cells products and start the conditioning regimen only after the product be received at the receptor service. There are conflicting data on how the procedure of cryopreservation (and thaw) of bone marrow affects outcomes. In this study we evaluated the primary graft failure incidence using cryopreserved bone marrow grafts compared to non-cryopreserved bone marrow grafts. We also evaluated transfusion needs during the first 30 days after transplant and the occurrence of severe adverse events (AEs) related to the product infusion, during covid-19 pandemic in a single Brazilian center. Methods: From January 2020 to 30 June 2022, we retrospectively analyzed clinical and laboratory data from 9 cryopreserved (cryo) (all of them unrelated) and 15 non-cryopreserved (non-cryo) bone marrow grafts (2 of them unrelated), here defined as control group. Haploidentical transplants were excluded due to a higher risk of primary graft failure reported on this type of transplant. Children and adults with malignant and non-malignant disorders were included. We evaluated engraftment, red blood cells (RBC) and platelets (Pl) units transfused during the first 30 days, and adverse effects associated with product infusion. Neutrophil engraftment was defined as the first of 3 consecutive days with absolute neutrophil counts of ≥ 500/µL and primary graft failure was defined as failure to achieve a neutrophil engraftment by day 28 after bone marrow infusion. Results were given as median (range) and comparison between the groups was performed employing the Mann-Whitney U test. Percentages were compared with the chi-square or the Fisher exact test. Results: Only two cryopreserved grafts have not been used (one patient died before the start of the conditioning regimen and the other did not receive the graft yet). Baseline characteristics were similar in both groups. The median (range) age was 21 (1-37) and 20 (6-32) years, with 42.86% and 40% under 18 years of age in the cryo and non-cryo groups, respectively. Also, 85.7% and 53.3% were male, and 14.3% and 13.3% had malignant disease (acute leukemia) in the cryo and non-cryo groups, respectively (Table 1). The numbers of infused total nuclear and CD34+ cells/kg in the cryo and non-cryo groups were not significantly different (p= 0.14, and p= 0.41, respectively) (Table 1). Three (42.8%) patients from the cryo group, all of them children (with osteopetrosis, aplastic anemia, and acute leukemia) did not achieve neutrophil engraftment by day 28, whereas all 15 patients from non-cryo group had hematologic recovery (RR= 4.75, 95% CI: 1.61-11.75; p= 0.02). Furthermore, there was a slight tendency toward delayed neutrophil engraftment in the cryo group: 21.5 (19-23) and 19.0 (15-28) days (p= 0.20). The numbers of RBC and Pl units transfused during the first 30 days after product infusion were higher in the cryo than non-cryo group: RBC 5.0 (3-9) and 1.0 (0-4) (p< 0.0001); Pl 15.0 (7-18) and 5.0 (1-20) (p= 0.0014), respectively (Figure 1). Three patients (42.8%) had severe AEs (seizure, hypertension urgency associated with second-degree atrioventricular block: Mobitz type 1, and acute renal failure) during or right after bone marrow infusion, all of them in the cryo group (relative risk= 4.75, 95% CI: 1.61-8.43, p= 0.02). All of them recovered completely from AE. Conclusions: Transplantation of cryopreserved bone marrow grafts seems to be associated with a higher risk of primary engraftment failure and, consequently, a higher transfusion needs, and with more severe adverse events related to the product infusion. Because of these findings and in spite of the ongoing pandemic, we recommend a careful evaluation of the necessity to adopt this practice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fluorescent Visualization of the Distribution of GFP+ Donor Cells in Mouse Organs after Transplantation of Native or Cryopreserved Bone Marrow

Fluorescent Visualization of the Distribution of GFP+ Donor Cells in Mouse Organs after Transplantation of Native or Cryopreserved Bone Marrow

Deep Profiling of the Immune Microenvironment throughout Myeloma Disease Stages

Background The immune system is altered in multiple myeloma (MM) and contributes to therapy resistance. The availability of novel immunotherapies necessitates understanding the influence of the immune microenvironment on disease progression which may inform sensitivity to therapy. The objective of this study is to fully characterize the immune microenvironment in MM precursor diseases and MM and identify any immune contribution to progression. To accomplish this we used high-dimensional mass cytometry (CyTOF) to investigate immune alterations associated with progression in pre-malignant and malignant stages of MM.Methods Cryopreserved bone marrow mononuclear cells (BMMCs) from healthy donors (HD, n=13), MGUS (n=21), SMM (n=19), newly diagnosed MM (NDMM, n=17), and ~3 months post- first autologous stem cell transplant (ASCT, n=21) were assessed using a panel of 35 cell surface and 3 intracellular antibodies that includes cell lineage markers for identification of immune populations and functional markers indicative of positive or negative immune regulation. BMMCs were thawed, stained with antibodies, and analyzed on a Helios mass cytometer. Data were normalized using bead normalization, transformed using the inverse hyperbolic sine function with a cofactor of 5 and gated for 45+ live, intact, singlets for global analysis by gating in FCS express and clustering by viSNE for visualization. Differences in population abundance were identified in an unbiased manner by FlowSOM and in marker intensity by CITRUS. Marker intensity analysis was performed using the multiple testing permutation procedure (SAM), with an FDR of 1% and minimum population size of 0.5%.Results To identify changes in the immune microenvironment associated with progression we compared immune population abundance and marker intensity indicative of immune status including activation, exhaustion, or senescence. MGUS was distinguished from HD by increased abundance of CD4 central memory (CM, p<0.001), effector memory (EM, p<0.001) and plasmacytoid and monocyte-derived dendritic cells (DC, p< 0.01). In MGUS, TIM3 and CD57 were elevated on NK cells and NKT cells, respectively, compared to HD suggesting reduced activity. In SMM increased abundance of B regulatory cells (3.0 vs 5.9 %, p<0.01) but reduced inhibitory markers on T cells including PD1, CTLA4 CD55, FOXP3 and TIGIT was observed compared to MGUS. NDMM was distinguished from SMM by reduced abundance of CD4 EM (p<0.01), CD8 early EM (p< 0.001), and B regulatory cells (p<0.01) and increased abundance of active Tregs (CD38+, P<0.01) and total NK cells (p<0.01) which had increased CD55, a complement inhibitory protein. Post-ASCT changes in immune abundance include increased total CD8 and CD8 terminal effectors (CD57 +, p< 0.0001), B regulatory cells (p<0.0001), and reduced total CD4 and CD4 CM (p<0.0001), compared to NDMM. CD4 T cells post-ASCT were characterized by reduced CD127 and CCR7 and increased CD28, CTLA4, FOXP3 and TIGIT and CD8 T cells had reduced CD28, CD127 and CCR7 and increased CD57 and TIGIT compared to NDMM. Interestingly, significant difference in NK cells were not observed but post-ASCT NK cells may be active as suggested by reduced CD59 and TIM3 compared to NDMM. To determine whether the immune microenvironment had normalized by 3 months post-ASCT we compared population abundance to HD, MGUS, and SMM cases. Immune abundance post-ASCT revealed a significantly lower percentage of CD4 CM, 4 -8 - T cells, normal PCs, and post-switch B cells (25+) and elevated CD8 terminal effector (57+) and B regulatory cells than all 3 other groups. Overall major differences in abundance of total T and B cells and their subsets were observed with differences in NK cells between stages primarily reflected in marker expression (e.g. CD161+ subset) rather than abundance.Conclusions Early changes in the immune microenvironment observed in MGUS/SMM lead to immune suppression and eventually immune evasion allowing MM to emerge. In this study the immune ME did not appear to normalize 3 months post-therapy indicated by an increase in B regulatory cells and markers of inactive effector cells. Profiling of the immune microenvironment throughout MM treatment may allow us to identify novel therapeutic targets and optimal timing of administration of novel immunotherapies and patients that would most benefit from these therapies. DisclosuresWalker: Sanofi: Speakers Bureau; Bristol Myers Squibb: Research Funding. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees.

Preclinical Evaluation of the Safety and Efficacy of Cryopreserved Bone Marrow Mesenchymal Stromal Cells for Corneal Repair.

PurposeMesenchymal stromal cells (MSCs) have been shown to enhance tissue repair as a cell-based therapy. In preparation for a phase I clinical study, we evaluated the safety, dosing, and efficacy of bone marrow–derived MSCs after subconjunctival injection in preclinical animal models of mice, rats, and rabbits.MethodsHuman bone marrow–derived MSCs were expanded to passage 4 and cryopreserved. Viability of MSCs after thawing and injection through small-gauge needles was evaluated by vital dye staining. The in vivo safety of human and rabbit MSCs was studied by subconjunctivally injecting MSCs in rabbits with follow-up to 90 days. The potency of MSCs on accelerating wound healing was evaluated in vitro using a scratch assay and in vivo using 2-mm corneal epithelial debridement wounds in mice. Human MSCs were tracked after subconjunctival injection in rat and rabbit eyes.ResultsThe viability of MSCs after thawing and immediate injection through 27- and 30-gauge needles was 93.1% ± 2.1% and 94.9% ± 1.3%, respectively. Rabbit eyes demonstrated mild self-limiting conjunctival inflammation at the site of injection with human but not rabbit MSCs. In scratch assay, the mean wound healing area was 93.5% ± 12.1% in epithelial cells co-cultured with MSCs compared with 40.8% ± 23.1% in controls. At 24 hours after wounding, all MSC-injected murine eyes had 100% corneal wound closure compared with 79.9% ± 5.5% in controls. Human MSCs were detectable in the subconjunctival area and peripheral cornea at 14 days after injection.ConclusionsSubconjunctival administration of MSCs is safe and effective in promoting corneal epithelial wound healing in animal models.Translational RelevanceThese results provide preclinical data to support a phase I clinical study.

Liquid Biopsy for the Identification of Intravascular Large B-Cell Lymphoma

[Backgrounds] Intravascular large B cell lymphoma (IVLBCL) is a rare subtype of extranodal diffuse large B-cell lymphoma (DLBCL) that features lymphoma cells lodged in lumina of small vessels. Because of the lack of mass formation, diagnosis by tumor biopsy is often difficult, and thus, cell-free DNA (cfDNA) analysis might be useful for assisting diagnosis of IVLBCL. In this study, we first aimed at investigating the prevalence of mutations in genes involved in the B-cell receptor, Toll-like receptor/interleukin-1 receptor, and nuclear factor kappa B pathways. We then evaluated the liquid biopsies for detection of hot-spot mutations by droplet digital polymerase chain reaction (ddPCR).[Patients and Methods] Twenty-seven IVLBCL patients whose archived samples were available were included in this study. The patients were diagnosed as IVLBCL from January 2005 to May 2017 in University of Tsukuba Hospital, JA Toride Medical Center, and Kameda Medical Center. The study was approved by the review board in each institution, and conducted according to the Declaration of Helsinki. Tissue-derived DNA (tdDNA) was extracted from formalin-fixed paraffin-embedded specimens and/or cryo-preserved bone marrow samples, in which the lymphoma cell infiltration had been pathologically confirmed. cfDNA was extracted from 1 ml of serum or plasma obtained prior to chemotherapy. We performed targeted sequence for 8 genes (B2M, BTG2, CARD11, CD79B, MYD88, PIM1, PRDM1, and TNFAIP3) in paired tdDNA and cfDNA from 9 IVLBCL subjects by Ion Torrent Personal Genome Machine (Thermo Fisher Scientific). MYD88 L265P (c.794T>C) mutational analysis was performed by ddPCR assays (BioRad) for 46 samples in total collected from 27 subjects. Statistical analysis was performed with EZR version 1.3.2. Paired t-test was used to compare variant allele frequencies (VAFs) in paired samples.[Results] All cases were categorized as the non-GCB by Hans classification. Random skin biopsies (RSB) and bone marrow biopsies (BMB) were performed in 21 and 27 patients. The diagnostic yield of RSB was 81.0% (17 of 21). Initial BMB detected lymphoma cells in 48.1% (13/27) patients, but repeated BMB increased successful detection up to 66.7% (18/27). tdDNA was available in 26 out of 27 subjects, and in 9 of which paired cfDNA was also available (Figure 1). In one subject, only cfDNA but not tdDNA was available. Targeted sequencing for 8 genes demonstrated at least one mutation in CD79B, MYD88, PIM1, or PRDM1 in both tdDNA and cfDNA, or only in cfDNA, in 8 of 9 subjects (Figure 2). We identified 3 missense CD79B mutations in 6 (66.7%; Y196C in 2, Y196H in 3, and L199P in 1). All MYD88 mutations were L265P, which were found in 5 of 9 (55.6%). Together, either Y196 CD79B or L265P MYD88 mutation was found in 7 of 9 (77.8%). PIM1 mutations and PRDM1 mutations were seen each in 3 (33.3%). VAFs were higher in cfDNA than in tdDNA (mean, 45.6% vs. 3.7%; P=0.0006, Figure 3). Four mutations in 3 patients were detected only in cfDNA. On the other hand, all variants detected in tdDNA were confirmed in cfDNA. ddPCR assay for the L265P MYD88 mutation with all 46 samples including tdDNA and cfDNA revealed the L265P MYD88 mutation in 24/46 (52.2%) samples from 16/27 (59.3%) IVLBCL patients. Furthermore, sequential analysis of cfDNA was performed in one of the MYD88 -mutated patients. The L265P MYD88 mutation was detected (VAF=30%) in cfDNA collected 17 weeks before the diagnosis of IVLBCL, whereas the mutation was not detected in DNA derived from the bone marrow sample that was collected at the same timing and resulted in the negative pathological finding.[Conclusion] Mutations in MYD88 and CD79B are frequent in IVLBCL . Targeted sequencing suggested that tumor cell-derived DNA is abundant in serum/plasma. Liquid biopsy to detect L265P MYD88 and Y196 CD79B may provide a powerful tool for approaching diagnosis of IVLBCL. [Display omitted] DisclosuresChiba:Nippon Shinyaku: Honoraria, Research Funding.

The Implications of Endoplasmic Reticulum Stress Response in the Prognosis and Pathogenesis of Myelodysplastic Syndrome

Background and PurposeMyelodysplastic syndrome (MDS) is a group of heterogeneous disorders characterized by ineffective hematopoiesis and risk of leukemia transformation. The exact pathophysiology of MDS remains incompletely defined. Endoplasmic reticulum (ER) stress response is induced when environmental stimuli and metabolic changes of cells cause derangement of normal ER functions and accumulation of unfolded proteins within it. ER stress response has been shown to be activated in many types of cancer including leukemia. Its activation is thought to confer survival benefit to cancer cells and is related to resistance to anticancer therapies. However, whether ER stress response plays a role in the pathogenesis or affects the clinical and genetic presentations of MDS is still unknown.Material and MethodsA total of 176 adult patients with newly diagnosed primary MDS at the National Taiwan University Hospital and 20 healthy marrow donors who had enough cryopreserved bone marrow cells and complete clinical and genetic information for analysis were enrolled. Diagnosis and classification of MDS were made according to the WHO 2008 classification. We used Affymetrix Human Transcriptome Array 2.0 array of RNA to profile the global gene expression. The probe intensity data (CEL files) and probe set summarization files (CHP files) were generated and analyzed by Affymetrix GeneChip Command Console (AGCC) and Transcriptome Analysis Console (TAC) software. We used the Gene Set Enrichment Analysis (GSEA) tool to comprehensively test the roles of the ER stress pathways in MDS.ResultsAmong the 176 MDS patients, 42 had refractory cytopenia with unilineage dysplasia (RCUD, 23.9%), 13 refractory anemia with ring sideroblasts (RARS, 7.4%), 36 refractory cytopenia with multilineage dysplasia (RCMD, 20.5%), 9 refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS, 5.1%), 32 refractory anemia with excess blasts-1 (RAEB1, 18.2%), and 44 refractory anemia with excess blasts-2 (RAEB2, 25.0%). Patients with each subtype of MDS had significantly different expression in more than 8000 probe sets of coding genes compared with healthy donors. In contrast, the differences were much less remarkable among the subgroups of MDS, implying a common theme of global gene expression profile among subtypes of MDS and an intrinsic genetic difference between MDS and normal controls. The differentially expressed probe sets between MDS patients and healthy donors were explored by database search and literature review. Among the perturbed biological pathways, we focused on the ER stress response for further study. GSEA revealed a significant dysregulation of ER stress-associated genes in MDS (GSEA enrichment score=0.58; P=0.035; Figure 1), indicating a crucial role of ER stress in the pathogenesis of MDS. We noted 17 genes which were up-regulated in MDS patients in our array data, including BAG5, ATG17, BECN1, CALR, CANX, TRAF2, EIF2S1, PKR, SIAH1, SIAH2, S1P, S2P, EDEM3, PDIA6, GRP94, AMPK, and XBP-1 . On the contrary, GADD34, which inhibits ER stress response, was down-regulated in the MDS patients.By univariate analysis, 10 (BAG5, CALR, CANX, GRP94, EDEM3, PDIA6, SIAH1, S1P, S2P, and XBP1) of the 17 up-regulated genes in MDS patients were associated with shorter overall survival, and 7 (CALR, CANX, S2P, EDEM3, PDIA6, XBP1, and TRAF2)of them were associated with shorter time to leukemia transformation (all with P<0.05). We used the sum of the expression levels of the five genes with the strongest associations with shorter overall survival, BAG5, CALR, PDIA6, EDEM3, and XBP1 (Figure 2), to establish an “ER stress score”. Higher ER stress score was associated with shorter overall survival (Hazard ratio = 2.208, P= 0.003) and shorter time to leukemia transformation (Hazard ratio = 3.176, P= 0.004) in multivariate analysis independent of other well known prognostic factors. (Figure 3A, 3B, and Table 1). The prognostic significance of the score was also confirmed in another 31 MDS patients. (P=0.046, Figure 3C)ConclusionSeveral important genes related to the ER stress response have higher expression levels in MDS patients compared with healthy donors and their up-regulations are associated with shorter overall survival and time to leukemia transformation. This finding indicated that deregulation of ER stress response can predict MDS prognosis and it may be involved in the pathogenesis of MDS. [Display omitted] DisclosuresTang:Celgene International Sàrl: Research Funding. Yao:Celgene International Sàrl: Research Funding. Li:Celgene International Sàrl: Research Funding. Huang:Celgene International Sàrl: Research Funding. Chen:Celgene International Sàrl: Research Funding. Ko:Celgene International Sàrl: Research Funding. Hsu:Celgene International Sàrl: Research Funding. Wu:Celgene International Sàrl: Research Funding. Lin:Celgene International Sàrl: Research Funding. Tsai:Celgene International Sàrl: Research Funding. Tien:Celgene International Sàrl: Research Funding.

Bone Marrow Infiltrating T-Cell Signatures Following Allo-HCT for Adult Acute Lymphoblastic Leukaemia Differentiate between Patients in Continuous CR and Those with Persistent Minimal Residual Disease or Relapse

Allogeneic stem cell transplantation (allo-HCT) has a proven survival benefit in adult acute lymphoblastic leukaemia (ALL) and remains the most commonly applied immunotherapy. The mechanism of graft-versus-leukaemia (GVL) in allo-HCT relies upon donor T cell immunity. After contributing to initial control of ALL, allo-reactive anti-tumour T cells may either persist, thus providing continuing immune surveillance, or undergo deletion/loss of function as a result of both extrinsic regulation and/or exhaustion. Therefore, there is a clear need to identify and track T cells with potential anti-ALL reactivity. UKALL14 (ISRCTN66541317) is an ongoing clinical trial for adults with de novo ALL in the UK in which all patients over 40 years are regarded as ‘high risk’ and are assigned to reduced intensity-conditioned allo-HCT. Minimal residual disease (MRD) monitoring - by immunoglobulin heavy chain/ T cell receptor quantification - of bone marrow and peripheral blood multilineage chimerism are assessed at regular intervals for 2 years post allo-HCT.We hypothesised that it would be possible to differentiate bone marrow infiltrating T-cell (BiL-T) signatures in patients in complete molecular remission versus those with MRD or frank clinical relapse. We have evaluated the feasibility of performing gene expression profiling on BiL-T cells from cryopreserved bone marrow samples of patients following allo-HCT and correlated this information with clinical outcome data.BiL-T were flow-sorted using the BD FACSAria ™ Fusion into CD3+,TCRab+, CD4+ and CD3+,TCRab+, CD8+. After extraction of total mRNA, samples with an RNA integrity index (RIN) >7 were subjected to RNA sequencing using the Illumina Hiseq. Data were analysed using differential expression gene analysis (DeSeq 2) and gene set enrichment analysis (GSEA). The Human Immunology V2 panel on the NanoString nCounter® SPRINT Profiler was used for gene expression profiling of samples with adequate RNA but a RIN <7. Gene expression data was correlated with molecular MRD data and other clinical parameters, including T cell chimerism where available.Both CD4+ and CD8+ subsets of BiL-T were successfully sorted from all 15 cryopreserved samples. DeSeq2 and GSEA of the RNAseq data was performed for CD4+TCRab+ BiL (n=5; 3 MRD- and 2 MRD+/relapse) (Figure 1) and CD8+TCRab+ (n=6; 4 MRD- and 2 MRD+/relapse). Despite the modest number of samples in this feasibility study, CD4+ T cells from patients with relapsing or MRD+ ALL showed significantly increased expression of genes associated with terminal differentiation (e.g. LAG3, GZMK); by contrast, there was lower expression of genes associated with memory differentiation and proliferative fitness (e.g. MYC, CCR7), a finding linked to increased pathway enrichment for PD-1 and telomere maintenance. CD8+ T cells from patients with relapsing or MRD+ ALL also demonstrated a terminally differentiated signature (e.g. increased expression of KLRC1 encoding NKG2A and lower expression of CCR7). GSEA showed enrichment for pathways linked to oxidative phosphorylation and interaction with non-haematopoetic stroma). We also analysed n=9 CD8+TCRab+ samples (n=4 MRD- and n=5 MRD+ or relapse) by Nanostring. These groups could also be segregated on the basis of genes representing pathways related to T cell survival, death receptor signalling and TCR signalling; in each case, CD8+ T cells from MRD+ or relapsing patients were characterised by a hypo-functional and apoptosis-prone signature.Our data show features suggestive of T cell exhaustion and altered metabolomes in patients with residual ALL post-allo-HCT, whether they are MRD+ or in frank clinical relapse; these features were not present in patients in continuous remission. To further test the hypothesis that T cell dysfunction significantly compromises GVL efficacy, we intend to perform transcriptional profiling of BiL-T in a wider cohort of UKALL14 patients who underwent allo-HCT. Since clinical strategies for reversal of T cell exhaustion including donor lymphocyte infusion (DLI) and potential PD-1 blockade already exist, we hope the impact of this project will be to determine precisely when and in whom to use these strategies during therapy for adult ALL. We expect that our findings will have relevance to the wider field of immunotherapy for ALL including blinatumomab and CAR-T approaches. [Display omitted] DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.