scholarly journals Single-Cell Analyses Identify Transcriptional Characterizations of Subsets Associated with Efficacy and Toxicity for CAR-T Immunotherapy in B-ALL Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4815-4815
Author(s):  
Mengyi Du ◽  
Heng Mei ◽  
Chenggong Li ◽  
Yinqiang Zhang ◽  
Lu Tang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Single Cell ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Mrna Sequencing ◽  
Car T ◽  
Mononuclear Bone Marrow Cells ◽  
Marrow Cells

Abstract Background The development of mRNA sequencing has contributed greatly to the mechanism exploration in hematologic malignancies disease. With the advent of revolutionized single-cell mRNA sequencing (scRNA-seq), it is now possible to characterize every subset of expression programs and functional states in a comprehensive and unbiased manner. Here, we present a systematic evaluation of engineered chimeric antigen receptor T (CAR-T) products and patient bone marrow profiles in terms of primary resistance and severe cytokine release syndrome (CRS) at the single-cell level. Methods Using single-cell mRNA sequencing in conjunction with flow cytometry (FCM), we performed characterization of CD19-targeted CAR-T and mononuclear bone marrow cells from 4 on-trial B acute lymphoblastic leukemia (B-ALL) patients (NCT02965092). Bioinformatics analysis was utilized to explore diversity between patients with different grades of response or CRS. Basing on marker genes, CAR-T products were divided into four groups, which were double-positive T (DPT), CD4 positive T (CD4), CD8 positive T (CD8), and double-negative T (DNT) cells. Meanwhile, both the mononuclear bone marrow cells before and after CAR-T infusion were grouped into six clusters, which were B-ALL, stem, progenitor, B, T, and myeloid cells. The expression and enrichment analyses results were calculated by R (version 3.6.3) and then verified in a 22-sample conventional transcription sequencing cohort of the same clinical trial. Patient efficacy was assessed by the national comprehension cancer network guidelines version 2.2020 for acute lymphoblastic leukemia, and CRS was graded by CTCAE 5.0. Results By FCM detection, the variances of CAR-T infusion products between patients with different clinical outcomes were limited, and nor did mononuclear bone marrow cells. The scRNA sequencing results showed that distinct CAR-T and bone marrow cell subsets indicated differentiated expression in proliferation, cytotoxicity, and intercellular signaling pathways. Expression differentiation variances in CAR-T infusion products were minor than in mononuclear bone marrow cells. CD8+ CAR-T products of complete response (CR) patients were still significantly enriched in pathways such as cell killing (p adjust=0.0012), antigen processing and presentation (p adjust=0.0027), and cell cycle (p adjust=0.0231), exhibiting greater immune function when compared with no response patients. Also, DPT CAR-T products of the non-CRS patients were meaningfully enriched in negative regulation of cytokine production pathway (p adjust=0.0127) when compared with CRS ones. In mononuclear bone marrow cells, B-ALL cells before CAR-T treatment of CR patients presented negatively in cell-cycle (p adjust=0.0019), leading to a low malignant cell proliferation level; and stem-progenitor cells after CAR-T treatment of CR patients showed a stronger ability of neutrophil activation (p adjust<0.0001). As with comparisons between CRS and non-CRS, B-ALL cells before infusion manifested a cell cycle arrest profile (p adjust<0.0006) in non-CRS patients, whereas the immune cells at the same time point were enriched in positive regulation of cell cycle process (p adjust=0.0002). Conclusions Through single-cell RNA-seq profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the cell cycle, immune phenotype, and metabolic profiles of subsets during CAR-T therapy, providing a mechanistic basis for ameliorating clinical outcomes and individualized management. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Bafilomycin A1 Targets Both Autophagy and Apoptosis Pathways in Pediatric t(1;19) Pre-B Acute Lymphoblastic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3699-3699
Author(s):  
Na Yuan ◽  
Lin Song ◽  
Suping Zhang ◽  
Weiwei Lin ◽  
Yan Cao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Western Blotting ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Primary Cells ◽  
Bafilomycin A1 ◽  
Low Concentration

Abstract The t (1; 19) subtype leukemia accounts for a quarter of pre-B acute lymphoblastic leukemia (ALL) and up to 5% of all ALL patients. Despite plausible remission rate, current treatment regimen on the pediatric pre-B ALL is associated with side effects and CNS relapse, which poses the need for more effective and safer drugs. Bafilomycin A1 (Baf-A1) is known as an inhibitor of late phase of autophagy by inhibiting fusion between autophagosomes and lysosomes as well as by inhibiting lysosomal degradation. Here we show that Baf-A1 of low concentration (1 nM) effectively and specifically inhibits and kills the pre-B ALL cells. E2A/Pbx1 fusion gene positivepre-B ALL 697 cells were used for In vitro experiments. The results of flow cytometry analysis and western blotting showed that Baf-A1 induced cell cycle arrest and proliferation inhibition of ALL cells by upregualting cell cycle negative regulators and downregulating cell cycle positive regulators. In contrast, AML and CML cell lines were insensitive to Baf-A1 treatment. Western blotting and confocal observation on protein LC3 also showed that Baf-A1 at 1 nM blocked basal autophagic flux. Baf-A1 treatment activated mTOR signaling and induced the formation of Becn1–Bcl-2 complex to inhibit the induction of autophagy. Furthermore, apoptosis was induced in ALL cells treated with Baf-A1 for 72 h. However, procaspase-3 and poly-(ADP-ribose) polymerase (PARP) were not cleaved in these cells. We observed that AIF relocalized to the nucleus after 72h Baf-A1 treatment by confocal and immunoblotting. Knockdown of AIF significantly attenuated apoptosis induced by Baf-A1. These data suggest that Baf-A1 targets mitochondria membrane to trigger apoptosis via AIF pathway. In the in vivo experiment, Baf-A1 treatment extended survival and improved pathology of 697 xenograft mice, and significantly reduced the E2A/PBX1 positive leukemia cells in the bone marrow of mice. In vivomouse toxicity assay confirms Baf-A1 as a safe compound. The bone marrow cells of pre-B ALL leukemia patients were sorted against CD133+CD19+ markers, and treatment with Baf-A1 induced a clear inhibition on the CD133+CD19+ primary cells with a significant increased cell death in the sorted B-ALL patient samples. Conversely, Baf-A1 had no inhibitory effect on the bone marrow cells isolated from acute myeloid leukemia patients and healthy people. In summary, Baf-A1 treatment at low concentration effectively and specifically inhibited autophagy by activating mTOR and inducing beclin1-Bcl-2 interaction and induced AIF-dependent apoptosis in t (1; 19) pre-B ALL 697 cells. In the pre-B ALL xenograft mouse model, Baf-A1 specifically targets the leukemia cells while sparing normal cells. More importantly, Baf-A1 potently inhibits and kills the primary cells from pediatric pre-B ALL patients both at initial diagnosis and relapse without compromising normal human hematopoietic cells, all proposing Baf-A1 as a promising drug candidate for this pre-B ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals DNA Methylation Analysis of Bone Marrow Cells at Diagnosis of Acute Lymphoblastic Leukemia and at Remission

PLoS ONE ◽  
2012 ◽  
Vol 7 (4) ◽  
pp. e34513 ◽  
Author(s):  
Jessica Nordlund ◽  
Lili Milani ◽  
Anders Lundmark ◽  
Gudmar Lönnerholm ◽  
Ann-Christine Syvänen
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Methylation Analysis ◽  
Dna Methylation Analysis ◽  
Marrow Cells

scholarly journals Immunoglobulin gene rearrangements in remission bone marrow specimens from patients with acute lymphoblastic leukemia

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 835-838 ◽  
Author(s):  
BA Zehnbauer ◽  
DM Pardoll ◽  
PJ Burke ◽  
ML Graham ◽  
B Vogelstein
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Clinical Remission ◽  
Recombinant Dna ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Chain Gene ◽  
Immunoglobulin Gene ◽  
Marrow Cells

Abstract Recombinant DNA probes for the joining (JH) segment of the immunoglobulin heavy chain gene were used to detect molecular rearrangements of this gene in the DNA of bone marrow cells obtained during remission of acute lymphoblastic leukemia (ALL). This molecular approach was optimized and found to exceed the sensitivity of conventional morphologic screening for detecting residual leukemia cells; one leukemic cell in 500 normal nucleated bone marrow cells was easily detected using this approach. In the present study, bone marrow from three of seven patients in complete clinical remission (defined morphologically) contained leukemic cells in these proportions. This analysis may be of use in evaluating the status of clinical remission in selected ALL patients.

scholarly journals Identification of the Novel Fusion Gene, SPAG9-JAK2, in Adolescent Acute Lymphoblastic Leukemia (ALL) with t(9;17)(p24;q21), and Its Association with Gene Alterations Involved in Ph1/BCR-ABL1-like ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5356-5356
Author(s):  
Machiko Kawamura ◽  
Tomohiko Taki ◽  
Kentarou Ohki ◽  
Yasuhide Hayashi
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Tyrosine Kinase ◽  
Bone Marrow Cells ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Snp Array ◽  
Fusion Transcript ◽  
Marrow Cells

Abstract Introduction: JAK2, which is located on chromosome 9p24, encodes a cytoplasmic tyrosine kinase involved in cytokine-mediated signal transduction, and is fused to 10 partner genes in hematological malignancies, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia, atypical chronic myelogenous leukemia, myelodysplastic syndrome/ myeloproliferative neoplasms (MPN), and Hodgkin lymphoma with 9p24 translocations. Furthermore, a somatic acquired activating mutation of JAK2 (V617F) was noted in a majority of patients with MPN, and mutations at R683 of JAK2 was detected not only in ALL with Down syndrome, but also in high-risk pediatric B-cell progenitor (BCP)-ALL. Case presentation: A 14-year-old boy presenting with a persistent fever was admitted to our hospital because of hyperleukocytosis and thrombocytopenia. Cytogenetic analysis demonstrated the 46,XY,t(9;17)(p24;q21) in 17 of 20 bone marrow cells. He was classified as extremely high risk BCP-ALL with myeloid marker and treated on TCCSG L95-14 HEX/SCT protocol. He obtained a complete remission after the induction therapy, relapsed after 7 months. Although he underwent an auto bone marrow transplant because he had no matched donor, 19 months after the initial diagnosis, he died due to progressive disease. Materials&Methods: To characterize the breakpoints, FISH analysis was performed on metaphase chromosome using BAC clones closely flanking JAK2. Genomic DNA and total RNA were isolated from bone marrow cells at diagnosis and relapse. For the mRNA-Seq sample preparation, sequencing libraries were generated according to the standard Illumina protocol for high-throughput sequencing. Using a HiSeq 2000 sequencer, 100 bp-paired end reads were obtained. Mapping reads to genes were performed by using a Bowtie software and fusion transcript discovery was done by a deFuse data analysis software. We evaluated DNA copy number changes by multiplex ligation-dependent probe amplification (MLPA) analysis and Single nucleotide polymorphism (SNP) array analysis. Results: FISH showed a fusion signal on normal chromosome 9, and split signals on der(9) and der(17). We identified a novel JAK2 fusion gene by paired-end mRNA-seq. This fusion transcript was identified. RT-PCR followed by direct sequencing confirmed the in-frame fusion of SPAG9 exon 26 and JAK2 exon 19. Moreover, the homozygous deletion of CDKN2A, TRG, TRA/D, and IGH and hemizygous deletion of CDKN2B, PAX5, BTG1, CDK6, TRB, ADARB2, IGL and IKZF1were identified by MLPA and SNP array. Discussion: This ALL having both rearrangement activating tyrosine kinase and genomic lesions affecting lymphoid transcription factors is similar to the Philadelphia chromosome (Ph1)/BCR-ABL1 like ALL subgroup. In conclusion, we have identified SPAG9 as a novel fusion partner of JAK2 gene in adolescent BCP-ALL as a consequence of a t(9;17)(p24;q21). The further examination of this fusion gene may be worthwhile to consider that JAK2 would be a good target for treatment in refractory ALL patients with rearrangements in JAK2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Interrelationship of immunologic characteristics, proliferation pattern, and prednisone sensitivity in acute lymphoblastic leukemia of childhood

Blood ◽  
1979 ◽  
Vol 53 (5) ◽  
pp. 883-891
Author(s):  
JA Bakkeren ◽  
GA de Vaan ◽  
HF Hillen
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Phase Distribution ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Sheep Erythrocyte ◽  
Cycle Phase ◽  
Cell Cycle Phase Distribution ◽  
Wbc Count ◽  
Marrow Cells

In children with acute lymphoblastic leukemia (ALL) the effect of prednisone therapy on the cell-cycle phase distribution of leukemic bone marrow cells was determined with pulse cytophotometry at the time of diagnosis. Also, the interrelationship with the presence of the sheep erythrocyte receptor as a marker for T cells was investigated. In 17 or 21 patients prednisone treatment caused a decrease in the percentage of cells in the S+G2+M phases. In 11 of 12 adult patients with ALL the same result was obtained. In a group of 31 children with ALL, 6 cases of T-cell ALL occurred in combination with intermediate or high WBC counts. The 3 patients with the highest E-rosetting percentages had rather high proportions of bone marrow cells in the S+G2+M phases. No correlation could be established between the WBC count or the E-rosetting percentage and the chance for complete remission.

scholarly journals Interrelationship of immunologic characteristics, proliferation pattern, and prednisone sensitivity in acute lymphoblastic leukemia of childhood

Blood ◽  
1979 ◽  
Vol 53 (5) ◽  
pp. 883-891 ◽  
Author(s):  
JA Bakkeren ◽  
GA de Vaan ◽  
HF Hillen
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Phase Distribution ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Sheep Erythrocyte ◽  
Cycle Phase ◽  
Cell Cycle Phase Distribution ◽  
Wbc Count ◽  
Marrow Cells

Abstract In children with acute lymphoblastic leukemia (ALL) the effect of prednisone therapy on the cell-cycle phase distribution of leukemic bone marrow cells was determined with pulse cytophotometry at the time of diagnosis. Also, the interrelationship with the presence of the sheep erythrocyte receptor as a marker for T cells was investigated. In 17 or 21 patients prednisone treatment caused a decrease in the percentage of cells in the S+G2+M phases. In 11 of 12 adult patients with ALL the same result was obtained. In a group of 31 children with ALL, 6 cases of T-cell ALL occurred in combination with intermediate or high WBC counts. The 3 patients with the highest E-rosetting percentages had rather high proportions of bone marrow cells in the S+G2+M phases. No correlation could be established between the WBC count or the E-rosetting percentage and the chance for complete remission.

scholarly journals Classification of bone marrow cells in the diagnosis of acute lymphoblastic leukemia

2021 ◽  
Vol 2058 (1) ◽  
pp. 012043
Author(s):  
V G Nikitayev ◽  
A N Pronichev ◽  
N N Tupitsin ◽  
V Yu Selchuk ◽  
V V Dmitrieva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Automated Classification ◽  
Classification Methods ◽  
Minimal Residual ◽  
Marrow Cells

Abstract The paper presents approaches to automated classification of bone marrow cells in the diagnosis of acute lymphoblastic leukemia and minimal residual disease using image recognition procedures. The classification methods that show the best accuracy in the recognition of eight types of bone marrow cells were experimentally determined. Recommendations for their use are given.

scholarly journals Immunoglobulin gene rearrangements in remission bone marrow specimens from patients with acute lymphoblastic leukemia

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 835-838 ◽  
Author(s):  
BA Zehnbauer ◽  
DM Pardoll ◽  
PJ Burke ◽  
ML Graham ◽  
B Vogelstein
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Clinical Remission ◽  
Recombinant Dna ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Chain Gene ◽  
Immunoglobulin Gene ◽  
Marrow Cells

Recombinant DNA probes for the joining (JH) segment of the immunoglobulin heavy chain gene were used to detect molecular rearrangements of this gene in the DNA of bone marrow cells obtained during remission of acute lymphoblastic leukemia (ALL). This molecular approach was optimized and found to exceed the sensitivity of conventional morphologic screening for detecting residual leukemia cells; one leukemic cell in 500 normal nucleated bone marrow cells was easily detected using this approach. In the present study, bone marrow from three of seven patients in complete clinical remission (defined morphologically) contained leukemic cells in these proportions. This analysis may be of use in evaluating the status of clinical remission in selected ALL patients.

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