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.

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 Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽  
1978 ◽  
Vol 52 (4) ◽  
pp. 712-718 ◽  
Author(s):  
SD Smith ◽  
EM Uyeki ◽  
JT Lowman
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Lymphoid Cells ◽  
Assay System ◽  
Leukemic Cells ◽  
Specific Cell ◽  
Specific Cell Surface

Abstract An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

scholarly journals Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽  
1978 ◽  
Vol 52 (4) ◽  
pp. 712-718 ◽  
Author(s):  
SD Smith ◽  
EM Uyeki ◽  
JT Lowman
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Lymphoid Cells ◽  
Assay System ◽  
Leukemic Cells ◽  
Specific Cell ◽  
Specific Cell Surface

An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

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.

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

Development of a Myeloproliferative Disease or a T Cell Lymphoblastic Leukemia in a Murine Bone Marrow Transplant Model of NUP214-ABL1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 618-618
Author(s):  
Jennifer L. Rocnik ◽  
Melanie Cornejo ◽  
Benjamin H. Lee ◽  
Rachel Okabe ◽  
Elizabeth McDowell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Leukemic Cells ◽  
Histopathological Analysis ◽  
Myeloproliferative Disease ◽  
Marrow Cells

Abstract Leukemias are often associated with aberrant tyrosine kinase activity that occurs as a result of chromosomal translocations. These mutations are able to confer a proliferative and survival advantage to leukemic cells, as well as cooperate with other mutations that impair cell differentiation, thus leading to the development of leukemia. NUP214-ABL1 is one such recently identified fusion gene that is generated by episomal amplification. The presence of the fusion was recently identified in approximately 6% of patients with T-cell acute lymphoblastic leukemia (T-ALL). By the use of a murine retroviral bone marrow transplantation model we have demonstrated that mice transplanted with NUP214-ABL1 transduced bone marrow cells developed either a myeloproliferative disorder (MPD) with a disease latency of 70 to 118 days or a T cell lymphoblastic leukemia with a disease latency of 115 to 124 days. The myeloproliferative phenotype was characterized by splenomagaly and leukocytosis, and analysis of the histopathology revealed extramedullary hematopoiesis in the liver, lung, kidney and Peyer’s patches, and an increase of peripheral blood neutrophils. Flow cytometry of single cell suspensions from spleen and bone marrow samples of mice with a myeloproliferative phenotype demonstrated an increase of Gr-1+/Mac-1+ cells (approximately 70%). Two of the mice that were transplanted with NUP214-ABL1 transduced bone marrow cells developed T cell lymphomas that were characterized by large thymomas, a phenotype that is consistent with other models of activated tyrosine kinases over long disease latencies. Histopathological analysis of the thymi revealed effacement of normal thymic architecture as well as T cell infiltrate into the surrounding skeletal muscle. In addition, flow cytometric analysis revealed a significant increase in the CD4+/CD8+ T cell population in the thymi of these animals. No disease was observed in secondary transplant recipients following 60 days of observation. In conclusion, these results indicate that NUP214-ABL1 is able to cause either a myeloproliferative disease or a T cell lymphoma over longer latencies in mice, the latter being similar to the phenotype observed in humans with expression of the NUP214-ABL1 fusion. These findings provide a useful model for future experiments to determine if there is a contribution of other mutations together with the NUP214-ABL1 fusion towards the development of a T-ALL phenotype in mice.

Clonal Profile Analysis of Leukemic Progenitors and Diagnosis Blast Cells in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4879-4879
Author(s):  
Marinella Veltroni ◽  
Maddalena Paganin ◽  
Chiara Frasson ◽  
Giulia Fabbri ◽  
Antonio Marzollo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Gene Rearrangements ◽  
Healthy Children ◽  
Cell Precursor ◽  
Clonal Rearrangement

Abstract Recent studies suggest that the majority of malignant cells found in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) arise from a rare population of leukemic progenitors. Little information is available on the presence of clonal rearrangements in cells at the stage of early precursor. To address this issue we analyzed clonality profile of early leukemic precursors sorted by flow-cytometry. Leukemic cells were obtained from bone marrow samples collected at diagnosis from 6 patients with childhood BCP-ALL. Furthermore, bone marrow cells were collected from 3 healthy children who were harvested for bone marrow donation. Three subpopulations of leukemic cells were investigated: total unsorted blasts, the sorted CD34+/CD38−/CD19+, and the sorted CD34+/CD38−/CD19− cells. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements were screened by polymerase chain reaction (PCR) in the sorted populations and in the bulk leukemic cells in order to identify molecular markers of clonal evolution. Sequence analysis was then performed on the N-region. Overall, a total of 38 different Ig/TCR gene rearrangements were identified in the 3 cell populations under study (total blasts, CD34+/CD38−/CD19+, and CD34+/CD38−/CD19−). Of them, 13 (34%) were found in the three populations; 12 (31%) were found in two of the three populations: 7 in total blasts and CD19+ subset, 3 in total blasts and CD19−, 2 in CD19− and CD19+; finally, 13 were found only in one subpopulation: 4 in total blast cell, 5 in CD19+, 4 in CD19−. In all the six patients studied, BCP-ALL progenitors CD34+/CD38−/CD19− and CD19+ and the bulk tumor blasts shared at least one Ig/TCR gene clonal rearrangement with the same N-region. In 5 out of 6 patients at least one rearrangement detected in the BCP-ALL progenitors was undetectable in total blasts. Conversely, in 3 patients the clonal rearrangement observed in the bulk leukemic cells was not identified in any of the two sorted ALL precursor populations. Clonal rearrangement was never detected in the samples from healthy bone marrow donors. Our findings confirm that clonal rearrangement may be detected at the stage of early B-lineage precursor CD34+/CD38−/CD19−, suggesting that leukemic transformation may occur at this stage or even before in BCP-ALL. We plan to extend this observation by repopulating studies in NOD/SCID mice.

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.

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