scholarly journals Igh-V(D)J NGS-MRD Measurement in Pediatric B-Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4923-4923
Author(s):  
Suying Lu ◽  
Jia Zhu ◽  
Junting Huang ◽  
Juan Wang ◽  
Feifei Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Induction Therapy ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Clinicopathological Characteristics ◽  
Marrow Cells

Abstract Introduction B-acute lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. Early response to induction chemotherapy is one of the important prognostic factors in B-ALL. However, the analytic sensitivity for flow cytometry (FC) is only 10 -4. The feasibility of using next-generation sequencing (NGS) of immunoglobulin for the determination of minimal residual disease (MRD) in B-ALL has been demonstrated. This study aimed to investigate the performance of NGS techniques measuring immunoglobulin heavy chain (IgH)-variable, diversity, and joining (V[D]J) clonal rearrangements compared with FC in detecting MRD for children with B-ALL and to predict the clinical outcome of B-ALL patients. Methods Newly diagnosed younger than 18 years old B-ALL patients who received the treatment strategy of South China children's leukemia Group (SCCLG)-ALL 2016 were recruited. DNA extracted from bone marrow cells at all available time points for each patient was submitted to Simcere diagnostics for sequencing using Illumina NovaSeq platform. We performed IgH V(D)J NGS and FCM on the bone marrow serially obtained at diagnosis (D0), 15 days at induction therapy (D15), 33 days at induction therapy (D33) and then at the end of induction therapy (EOI). We defined MRD positive (MRD +) by IgH V(D)J NGS and FCM as more than 1 blast cell among 10 4 and 10 6 bone marrow cells, respectively. The sensitivity of MRD detection by IgH V(D)J NGS and FCM, and the association of MRD status with clinicopathological characteristics were investigated. Statistical analysis was performed through SPSS Statistics 22. Enumeration data and correlation between MRD data and clinicopathological characteristics were compared by Chi-square test or Fisher's exact test. This trial was registered at www.clinicaltrials.gov as # NCT04977895. Results As of July 27, 2021, 22 patients (median age, 4.5 years; range, 3.0-7.3) were enrolled in the study. Three patients (13.6%) had a t (9;22) translocation consistent with Philadelphia chromosome positive disease. According to risk stratifications, 8 (36.4%), 8 (36.4%), and 2 (9.1%) patients were classified as low risk (LR), intermediate risk (IR), and high risk (HR) groups, respectively. The remaining 4 patients are still under treatment and have not been classified. We identified leukemic IgH clones in 100% of the diagnostic samples and 68.2% (15/22) of the patients were polyclonal. In 11 patients whose samples of all the four timepoints (D0, D15, D33, EOI) have been tested in parallel by FCM and IgH V(D)J NGS, the frequencies of patients with MRD + were 30.4% vs. 90.9% at D15 (P＜0.05) by FCM and IgH V(D)J NGS. IgH V(D)J NGS MRD monitoring could identify MRD + patients with frequency of 45.5% and 18.2% among patients achieved MRD negativity by FCM at D33 (P＜0.05) and EOI (P = 0.46). With an MRD detection limit of 10 -6, 90.9% (10/11), 36.4% (4/11) and 18.2% (2/11) patients were MRD negative by FCM but positive by the NGS test at D15, D33 and EOI, respectively. This suggested that the sensitivity of IgH V(D)J NGS was significantly higher than that of FCM. Correlation of the measured MRD between the two methods in the entire cohort (r = 0.7934, P < 0.0001) as well as in the concordant cases (r = 0.5558, P = 0.0032) was very high. There was a high discordant rate with NGS identifying more patients MRD + at this threshold. Furthermore, NGS MRD was positive but the FCM MRD was negative in 13 samples (P < 0.0001). In addition, positive MRD status of D33 by NGS was significantly associated with the age of B-ALL patients, patients under 6 years more frequently harbored detectable MRD compared with those ≥ 6 years old (87.5% vs. 11.1%, P < 0.01). There was no patient relapsed after a medium follow-up of 10.5 months. Conclusions We demonstrated the higher sensitivity of IgH-V(D)J NGS in MRD detection of B-ALL, which implies that NGS MRD monitoring could be helpful for more accurate risk stratifications and more precise treatment according to risk stratifications. Further study with a larger sample size and a longer follow-up period is need. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Blinatumomab + Ponatinib for Relapsed Ph1-Positive Acute Lymphoblastic Leukemia: The French Experience

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4014-4014 ◽  
Author(s):  
Marie-Anne Couturier ◽  
Xavier Thomas ◽  
Francoise Huguet ◽  
Céline Berthon ◽  
Célestine Simand ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Disease Risk ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Philadelphia Chromosome ◽  
New Drugs ◽  
Molecular Response ◽  
Complete Molecular Response

Abstract Prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph1+ ALL) has been considerably improved since the beginning of the BCR-ABL1 tyrosine kinase inhibitors (TKI) era 20 years ago. However, the prognosis of patients with a refractory/relapsed (including molecular relapse) disease is still very dismal. New drugs or combination of new drugs may improve outcomes of these patients. For example, ponatinib, a 3rd-generation oral TKI, known to have activity against BCR-ABL1 T315I mutations, has shown some efficacy in this context. Similarly, a recent study has reported very encouraging results of the bispecific anti-CD3/CD19 monoclonal antibody blinatumomab as single-agent in refractory/relapsed Ph1+ ALL. Data regarding the efficacy and tolerance of a combination of blinatumomab+ponatinib (blina/pona) are still scarce. This was a retrospective study with the aim to report outcomes of patients receiving a combination of blina/pona for refractory/relapsed Ph1+ ALL in France. Fifteen adults from 8 French centers were identified and data were collected by physicians of each centers, then gathered for the purpose of this study. There were 9 males and 6 females, with a median age of 53 years (range:17-72). All patients, but 2 with blast crisis of chronic myeloid leukemia, had de novo Ph1+ ALL. Four cases had BCR-ABL1 T315I mutation. Patients received the blina/pona combination, either after a first (n=8) or a second (n=7) cytologic relapse. There was no refractory patient in theses series. Previous allograft and autograft has been performed in 7 and 2 patients, respectively. The majority of patients (n=12) had previously received 2 or more lines of TKI. The median time between the first cycle of blina/pona and diagnosis was 14 months (range: 8-40). The median number of blinatumomab cycle (28 mg/day by continuous infusion for 28 days every 6 weeks) administered per patient was 3 (range: 1-6) while ponatinib was concomittantly administered continuously at an initial dose of 45 mg once daily in 11 pts (73%) and 30 mg in 4 pts (27%). Median duration of ponatinib administration was 4 months (range: 1.1-10.9) from first blinatumomab cycle. The toxicity profile was safe: all patients received a complete first cycle without grade 3-4 adverse events. After cycle 1, blinatumomab was stopped in 47% of cases because of neurologic events in 4 and infections in 3; ponatinib was stopped in 33% of cases because of neurologic events in 3, fluid retention in 1 and severe arteriopathy in 1 patient with other vascular disease risk factors. All neurologic events resolved after stopping blinatumomab or ponatinib. The majority of patients were evaluated after one cycle (n=11, after cycle 2 n=3, after cycle 3 n=1). All but one patients (93%) obtained a cytologic complete remission (CR), of whom 12/14 (86%) achieved a complete molecular response. However, 2 patients were documented with CNS relapse at the time of blina/pona evaluation although in bone marrow molecular remission. Both obtained clearance of leukemic blasts after intrathecal infusion of chemotherapy. Then, 5 patients underwent allogeneic transplant (including 2 patients already allotransplanted before blina/pona) and 1 patient received donor lymphocyte infusion. Seven cases pursued maintenance therapy with ponatinib as single agent after stopping blinatumomab. With a median follow-up of 8 months (range: 2.6-30.2) for alive patients, median overall and leukemia-free survivals from first cycle of blina/pona were 8.5 months (range: 1.7-30.2) and 8 months (range: 1.3-30.2), respectively. At last follow-up (July 2018), only 4 relapses had occurred at a median of 3.3 months (range: 1.3-8.3) from first blina/pona cycle and 6 patients had died (3 bacterial infections, 1 fungal infection, 1 secondary cancer and 1 ALL relapse). All alive patients (n=9) but one (cytologic CR but detectable minimal residual disease) are in complete molecular response. Four patients are still under ponatinib medication at last follow-up. The combination of blinatumomab+ponatinib appears effective and tolerable in relapsed Ph1+ALL patients and may replace chemotherapy salvage regimens. The combination should be tested in first-line therapy in the future. Our results have also to be confirmed prospectively on a larger cohort of patients. Disclosures No relevant conflicts of interest to declare.

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

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 Poor Outcome of CRLF2 Rearranged Philadelphia Negative Acute Lymphoblastic Leukemia Adults Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5290-5290 ◽  
Author(s):  
Yasser H Elnahass ◽  
Omar A Fahmy ◽  
Mohamed Abdel Mooti Samra ◽  
Fatma A Elrefaey ◽  
Mahmoud T Bokhary ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Disease Free Survival ◽  
Bone Marrow Aspiration ◽  
Poor Outcome ◽  
Egyptian Patients ◽  
Diagnostic Work

Abstract BACKGROUND: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph'-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1 positive ALL and a poor outcome. Rearrangements of cytokine receptor like factor 2 (CRLF2) are identified in approximately 50% of Ph'-like and 10% of B-other ALL patients. AIM: To identify the incidence of CRLF2 rearrangements and the frequency of relapse in a cohort of B-other Precursor B-ALL adults Egyptian patients who were treated with conventional therapy at National Cancer Institute, Cairo University. METHODS: The routine diagnostic work-up at diagnosis included Bone marrow aspiration, immunophenotyping, karyotyping, fluorescent in situ hybridization (FISH) for BCR-ABL1 and KMT2A (MLL) and CRLF2 rearrangements (IGH-CRLF2 or P2RY8-CRLF2), and molecular analyses of BCR-ABL1 translocations. Patients within the age group 18-25 years received Total XV protocol while patients >25 years received Hoelzer's protocol. RESULTS: Forty patients were included (22 males, 18 females). Median age at diagnosis was 25 years (18 -65). Median TLC was 14.9 x109/L (1.1 - 201), median Hb was 9.9 gm/dl (5-12.9), median platelet count was 47 x 109/L (19-359) and median BM blasts count was 90% (30-100). CRLF2 rearrangement was detected in 4/40 (10%) patients. All CRLF2 positive patients (100%) relapsed at 6 months vs. 11/36 (30%) CRLF2 negative (p<0.001). At 6 months follow up, disease free survival (DFS) was 70% in CRLF2 negative patients vs. 0% in CRLF2 rearranged (p=0.04). At 1 year follow up, overall survival (OS) was 30 (75%) for CRLF2 negative vs. 0 (0%) for CRLF2 positive patients (p=0.01). CONCLUSION: CRLF2 rearrangements constitute a high risk independent prognostic factor in adult precursor B-ALL patients denoting a poor outcome and should be studied in ALL Ph' negative patients. Additional TKI therapy should be included for this category of patients in our protocols to improve outcome. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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.

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