MHH-TALL1, a T-ALL Cell Line Expressing a Novel BCR-ABL Transcript.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4390-4390
Author(s):  
Hilmar Quentmeier ◽  
Jan Cools ◽  
Roderick A.F. MacLeod ◽  
Peter Marynen ◽  
Cord C. Uphoff ◽  
...  
Keyword(s):  
Cell Line ◽  
Kinase Inhibitors ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Weak Band ◽  
Fusion Transcripts ◽  
Pcr Conditions

Abstract The Philadelphia chromosome (Ph) is the result of the chromosomal translocation t(9;22)(q34;q11), leading to the BCR-ABL fusion gene. The Ph chromosome is the hallmark of chronic myeloid leukemia, but is also detected in acute lymphoblastic leukemia (ALL), particularly in adults. The majority of Ph-positive ALL cases belong to the category of B-cell precursor ALL, whereas Ph-positive T-ALL cases are rather rare. The MHH-TALL1 cell line was established from the peripheral blood of an 11-year-old boy with T-ALL in 1993. PCR analysis of primary tumor cells failed to reveal the existence of a BCR-ABL fusion. Interestingly however, conventional cytogenetics and fluorescence in situ hybridization performed on the cell line showed a 3-way t(1;9;22)(q32;q34;q11) rearrangement effecting Ph formation. As with the patient, standard RT-PCR of the various known BCR-ABL fusion transcripts was negative in the cell line. However, a weak band, about 600 bp larger than the usual e1-a2 BCR-ABL transcript was detected, and subsequently confirmed on reanalysis after optimizing PCR conditions. Sequencing of the RT-PCR product showed that MHH-TALL1 expressed an e6-a2 BCR-ABL fusion transcript. Northern and Western blot analyses demonstrated that the BCR-ABL gene products were expressed at very low levels only. It may be speculated that the presence of this novel BCR-ABL variant has been overlooked in previous analyses, because (i) PCR conditions used to screen for BCR-ABL fusion transcripts were not optimal to detect this variant and (ii) a weak signal running at the “wrong” size might have been neglected. In summary, we report a novel BCR-ABL fusion variant expressed in a T-ALL cell line. Our data raise the intriguing possibility that some BCR-ABL negative cases may express the novel e6-a2 transcript described herein. This might have an impact on treatment of the respective patients with ABL kinase inhibitors.

scholarly journals Philadelphia Chromosome-Positive Leukemia in the Lymphoid Lineage—Similarities and Differences with the Myeloid Lineage and Specific Vulnerabilities

2020 ◽  
Vol 21 (16) ◽  
pp. 5776 ◽  
Author(s):  
Lukasz Komorowski ◽  
Klaudyna Fidyt ◽  
Elżbieta Patkowska ◽  
Malgorzata Firczuk
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Great Majority ◽  
Metabolic Reprogramming ◽  
Chromosome 9 ◽  
Treatment Protocols ◽  
Resistant Disease

Philadelphia chromosome (Ph) results from a translocation between the breakpoint cluster region (BCR) gene on chromosome 9 and ABL proto-oncogene 1 (ABL1) gene on chromosome 22. The fusion gene, BCR-ABL1, is a constitutively active tyrosine kinase which promotes development of leukemia. Depending on the breakpoint site within the BCR gene, different isoforms of BCR-ABL1 exist, with p210 and p190 being the most prevalent. P210 isoform is the hallmark of chronic myeloid leukemia (CML), while p190 isoform is expressed in majority of Ph-positive B cell acute lymphoblastic leukemia (Ph+ B-ALL) cases. The crucial component of treatment protocols of CML and Ph+ B-ALL patients are tyrosine kinase inhibitors (TKIs), drugs which target both BCR-ABL1 isoforms. While TKIs therapy is successful in great majority of CML patients, Ph+ B-ALL often relapses as a drug-resistant disease. Recently, the high-throughput genomic and proteomic analyses revealed significant differences between CML and Ph+ B-ALL. In this review we summarize recent discoveries related to differential signaling pathways mediated by different BCR-ABL1 isoforms, lineage-specific genetic lesions, and metabolic reprogramming. In particular, we emphasize the features distinguishing Ph+ B-ALL from CML and focus on potential therapeutic approaches exploiting those characteristics, which could improve the treatment of Ph+ B-ALL.

Establishment of a NOD/SCID/γc−/−-Dependent Lymphoid Leukemia Cell Line, D593, with Novel RUNX1-LAF4 Fusion Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4276-4276
Author(s):  
Akihiro Abe ◽  
Manabu Ninomiya ◽  
Shizuka Imagama ◽  
Momoko Suzuki ◽  
Fumihiko Hayakawa ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Cell Receptor ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Pcr Analysis ◽  
Lymphoid Leukemia

Abstract We established a NOD/SCID/γc−/−(NOG mouse)-dependent human lymphoid leukemia cell line, D593, by repeated xenotransplantation of pediatric T-cell acute lymphoblastic leukemia cells with the translocation t(2;21). The cell line, D-593, could be serially transplanted from mouse to mouse over a 2-year period. D593 had the same immuno-phenotype as the original leukemia cells: positive for CD2, 5, 7, 14, and 34, and negative for CD3, 4, 8, 19, and 41a. Cytoplasmic CD3 was positive and the rearrangement of T-cell receptor was detected by Southern blot analysis. A previously unreported translocation of t(2;21)(q11;q22) was observed in both the original patient sample and D593. The split signal of RUNX1 was detected by fluorescence in site hybridization in D593 indicating the involvement of RUNX1. Using 3′-RACE and RT-PCR analysis, we identified novel chimeric transcripts of RUNX1-LAF4 joining exon 7 of RUNX1 to exon 4 of LAF4. In the transplanted NOG mice, D593 homed into the trabecular endosteal region of bone marrow (BM), and proliferated from the endosteum to medulla. At the late stage of engraftment, the BM was filled with human lymphoblasts and metastases into the trabecular of the spleen and Glisson’s sheath of the liver were also observed. These findings suggest that D593 is a useful cell line to study not only the leukemia-related biology of RUNX1-LAF4 but also the novel therapeutic model against core-binding factor (CBF) leukemia.

Prospective Multicentric Molecular Study for Poor Prognosis Fusion Transcripts at Diagnosis in Adult ALL Patients - The LALA94 Experience.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4476-4476
Author(s):  
Jean A. Gabert ◽  
Christophe Picard ◽  
Sandrine Hayette ◽  
Christelle Bilhou-Nabera ◽  
Jean-Michel Cayuela ◽  
...  
Keyword(s):  
Clinical Data ◽  
Kinase Inhibitor ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Molecular Study ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Multicentric Study ◽  
Gene Transcripts ◽  
New Guidelines

Abstract From 1994 to 2000, 984 adults aged from 15 to 55 years with newly diagnosed Acute Lymphoblastic Leukemia (ALL) were eligible for randomization in the multicentric LALA-94 clinical protocol. The t(9;22), t(1;19) and t(4;11) translocations corresponding to BCR-ABL, E2A-PBX1 and MLL-AF4 fusion gene transcripts respectively, were considered as independent poor prognostic factors. Standardized RT-PCR analysis of these fusion gene transcripts were performed by 17 laboratories in order to provide data before the second randomization (J35) on 787 patients. In this multicentric study, validated data were available for therapeutic stratification for 91% of these analysed patients. No false positive RT-PCR was reported. Secondarily to retrospective BCR-ABL FISH, few false BCR-ABL negative RT-PCR were identified, leading to the design of new BIOMED-1 primers for b3-a3 junctions detection. Moreover, the LALA-94 study allowed to define new guidelines for molecular analysis at diagnosis. Like in other studies, the BCR-ABL transcript was found to be the most frequent molecular abnormality in B-ALL (24%) whereas MLL-AF4 and E2A-PBX1 were detected in 5% and 3.5% of B-ALL, respectively. Epidemiological and clinical data of MLL-AF4 and E2A-PBX1 were concordant with previous publications. Interestingly, because of the large number of reviewed patients, the different BCR-ABL subtypes (M-BCR and m-BCR) were statistically characterized by few clinical data. M-BCR subgroups had a higher age than m-BCR (p= 0.016) and occurs especially during the second semester (p= 0.034). Moreover, the comparison of clinical data at diagnosis of M-BCR variants showed that median age of b3a2 was statistically younger than b2a2 (p= 0.04) and that b3a2 occurs more frequently in man (p= 0.02). For the first time, these data suggest that these BCR-ABL breakpoints: m-BCR and M-BCR and also b2a2 and b2a3, are secondary to different physio-oncologic mechanisms even if therapeutic regimens including the same targeted therapy (tyrosine kinase inhibitor) for all BCR-ABL variants is the rule today.

Molecular Heterogeneity of the FIP1L1-PDGFRA Fusion Gene in Chronic Eosinophilic Leukemia (CEL) and Systemic Mastocytosis with Eosinophilia (SME): A Study of 43 Cases.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2428-2428 ◽  
Author(s):  
Christoph Walz ◽  
Daniela Cilloni ◽  
Simona Soverini ◽  
Catherine Roche ◽  
Emanuela Ottaviani ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Fusion Gene ◽  
Systemic Mastocytosis ◽  
Collaborative Study ◽  
Fusion Transcript ◽  
Pcr Analysis ◽  
Molecular Heterogeneity ◽  
Rt Pcr ◽  
Fusion Transcripts ◽  
Chronic Eosinophilic Leukemia

Abstract The FIP1L1-PDGFRA fusion gene results from a cytogenetically invisible interstitial chromosomal deletion on chromosome 4q12 and was recently identified as a recurrent molecular abnormality in patients with chronic eosinophilic leukemia (CEL) and systemic mastocytosis with eosinophilia (SME). The pathogenesis of FIP1L1-PDGFRA positive CEL/SME is similar to BCR-ABL positive chronic myeloid leukemia (CML) with constitutively increased tyrosine kinase activity of the fusion protein and excellent response to treatment with imatinib. The breakpoints within FIP1L1 are variable and a number of different exons are fused to a truncated PDGFRA exon 12. However, the numbers of sequenced fusion transcripts in single reports have been too small for a more detailed analysis of the anatomy and relative frequency of the different fusion transcripts. We therefore sought to collect data from FIP1L1-PDGFRA positive patients from several laboratories across Europe (France, Germany, Italy, UK) in a collaborative study within the workpackage "Minimal residual disease" of the European LeukemiaNet. A total number of 43 FIP1L1-PDGFRA positive cases were identified by RT-PCR. For yet unknown reasons a considerable number of cases were only found to be positive after nested RT-PCR despite adequate sample quality, high leukocyte counts and marked eosinophilia. Possible reasons might be a relatively low proportion of FIP1L1-PDGFRA positive cells, relatively low expression of the fusion transcript and/or relatively rapid fusion transcript degradation; although FIP1L1-PDGFRA was found to be expressed at a level comparable to the ABL control gene in RQ-PCR analysis of EOL-1 cell line. Sequence analysis revealed that all PDGFRA breakpoints fell exclusively within exon 12, thus retaining the entire kinase domain of PDGFRA in all cases. The truncated PDGFRA (p) exon 12 was fused to FIP1L1 (f) exons 9 to 13 (formerly described as 7a, 8, 8a, 9 and 10 - Cools et al., NEJM. 2003;348: 1201–1214): f9p12 (n=1; 2%), f10p12 (n = 10; 22%), f11p12 (n=15; 33%), f12p12 (n=7; 15%), f13p12 (n=10; 22%). An insertion of additional sequences of up to 107 bp was found in 24 patients (56%) leading to an open reading frame in all cases. These sequences were derived from introns of FIP1L1 in 14 cases, from FIP1L1 exon 13 in one case and from 4q33 in one case, the latter indicating a more complex rearrangement. Eight inserts of 2 – 6 bp could not be matched to known sequences because they were too short. An entirely identical fusion transcript resulting from an identical PDGFRA sequence fused to the same FIP1L1 exon without insert was found in 6 patients with a f12p12 fusion transcript and 4 patients with a f10p12 fusion transcript. We conclude that the combination of a great variability of breakpoints within FIP1L1 and PDGFRA plus the insertion of sequences which are variable in length and origin lead to unique FIP1L1-PDGFRA fusion sequences in the majority of patients. This carries important implications for strategies for molecular detection and development of RQ-PCR assays to determine response to imatinib or alternative tyrosine kinase inhibitors.

The Use of ABL and GUS as Control Genes for Normalizing Quantification of Fusion Transcripts by Real Time PCR: Our Experience of More Than 1000 Samples.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4727-4727
Author(s):  
Mohamed Amin Ben Lassoued ◽  
Nathalie Beaufils ◽  
Anderson Dieudonné Loundou ◽  
Christiane Arnaud ◽  
Jean Gabert
Keyword(s):  
Myeloid Leukaemia ◽  
Real Time ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Hypereosinophilic Syndrome ◽  
Myelogenous Leukemia ◽  
Pcr Analysis ◽  
Routine Practice ◽  
Rt Pcr

Abstract Abstract 4727 Introduction Real-time quantitative PCR (RT-PCR) is a sensitive and accurate tool for monitoring leukemic patients by amplification of fusion gene (FG) transcripts. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript. In our laboratory two CG are used: Abelson (ABL) and beta-glucuronidase (GUS). Clinical application for monitoring the treatment of chronic myelogenous leukemia (CML) patients with tyrosine kinase inhibitor makes the quantification of which CG to use very important into the clinical practice today. Objective The aim of this study was to assess the degree of agreement between the GC, compare the expression of GC (type and time of sampling and pathology), try to redefine the limits between the 4 groups of quality (very good, good, poor and very poor quality samples) and to focus on CML patients. Materials and methods A total of 1087 samples (peripheral blood and bone marrow) were obtained from 475 patients from January 2005 to December 2007. This study includes patients with acute myeloid leukaemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukaemia (CML) and Hypereosinophilic syndrome (HES). We collected 366 samples of 41 CML patients between 2002 and March 2009 including 32 CML at diagnosis. RT-PCR analysis was performed using the EAC protocols. Results Comparison between CML, AML, ALL and HES patients showed that ABL and GUS expression did not differ significantly (Bland and Altman method). ABL and GUS gene expressions did not differ significantly between CML patients at diagnosis and the others. Conclusion Based on the literature and our local experience of more than 1000 samples, we propose to use only ABL as CG in routine practice in onco-hematology particularly for CML patients at diagnosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Early Detection of BCR-ABL Fusion Gene of Cerebrospinal Fluid (CSF) by RT-PCR in Relapsed Acute Lymphoblastic Leukemia with Philadelphia Chromosome

2012 ◽  
Vol 43 (suppl 2) ◽  
pp. e33-e37 ◽  
Author(s):  
Na Eun Jang ◽  
Sun Kyung Baek ◽  
Jae-heon Jeong ◽  
Si-Young Kim ◽  
Hwi-Joong Yoon ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Acute Lymphoblastic Leukemia ◽  
Early Detection ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Rt Pcr

Effect of Dasatinib on Genes Related to Mitotic Catastrophe Pathway in Chronic Myeloid Leukemia Cells

2020 ◽  
Vol 20 ◽  
Author(s):  
Sezgi Kipcak ◽  
Buket Ozel ◽  
Cigir B. Avci ◽  
Leila S. Takanlou ◽  
Maryam S. Takanlou ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Fusion Gene ◽  
Philadelphia Chromosome ◽  
K562 Cells ◽  
Mitotic Catastrophe ◽  
Control Group ◽  
Cancer Therapies ◽  
Apoptosis Pathways

Background: Chronic myeloid leukemia (CML), is characterized by a reciprocal translocation t(9;22) and forms the BCR/ABL1 fusion gene, which is called the Philadelphia chromosome. The therapeutic targets for CML patients which are mediated with BCR/ABL1 oncogenic are tyrosine kinase inhibitors such as imatinib, dasatinib, and nilotinib. The latter two of which have been approved for the treatment of imatinib-resistant or intolerance CML patients. Mitotic catastrophe (MC) is one of the non-apoptotic mechanisms which frequently initiated in types of cancer cells in response to anti-cancer therapies; pharmacological inhibitors of G2 checkpoint members or genetic suppression of PLK1, PLK2, ATR, ATM, CHK1, and CHK2 can trigger DNA-damage-stimulated mitotic catastrophe. PLK1, AURKA/B anomalously expressed in CML cells, that phosphorylation and activation of PLK1 occur by AURKB at centromeres and kinetochores. Objective: The purpose of this study was to investigate the effect of dasatinib on the expression of genes in MC and apoptosis pathways in K562 cells. Methods: Total RNA was isolated from K-562 cells treated with the IC50 value of dasatinib and untreated cells as a control group. The expression of MC and apoptosis-related genes were analyzed by the qRT-PCR system. Results: The array-data demonstrated that dasatinib-treated K562 cells significantly caused the decrease of several genes (AURKA, AURKB, PLK, CHEK1, MYC, XPC, BCL2, and XRCC2). Conclusion: The evidence supply a basis to support clinical researches for the suppression of oncogenes such as PLKs with AURKs in the treatment of types of cancer especially chronic myeloid leukemia.

scholarly journals Crizotinib acts as ABL1 inhibitor combining ATP-binding with allosteric inhibition and is active against native BCR-ABL1 and its resistance and compound mutants BCR-ABL1T315I and BCR-ABL1T315I-E255K

2021 ◽  
Author(s):  
Afsar Ali Mian ◽  
Isabella Haberbosch ◽  
Hazem Khamaisie ◽  
Abed Agbarya ◽  
Larissa Pietsch ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Binding Pocket ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Lung Cancer Patients

AbstractResistance remains the major clinical challenge for the therapy of Philadelphia chromosome–positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common “gatekeeper” mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph− cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia–like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.

scholarly journals Chimeric BCR-abl messenger RNA as a marker for minimal residual disease in patients transplanted for Philadelphia chromosome-positive acute lymphoblastic leukemia

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 458-465 ◽  
Author(s):  
GB Gehly ◽  
EM Bryant ◽  
AM Lee ◽  
PG Kidd ◽  
ED Thomas
Keyword(s):  
Clinical Remission ◽  
Messenger Rna ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Fusion Transcript ◽  
Lymphocytic Leukemia ◽  
Pcr Analysis ◽  
Ph Chromosome ◽  
Transplant Complications

We correlated polymerase chain reaction (PCR)-detectable BCR-abl fusion transcripts with cytogenetic status in 24 patients with acute lymphocytic leukemia (ALL). Of 10 Philadelphia chromosome negative (Ph- ) patients, only one was found to exhibit a BCR-abl fusion transcript. Fourteen patients with Ph+ ALL, including eight in clinical remission, exhibited PCR-detectable BCR-abl rearrangements. A detectable Ph chromosome was present in only five of the eight patients in clinical remission. Of the three cytogenetically negative, BCR-abl-positive patients, two eventually succumbed to post-bone marrow transplantation (BMT) relapse. The third died of early transplant complications. Serial PCR analyses were performed on four Ph+ ALL patients in clinical remission who underwent allogeneic BMT. One patient who was PCR negative on post-BMT days 21 and 75 became PCR-positive on day 116 and died in relapse on day 154. One patient was weakly positive for BCR-abl on day 23, negative on day 56, but died of transplant complications on day 124. Two patients exhibited no post-BMT BCR-abl rearrangements and remain well on days 279 and 371. Our findings suggest that PCR analysis may be useful in the early identification of relapse in patients transplanted for Ph+ ALL.

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