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1
Gastier-Foster, Julie M., Andrew J. Carroll, Denise Ell, Richard Harvey, I.-Ming Chen, Rhett Ketterling, Aurelia Meloni-Ehrig, et al. "Two Distinct Subsets of dic(9;12)(p12;p11.2) among Children with B-Cell Precursor Acute Lymphoblastic Leukemia (ALL): PAX5-ETV6 and ETV6-RUNX1 Rearrangements: A Report from the Children’s Oncology Group." Blood 110, no. 11 (November 16, 2007): 1439. http://dx.doi.org/10.1182/blood.v110.11.1439.1439.
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Abstract The dic(9;12)(p12;p11.2) has been described as a rare cytogenetic abnormality in pediatric precursor B-cell ALL. Initial studies suggested that the rearrangement is associated with a favorable outcome, and recent studies demonstrated the presence of a PAX5-ETV6 fusion gene was associated with this cytogenetic abnormality. Twenty cases with a cytogenetic dic(9;12) were identified in the Children’s Oncology Group (COG) cytogenetics databases. FISH analysis with the ETV6-RUNX1 (TEL-AML1) probes was done on 12 of these samples. Five cases were positive for fusion, indicating a cryptic t(12;21)(p13;q22), and also had loss of the ETV6 probe from the chromosome 12 not involved in the t(12;21). Seven cases were negative for fusion and had loss of an ETV6 signal, although one of the latter had a diminished ETV6 signal identified. To determine whether both PAX5-ETV6 and ETV6-RUNX1 rearrangements occurred in some patients, a diagnostic sample from each patient was analyzed by RT-PCR for the PAX5-ETV6 and ETV6-RUNX1 fusion genes. Primers from exon 3 of PAX5 and exon 3 of ETV6 were used for the PAX5-ETV6 analysis and from exon 5 of ETV6 and exon 4 of RUNX1 for the ETV6-RUNX1 analysis. Of the 20 cases, only 8 were RT-PCR positive for the PAX5-ETV6 fusion with the above primers; however, an additional 2 were RT-PCR positive with alternate primers, and all 10 of these were negative for the ETV6-RUNX1 fusion by RT-PCR. Of the remaining 10 patients, 9 were RT-PCR positive for the ETV6-RUNX1 fusion, including all of the ETV6-RUNX1 cases positive by FISH. The gene rearrangement associated with the dic(9;12) in these cases is not known. One patient was negative for both fusions by RT-PCR, negative by FISH for ETV6-RUNX1 rearrangement, yet had loss of an ETV6 signal. No cytogenetic differences could be seen between the 2 groups, either in the appearance of the dic(9;12) or in the other abnormalities identified. These results demonstrate the presence of two mutually exclusive dic(9;12) rearrangements in pediatric ALL; one associated with ETV6-RUNX1 rearrangement and one resulting in PAX5-ETV6 fusion. Both PAX5-ETV6 and ETV6-RUNX1 rearrangements are associated with a favorable prognosis. However, molecular analysis of the dic(9;12) patients must be performed to determine whether the dicentric chromosome results in PAX5-ETV6 fusion or whether the case has ETV6-RUNX1 fusion.
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Taki, Tomohiko, Yasuhide Hayashi, Takeshi Taketani, Masahiro Sako, and Masafumi Taniwaki. "Identification of Novel Type of ETV6-TTL Fusion Gene in Pediatric Myeloid/NK Cell Precursor Acute Leukemia Having t(12;13)(p13;q14)." Blood 106, no. 11 (November 16, 2005): 4520. http://dx.doi.org/10.1182/blood.v106.11.4520.4520.
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Abstract The t(12;13)(p11-13:q11-14) is a recurring chromosomal abnormality found in different types of hematological malignancies. Three genes on 13q12-14, CDX2, FLT3, and TTL have been identified as fusion partners of ETV6(TEL) on 12p13. These genes create different forms of fusion transcripts with ETV6. In the present study, we identified novel types of ETV6-TTL fusion transcripts in a case of a 9 year-old female who developed myeloid/NK cell precursor acute leukemia with t(12;13)(p13;q14). Cytogenetic analyses of the leukemic cells of the patient using a regular G-banding revealed 47, XX, del(2)(q?), -9, der(12)t(12;13)(p13;q14), add(13)(q1?2), +mar1, +mar2 [13/20]. Fluorescence in situ hybridization using YAC clone revealed that ETV6 gene was split in metaphase chromosomes of patient’s leukemic cells. We performed reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis to detect the fusion transcripts of ETV6-CDX2, ETV6-FLT3 or TTL-ETV6, however, no fusion transcripts of previously described types were detected. We next performed RT-PCR analysis using various sets of primers to detect unknown types of fusion transcripts involving these genes, and detected novel types of fusion transcripts of ETV6-TTL. These fusion transcripts consisted of exons 1 to 5 of ETV6 and exons 5 to 8 of TTL, and exons 1 to 6 of ETV6 and exon 9 or exons 8 to 9 of TTL. No reciprocal fusion transcripts were detected. Predicted fusion proteins consisted of N-terminal ETV6 lacking whole or part of ETS-binding domain and C-terminal TTL. Previous report showed major type of TTL-ETV6 fusion transcript consisted of exons 1 to 5 of TTL and exons 2 to 8 of ETV6 which contained both helix-loop-helix and ETS-binding domains. These results suggested that novel types of ETV6-TTL act as different fusion proteins from previously reported TTL-ETV6 in leukemogenesis. At the cytogenetic level, it may be difficult to distinguish ETV6-CDX2, ETV6-FLT3, and ETV6-TTL. Further accumulation of the patients with t(12;13) and further analysis of these novel types of fusion transcripts may clarify the pathogenesis of t(12;13)-leukemia.
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Cools, Jan, Chrystèle Bilhou-Nabera, Iwona Wlodarska, Christine Cabrol, Pascaline Talmant, Philippe Bernard, Anne Hagemeijer, and Peter Marynen. "Fusion of a Novel Gene, BTL, to ETV6 in Acute Myeloid Leukemias With a t(4;12)(q11-q12;p13)." Blood 94, no. 5 (September 1, 1999): 1820–24. http://dx.doi.org/10.1182/blood.v94.5.1820.
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Abstract The ETV6 gene (also known as TEL) is the main target of chromosomal translocations affecting chromosome band 12p13. The rearrangements fuse ETV6 to a wide variety of partner genes in both myeloid and lymphoid malignancies. We report here 4 new cases of acute myeloid leukemia (AML) with very immature myeloblasts (French-American-British [FAB]-M0) and with a t(4;12)(q11-q12;p13). In all cases, ETV6 was found recombined to a new gene, homologous to the mouse Brx gene. The gene was named BTL (Brx-likeTranslocated in Leukemia). Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments indicate that the expression of the BTL-ETV6 transcript, but not of the reciprocal ETV6-BTL transcript, is a common finding in these leukemias. In contrast to the majority of other ETV6 fusions, both the complete helix-loop-helix (HLH) and ETS DNA binding domains of ETV6 are present in the predicted BTL-ETV6 fusion protein, and the chimeric gene is transcribed from theBTL promoter.
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Cools, Jan, Chrystèle Bilhou-Nabera, Iwona Wlodarska, Christine Cabrol, Pascaline Talmant, Philippe Bernard, Anne Hagemeijer, and Peter Marynen. "Fusion of a Novel Gene, BTL, to ETV6 in Acute Myeloid Leukemias With a t(4;12)(q11-q12;p13)." Blood 94, no. 5 (September 1, 1999): 1820–24. http://dx.doi.org/10.1182/blood.v94.5.1820.417k09_1820_1824.
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The ETV6 gene (also known as TEL) is the main target of chromosomal translocations affecting chromosome band 12p13. The rearrangements fuse ETV6 to a wide variety of partner genes in both myeloid and lymphoid malignancies. We report here 4 new cases of acute myeloid leukemia (AML) with very immature myeloblasts (French-American-British [FAB]-M0) and with a t(4;12)(q11-q12;p13). In all cases, ETV6 was found recombined to a new gene, homologous to the mouse Brx gene. The gene was named BTL (Brx-likeTranslocated in Leukemia). Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments indicate that the expression of the BTL-ETV6 transcript, but not of the reciprocal ETV6-BTL transcript, is a common finding in these leukemias. In contrast to the majority of other ETV6 fusions, both the complete helix-loop-helix (HLH) and ETS DNA binding domains of ETV6 are present in the predicted BTL-ETV6 fusion protein, and the chimeric gene is transcribed from theBTL promoter.
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Wildenhain, Sarah, Julia Hauer, Silija Roettgers, Hartmut Engelmann, Astrid Novosel, Vera Binder, Robert K. Slany, Wolf-Dieter Ludwig, Jochen Harbott, and Arndt Borkhardt. "HOX-Gene Expression and Inhibition of the Non-Canonical NF-KappaB Pathway in t(7;12) Leukemias." Blood 112, no. 11 (November 16, 2008): 3110. http://dx.doi.org/10.1182/blood.v112.11.3110.3110.
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Abstract The chromosomal translocation t(7;12) was described recently in MLL negative acute myeloid leukemias of infancy associated with a very poor clinical outcome. The rearrangement involves the genes HLXB9 (7q36) and ETV6 (12p13) with a fusion transcript of exon1/HLXB9 and exon3/ETV6. An alternative in frame splicing variant of exon1/HLXB9 to exon2/ETV6 is also detectable. Leukemic bone marrow samples of 42 infants, diagnosed in Germany with AML, were screened for the fusion transcript HLXB9/ETV6 with RT-PCR. Inclusion criteria were diagnosis of AML, age<2 years, no MLL/AF9, MLL/AF4 or MLL/ENL rearrangement in RT-PCR, no chromosomal 11q23 rearrangement and no trisomy 21. Positive samples were cloned and sequenced. A fusion transcript was found in approx. ~17% of patients. Common features of HLXB9/ETV6 positive leukemias in this cohort are poorly differentiated FAB subtypes, trisomy 19 karyotype of the leukemic cells, coexpression of the T-cell markers CD4 and CD7 as well as high expression of the un-rearranged HLXB9 gene. Quantitative PCR showed no upregulation of HOXA9 and MEIS1 in HLXB9/ETV6 positive patients as reported for MLL+ leukemias. The mechanism of HLXB9/ETV6 induced leukemogenesis remains unknown. We show that in vitro retroviral transduction of murine hematopoietic precursor cells with either HLXB9/ETV6 or HLXB9 or ETV6/RUNX1 was not transforming. Furthermore altered activity of the transcription factor family NF-kappaB has been demonstrated for various types of cancer including acute myeloid leukemias. Therefore we analysed the impact of the HLXB9/ETV6 fusion on the NF-kappaB signaling pathway in a 293T cell-system. HLXB9/ETV6 and HLXB9 showed inhibition of the NF-kappaB dependent transcription signal in a reporter assay, whereas the typical ALL-fusion ETV6/RUNX1 showed no influence. Analysis of nuclear extracts by western blot confirmed, that the inhibition of the NF-kappaB signal is due to a reduced nuclear translocation of the non-canonical NF-kappaB components p52 and RelB, whereas the canonical pathway remains not influenced. Conclusion: The HLXB9/ETV6 fusion transcript can be found in 17% of infants with MLL-negative AML in a German cohort. HLXB9/ETV6 positive leukemias show no increased expression on HOXA9 and MEIS1, but coexpression of T-cell markers and inhibition of the non-canonical NF-kappaB pathway on protein level. HLXB9/ETV6 did not induce malignant transformation in murine hematopoietic precursor cells.
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Mayumi, Azusa, Hideki Yoshida, Kouhei Mitsuno, Nozomi Nishida, Shinya Osone, Takahiko Yasuda, and Toshihiko Imamura. "Leukemic Cells Expressing ETV6-Frk Identified in a Refractory B-ALL Patient Are Sensitive to Dasatinib in Vitro." Blood 138, Supplement 1 (November 5, 2021): 4335. http://dx.doi.org/10.1182/blood-2021-149926.
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Abstract [Background] ETV6-FRK is a rare kinase-related fusion gene which was identified only in acute myeloid leukemia (Hosoya N, et al. Genes Chromosomes Cancer. 2005). Herein, we firstly identified ETV6-FRK fusion gene in a patient with high-risk pediatric B cell precursor ALL (B-ALL). Because FRK is Src family tyrosine kinase, we performed functional analysis of ETV6-FRK to establish molecular targeting therapy. [Patient] A 11-year-old boy with B-ALL was refractory to conventional chemotherapy and received allogeneic bone marrow transplantation (allo-BMT) following two courses of blinatumomab. This patient maintains complete remission for three months after allo-BMT. Cytogenetic analysis demonstrated t(6;12)(q21;p13) as a part of complex karyotype. Targeted capture mRNA sequencing identified ETV6-FRK fusion transcript in this patient. [Materials and methods] ETV6-FRK fusion was validated by RT-PCR of the diagnostic leukemic sample from this patient. Full length of ETV6-FRK cDNA was cloned into retroviral vector with Tet-On system. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing ETV6-FRK (Ba/F3-ETV6-FRK) under doxycycline (DOX) dependent manner. Ba/F3-ETV6-FRK was analyzed whether IL-3 independent growth was achieved. To determine whether aberrant activation of FRK-STAT pathway as the downstream effects of ETV6-FRK, activation of FRK-STAT pathway was evaluated by western blot. Finally, in cytotoxic assay, proliferation of Ba/F3-ETV6-FRK was assessed under the media with various concentrations of dasatinib, a tyrosine kinase inhibitor. [Results and discussions] Sequencing of RT-PCR product revealed that ETV6 exon 4 was fused in-frame to FRK exon 3, creating an ETV6-FRK fusion gene. The ETV6-FRK fusion gene produced a chimeric protein consisting of the entire pointed (PNT) oligomerization domain of ETV6 and the kinase domain of FRK (Fig 1). The expression of ETV6-FRK in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-ETV6-FRK proliferated without IL-3 in contrast to Ba/F3 cells not expressing ETV6-FRK (p<0.01), suggesting ETV6-FRK had proliferation activity. Western blot analysis revealed constitutive phosphorylation of tyrosine residues of ETV6-FRK and STAT5/STAT3/STAT1, suggesting constitutive activation of FRK-STATs pathway was associated with IL-3 independent proliferation activity of ETV6-FRK. Considering that dasatinib, which is Src-kinase inhibitor, could block constitutive phosphorylation of ETV6-FRK, we hypothesized that dasatinib might block the IL-3 independent proliferation of Ba/F3-ETV6-FRK. In vitro killing assay showed that dasatinib suppressed efficiently the proliferation of Ba/F3-ETV6-FRK with 50% inhibitory concentration (IC50) 1.64 ± 0.02 nM, although dasatinib didn't show any effect on Ba/F3 cells not expressing ETV6-FRK (Fig. 2). Annexin V assay determined that 35.8 ± 6.9 % of Ba/F3-ETV6-FRK with dasatinib (10nM, 48hrs) were apoptotic than Ba/F3 cells not expressing ETV6-FRK (Fig. 3, p<0.01). These findings suggested that dasatinib abolished the proliferation activity of ETV6-FRK selectively. [Conclusion] We identified the first patient of pediatric high-risk B-ALL harboring ETV6-FRK fusion by targeted capture mRNA sequencing, who was refractory to the conventional chemotherapy. We also provide the first evidence that dasatinib could abrogate proliferation activity of ETV6-FRK in vitro, suggesting that dasatinib might be effective for the patient with B-ALL carrying a ETV6-FRK fusion. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Xiong, Xiaofang, Zheng Yan, Wei Jiang, and Xuejun Jiang. "ETS variant transcription factor 6 enhances oxidized low-density lipoprotein-induced inflammatory response in atherosclerotic macrophages via activating NF-κB signaling." International Journal of Immunopathology and Pharmacology 36 (January 2022): 205873842210764. http://dx.doi.org/10.1177/20587384221076472.
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Objectives: Macrophages play a critical role in atherosclerosis by contributing to plaque development, local inflammation, and thrombosis. Elucidation of the molecular cascades in atherosclerotic macrophages is important for preventing and treating atherosclerosis. This study aims to deepen the understanding of the mechanisms that regulate the function of aorta macrophage in atherosclerosis. Methods: In the current study, the expression and function of ETS variant transcription factor 6 (ETV6) in aorta macrophages in a mouse atherosclerosis model. Aorta macrophages were enriched by flow cytometry. ETV6 expression was analyzed by quantitative RT-PCR. The role of ETV6 in macrophage-mediated pro-inflammatory response was evaluated both in vitro and in vivo after ETV6 silencing. Results: A remarkable elevation of ETV6 in aorta macrophages of atherosclerotic mice was observed. In addition, in vitro analysis indicated that oxidized low-density lipoprotein (oxLDL) up-regulated ETV6 in macrophages via the NF-κB pathway. ETV6 silencing suppressed oxLDL-induced expression of IL-1β, IL-6, and TNF-α in macrophages in vitro. However, ETV6 silencing did not impact the uptake of either oxLDL or cholesterol by macrophages. Furthermore, ETV6 silencing suppressed oxLDL-induced activation of the NF-κB pathway in macrophages, as evidenced by less phosphorylation of IKKβ and NF-κB p65, more cytoplasmic IκBα, and lower nuclear NF-κB p65. Moreover, ETV6 silencing inhibited the production of IL-1β and TNF-α in aorta macrophages in vivo. Conclusion: ETV6 supports macrophage-mediated inflammation in atherosclerotic aortas. This is a novel mechanism regulating the pro-inflammatory activity of atherosclerotic macrophages.
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Hart, Stephen, Veronique Duke, Richard Pannell, Terence Rabbitts, Kikkeri Naresh, and Letizia Foroni. "ETV6: Its Role in Mouse Development and Leukaemogenesis Using Knock-Out and Knock-In Models." Blood 110, no. 11 (November 16, 2007): 461. http://dx.doi.org/10.1182/blood.v110.11.461.461.
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Abstract Disruption of the transcription factor ETV6 (TEL) is believed to play a pivotal role in the development of many types of leukaemia. As well as there being more than 15 ETV6 fusion gene partners described to date, there are other genetic alterations of ETV6 including deletions, point mutations and disruption in the ETV6 promoter region that may contribute to the malignant phenotype. We generated two Etv6 knock-out mouse models (Met1 and Met43) to investigate the role of Etv6 in mouse embryonic development and two knock-in models carrying the t(12;21) and t(9;21) fusion genes to investigate the role of the Etv6-RUNX1 and Etv6-ABL fusion oncoproteins in leukaemogenesis. Etv6-Met1−/− was compatible with full embryonic development. However, from a total of 382 animals generated from the Etv6-Met1+/− cross only 39 were identified as homozygous suggesting some embryonic lethality. A second knock-out construct (Etv6-Met43) was generated in which the second transcription start site at AA position 43 was removed. Of the 235 offspring genotyped from the Etv6-Met43+/− cross, no homozygous animals were identified. Further investigation revealed that Etv6-Met43−/− foetuses lacked definitive haematopoiesis and died between dE10.5–11.5. These data indicate that an Etv6 transcript generated from the methionine at AA position 43 can partially rescue the Etv6-Met1−/− phenotype. No mice homozygous for the Etv6-RUNX1 were identified from the 628 offspring genotyped from the Etv6-RUNX1+/− cross. Further investigation revealed that Etv6-RUNX1−/− foetuses displayed a similar phenotype to the Etv6-Met43−/− animals and died in-utero at a similar time. Etv6-RUNX1+/− animals were fertile and remained well for the first year of life. However, after this time in a group of 91 animals it was noticed that among 41 Etv6-RUNX1+/− animals, the majority were dying from a blastic haematological tumour formation best identified in spleens, at a higher incidence than in the 51 wild-type littermates. Chimeric mice targeted with the Etv6-ABL construct remained well for a short period after birth but all subsequently sickened and died within the first year of life. Upon examination peripheral blood as well as post-mortem organs showed a homogeneous infiltration of granulocytic cells resembling a chronic myeloid proliferation. RT-PCR on RNA isolated from the peripheral blood, bone marrow and spleen of three chimeric animals showed the presence of the fusion gene. Our study demonstrates that Etv6 is essential for mouse embryonic development. The role of Etv6 in malignant transformation is complex and would appear to be dependent on the function of the partner fusion gene rather than on the common Etv6 part of the protein.
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Cazzaniga, Giovanni, Sabrina Tosi, Alessandra Aloisi, Giovanni Giudici, Maria Daniotti, Pietro Pioltelli, Lyndal Kearney, and Andrea Biondi. "The Tyrosine Kinase Abl-Related Gene ARG Is Fused toETV6 in an AML-M4Eo Patient With a t(1;12)(q25;p13): Molecular Cloning of Both Reciprocal Transcripts." Blood 94, no. 12 (December 15, 1999): 4370–73. http://dx.doi.org/10.1182/blood.v94.12.4370.
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Abstract The Ets variant gene 6 (ETV6/TEL) gene is rearranged in the majority of patients with 12p13 translocations fused to a number of different partners. We present here a case of acute myeloid leukemia M4 with eosinophilia (AML-M4Eo) positive for the CBFb/MYH11 rearrangement and carrying a t(1;12)(q25;p13) that involves the ETV6 gene at 12p13. By 3′rapid amplification of cDNA ends-polymerase chain reaction (3′RACE-PCR), a novel fusion transcript was identified between the ETV6 and the Abelson-related gene (ARG) at 1q25, resulting in a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the SH2, SH3, and protein tyrosine kinase (PTK) domains of ARG. The reciprocal transcript ARG-ETV6 was also detected in the patient RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), although at a lower expression level. The ARG gene encodes for a nonreceptor tyrosine kinase characterized by high homology with c-Abl in the TK, SH2, and SH3 domains. This is the first report on ARGinvolvement in a human malignancy.
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Cazzaniga, Giovanni, Sabrina Tosi, Alessandra Aloisi, Giovanni Giudici, Maria Daniotti, Pietro Pioltelli, Lyndal Kearney, and Andrea Biondi. "The Tyrosine Kinase Abl-Related Gene ARG Is Fused toETV6 in an AML-M4Eo Patient With a t(1;12)(q25;p13): Molecular Cloning of Both Reciprocal Transcripts." Blood 94, no. 12 (December 15, 1999): 4370–73. http://dx.doi.org/10.1182/blood.v94.12.4370.424k34_4370_4373.
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The Ets variant gene 6 (ETV6/TEL) gene is rearranged in the majority of patients with 12p13 translocations fused to a number of different partners. We present here a case of acute myeloid leukemia M4 with eosinophilia (AML-M4Eo) positive for the CBFb/MYH11 rearrangement and carrying a t(1;12)(q25;p13) that involves the ETV6 gene at 12p13. By 3′rapid amplification of cDNA ends-polymerase chain reaction (3′RACE-PCR), a novel fusion transcript was identified between the ETV6 and the Abelson-related gene (ARG) at 1q25, resulting in a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the SH2, SH3, and protein tyrosine kinase (PTK) domains of ARG. The reciprocal transcript ARG-ETV6 was also detected in the patient RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), although at a lower expression level. The ARG gene encodes for a nonreceptor tyrosine kinase characterized by high homology with c-Abl in the TK, SH2, and SH3 domains. This is the first report on ARGinvolvement in a human malignancy.
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Wang, Tong, Xue Chen, Shuang Hui, Jingbo Ni, Ying Yin, Wei Cao, Yan Zhang, et al. "Myeloid Neoplasms with t(12;22)(p13;q12) and MN1 Ectopia Are Refractory to Chemotherapy and Can Benefit from Hematopoietic Stem Cell Transplantation." Blood 134, Supplement_1 (November 13, 2019): 2721. http://dx.doi.org/10.1182/blood-2019-131610.
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Translocation of t(12;22)(p13;q12) is a rare but recurrent chromosomal (chr) abnormality in hematologic malignancies involving ETS variant 6 (ETV6) and meningioma 1 (MN1) genes. The pathogenic mechanism of t(12;22)(p13;q12) and the fact that nearly half of the cases lack fusion genes remain mysterious. We focused here on AML cases with t(12;22)(p13;q12) to elucidate their molecular etiology and outcomes of allogeneic hemopoietic stem cell transplantation (allo-HSCT). A total of eight cases with t(12;22)(p13;q12) and additionally one with t(12;17;22)( p13;q21;q13) (six males and three females) were detected in 2782 newly diagnosed AML and MDS patients by G-banding karyotyping (Table 1). The median age of onset was 45 years (range 4-60 years). The cases include three AML-M5, one AML-M2, one AML progressed from MDS (MDS-AML), one MDS with excess blasts 2 (MDS-EB-2), one chronic myelomonocytic leukemia (CMML), and two myeloid/T mixed-phenotype acute leukemia (MPAL) (Table 1). All cases underwent standard or intensive chemotherapy for myeloid malignancies, and six patients underwent allo-HSCT (Fig. 1a). MN1-ETV6, ETV6-MN1, and MN1 expression were analyzed by RT-PCR. Mutations of 58 commonly mutated genes in hematologic malignancies were analyzed by next-generation sequencing in seven cases (Table 1). Fluorescence in situ hybridization (FISH) was performed using the MN1 dual-color break-apart probe on cases P4 and P9, who were negative for MN1-ETV6. 30 × whole-genome sequencing (WGS) was performed on diagnostic bone marrow (BM) samples from the four cases which were negative for MN1-ETV6, and structure variations including translocations (Fig. 1b) were analyzed. 5/9 cases were positive for both MN1-ETV6 and ETV6-MN1fusions (all type I), and the expression of MN1 was significantly up-regulated in 3/4 fusion negative cases (Table 1). FISH analysis showed split signals in both P4 and P9, indicating a fracture within or adjacent to MN1. WGS analysis showed ectopia of intact MN1 in case P1, P3, and P4 (Fig. 1b), including translocated adjacent to the super enhancers (SEs) of the cracked ETV6. A novel MN1-STAT3 fusion was identified and confirmed by RT-PCR in case P9. We observed overexpression of MN1 in MN1-ETV6 negative cases in our study, which strongly indicates that the contribution of the SEs within ETV6 made on the overexpression of MN1 or aberrant activation of the chimeric MN1-ETV6. Both type I/II MN1-ETV6, and the novel MN1-STAT3 in this study retains the main functional domains of MN1 (Fig. 1c). The MN1-STAT3 contains only 69 amino acids of the STAT3 residual without functional domains, and STAT3-MN1 was absent due to the triadic translocation. Thus, the fusion protein will retain the primary function of MN1 but not STAT3 in case P9, which also indicates that it is the functionally retained MN1 within MN1-ETV6 rather than the fusion, that plays the essential role in leukemogenesis. The genomic evidence of ETV6 disruption was observed in 8/9 cases in our study. ETV6 is a tumor suppressor which is essential in hemopoietic stem cell differentiation and multilineage blood cell development. It is conceivable that the MN1 overactivation and the ETV6 disruptive haploinsufficiency synthetically contribute to the leukemogenesis in t(12;22)(p13;q12) malignancies. This scenario is partially similar to the mechanism of GATA2-MECOM in inv(3)(q21q26) AML, within which that the central pathogenesis is the ectopia of the GATA2 SE that activates MECOM expression and confers the GATA2 haploinsufficiency simultaneously. The present study confirmed that myeloid neoplasms with t(12;22)(p13;q12) were rare and responded poorly to conventional chemotherapy, and provide evidence that these patients can benefit from allo-HSCT. Our investigation especially highlights the evidence of a SE-associated orchestrated mechanism of MN1 overexpression and ETV6 haploinsufficiency in t(12;22)(p13;q12) myeloid neoplasms in a spatiotemporally specific manner, rather than the conventional thought of MN1-ETV6/ETV6-MN1 fusion formation. Figure 1 Disclosures No relevant conflicts of interest to declare.
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Jones, Courtney L., Gregory Kirkpatrick, Courtney Fleenor, Welsh Seth, Leila J. Noetzli, Susan Fosmire, Dmitry Baturin, et al. "ETV6 Regulates Pax5 Expression in Early B Cell Development." Blood 128, no. 22 (December 2, 2016): 2655. http://dx.doi.org/10.1182/blood.v128.22.2655.2655.
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Abstract Recent studies from our group and others have revealed a role for ETV6 germline mutations in the predisposition to ALL. Although ETV6 is among the most commonly mutated genes in ALL, its mechanistic role in leukemogenesis remains unclear. ETV6 is an ETS family transcription factor. ETV6 regulates gene transcription through homo- and hetero- oligomerization with other ETS family members and transcriptional repressors. The germline mutation (P214L amino acid change) identified by our group and others impairs the transcriptional activity and nuclear localization of ETV6 in a dominant negative fashion. The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia. To identify functions of ETV6 in B cell development, we queried the gene expression commons database for evidence of Etv6 expression during B cell development. Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (P<0.0001). To confirm relative patterns of Etv6 and Pax5 expression in developing B cells, we isolated bone marrow (BM) from wild type (WT) mice and fractionated cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19-), Fraction B (CD19+, CD24+, BP1-) and Fraction C (CD19+ CD24+ BP1+). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=.9993; P= 0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19-, and CD20-) compared to the preB cell fraction (CD10+, CD34-, CD19+, CD20-). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared to the early B cell fraction. To determine if a function relationship exists between ETV6 and Pax5 we overexpressed an empty vector (MiG), wild type (WT) ETV6 and ETV6 P214L in a murine lymphoid progenitor line (Ba/F3). ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). To further interrogate the role of ETV6 in regulating Pax5 transcription we measured the association of ETV6 with putative ETS factor binding sites (GGAA sequence) within the Pax5 transcription start site (TSS) using ChIP-PCR. ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We next determined the consequences of ETV6 mutation on the recruitment of ETV6, SIN3A, and HDAC3 to the Pax5 locus by performing ChIP-PCR in Ba/F3 cells that express a FLAG-tagged WT ETV6 or ETV6 P214L. We detected association of ETV6, SIN3A and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. We conclude that ETV6, SIN3A and HDAC3 are responsible for the repression of Pax5 transcription. Moreover, mutant ETV6 inhibits the ability of normal ETV6 to bind and recruit SIN3A and HDAC3 to the Pax5 locus. Finally, we determined if the recruitment of SIN3A and HDACs to the Pax5 locus was essential to repression of Pax5 by WT ETV6 by knocking out SIN3A and inhibiting HDACs using pan HDAC inhibitor, SAHA and measuring Pax5 expression by RT-PCR. We found that upon SIN3A knockout or HDAC inhibition Pax5 expression was no longer repressed upon WT ETV6 overexpression. To determine the consequences of ETV6 P214L expression on B cell development, we generated a transgenic mouse expressing the P214L mutation in the endogenous ETV6 gene. Preliminary data suggests that these mice have thrombocytopenia, similar to patients with germline ETV6 mutation. In addition, mice with the ETV6 P214L mutation displayed reduced level of cKIT expression on the FrA B cell population. Further studies will be necessary to understand the consequences of reduced cKIT expression to overall B cell development and if this cKIT reduction is linked to aberrant Pax5 expression. In conclusion, ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 misregulation results in a B cell development halt, lineage infidelity and leukemogenesis. Disclosures No relevant conflicts of interest to declare.
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Zemanova, Zuzana, Kyra Michalova, Libuse Babicka, Lenka Pavlistova, Marie Jarosova, Milena Holzerova, Alexandra Oltova, et al. "Clinical Relevance of Complex Chromosomal Aberrations in Bone Marrow Cells of 107 Children with ETV6/RUNX1 Positive Acute Lymphoblastic Leukemia (ALL)." Blood 108, no. 11 (November 1, 2006): 2278. http://dx.doi.org/10.1182/blood.v108.11.2278.2278.
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Abstract Cryptic translocation t(12;21)(p13;q22) which give origin to the ETV6/RUNX1 hybrid gene can be found by I-FISH in approximately 20–25% of children with B precursor ALL as the most frequent specific aberration. This translocation is generally associated with good outcome. Despite of its favorable prognostic value, late relapses may occur within this group of patients. One of the reasons could be the high instability of the genome of leukemic cells, which is manifested at the chromosomal level by additional aberrations and/or complex chromosomal rearrangements. The aim of the study was to evaluate the significance of the additional chromosomal aberrations for prognosis of children with ETV6/RUNX1 positive ALL. For the assessment of ETV6/RUNX1 fusion gene RT-PCR and/or double target interphase FISH with locus-specific probe (Abbott-Vysis, Des Plaines, Illinois, USA) were used (200 interphase nuclei analyzed, cut-off level 2.5% tested on controls, standard deviation ≤0.5%). Karyotypes were analyzed by conventional and molecular cytogenetic methods. Structural and/or complex chromosomal aberration were verified by FISH with whole chromosome painting probes (Cambio, Cambridge, UK) and/or by mFISH with the "24XCyte" probe kit (MetaSystems GmbH, Altlussheim, Germany). We performed prospective and retrospective study of 107 children with ALL and ETV6/RUNX1 fusion gene detected by RT-PCR and/or I-FISH. Patients were diagnosed between 1995 and 2006, age ranged between 15 months and 16.9 years (median 4.2 years). Relapse appeared in 19 children (17.8%), four of them died. In 64 children (59.8%) we found besides t(12;21)(p13;q22) additional chromosomal aberrations, the most frequently trisomy or tetrasomy of chromosome 21 (20 cases), deletion of non-translocated ETV6 allele (24 cases), deletion of 6q (7 cases) and/or rearrangements of the long arm of chromosome X (6 cases). Complex karyotypes were identified in 38 children (35.5%). In twelve of them variant translocations of chromosomes 12 and 21 with other partners were observed. Event-free survival (EFS) was significantly shorter in patients with additional structural and/or complex aberrations in ETV6/RUNX1 positive cells (p=0.01). In our cohort complex karyotypes indicated poor prognosis. Finding of complex chromosomal aberrations in leukemic cells is accompanied by higher risk of relapse even in those cases where the prognostically positive aberration is primarily present.
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Penas, Eva Maria Murga, Marianne Klokow, Petra Behrmann, Snjezana Janjetovic, Georgia Schilling, Carsten Bokemeyer, and Judith Dierlamm. "Incidence of ETV6 Cryptic Translocations In Myeloid Disorders." Blood 116, no. 21 (November 19, 2010): 2733. http://dx.doi.org/10.1182/blood.v116.21.2733.2733.
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Abstract Abstract 2733 Rearrangements affecting band 12p13 are found as recurring chromosomal changes in myeloid malignancies. The main target of these rearrangements is the ETV6 gene, a member of the ETS family of transcription factors that is required for hematopoiesis within the bone marrow. To date, more than 41 translocations affecting ETV6 have been described, most of them encoding chimeric proteins with oncogenic properties. However, some of the translocations do not produce a functional fusion protein, but may induce ectopic expression of oncogenes located in the neighbourhood of the breakpoint. In order to investigate the incidence of ETV6 aberrations in myeloid disorders and to search for novel ETV6 translocations, we performed fluorescence in situ hybridization (FISH) screening with a specific breakapart probe for the ETV6 gene (Abbott Vysis) on 134 acute myeloid leukemia (AML) cases, of which 20 cases were secondary AML evolved from myelodysplatic (MDS) or myeloproliferative syndromes (MPS), 10 MDS, and 3 MPS. Conventional cytogenetic analysis of the cases identifed abnormal karyotypes in 72 cases, of which 20 cases presented del(12p), 5 cases add(12p), and 8 cases a translocation involving 12p. The remaining 39 abnormal karyotypes did not present alterations in the short arm of chromosome 12. Normal karyotypes were detected in 53 cases and cytogenetic analyses failed in 22 cases. FISH detected 22 cases with heterozygous deletions of ETV6, of which 15 correlated with previous cytogenetic findings, 4 were detected in patients without evaluable mitoses, 2 were associated with add(12p), and one resulted from an unbalanced translocation involving chromosomes × and 12. In the remaining 7 translocations, FISH confirmed the t(3;12)/EVI1-ETV6, t(4;12)/CHIC2-ETV6, and t(5;12)/PDGFRβ-ETV6 in 4 cases, excluded an ETV6 involvement in 2 translocations, and revealed in one patient a novel t(3;12;17)(q21;p13;q25) with breakpoint in ETV6, previously described as t(3;12)(q21;p13). Interestingly, cryptic translocations involving ETV6 were revealed in 3 patients with cytogenetically described 12p deletions: the t(12;17)(p13;p12–13) in a secondary AML1; inv(12)(p13q24) in a secondary AML; and t(2;12)(p16.1;p13) in an AML FAB subtype M1. In one additional patient with del(12p), FISH revealed a cryptic interstitial deletion with breakpoint in ETV6, del(12)(pterp13). In the latter patient, hybridization signals centromeric to ETV6 were deleted, whereas the probe hybridizing telomeric to ETV6 was retained on the derivative chromosome 12. Finally, one further ETV6 cryptic translocation, t(1;12)(p36;p13), was found in patient with MDS and an apparently normal karyotype. The ETV6 translocation partner could be identified in the t(2;12)(p16.1;p13) and showed a novel fusion transcript derived from the ETV6 gene and the BAC clone RP11-440P5, on chromosome 2p16.1. DNA sequencing revealed an in-frame fusion of exon 7 of ETV6 to sequences derived from BAC 44OP5. We further investigated whether the ETV6 translocation partner in the t(1;12) was the recently described MDS2 gene of the t(1;12)(p36;p13).2 However, RT-PCR with specific primers for ETV6 and MDS2 failed to amplified an ETV6-MDS2 fusion transcript. The molecular cloning of the inv(12) and the t(3;12;17) is in progress. In conclusion, we have identified 6 new ETV6 rearrangements in patients with myeloid neoplasias, of which 5 were not seen by standard cytogenetic analyses. The prevalence of ETV6 rearrangements assessed by FISH (10/147, 6,8%) was 50% higher than by conventional cytogenetic (5/147, 3,4%). This study shows the relatively high frequency of cryptic translocations involving ETV6 on 12p13 and underlines the importance of FISH to detect and characterize these translocations. This work was supported by the Deutsche José Carreras Leukämie-Stiftung eV. References: 1. Murga Penas EM, Cools J, Algenstaedt P, et al. A novel cryptic translocation t(12;17)(p13;p12-p13) in a secondary acute myeloid leukemia results in a fusion of the ETV6 gene and the antisense strand of the PER1 gene. Genes Chromosomes Cancer. 2003;37(1):79–83. 2. Odero MD, Vizmanos JL, Román JP, et al. A novel gene, MDS2, is fused to ETV6/TEL in a t(1;12)(p36.1;p13) in a patient with myelodysplastic syndrome. Genes Chromosomes Cancer. 2002;35(1):11–19. Disclosures: No relevant conflicts of interest to declare.
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Emerenciano, Mariana, Silvia Bungaro, Giovanni Cazzaniga, Maria Dolores Fonseca Dorea, Virginia Maria Coser, Isis Quezado Magalhaes, Andrea Biondi, and Maria S. Pombo-de-Oliveira. "Short Time Latency in Infant Leukemia with ETV6/RUNX1 fusion Gene." Blood 112, no. 11 (November 16, 2008): 4882. http://dx.doi.org/10.1182/blood.v112.11.4882.4882.
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Abstract Acute lymphoblastic leukemia (ALL) in infants is characterized by a high frequency of MLL gene rearrangements. By contrast, the ETV6/RUNX1 (TEL/AML1) fusion gene is usually detected in children older than 2 years. In a series of Brazilian infant leukemia cases collected in an epidemiological study, a screening was performed to identify fusion genes found in ALL, we have identified four cases harboring ETV6/RUNX1 in blast cells of infants with 2, 3, 5 and 7 months-old, and common-ALL. To underlie additional genomic hits required to accelerate the emergence of a frank leukemia in these c-ALL cases, we applied the SNP-based genomic copy number analysis, comparing t(12;21) infants with other children with c-ALL in older age-range groups. The presence of ETV6/RUNX1 fusion gene was demonstrated by reverse transcription polymerase chain reaction (RT-PCR) and confirmed by fluorescence in situ hybridization (FISH). All cases were negative for MLL rearrangements, as assessed both by RT-PCR and FISH analyses. As control group (non-leukemic children), we selected 5 samples from children younger than 2 years-old already included in a previous epidemiological study. Infant cases described here present high WBC count (on average 131 × 109/L), and adverse prognosis: two of them relapsed, and one patient only (relapsed) is still alive four years after diagnosis. Recurrent deletions, including 9p21.3 (CDKN2A/2B and MTAP), 11p13 (CD44), 12p13.2 (ETV6) and regions affected by copy number neutral loss of heterozigosity (LOH) were observed. The frequency of genomic deletions and amplifications detected by 100K SNP arrays varied significantly between age-related c-ALL subgroups. The infant leukemia group showed the lower number of chromosome imbalances, whereas the mean number of deletions was similar in the <2 years and 2–10 years age groups, consistent with the observation that the number of gains was largely increasing in older children. It has been postulated that infant leukemia might be caused by transplacental exposures during pregnancy. Then, with these unusual results we have reviewed the data of their mothers’ interviews. Mothers of infants were effectively exposed to a non-steroidal anti-inflammatory drug, and other chemical substances. It may be hypothesize that the pre-leukemic phase in these age-related subgroups of ETV6/RUNX1 positive patients may be evolved with different latencies due to the occurrence of a combination of genetic and environmental factors.
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Contento-Gonzalo, Alejandro, Antonio Jimenez-Velazco, Alcala-Peña Magdalena, Manuel Barrios, Katie Hurst, Dana Diaz-Canales, Soraya Lorente de uña, Jose Coin-Ruiz, Maria Elvira Gonzalez-valentin, and Ana Isabel Heiniger Mazo. "Influence of the fusion gene ETV6-RUNX1 in the prognosis of the Pediatric B- Precursor Acute Lymphoblastic Leukemia." Blood 118, no. 21 (November 18, 2011): 2515. http://dx.doi.org/10.1182/blood.v118.21.2515.2515.
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Abstract Abstract 2515 INTRODUCTION AND OBJECTIVES: The ETV6-RUNX1 (TEL-AML1) rearrangement comes from the translocation of two chromosomes t(12;21)(p12;q22), and represents one of the most frequently detected anomalies (15–30%) in the B-precursor Acute Lymphoblastic Leukemia (B-ALL). It must be identified by polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) methods, since this translocation is not detected by conventional cytogenetic techniques. The prognostic value of ETV6-RUNX1 is still a matter of controversy. Recently, the group of the St Jude Children's Hospital reported an excellent outcome in patients carrying the translocation, whereas the BMF group did not find any significant difference in the survival of ETV6-RUNX1 positive patients when compared to ETV6-RUNX1 negative. The aim of our study has been to determine the prognostic impact of the ETV6-RUNX1 rearrangement in patients diagnosed of B-ALL in our Hospital, after a long period of follow-up and with the same Spanish treatment protocols (from PETHEMA and SHOP groups). PATIENTS AND METHODS: All patients with B-ALL diagnosis from January 1997 to May 2011 were included in the study: in total, 114 patients with a mean of age of 6 ys (0.3–14). The type of leukemia was ALL common (83 patients), pro-B (17 patients), pre-B (13 patients) and mature (1 patient). All the children over 1 yr received treatment according to PETHEMA group protocols, adjusted to the risk. Children under 1 yr were treated following SHOP group protocols. Seventeen patients received an allogeneic transplantation. The main clinical features of the positive and negative patients for ETV6-RUNX1 are detailed in table 1. ETV6-RUNX1 assay was performed in our laboratory by RT-PCR, according to the European BIOMED project methodology. RESULTS: ETV6-RUNX1 was found in 31 of the 114 patients (27.2%). These patients showed a significantly higher frequency of myeloid antigens (p<0.001), and were always positive for CD10 (p=0.006). All cases of positive ETV6-RUNX1 were over 2 years old. No significant differences between positive and negative ETV6-RUNX1 were obtained when complete remissions (100 vs 80%), relapse (16 vs 20%) or deaths (10 vs 13%) were analyzed. Furthermore, estimation of disease free survival (DSF) at 14 ys for both groups were similar: 80 ± 8% for positive vs 66 ± 7% for negative (p=0.21, log-rank test). And the same happened for overall survival (OS): 87 ± 7% for positive vs 83 ± 5% for negative (p=0.4, log-rank test). DISCUSSION: In our series, including patients with B-ALL treated with similar protocols with long periods of follow-up, we could not find differences between positive and negative ETV6-RUNX1 patients. It is well known that the intensity of the chemotherapy regimen and the age of inclusion in different protocols may influence the prognosis. Therefore, at present, it is still a matter of discussion if previous reported differences in the B-ALL ETV6-RUNX1 positive group could be explained by a different stratification in risk groups or by different chemotherapy regimens. This work has been funded by a grant from AECC, Carmen Lavigne Prize 2010 Disclosures: No relevant conflicts of interest to declare.
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Chen, Suning, Bjoern Schneider, Stefan Nagel, Robert Geffers, Maren Kaufmann, Hilmar Quentmeier, Hans G. Drexler, and Roderick A. F. MacLeod. "Spliceosomal Targeting in Acute Myeloid Leukemia Cells with ETV6-NTRK3 Fusion." Blood 114, no. 22 (November 20, 2009): 5042. http://dx.doi.org/10.1182/blood.v114.22.5042.5042.
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Abstract Abstract 5042 Background In acute myeloid leukemia (AML) a recurrent chromosome abnormality t(12;15)(p13;q25) fuses ETV6 with NTRK3. This rearrangement uniquely occurs in both solid tumors – including secretory breast cancer where it has been recently shown to target WNT signalling (Li et al., Cancer Cell 2007, 12: 542) - and leukemia, but has yet to be characterized in the hematologic setting. Tyrosine receptor kinases (TRK) play key roles in leukemogenesis and already serve as therapeutic targets. We set out to characterize potential downstream targets of ETV6-NTKR3 in AML cells. Methods and Cells By applying molecular cytogenetics, rapid amplification of c-DNA ends, microarray transcriptional profiling, reverse transcriptase quantitative-PCR, sequencing technology, and pathway analysis we defined and characterized the transcriptosome of a t(12;15) cell line (AP-1060) recently established from a patient with acute promyelocytic leukemia. We also investigated the transcriptional responses of AP-1060 cells to TRKi(nhibitor). For comparison we used, firstly a panel of 12 AML cell lines lacking ETV6-NTRK3 or PML-RARA, followed by NB-4 cells with solo PML-RARA. Results FISH confirmed ETV6 rearrangement, while 3′-RACE and RT-PCR identified and confirmed ETV6-NTRK3 fusion transcripts. Sequencing revealed both ETV6 exon-4 / NTKR3 exon-14, and ETV6 exon-2 / exon-18 of NTKR3 (hematopoietic) transcripts - the former dominating. Comparative transcriptional profiling of AP-1060 and control AML cells with or without PML-RARA showed upregulation of RAS-MAPK and PI3K-AKT related genes, highlighting the involvement of both TRK physiological signaling pathways via ETV6-NTRK3. Top genes upregulated in AP-1060 confer signatures both for AML - CCNA1, CD96, DSU, EVI1, HGF, IL32, LGALS3, MDS1, TLE1, TSPAN2; and lymphocyte development - BSPRY, BST1, CCR6, EMP1, GIMAP1, GZMA, PLEKHG1. Several primitive hematopoietic or stem cell mRNAs were also overexpressed, including PRSS2, CD96, SIPA1L2, and PYHIN1. Prominent downregulated genes also included: ADD3, CD36, HOXA-9/10, LGALS9, MALAT1, PGDS, PLA2G4A (AML signature); HOXB4, KIAA1949, NR2F6, TEAD4 (stem cell); and LY6E, TRIM44 (lymphocyte signature). Growth and proliferation of ETV6-NTKR3 cells was exquisitely sensitive to TRKi treatments which spared control AML and to which NB-4 cells were highly resistant. Accordingly we used pharmacologic modulation of conspicuously expressed genes by small molecule TRKi treatment to highlight likely kinase signaling targets among conspicuously expressed genes. Several candidate target genes thus emerged, notably AWNT1, IL32, and the MDS-EVI1 fusion transcript. Salient pharmacologically unmodulated genes were preferentially stem cell in character highlighting this setting for t(12;15) formation in AP-1060 cells. Bioinformatic pathway analysis (http://david.abcc.ncifcrf.gov/) of both up- and down- conspicuously regulated genes identified “Alternative Splicing” as top category, with respectively 743 and 373 alternate spliceform genes up- and down-regulated. These included several genes whose spliceforms may be differentially expressed in oncogenesis, including MDS1-EVI1/EVI1, MALAT1, and WT1/AWT1. Interestingly, a key pre-mRNA splicing gene, MBNL2 was conspicuously downregulated, while another spliceosomal component THOC5 (C22orf19), recently identified as a leukemic kinase signalling target (Pierce et al., Br J Haematol 2008;141:641), is upregulated. Conclusions We present a human leukemia model and resource for ETV6-NTRK3. Taken together, our findings support spliceosomal targeting by ETV6-NTRK3 and suggest a possible underlying mechanistic framework. Additional targets, e.g. WNT signaling, seem to be shared with solid tumors bearing the same oncogene fusion. Perspectives: Future work includes transcriptosomal analysis of AP-1060 cells after knockdown of ETV6-NTRK3 and key splicesomal genes, such as THOC5, by short-hairpin RNAs, and novel, highly selective 4-aminopyrazolylpyrimidine TRKi (Thress et al., Mol Cancer Therapy 2009;8:1818). Disclosures No relevant conflicts of interest to declare.
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Aljamaan, Khalid, Talal Khalid Aljumah, Saleh Aloraibi, Muhammad Absar, Giuseppe Saglio, and Zafar Iqbal. "Low Representation of ETV6-RUNX1 Fusion Oncogene in Pediatric Acute Lymphoblastic Leukemia Patients from Saudi Arabia and Strong Association with Clinical Response to Day-14 of Remission Induction Therapy." Blood 126, no. 23 (December 3, 2015): 4987. http://dx.doi.org/10.1182/blood.v126.23.4987.4987.
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Abstract Abstract : Introduction: t(12; 21) leading to ETV6-RUNX1 fusion oncogene is associated with better prognosis and excellent treatment outcome in pediatric ALL (pALL) patients have good prognosis as compared to other genetic abnormalities1. Accordingly, frequency of ETV6-RUNX1 has a huge implication in treatment strategies of pALL in a given population. Different ethnic groups from various geographical regions have different frequencies of ETV6-RUNX1 ranging from 10% (Southeast Asia) to 33% (Australia) 2,3. Therefore, aim of this study was to determine ETV6-RUNX1 status of Saudi Arabian pALL patients and its association with clinical parameters and early remission. Patients & Methods: Pediatric Acute Lymphoblastic Leukemia (pALL) patients at King Abdulaziz Medical City represent pure Saudi Arabian population. Clinical parameters and ETV6-RUNX1 status (using FISH technique) of pALL patients attending Pediatric Oncology Clinic, King Abdulaziz Medical City Riyadh from 2006 to 2011 were studied using Vysis ETV6/RUNX1 DF FISH Probe Kit (Abbot Laboratories, Illinois, USA)4. CCG1991 protocol was used for standard risk patients while CCG1961 protocol was used for high risk patients. Number of blasts at day 14 and day 29 of the treatment were also calculated as a part of routine clinical follow-up.Comparison between ETV6-RUNX1 positive and negative groups done using chi-square test or Fisher's exact test. All Statistical analysis was performed using SAS version 9.2 (SAS Institute, Inc., Cary, NC). Results: Out of 54 patients, 33 were male and 21 were females (ration1.57:1). B- and T-cell lineage was found in 47 (87%) and 7 (13%) patients respectively. Only 5 (9.3%) patients with ETV6-RUNX1 positive while 49(80.7%) were ETV6-RUNX1 negative. All ETV6-RUNX1 patients (100%) were of B-cell lineage and 80% (4/5) were in 3-7 year age group. None of ETV6-RUNX11 patients had ≥5% blasts (no remission) at Day 14 as compared to 9% patients from ETV6-RUNX1 negative group (Table 1). Discussion: Frequency of ETV6-RUNX1 positive patients (less than 10%) in our pALL patients is much lower than reported in most of the European countries, North America, Australia and Japan while it is in accordance with ETV6-RUNX1 frequencies from Egypt (11.6%), Pakistan (10%), and India (5-7%) 2,3,5,6. This diversity in frequencies of ETV6-RUNX1 among pALL can be attributed to level of industrialization and/or westernized lifestyle. Moreover, ethnic differences in frequencies of this and other prognostically important genetic abnormalities can have a significant bearing on global pediatric ALL management strategies7 which necessitates further large scale studies in this regard. References: 1. Cooper SL, Brown PA. Treatment of pediatric acute lymphoblastic leukemia. Pediatr Clin North Am. 2015 Feb;62(1):61-73. 2. Iqbal Z. Molecular genetic studies on 167 pediatric ALL patients from different areas of Pakistan confirm a low frequency of the favorable prognosis fusion oncogene TEL-AML1 (t 12; 21) in underdeveloped countries of the region. Asian Pac J Cancer Prev. 2014;15(8):3541-6. 3. Amor DJ, Algar EM, Slater HR, Smith PJ. High frequency of t(12;21) in childhood acute lymphoblastic leukemia detected by RT-PCR. Pathology. 1998 Nov;30(4):381-5. 4. Vysis ETV6/RUNX1 DF FISH Probe Kit. https://www.abbottmolecular.com/vysis-etv6runx1-df-fish-probe-kit.html 5. Harbott J, Viehmann S, Borkhardt A, Henze G, Lampert F. Incidence of TEL/AML1 fusion gene analyzed consecutively in children with acute lymphoblastic leukemia in relapse. Blood. 1997 Dec 15;90(12):4933-7. 6. Shurtleff SA, Buijs A, Behm FG, Rubnitz JE, Raimondi SC, Hancock ML, Chan GC, Pui CH, Grosveld G, Downing JR. TEL/AML1 fusion resulting from a cryptic t(12;21)is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis. Leukemia. 1995 Dec;9(12):1985-9. 7. Weso³owska-Andersen A, Borst L, Dalgaard MD, Yadav R, Rasmussen KK, Wehner PS,et al. Genomic profiling of thousands of candidate polymorphisms predicts risk of relapse in 778 Danish and German childhood acute lymphoblastic leukemia patients. Leukemia. 2015 Feb;29(2):297-303. Disclosures Saglio: Novartis: Consultancy, Honoraria; BMS: Consultancy.
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Roberts, Kathryn G., Olga Bridges, Laura J. Janke, Kevin Ebata, Brian B. Tuch, Nisha Nanda, and Charles Mullighan. "Genetic Modeling and Therapeutic Targeting of ETV6-NTRK3 with Loxo-101in Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 278. http://dx.doi.org/10.1182/blood.v128.22.278.278.
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Abstract Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype characterized by kinase-activating alterations. One recurrent alteration is the ETV6-NTRK3 fusion, which results in constitutive activation of NTRK3, a member of the neurotrophic receptor kinase family. ETV6-NTRK3 has been identified in a range of malignancies, including breast cancer, pediatric glioma and infantile fibrosarcoma. The oncogenic role of ETV6-NTRK3 in B-cell ALL has not been investigated. The goals of this study were to assess the development of leukemia in genetically engineered models of ETV6-NTRK3, and to investigate efficacy of the specific TRK A, B and C inhibitor, LOXO-101, currently in clinical trials for the treatment of solid tumor patients who harbor NTRK fusions. Methods: For in vitro studies, kinase fusions were expressed in IL3 dependent Ba/F3 cells. To generate a genetically engineered mouse model, we used a previously reported conditional knockin model of Etv6-NTRK3 (Cancer Cell 2007;12:542-558), whereby the human portion of NTRK3 cDNA encoding the tyrosine kinase domain was inserted into exon 6 of the mouse Etv6 locus, downstream of a floxed transcriptional terminator sequence. Expression of the Etv6-NTRK3 protein was accomplished using Cre-recombinase driven by the B-lineage promoter CD19. A patient derived xenograft (PDX) model of ETV6-NTRK3 was established by engrafting primary human ALL cells expressing luciferase into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Phosphoflow cytometry analysis and sensitivity to LOXO-101 was assessed in vitro and in vivo. Results: Etv6-NTRK3/+, CD19-Cre mice developed aggressive disease with 100% penetrance and a median latency of 38 days (n=27). The average body weight of Etv6-NTRK3/+, CD19-Cre mice was significantly reduced compared to age-matched Etv6-NTRK3/+ controls (13.9 vs 20.2g, p<0.001). We observed increased spleen weight in Etv6-NTRK3/+, CD19-Cre mice compared to controls (142 vs 71mg, p=0.02), but no difference in peripheral white blood counts (9.7 vs 13.4 x 109/L, p=0.3). Presence of the Etv6-NTRK3 fusion was confirmed in bone marrow samples by RT-PCR. Immunophenotyping of bone marrow indicated arrest at the pre-B stage (Hardy stage C: B220+, CD19+, CD43+, BP1+, IgM-), recapitulating human ALL. Pathological analysis using hematoxylin and eosin and B220 staining showed infiltration of leukemic cells into the bone marrow, spleen, liver and lung. Interestingly, we observed extensive infiltration of leukemic cells into the central nervous system, specifically ventral to the thoracic and lumbar vertebrae, and the meninges within the brain. Copy number alteration and sequence mutation analysis is currently being performed to determine additional genetic lesions. Leukemia cells from the bone marrow displayed constitutive activation of the MAPK pathway via pERK1/2. We next assessed the in vitro efficacy of the TRK inhibitors crizotinib, which also inhibits ALK, and a more specific inhibitor, LOXO-101. Compared to crizotinib (IC50 205 nM), LOXO-101 was 10 times more potent against BaF3-ETV6-NTRK3 cells (IC5017 nM), and had no effect on other kinase fusions (ABL1, ABL2, CSF1R, FLT3, JAK2) up to 10µM. In addition, LOXO-101 was remarkably selective for TRK A, B and C in a cytotoxicity screen of 77 human cancer cell lines as compared to crizotinib. Using a PDX model of ETV6-NTRK3, we demonstrate that treatment with LOXO-101 (200mg/kg/day p.o for six weeks) reduced leukemic infiltration to undetectable levels in the bone marrow (0 vs 75.8% human CD45/CD19 bone marrow blasts, n=5 each group) and spleen compared to vehicle-treated mice (splenic weight 316 vs 20mg, p<0.001). Notably, treatment with dexamethasone had a modest effect against this tumor (average 55.3% bone marrow blasts and spleen weight 134mg, n=5). Mice treated with LOXO-101 were still alive and leukemia-free four weeks after the cessation of treatment, as determined by Xenogen imaging. Conclusion: We have described the first genetically engineered mouse model of Ph-like ALL with an ETV6-NTRK3 fusion, and reported remarkable efficacy of LOXO-101 against the NTRK3 fusion, with complete suppression of leukemic cell proliferation when administered as a monotherapy. These findings warrant screening for ETV6-NTRK3 in newly diagnosed ALL patients, and testing the efficacy of LOXO-101 in combination with chemotherapy regimens. Disclosures Ebata: Loxo Oncology: Employment, Other: Shareholder. Tuch:Loxo Oncology: Employment, Other: Shareholder. Nanda:Loxo Oncology: Employment, Other: Shareholder. Mullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding.
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Hahm, Chorong, Sung Hee Han, Yeung Chul Mun, Chu Myong Seong, Wha Soon Chung, and Jungwon Huh. "ETV6/RUNX1 Rearrangement Identified by RT-PCR without Evidence on FISH." Acta Haematologica 132, no. 1 (2014): 122–24. http://dx.doi.org/10.1159/000356778.
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Vu, Hoang Anh, Phan Thi Xinh, Seiko Shimizu, Michihiko Masuda, Toshiko Motoji, Katsushi Tokunaga, and Yuko Sato. "FLT3 Is Fused to ETV6 in a Myeloproliferative Disorder with a t(12;13)(p13;q12) Translocation." Blood 104, no. 11 (November 16, 2004): 2901. http://dx.doi.org/10.1182/blood.v104.11.2901.2901.
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Abstract The FLT3, located at band 13q12 and encoding a receptor tyrosine kinase (RTK), is one of the most frequently mutated genes in hematologic malignancies including ALL, MDS, and AML. The most common mutation of the FLT3 is an internal tandem duplication in exons 14 and 15, whereas other mutations have also been found at and around codon 835 of exon 20. These activating mutations promote constitutive RTK activity in the absence of ligand, proposing FLT3 as an attractive therapeutic target for directed inhibition. However, many questions with regards to the biology of FLT3 and its role in leukemogenesis remain to be clarified. Despite its highly frequent mutations, FLT3 has never been reported to fuse to any other genes, a phenomenon usually observed in other RTKs. Here, we report a case of a novel fusion gene between FLT3 and ETV6 at 12p13, a well-known target for a number of translocations. The patient, a 68-year-old female, was diagnosed as myeloproliferative disorder with hypereosinophilia in May 2002. Peripheral blood showed WBC 33.6x106/L (3% myelocytes, 33.5% neutrophils, 54% eosinophils, 1.5% basophils, 1.0% monocytes and 7% lymphocytes), Hb 119g/L and platelet counts 5,450x106/L. The bone marrow (BM) was marked hypercellular with 0.9% blasts, 6.0% promyelocytes, 15.6% myelocytes, 8.1% immature eosinophils and 19.2% mature eosinophils. Karyotype of BM cells was 46, XX, t(12;13)(p13.1;q12.3–13)[28]/46, XX[2]. Under the suspicion of Ph-negative CML, she was treated with IFNα with no response. Then, HU was started and her WBC decreased to 30x106/L. FISH analysis showed that the breakpoint at 12p13 occurred within ETV6, while the breakpoints at 13q12 occurred at two locations, within FLT3 or CDX2. To identify the fusion partner of ETV6, 3′-RACE PCR was performed. Sequence analysis of PCR-products revealed 4 types of ETV6/FLT3 transcripts. These fusion transcripts were confirmed by Northern blot analysis. Each ETV6/FLT3 transcript contained the entire helix-loop-helix domain of ETV6 (exons 1 to 4 or 5) and almost all of the functional domains of FLT3 including the tyrosine kinase domain (from exons 14, 16 or 17), suggesting that the resultant chimeric protein would be constitutively activated FLT3 kinase. Of them, three are in-frame fusion, presumably encoding for the approximately 58, 62, and 83 kD fusion proteins. However, Western blot analysis showed only expression of the 58 and 83 kD proteins. RT-PCR detected the reciprocal FLT3/ETV6 transcript, comprising the FLT3 exons 1 to 13 frameshifly fused to the ETV6 exons 6 to 8, within which a stop codon appeared at codon 33- downstream from the fusion point. Functional studies to assess the oncogenic properties of these fusion proteins are now in progress. Our findings provide an evidence that FLT3 is also involved in hematologic malignancies as a fusion gene.
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Haferlach, Claudia, Niroshan Nadarajah, Manja Meggendorfer, Nadine Dicht, Anna Stengel, Wolfgang Kern, and Torsten Haferlach. "Targeted RNA Sequencing Is Capable of Identifying Thus Far Unknown Partner Genes in Leukemias with Rare Translocations and Provides Important Clinical and Therapeutical Information." Blood 128, no. 22 (December 2, 2016): 2857. http://dx.doi.org/10.1182/blood.v128.22.2857.2857.
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Abstract Background: The genomic landscape of hematological malignancies has been resolved mainly based on whole exome and whole genome sequencing, primarily targeting gene mutations. Beside mutations also gene fusions function as therapeutic targets, impressively shown for e.g. BCR-ABL1 and ETV6-PDGFRB. Hence, the need for a comprehensive genetic analysis is increasing, as it is the basis for precision medicine, selecting treatment based on genotype and providing markers for disease monitoring. Aim: To test the value of targeted RNA sequencing in a routine diagnostic work up. Patients and Methods: 38 cases were selected in which rearrangements involving KMT2A (n=8), RUNX1 (n=19), ETV6 (n=9), RARA (n=1) and JAK2 (n=1) had been identified by chromosome banding analysis (CBA) complemented by FISH analysis. In all cases the partner gene could not be identified using standard methods. Targeted RNA sequencing was performed using the TruSight RNA Fusion panel (Illumina, San Diego, CA) consisting of 7690 probes covering 507 genes known to be involved in gene fusions. Library was prepared according to manufacturer's protocol with ~50ng DNA extracted from fresh/frozen samples. This assay allows the capture of all targeted transcripts. Sequencing was performed on two NextSeq runs (Illumina, San Diego, CA) with 20 multiplexed samples including two samples with known fusions as positive control samples. Analysis was performed with the RNA-Seq Alignment App (BaseSpace Sequence Hub) using Star for Alignment and Manta for gene fusion calling with default parameters (Illumina, San Diego, CA). Results: In 22/38 cases with rearrangements involving KMT2A (n=8), RUNX1 (n=8), ETV6 (n=4), RARA (n=1) or JAK2 (n=1) this approach led to important new information: The following partner genes for KMT2A were identified: MLLT10 (n=2), MLLT1 (n=2), ITPR2, FLNC, ASXL2 and ARHGEF12. MLLT10 and MLLT1 are two of the most frequent partner genes of KMT2A, while KMT2A-ARHGEF12 fusions are rare. Fusion of KMT2A to ITPR2, FLNC, or ASXL2 have not been reported yet. Seven different partner genes were identified in RUNX1 translocated cases. These were PLAG1 (n=2), PRDM16, MECOM, ZFPM2, MAN1A2, N6AMT2, and KIAA1549L. PRDM1, MECOM and ZFPM2 have previously been described in the literature as RUNX1 partner genes but were not suspected in our cases as partner genes due to complex cytogenetic rearrangements in CBA. The other identified partner genes have not been described so far. Interestingly, PRDM1, MECOM, ZFPM2 and the newly identified PLAG1 are all members of the C2H2-type zinc finger gene family. Four different partner genes were identified in ETV6 rearranged cases: ABL1, CCDC126, CLPTM1L, and CFLAR-AS1. Most strikingly was the identification of the ETV6-ABL1 fusion, which could not be suspected by cytogenetics as the 5' ETV6 FISH signal was located on chromosome 7. This ETV6-ABL1 fusion was confirmed by conventional RT-PCR. In an ALL patient a JAK2-PPFIBP1 fusion was identified leading to classification as a BCR-ABL1-like ALL. In an APL patient showing an ins(17;11)(q12;q14q23) in chromosome banding analysis a ZBTB16-RARA fusion was identified and thus resistance to all-trans retinoic acid, arsenic trioxide, and anthracyclines can be predicted. All these fusions were not detectable by our routine RT-PCR analyses as these assays cover only the most frequently occurring breakpoints in fusions with known partner genes, but might miss very rare variants. For all yet undescribed fusion partners routine assays are not available. Based on the results of targeted RNA sequencing quantitative PCR assays for MRD monitoring can now be established. In 11 cases with a RUNX1 rearrangement and 5 cases with an ETV6 rearrangement no fusion transcript was identified. Further analyses will have to clarify whether in these cases no transcript was derived from the genomic rearrangement. Conclusions: 1) Targeted RNA sequencing was able to identify and characterize rare gene fusions and thus provided the basis for the design of RT-PCR based assays for monitoring MRD. 2) Targetable genetic aberrations were identified, which were not identifiable by chromosome banding analysis but would now lead to more individualized treatment. 3) Thus, targeted RNA sequencing may be a valuable tool in routine diagnostics for patients with rearrangements unresolved by standard techniques, also paving the way to precision medicine in a considerable number of patients. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Dicht:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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Gandemer, Virginie, Marie-Françoise Auclerc, Arnaud Petit, Benoît Brethon, Paola Ballerini, Gerard Michel, Yves Perel, et al. "Excellent Prognosis of Children with ETV6-RUNX1 Positive (+) Acute Lymphoblastic Leukemia (ALL) in the FRALLE 2000 Protocol." Blood 114, no. 22 (November 20, 2009): 1628. http://dx.doi.org/10.1182/blood.v114.22.1628.1628.
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Abstract Abstract 1628 Poster Board I-654 Since the cloning of the t(12 ;21) in 1995 the prognosis of children with ETV6-RUNX1(+)ALL seems to further increase in the current era. From december 2000 to july 2008, 1461 children and adolescents aged from 1 to 20 years with B cell lineage ALL have been treated according the FRALLE protocols: one for standard-risk (SR) ALL (age 1-9 y, WBC < 50 G/L, no extra medullary involvement) F2000-A; the other one FRALLE 2000-B for high and very high risk ALL (HR) (all other pts). The two protocols mainly differ by a larger use of dexamethasone and a reduced use of anthracyclins in F2000A and the use of HDMTX and a double delayed intensification in FRALLE 2000B. ETV6-RUNX1 presence has been assessed by RT-PCR in 1392 pts (i.e. 96%) and found positive in 321 pts, i.e. 23%. Initial features include a median age of 4.2 y (1.2-15.6), a sex ratio (M/F) of 1.2, a median WBC of 10 G/L(1-293). Both peripheral blood D8 response to prednisone (PRED) and D21 marrow response to chemotherapy were evaluable in 316 pts: 94% of the pts have a rapid early response at D8 and D21. Only 3% of the pts were qualified as D8 poor PRED responders and 3% slow marrow responders at D21. The D35-42 CR rate is 100%. End of induction (EOI) minimal residual disease (MRD) using Ig-TCR methods is known in 259 out of 321 pts (81%) pts. Only 4 pts (2%) had a very high (≥10-2) EOI-MRD. Five year EFS, DFS and OS are 95±2%, 95±2%, 98±1%, respectively. The 5y EFS is 96±2% for the 252 SR pts and 90±7% for the 69 HR pts, p=0.30. The 5y EFS is significantly better for children with ETV6-RUNX1(+)ALL compared to those with ETV6-RUNX1(-) B-lineage ALL: 95±2% vs 84±2%, respectively (p=.001). Nine relapses have been reported in the bone marrow (4) the testis (4), the CNS (1) after a median time of 44 m (25-68), i.e. significantly longer than in the ETV6-RUNX1(-) cases (28m (1-92), p=.01). More testis relapses are observed in boys with ETV6-RUNX1(+)ALL (4 out 7 relapses vs 2 out of 62 in boys with ETV6-RUNX1(-) ALL). These results represent a significant progress compared to the previous protocol F 93 (191 children) both in terms of EFS and overall survival (5y EFS: 95±2% vs 78±3%, p=.001; 5 y OS: 98±1% vs 92±2%, p=.001). Conclusion an excellent prognosis of children with t(12;21)/ETV6-RUNX1 positive acute lymphoblastic leukemia is now observed in the FRALLE 2000 protocol. The question of a cautious de-escalation in this subgroup will be envisaged in the next protocol. Disclosures No relevant conflicts of interest to declare.
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Gosenca, Darko, Philipp Erben, Claudia Haferlach, Rainer Schwerdtfeger, Herrad Baurmann, Georg Bolz, Juliana Popa, et al. "“Clinical and Molecular Heterogeneity of Eosinophilia-Associated Myeloproliferative Neoplasms with a Translocation t(5;12)”." Blood 114, no. 22 (November 20, 2009): 4983. http://dx.doi.org/10.1182/blood.v114.22.4983.4983.
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Abstract Abstract 4983 Chromosomal or molecular aberrations in eosinophilia-associated myeloproliferative neoplasms (Eos-MPN) often involve receptor (e.g. PDGFRA at 4q12, PDGFRB at 5q31, FGFR1 at 8p11) or intracellular (e.g. ABL1 at 9q34, JAK2 at 9p24) tyrosine kinases (TK). Fusion genes with rearrangement of PDGFRA or PDGFRB are known to be associated with rapid and durable complete hematological and clinical remission on treatment with imatinib; many of these cases also achieve complete molecular remission (undetectable fusion gene transcripts by nested reverse transcriptase polymerase chain reaction, RT-PCR). The most common fusion genes in Eos-MPN are FIP1L1-PDGFRA caused by a cytogenetically invisible interstitial deletion on 4q12, and ETV6-PDGFRB as consequence of a reciprocal translocation t(5;12)(q31-33;p13). Due to restricted availability of molecular diagnostic tools, a substantial proportion of Eos-MPN patients with t(5;12) are treated with imatinib based on karyotype but without confirmation of the underlying fusion gene by fluorescence-in-situ-hybridization (FISH) or RT-PCR. We report here on three patients (male, n=2; female, n=1) (median age 58 years, range 38-71) with Eos-MPN (median absolute eosinophil numbers in peripheral blood 8,100/μL, range 2,300-62,000) in chronic (n=2) or secondary acute myeloid leukemia (AML)/blast phase (n=1). Cytogenetic analysis revealed a t(5;12) (n=2) or a complex karyotype with involvement of chromosome arms 5q and 12p (n=1). Despite the fact that the clinical and hematological phenotype was resembling ETV6-PDGFRB positive disease (e.g. leukocytosis, eosinophilia, hypercellular marrow, splenomegaly), all three cases tested negative for ETV6-PDGFRB. A fourth patient with t(5;12)(q31;p13) but negative for ETV6-PDGFRB had identical clinical characteristics except basophilia of 7% without eosinophilia. Further molecular analyses revealed an ETV6-ACSL6 fusion gene in all four patients. Two patients received imatinib (400 mg/d) without knowledge of the molecular status and two patients were treated with sorafenib (400-800 mg/d) due to its multitargeted activity towards signal transduction molecules. No responses were observed to imatinib or sorafenib. Two male patients received an allogeneic stem cell transplantation (SCT) from a related or a matched unrelated donor, respectively. The first patient died on day +67 due to relapse of secondary AML/blast phase while the second patient died on day +64 due to transplant related complications. After failure to imatinib, the female patient with secondary AML/blast crisis only received supportive care because of comorbidity and died 7 months after diagnosis due to cytopenia-related complications. The remaining chronic phase male patient is alive 5 months after diagnosis. In addition to patients known from the literature (n=6), primary AML or rapid progression to secondary AML/blast phase has been observed in 7 of overall 10 patients with t(5;12) and an ETV6-ACSL6 fusion gene indicating a potentially aggressive clinical course. Consistent with the lack of involvement of a TK, the disease is primarily resistant to currently available TK-inhibitors and allogeneic SCT should be considered in eligible patients. In conclusion, treatment with TK-inhibitors in patients with myeloproliferative neoplasms and a t(5;12) should only be initiated if involvement of PDGFRB is confirmed by FISH or PCR analysis. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
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Piette, Caroline, Stefan Suciu, Emmanuelle Clappier, Yves Bertrand, Genevieve Plat, Christophe Chantrain, Vitor Costa, et al. "t(12;21)/ETV6 -RUNX1 Confers a Specific Pattern of In Vivo Sensitivity to Treatments in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL): Results of the Randomized Trials 58881 and 58951 of the EORTC Children Leukemia Group." Blood 126, no. 23 (December 3, 2015): 793. http://dx.doi.org/10.1182/blood.v126.23.793.793.
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Background In childhood BCP-ALL, the presence of t(12;21)/ETV6 -RUNX1 defines one of the most prevalent oncogenic subgroup and is usually associated with a favorable outcome. Nevertheless, an excellent prognosis has not been reported by all collaborative groups, suggesting that the outcome of ETV6 -RUNX1 patients (pts) could be influenced by the treatment. To address this issue, the long-term outcome of ETV6 -RUNX1 pts was investigated into the EORTC 58881 and 58951 studies, with particular attention to the effect of the randomized treatments. Methods The 58881 study (1989-1998) used a BFM backbone without cranial irradiation, and aimed to compare E-Coli (Medac®) Asparaginase (A'ase) with Erwinia A'ase and to assess the value of 6-Mercaptopurine (6-MP) i.v. (1 g/m²/month) when added to classic maintenance. The subsequent 58951 study (1999-2008) used the best arm of the trial 58881, i.e. E-Coli A'ase and classic maintenance. The aims of this study were to compare dexamethasone (Dexa) (6 mg/m²/day) with prednisone (Pred) (60 mg/m²/day) during induction and maintenance; to evaluate increased number of A'ase administrations (24 vs 12) for non-very high risk pts; and to assess the value of vincristine/corticosteroid pulses during maintenance for average risk pts. Detection of ETV6 -RUNX1 by FISH and/or RT-PCR was centralized. Pts less than 1 year (yr) or with t(9;22)/BCR-ABL were excluded from the analysis. Results An ETV6 -RUNX1 was evidenced in 104/363 (28%) and 380/1493 (27%) newly diagnosed BCP-ALL pts enrolled in the 58881 and 58951 trial respectively. A majority of ETV6-RUNX1 pts were below the age of 10 yrs (93.3% in 58881 and 91.3% in 58951). The median follow-up was 11.8 yrs for the 58881 and 6.7 yrs for the 58951. In both studies, the 10-yr event-free-survival (EFS) rate was significantly higher for ETV6 -RUNX1 pts than for all BCP-ALL pts (82.5% vs 74.9% in 58881 and 90.8% vs 82.7% in 58951), and was similar to the 10-yr EFS of hyperdiploid pts. Noteworthy, very few EFS events were observed during treatment period or after 6 yrs from diagnosis. The main prognostic factors of the ETV6-RUNX1 subgroup in both studies were the white blood cell count (WBC) and the response to prephase. As shown in the table below, the analysis of the relationship between treatment modalities and outcome revealed that the in vivo drug sensitivity of ETV6-RUNX1 ALL was distinct from that of other BCP-ALL. In this subgroup, the benefit of a more potent A'ase (58881) or of intensified A'ase administrations (58951) was less pronounced as compared to other pts, and 6-MP i.v. during maintenance was particularly deleterious. Moreover, the overall benefit of vincristine/corticosteroid pulses was not observed in ETV6-RUNX1 average risk group pts, who already had an outstanding outcome. By contrast, the use of Dexa in place of Pred significantly improved the 10-yr EFS of ETV6-RUNX1 pts (95.0% vs 87.2%, hazard ratio (HR)=0.44, 95% CI 0.20-0.96) whereas no difference was observed in the remaining population (HR=1.01, 95% CI 0.77-1.33) (test for interaction: p=0.04). Table.5888158951All (n=363)ETV6-RUNX1 (n=104)Hyperdiploid (n=102)Others1 (n=155)All (n=1493)ETV6-RUNX1 (n=380)Hyperdiploid (n=484)Others1 (n=619)10-yr EFS ratesAll pts74.9%82.5%83.3%65.2%82.7%90.8%88.4%73.2%Pred ² (n=745)81.8%87.2%88.8%73.0%Dexa ² (n=748)83.6%95.0%88.0%73.5%Medac A'ase 2,3 (n=320)76.6%83.2%88.6%65.7%Other A'ase 2,3 (n=43)62.8%78.6%50.0%60.0%10-yr disease-free-survival ratesShort A'ase 4 (n=607)83.5%91.2%90.1%72.4%Long A'ase 4 (n=622)87.0%94.8%88.2%80.7%No 6-MP iv 2,5 (n=96)85.4%100%90.9%72.5%6-MP iv 2,5 (n=94)72.3%70.6%78.6%68.8%No Pulse 5,6 (n=148)87.5%96.1%88.5%71.1%Pulse 5,6 (n=153)82.8%95.1%91.3%82.3%1Others, MLL rearrangements excluded 2All risk groups 3Pts randomized or not for A'ase 4Non-very high risk pts 5Pts who started maintenance 6Average risk pts Conclusions Within the EORTC 58881 and 58951 trials, the use of Dexa rather than Pred allowed to further improved the long-term outcome for ETV6-RUNX1 pts. Our data also show that this excellent outcome can be jeopardized by slight changes in therapy, such as the addition of 6-MP i.v. to classic maintenance. Together, these results stress the importance of analyzing homogeneous oncogenetic subgroups when comparing different therapeutic schemes, to unmask specific drug effects that could be hidden when analyzing the whole group of patients. Disclosures Bertrand: ERYTECH Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees.
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Reiter, Andreas, Gunay Ahmadova, Mohamad Jawhar, Andres Virchis, Tim Somervaille, Mark Williams, Bernd Schönberger, et al. "Clinical Characteristics and Treatment with Various Tyrosine Kinase Inhibitors in Patients with ETV6-ABL1 positive Eosinophilia-Associated Myeloproliferative Neoplasms." Blood 128, no. 22 (December 2, 2016): 3114. http://dx.doi.org/10.1182/blood.v128.22.3114.3114.
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Abstract The ETV6-ABL1 fusion gene, as consequence of a t(9;12)(q34;p13), is a rare but recurrent genetic aberration found in chronic or blast phase of eosinophilia-associated myeloproliferative neoplasms (MPN-eo) and de novo acute B-cell lymphoblastic leukemias (B-ALL) or lymphoblastic T-cell lymphomas (T-LBL). Here, we sought to evaluate a) relevant clinical characteristics, b) treatment options and c) survival in 7 ETV6-ABL1 positive patients (male, n=4; median age 46 years; range 20-61). Cytogenetic analyses revealed a conventional reciprocal translocation t(9;12)(q34;p13) in 3 cases, an ins(12;9)(p13;q34q22) and a normal karyotype in one patient each, and a complex karyotype in 2 patients. In all cases, ETV6-ABL1 was confirmed by FISH analysis and/or RT-PCR. Histopathological diagnoses of the hypercellular bone marrow (BM) included atypical chronic myeloid leukemia (n=3), MPN-eo (n=3, concomitant T-LBL in one patient) or chronic myelomonocytic leukemia (n=1). In peripheral blood, all patients presented with left shifted leukocytosis (median 84 x 109/l, range 21-143) and significant (>1.5 x 109/l) eosinophilia (median 6.1 x 109/l, range 2.0-7.1) but without increased blast cells. Splenomegaly was present in 60% of patients. After a median of 3 months (range 0-6) from diagnosis, all patients were treated with a TKI (imatinib, n=5; dasatinib, n=1; nilotinib, n=1) at standard doses. On dasatinib (#2) or nilotinib (#3), 2 patients achieved a complete hematologic remission (CHR) within 3 months and complete cytogenetic remission (CCR) after 5 months (#3) or complete molecular remission (CMR) after 18 months (#2), respectively (Figure). On imatinib (n=5), 2 of 5 patients (#4 and #5) achieved a CHR within 3 months with loss of CHR after 9 (#4) and 5 months (#5), respectively. Patient #4 developed a myeloid sarcoma. After local radiation, he received an allogeneic stem cell transplant (SCT) but died 8 months later due to GvHD while in CMR. Patient #5 switched to nilotinib and achieved a CCR and CMR after 3 and 10 months, respectively. Patient #1 has not achieved a significant response after 4 months on imatinib. Patient #6 showed progressive disease within 2 months on imatinib, but achieved a rapid CHR and durable CCR and CMR on dasatinib after 13 and 14 months, respectively. Patient #7 presented with a classical myeloid/lymphoid (T-LBL) neoplasm with eosinophilia (MLN-eo) according to the WHO classification. On imatinib, a regression of lymphadenopathy, but persistence of leukocytosis and eosinophilia was observed. With increasing leukocytosis and reappearance of lymphadenopathy, the patient was switched to dasatinib (9 months), followed by nilotinib (2 months). The responses were only partial and transient and the patient died 23 months after diagnosis with myeloid blast phase (secondary acute myeloid leukemia). Overall, after a median treatment time of 22 months (range, 3-58), 4 patients are in CCR (n=1, patient #3) or CMR (n=3; patients #2, #5, #6) while on a second generation TKI and two patients (#4, #7) have died. On imatinib, none of 5 patients achieved a CCR or CMR. We conclude that a) cytogenetic analysis is an important tool for the identification of potential TK fusion genes such as ETV6-ABL1, b) ETV6-ABL1 is a candidate for incorporation into the WHO-defined subcategory ´MLN-eo´, c) patients with MPN-eo can achieve durable CHR, CCR or CMR on TKI with second generation TKI being more effective than imatinib d) close monitoring by cytogenetics, FISH and RT-PCR is recommended for early identification of inadequate response or resistance. Figure (A) responses and (B) overall survival in 7 patients with ETV6-ABL1 positive MPN-eo treated with various tyrosine kinase inhibitors.Abbreviation, CHR:complete hematologic response; CCR: complete cytogenetic response; CMR: complete molecular response; CP: chronic phase; BP: blast phase; allo SCT: allogeneic stem cell transplantation. Figure. (A) responses and (B) overall survival in 7 patients with ETV6-ABL1 positive MPN-eo treated with various tyrosine kinase inhibitors.Abbreviation, CHR:complete hematologic response; CCR: complete cytogenetic response; CMR: complete molecular response; CP: chronic phase; BP: blast phase; allo SCT: allogeneic stem cell transplantation. Disclosures Somervaille: Novartis: Consultancy, Honoraria; Imago Biosciences: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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Iqbal, Zafar, Tashfin Awan, Mudassar Iqbal, Noreen Sabir, Sultan Asad, Mohammad Hasan Siddique, Sumrin Sahar, Ammara H. Tahir, Aamer Aleem, and Tanveer Akhtar. "High frequency of BCR-ABL oncogene in pediatric acute lymphoblastic leukemia (ALL) patients as revealed by RT-PCR and interphase FISH: Association with disease biology and treatment outcome." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): 6612. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.6612.
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6612 Background: ALL is a complex genetic disease involving many fusion oncogenes (FGs)1 frequency of which can vary in different ethnic groups2,3 thus having implication in differential diagnosis, prognosis and treatment. Methods: We studied FGs in 101 pediatric ALL patients using RT-PCR1 at Day 0, Day 15 and Day 29 and Interphase FISH, and their association with clinical features and treatment outcome. Results: Five most common FGs i.e. BCRABL (t 22; 9), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (del 1p32) were found in 88.1% (89/101) patients. Frequency of BCR-ABL was 44.5% (45/101). Patients with BCR-ABL had significantly lower survival (43.733 weeks ±4.241) as compared to others except MLL-AF4 and significantly higher TLC count. Overall survival was lower (52.2±3.75) than the patients with ETV6-RUN X 1 (65.2± 9.9) which may be due to overall high frequency of poor prognostic FGs (71%) as compared to ETV6-RUNX 1 (17.1%) (p=0.01). Conclusions: This is the first study from Pakistan correlating molecular markers, disease biology and treatment response. It is the highest reported frequency of BCR-ABL in pediatric ALL and was associated with disease biology and survival. Some authors have reported BCR-ABL frequency higher than in West2,4,5 while others reported 45% frequency of ETV6-RUNX16. These and our data reflect strong interplay of genetic and environmental factors in biology of pediatric ALL and its correlation with disease biology and treatment2,3. Our data indicates immediate need for large clinical trials of imatinib, dasatinib and nilotinib in paediatric ALL treatment in our ethnic group. This study will lead to unravel the mechanisms of BCR-ABL Leukemogenesis and to find population-specific biomarkers and drug targets. References: 1) van Dongen JJ, et al., Leukemia. 1999 Dec; 13(12):1901-28. 2) Iqbal Z, et al. J Pediatr Hematol Oncol. 2007 Aug; 29(8):585. 3) Ariffin H, et al. J Pediatr Hematol Oncol. 2007 Jan; 29(1):27-31. 4) Ramos C, et al. 2011 Sep; 139(9):1135-42. 5) Artigas A CG, et al. Rev Med Chil. 2006 Nov; 134(11):1367-76. 6) Karrman K, et al. Br J Haematol. 2006 Nov; 135(3):352-4.
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Fisher, Kevin E., Jacquelyn Reuther, Hadi Sayeed, Erica Fang Tam, Vijetha Kumar, Lizmery Suarez Ferguson, Jyotinder Nain Punia, et al. "Leukemia Fusion Gene Detection in the Clinical Molecular Laboratory Using RNA-Based Targeted Next-Generation Sequencing." Blood 128, no. 22 (December 2, 2016): 4074. http://dx.doi.org/10.1182/blood.v128.22.4074.4074.
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Abstract Introduction: The diagnosis and risk stratification for patients with B-lymphoblastic leukemia (B-ALL) requires the accurate detection of fusion genes such as BCR-ABL1, ETV6-RUNX1,or Philadelphia-like (Ph-like) B-ALL kinase fusions, or gene rearrangements (e.g. KMT2A). No single assay can detect all relevant alterations, so costly and inefficient testing algorithms that combine karyotyping, fluorescent in situ hybridization (FISH), and reverse transcriptase PCR (RT-PCR) are often required. A comprehensive and sensitive RNA-based next-generation sequencing (NGS) assay that could consolidate diagnostic and prognostic B-ALL gene fusion and rearrangement testing into a single clinical test is an attractive alternative. Methods: We obtained RNA from 15 clinical specimens collected from 14 patients [11 bloods (2 from the same patient) and 4 bone marrows] with hematologic malignancies and known genomic alterations by RT-PCR, FISH, or cytogenetics, and 1 Ph-positive B-ALL cell line (SUP-B15). Samples harbored either B-ALL-associated alterations [BCR-ABL1 (3), ETV6-RUNX1 (2), intrachromosomal amplification 21 (iAMP21) (2), and KMT2A (2) or PDGFRB (1) rearrangements], or alterations detected in other hematologic malignancies [PML-RARA (2, same patient), RUNX1-RUNX1T1, t(5;9)(q33;q22), t(1;4)(p13;q12), and monosomy 7]. We prepared NGS libraries from extracted total RNA (100 ng) using the Archer® FusionPlex® Heme v2 anchored multiplex PCR-based NGS library with molecular barcoding protocol that targets 553 exons in 87 genes associated with B-ALL and other hematologic malignancies for detection of expressed fusions regardless of gene partner. Illumina paired-end indexed libraries were multiplexed (8 per run) and sequenced on a MiSeq (2x150 bp, v2) in two separate runs. Data were analyzed using vendor-provided virtual-machine based analysis pipelines and custom-developed scripts. ANNOVAR was used for breakpoint annotation, and fusion calls were compared to previous molecular results. Results: A mean total of 5.1x105 unique and 6.26x104 unique RNA paired-end reads were generated. One sample [t(1;4)(p13;q12)] failed QC due to inadequate RNA reads and was excluded from analysis. NGS detected the fusion or gene rearrangement in 10/11 samples (91%) with mean supporting split read counts of 175 reads (range: 6-1209). NGS detected both ETV6-RUNX1 fusions in samples with 4% and 5% blasts, respectively, defined binding partners for the KMT2A and PDGFRB gene rearrangements (KMT2A-MLLT10, KMT2A-MLLT3, and CCDC88C-PDGFRB), and detected novel P2RY8-CRLF2 fusions in both iAMP21 samples. NGS failed to detect a PML-RARA fusion in a post-treatment low-level RT-PCR-positive, FISH-, flow-, and morphology-negative blood sample, and no fusion was detected in the t(5;9)(q33;q22) sample. A low-level KMT2A-MLLT10 fusion was detected in an ETV6-RUNX1 sample suggesting a minor subclone or false-positive result. Conlcusions: In summary, RNA-based targeted NGS detects gene fusions and rearrangements comparable to conventional methods, accurately detects fusions in samples with ~5% blasts, and highlights previously unknown fusions and fusion partners. Additional studies are required to establish clinical specimen requirements, limit of detection, sensitivity, and specificity. Experiments are underway to confirm novel fusions, sequence additional recurrent B-ALL alterations (e.g. JAK2, TCF3, and PDGFRA fusions), and assess assay performance in formalin-fixed, paraffin-embedded tissue samples. RNA-based targeted NGS may be a clinically useful method to detect gene fusions and rearrangements in B-ALL and other hematologic malignancies. Disclosures No relevant conflicts of interest to declare.
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Alm, Sofie J., Charlotte Engvall, Julia Asp, Lars Palmqvist, Jonas Abrahamsson, and Linda Fogelstrand. "Minimal Residual Disease Monitoring in Childhood Precursor B Lymphoblastic Leukemia with t(12;21)(p13;q22); ETV6-RUNX1: A Comparison Between Quantitative RT-PCR and Flow Cytometry." Blood 124, no. 21 (December 6, 2014): 3774. http://dx.doi.org/10.1182/blood.v124.21.3774.3774.
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Abstract The translocation t(12;21)(p13;q22) resulting in the fusion gene ETV6-RUNX1, is the most frequent gene fusion in childhood precursor B lymphoblastic leukemia (pre-B ALL), affecting about one in four children with pre-B ALL. In the NOPHO ALL-2008 treatment protocol, treatment assignment in pre-B ALL is based on clinical parameters, genetic aberrations, and results from analysis of minimal residual disease (MRD) at day 29 and 79 during treatment (where MRD >0.1% leads to upgrading of treatment). For pre-B ALL, in this protocol MRD analysis is performed using flow cytometry as the method of choice. In this study, we also analyzed MRD in t(12;21)(p13;q22) cases with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the fusion transcript ETV6-RUNX1 in parallel with routine MRD analysis with flow cytometry, to determine if qRT-PCR of the ETV6-RUNX1 fusion transcript would be a reliable alternative to FACS. Bone marrow samples were collected at diagnosis and at day 15, 29 and 79 during treatment from 31 children treated according to the NOPHO ALL-2000 (n = 3) and NOPHO ALL-2008 (n = 28) protocols in Gothenburg, Sweden, between 2006 and 2013. Samples were analyzed in parallel with qRT-PCR for ETV6-RUNX1 fusion transcript and with FACS. For qRT-PCR, mRNA was isolated, cDNA synthesized, and qRT-PCR performed with GUSB as reference gene. MRD-qRT-PCR was defined as the ETV6-RUNX1/GUSB ratio at the follow-up time point (day 15/29/79) divided with the ETV6-RUNX1/GUSB ratio at diagnosis (%). MRD analysis with FACS was performed, after lysis of erythrocytes, using antibodies against CD10, CD19, CD20, CD22, CD34, CD38, CD45, CD58, CD66c, CD123, and terminal deoxynucleotidyl transferase, and when applicable also CD13 and CD33. Results of MRD-FACS were expressed as % of all cells. In total, 83 samples were analyzed with both methods in parallel; 31 from day 15 in treatment, 28 from day 29, and 24 from day 79. Overall, MRD-qRT-PCR showed good correlation with MRD-FACS. In total, 31 samples were positive with qRT-PCR and 24 with FACS, with concordant results (positive with both methods or negative with both methods) in 89% of samples, when the limit of decision (positive/negative MRD) was set to 0.1%. The concordance was especially high at the treatment stratifying time points, i.e. day 29 and 79; 89% and 100%, respectively. No samples at these time points were positive with FACS but negative with qRT-PCR. During the follow-up period (6-81 months), one patient relapsed (with negative MRD with both methods at stratifying time points), and two succumbed from therapy-related causes. Our results show that there is a significant relationship between the results of MRD analysis using FACS and MRD analysis using qRT-PCR of ETV6-RUNX1 fusion transcript. The high concordance between the methods indicates that negative MRD using qRT-PCR is as reliable as negative MRD using FACS, and that qRT-PCR could therefore be an alternative to FACS in cases where FACS is not achievable. In comparison to quantitative PCR of TCR/Ig gene rearrangements, which is the current backup MRD method for cases with pre-B ALL in NOPHO ALL-2008, qRT-PCR of ETV6-RUNX1 is much less time and labor consuming, making it appealing in a clinical laboratory setting. Disclosures No relevant conflicts of interest to declare.
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Bungaro, Silvia, Marta Campo Dell’Orto, Dario Basso, Tatiana A. Gorletta, Manoj Raghavan, Anna Leszl, Bryan D. Young, et al. "Identification of New Genomic Lesions in Childhood ALL without Known Genetic Aberrations: A Microarray Study of Gene Expression and Genotype Data." Blood 108, no. 11 (November 1, 2006): 2066. http://dx.doi.org/10.1182/blood.v108.11.2066.2066.
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Abstract Despite the risk stratification based on the prognostic relevant translocations, approximately 20–25% of childhood ALL patients cannot be classified according to genetic hallmarks. Recent studies based on single nucleotide polymorphism (SNP) analyses showed that Loss of Heterozygosity (LOH), with or without loss of genetic material, is frequently related to childhood ALL and AML. Moreover, there is an increasing interest in integrating the gene expression profiles with chromosomal localizations to identify new genetic subgroups. We applied an integrated approach composed of DNA index estimation, PCR (and/or RT-PCR) and cytogenetics, to exclude patients with known molecular and cytogenetics aberrations. Aim of the study was to identify criptic abnormalities in childhood ALL patients by performing an integrative analysis of gene expression and copy number changes (CNC) data. The 30 patients included in our study met the following inclusion criteria: a) B-cell precursor childhood ALL b) DNA index 1; c) negativity at t(4;11), t(12;21), t(9;22), t(1;19) RT-PCR screening; d) cytogenetics revealing normal karyotype or not technically feasible. Patients genomic DNA has been analyzed by the Affymetrix GeneChip Mapping 100K SNP arrays to identify genomic regions of LOH and CNC. In parallel, biotin-labeled cRNA has been synthesized and hybridized on HG-U133Plus2 Probe Arrays in accordance with the MILE (mircroarray innovation in leukemia) protocols; all samples were part of the B-ALL class without known genetic aberrations. The presence of del(9)(p21) was found in 9/30 (30%) patients, with an homozygous commonly deleted region involving CDKN2A and CDKN2B genes in 7/30. Hemizygous losses on 9p13 were found in two cases. Four patients (13,3%) showed the hemizygous deletion of chromosome 12p13.2, with a commonly deleted region involving the ETV6 gene. In one ETV6 deleted patient FISH analysis identified a translocation involving the first ETV6 exon and an unknown partner gene. Four patients, including three ETV6 deleted, showed the presence of the intrachromosomal amplification of chromosome 21 (iAMP21) with a common region of amplification between 31.3–43.5 Mb and a common region of deletion in 2/4 patients between 43.5–47 Mb. Four patients showed large regions of LOH not associated with CNC. Three patients did not show CNC and random microdeletions were found in single cases. Remission samples were analyzed for 5 patients carrying different aberrations, showing small regions of LOH in both the diagnosis and remission samples in 4 cases. No CNC was found in the remission samples. We found a statistically significant reduction for the expression of the CDKN2A transcript located within the (9)(p21) region. We are applying a locally adaptive statistical procedure to identify genes whose expression signals are specifically modulated by CNC. The integration of genomic variation and gene expression profiling might may be useful to identify new hidden genetic lesions, and to learn how critical regulators of tumor are linked to genomic alterations in cancer cells.
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Argani, Pedram, Michael K. Fritsch, Amy E. Shuster, Elizabeth J. Perlman, and Cheryl M. Coffin. "Reduced Sensitivity of Paraffin-Based RT-PCR Assays for ETV6-NTRK3 Fusion Transcripts in Morphologically Defined Infantile Fibrosarcoma." American Journal of Surgical Pathology 25, no. 11 (November 2001): 1461–63. http://dx.doi.org/10.1097/00000478-200111000-00021.
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Sorensen, Poul H. B., and Joan Mathers. "Reduced Sensitivity of Paraffin-Based RT-PCR Assays for ETV6-NTRK3 Fusion Transcripts in Morphologically Defined Infantile Fibrosarcoma." American Journal of Surgical Pathology 25, no. 11 (November 2001): 1464. http://dx.doi.org/10.1097/00000478-200111000-00022.
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Cazzaniga, Valeria, Marco Giordan, Cristina Bugarin, Chiara Palmi, Angela Savino, Geertruy te Kronnie, Giuseppe Gaipa, Andrea Biondi, Grazia Fazio, and Giovanni Cazzaniga. "PAX5 Fusion Genes Activate The STAT5 Signaling Pathway Through Lck Over-Expression." Blood 122, no. 21 (November 15, 2013): 3738. http://dx.doi.org/10.1182/blood.v122.21.3738.3738.
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Abstract PAX5 is a transcription factor acting both as an activator and a repressor, known to be frequently altered (30% of the cases) in B Cell Precursor Leukemia (BCP-ALL). PAX5 aberrations include point mutations, deletions, amplifications and translocations with several partner genes. All the originated fusion genes retain the PAX5 DNA binding domain but acquire the regulatory regions of the partner gene, showing a dominant negative mechanism on endogenous PAX5. However, the function of PAX5 fusion proteins and their common mechanisms of leukemogenesis are poorly understood. We have established an in vitro model in which wild-type murine pre-BI cells were transduced with the most recurrent PAX5 fusion gene, PAX5/ETV6. Gene expression profiling of transduced PAX5/ETV6 pre-BI cells showed a significantly altered transcription profile compared to empty vector control. Interestingly, pathway analyses revealed that many of the genes implicated in the B-cell receptor (BCR) assembly and signaling were repressed by PAX5/ETV6. In addition, the downstream signaling of the BCR was non-functional through a block in IgM heavy chain rearrangement. Aim of the current study has been to elucidate the signaling pathway activated either in PAX5/ETV6 transduced pre-BI cells and in primary cells from patients carrying different PAX5 translocations, in order to overcome the absence of a functional BCR expression. Interestingly, among the top-ranked over-expressed genes in PAX5/ETV6 transduced pre-BI cells, the PAX5 repressed target gene LCK was up-regulated both at basal level (mean fold change by RQ-PCR in 3 different pre-BI lines: 1.79, p<0.001) and at all time points (0, 24, 48 and 72 hours) after o.n. IL-7 starvation (mean FC: 8.0, range 3.8-15.9, p<0.01). LCK is a src family kinase involved in STAT5 signaling pathway and known to play an important role in T cell survival. Indeed, for the first time we found LCK as significantly up-regulated in primary samples of patients carrying different PAX5 translocations, including PAX5/AUTS2, PAX5/CHFR, PAX5/SOX5 and PAX5/POM121C, when compared to BCP-ALL cases with wild-type PAX5, as shown by microarray analyses and confirmed by RT RQ-PCR (mean fold increase: 6.3, p=0.027). Strikingly, in addition to LCK overexpression, PAX5/ETV6 induction in pre-BI cells dephosphorylates the inhibitory domain of LCK (LCK Y505) (mean difference in LCKY505/total LCK ratio: 63%, ranging from 38% to 81% in 3 different pre-BI lines), thus hyper-activating the LCK signaling. By phosphoflow analysis we also demonstrated LCK-dependent STAT5 hyper-phosphorylation after IL7 stimulation at all time points in time course experiments (mean fold change: 2.0, range 1.77-2.18). Downstream in the pathway, as a confirmation, we found by RQ-PCR the up-regulation of cMYC, a STAT5 target (FC: 2.65, p<0.01, at 30 minutes and 3.85, p<0.01, at 24h after IL-7 stimulation). Also CCND2, a cMYC target, was upregulated (FC: 2.27, p<0.001, at 30 minutes and 1.83, p<0.001, at 24h after IL-7 stimulation). Moreover, PAX5/ETV6 induced pre-BI cells show an increase in the replicative S phase cells at all time points after o.n. IL7 withdrawal (+58%, +39% and 61%, p<0.001, for PAX5/ETV6 after 0, 24 and 48 hours, respectively) and a faster proliferation rate, likely due to a different STAT5 activation profile. This mechanism is specific and reversible, as the LCK inhibitor BIBF1120 reduces STAT5 phosphorylation in PAX5/ETV6 cells (-11% and -46% after 24 and 48 hours, respectively), thus decreasing the fraction of S phase cells (-20%, p<0.01 and -31%, p<0.001, after 24 and 48 hours, respectively) and leading to a proliferation rate comparable to the control cells. These results further sustain the role of PAX5 fusion proteins as aberrant transcriptional factors, leading to the over-expression and hyper-activation of LCK, with a role in the activation of signaling pathways alternative to BCR (i.e. STAT5 hyper-phosphorylation) to sustain survival of leukemic cells. The further comprehension of the signaling mechanisms activated by PAX5 fusion genes will be fundamental for the development of new strategies to arrest tumor proliferation. Disclosures: No relevant conflicts of interest to declare.
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Lim, Jihyang, Myungshin Kim, Yonggoo Kim, Kyungja Han, Hee-Je Kim, Seok Lee, Woo-Sung Min, Nak-Gyun Chung, Bin Cho, and Hack-Ki Kim. "Analysis of Genetic Alterations In 843 Korean Acute Leukemia Patients by Multiplex RT-PCR Assay (HemaVision) and Karyotyping." Blood 116, no. 21 (November 19, 2010): 4845. http://dx.doi.org/10.1182/blood.v116.21.4845.4845.
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Abstract Abstract 4845 Background: The importance of molecular tests has been increased for diagnosis, classification and MRD monitoring in acute leukemia. Recently, multiplex RT-PCR assays (HemaVision) for simultaneous detection of 28 leukemia-associated translocations was introduced and has been used for molecular screening of leukemic patients. We analyzed the results of multiplex RT-PCR assay (HemaVision) and karyotyping in Korean acute leukemia patients. Methods: From April 2007 to March 2010, multiplex RT-PCR assay (HemaVision, DNA Technology A/S, Denmark) was performed in 843 Korean acute leukemia cases (AML 566 vs. ALL 277 and adults 596 vs. pediatrics 237) for screening leukemia-associated translocations. All results were compared with chromosome analysis results by conventional karyotyping. Results: Three hundreds fifty-one cases (41.6%) were positive in multiplex RT-PCR. In AML cases, 221 cases (39.0%) revealed positive results and RUNX1-RUNX1T1 85 cases (38.5%), PML-RARA 74 cases (33.5%), CBFB-MYH11 25 cases (11.3%) and MLL-related 19 cases (8.6%) were frequently detected. In contrast, 130 cases (46.9%) revealed positive results in 277 ALL cases and BCR-ABL1 68 cases (52.3%), ETV6-RUNX1 28 cases (21.5%), TCF3-PBX1 15 cases (11.5%), MLL-related 10 cases (7.7%) were frequently detected. In comparison to karyotyping, 337 cases (96.0%) revealed abnormal karyotype and 14 cases (4.0%) revealed normal karyotype in 351 cases that revealed positive results by multiplex RT-PCR assay. In 492 cases that revealed negative results by multiplex RT-PCR assay, 278 cases (56.5%) revealed abnormal karyotype and 214 cases (43.5%) revealed normal karyotype. Overall 629 cases (74.6%) were detected genetic alterations by multiplex RT-PCR assay and/or karyotyping. Conclusion: Most acute leukemias may have a genetic alteration in their leukemogenesis. Both multiplex RT-PCR assay and karyotyping are essential for screening leukemia-associated genetic alterations. Disclosures: No relevant conflicts of interest to declare.
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Taylor, Justin, Christina Marcelus, Dean Pavlick, Ryma Benayed, Akihide Yoshimi, Emiliano Cocco, Benjamin H. Durham, et al. "Characterization of Ntrk fusions and Therapeutic Response to Ntrk Inhibition in Hematologic Malignancies." Blood 130, Suppl_1 (December 7, 2017): 794. http://dx.doi.org/10.1182/blood.v130.suppl_1.794.794.
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Abstract Chromosomal rearrangements involving the neurotrophic receptor tyrosine kinases NTRK1-3 produce oncogenic fusions in a wide variety of adult and pediatric cancers. Although the frequency of NTRK fusions in most cancers is <5%, efficacy in solid tumors harboring these fusions is striking with a 76% durable response rate recently reported with the highly selective pan-TRK inhibitor larotrectinib (LOXO-101) in a cohort comprised of 17 unique tumor types. By contrast, the frequency of NTRK fusions is not well appreciated in hematologic malignancies and targeting of NTRK fusions has not been clinically tested. Herein, we describe the occurrence of NTRK fusions across >7,000 patients with hematologic malignancies and characterize their signal transduction, transforming properties, and response to larotrectinib in vitro and in an AML patient and corresponding patient-derived xenograft (PDX) in vivo . We performed targeted RNA sequencing using the Foundation One Heme sequencing panel across 7,311 cases of hematologic malignancies and discovered 8 patients (0.11%) harboring NTRK fusions. Fusions occurred in patients with histiocytic (LMNA-NTRK1, TFG-NTRK1) and dendritic cell (TPR-NTRK1) neoplasms (n=2/78), ALL (ETV6-NTRK3; n=1/659) as well as two with AML (n=2/1201). While previous case reports have reported ETV6-NTRK3 fusions in ALL and AML, our cohort also included an ETV6-NTRK2 fusion previously unreported in AML. In addition, we detected two multiple myeloma patients with NTRK3 fusions (UBE2R2-NTRK3 and HNRNPA2B1-NTRK3; n=2/1859) which represent the first description of NTRK fusions in myeloma. The fusion breakpoints are predicted to create in-frame fusions containing the tyrosine kinase domain of each of the NTRK genes and Sanger sequencing of RT-PCR on available tissues confirmed this. We next cloned 4 of these fusions and tested their transforming capacity in cytokine-dependent murine hematopoietic cells (Ba/F3 cells), which do not express endogenous Trk proteins. Despite equivalent levels of Trk expression, the transforming properties and auto-phosphorylation of each TRK fusion was distinct (A). The LMNA-NTRK1 and ETV6-NTRK2 fusions caused robust cytokine-independent growth. In contrast, additional NTRK fusions in which the 5' partner lacked classic oligomerization domains resulted in slower transformation (UBE2R2-NTRK3 fusion)or no transformation (HNRNPA2/B1-NTRK3). Consistent with these different growth properties, each fusion activated PI3K-AKT signaling to differing degrees after cytokine withdrawal (B) . Finally, the cells that gained cytokine-independence were exquisitely sensitive to treatment with larotrectinib. In contrast, Ba/F3 cells transformed by BRAF V600E mutation were unresponsive to Trk inhibition (C). The course of the above studies identified a patient with an ETV6-NTRK2 fusion AML. Using a PDX generated from this patient, we initiated treatment with larotrectinib (200mg/kg/day) after 8 weeks of transplantation when human myeloid leukemia engraftment reached a median of 15%. Larotrectinib treatment reduced human chimerism compared with mice receiving vehicle (although human myeloid leukemia cells persisted even with larotrectinib treatment- D). Consistent with the response of the AML PDX to Trk inhibition, treatment of the same patient with larotrectinib initiated under the FDA expanded access program resulted in clinical partial remission. This was due to eradication of the ETV6-NTRK2 mutant clone, which was sustained until outgrowth of a treatment refractory ETV6-MECOM clone resulted in progressive disease. FACS sorting and analysis of the AML revealed that each ETV6 fusion occurred in a distinct AML clone. Serial targeted RNA-seq analysis of bulk cells identified reduction of expression of the ETV6-NTRK2 fusion throughout the period of LOXO-101 treatment with concomitant increased expression of the ETV6-MECOM fusion (E). We herein describe that NTRK fusions occur across patients with a wide variety of hematologic malignancies and are amenable to Trk inhibition. Further studies to evaluate the clonality of NTRK fusions across cancers and whether this is predictive of therapeutic response to Trk inhibition will be critical based on the case here. Nonetheless, the clinical response here in a refractory patient argues for the need for systematic evaluation of NTRK fusions despite their rarity across hematologic neoplasms. Figure Figure. Disclosures Pavlick: Foundation Medicine: Employment. Watts: Jazz Pharmaceuticals: Consultancy, Speakers Bureau. Albacker: Foundation Medicine Inc.: Employment, Equity Ownership. Mughal: Foundation Medicine, Inc: Employment, Other: Stock. Ebata: LOXO Oncology: Employment. Tuch: LOXO Oncology: Employment. Ku: LOXO Oncology: Employment. Arcila: Archer: Honoraria; Raindance Tecnologies: Honoraria; Invivoscribe: Honoraria. Ali: Foundation Medicine, Inc: Employment, Other: Stock. Park: Amgen: Consultancy.
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Nilsson, Tina, Ahmed Waraky, Anders Östlund, Laleh Arabanian, Julia Asp, Linda Fogelstrand, and Lars Palmqvist. "Recreation of t(7;12)(q36;p13) in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Generates a Useful Model for Studying Acute Myeloid Leukemia." Blood 134, Supplement_1 (November 13, 2019): 2736. http://dx.doi.org/10.1182/blood-2019-123004.
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Introduction Acute myeloid leukemia (AML) is the result of aberrant hematopoietic processes, such as enhanced proliferation, blocked differentiation, and dysregulated apoptosis of hematopoietic stem and progenitor cells, and frequently these changes are initiated by chromosomal translocations in leukemia. Efficient methods for modelling leukemia and to recreate leukemia-associated genetic aberrations, such as chromosome translocations, are therefore crucial for investigating how leukemia is initiated. Today, most such models are murine and usually based on introduction of fusion gene transcripts of interest under the control of a constitutive active promoter using lenti- or retroviral transduction rather than the chromosomal translocation itself. The aim of the current project was to create a human cellular model of a chromosomal translocation that is typically found in AML in children under 24 months of age, the translocation t(7;12)(q36;p13). This translocation has been associated with poor prognosis, and leads to a gene fusion MNX1-ETV6 but also aberrant MNX1 gene expression. Its mechanism for leukemia initiation is so far unknown, mainly due to lack of a suitable experimental model. Material and methods CRISPR/Cas9 was used to reconstruct the genetics of the t(7;12)(q36;p13) rearrangement in human induced pluripotent stem cells (iPSC) while maintaining the genomic architecture and regulatory elements. Ribonucleoprotein (RNP) complex was delivered by lipofection (Nucleofection, Amaxa 4D system) into undifferentiated iPSC (ChiPSC 22, Cellartis). An ATTO550 tag on tracrRNA/RNP complex was used to sort out positive cells by flow cytometry and then seeded as single-cells in 96-well plates. Genomic DNA from the single-cell derived iPSC clones were screened by PCR for the presence of the translocation and positive clones were verified with a FISH probe specific for t(7;12)(q36;p13) (Double Fusion Break Apart probe, Metasystem). RT-qPCR was used to detect and quantify the expression of MNX1-ETV6 fusion and MNX1 transcripts. Differentiation potential was tested with the Trilineage Differentiation and Hematopoietic Kits (STEMdiff, STEMCELL Technologies). Results Using CRISPR/Cas9, we could successfully generate iPSC with the t(7;12)(q36;p13) translocation. The translocation was confirmed using conventional karyotyping and FISH and the mRNA expression of the fusion was confirmed with RT-qPCR. No additional chromosomal aberrations were seen. The t(7;12)(q36;p13) iPSC showed similar growth and differentiation properties as the parental iPSC. They showed propensity to differentiate into all three germ layers, confirming their pluripotent stem cell properties. The potential for differentiation into hematopoietic progenitor cells was shown by expression of CD34+, CD43+ and CD45+. In AML with t(7;12)(q36;p13), MNX1 mRNA expression is increased and this may play a role for leukemia development. In the t(7;12)(q36;p13) iPSC, RT-qPCR indeed showed increased expression of MNX1 expression compared with iPSC without the translocation. This increase of MNX1 was not seen in murine adult bone marrow or fetal liver cells transduced with retrovirus expressing the MNX1-ETV6 fusion. Further characterization of the t(7;12)(q36;p13) iPSC, e.g. whole exome and transcriptome sequencing and engraftment potential in immunocompromised mice (NSG-SGM), is ongoing. Conclusion In summary, we have using CRISPR/Cas9 successfully created a t(7;12)(q36;p13) iPSC line with potential to differentiate into hematopoietic progenitor cells and with gene expression pattern similar to what is seen in human AML samples with the t(7;12)(q36;p13). The introduction of the MNX1-ETV6 fusion in its correct genomic context could recapitulate local gene regulation, making it superior to models based on lenti- or retroviral introduction of fusion genes transcripts. In conclusion, this created cell line will be a valuable tool to study the mechanisms behind t(7;12)(q36;p13) AML, a severe form of AML associated with poor prognosis. Disclosures No relevant conflicts of interest to declare.
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Olsen, Marianne, Henrik Hjalgrim, Mads Melbye, Hans O. Madsen, and Kjeld Schmiegelow. "RT-PCR Screening for ETV6-RUNX1-positive Clones in Cord Blood From Newborns in the Danish National Birth Cohort." Journal of Pediatric Hematology/Oncology 34, no. 4 (May 2012): 301–3. http://dx.doi.org/10.1097/mph.0b013e3182332268.
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Ueno, Hiroo, Kenichi Yoshida, Yuka Yamashita, Yusuke Shiozawa, Tomomi Ishida, Hiroyuki Tsukamoto, Yuichi Shiraishi, et al. "Landscape of Driver Mutations and Their Clinical Impacts in Pediatric Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 912. http://dx.doi.org/10.1182/blood.v128.22.912.912.
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Abstract Introduction B-progenitor acute lymphoblastic leukemia (B-ALLs) accounts for 85% of pediatric ALL and categorized into several molecular subgroups according to their ploidy and recurrent translocations, such as ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL-rearrangements. In addition, recent genetic studies using high-throughput sequencing have disclosed landscapes of gene alterations in each subgroup, however, their clinical relevance have not fully been investigated in a large cohort of B-ALL patients who are uniformly treated and enrolled in an unbiased manner. Methods We enrolled a total of 515 pediatric B-ALL patients, who had been uniformly treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-02 protocol between 2002 and 2008. These patients were categorized into three risk groups, including standard-, high-, and extremely high-risk. Infantile ALL as well as BCR-ABL1-positive and Down syndrome-associated cases were excluded. A total of 158 known or putative driver genes in pediatric ALL were analyzed for somatic mutations by targeted-capture sequencing. IgH rearrangements were captured using 662 baits tiling the entire IgH enhancer locus. Finally, an additional 1205 baits was also designed to enable sequencing-based genome-wide copy number detection. Results The median age at diagnosis and observation period were 5.2 (1-18.5) and 4.2 (1.8-9) years, respectively. Sixty-six of the 515 patients (13%) had relapsed diseases and 47 patients (9%) had been died. Real-time RT-PCR and conventional cytogenetic analyses revealed subgroup-defining genetic lesions in 368/515 (71%) patients: 117 (23%) cases with ETV6-RUNX1, 48 (9%) with TCF3-PBX1, 13 (3%) with MLL rearrangements, together with those with hyper- (169 [33%]), and hypo- (6 [1%]) diploid. Remaining 162 patients (31%) had none of these abnormalities. The mean depth of the targeted sequencing was 569× across the entire cohort. In total, 823 driver mutations (median 1 per patient, range 0-7) and 954 focal deletions (median 2 per patient, range 0-13) were detected in 483 patients (92%). Among these, most frequently detected were mutations/deletions in CDKN2A (24%), ETV6 (21%), NRAS (18%), KRAS (18%), and PAX5 (15%). IgH-rearrangements were detected in 51 patients, including IGH-DUX4 (26 [5.0%]), IGH-EPOR (3 [0.6%]) and IGH-CRLF2(2 [0.3%]). Genetic alterations were enriched in several functional pathways, of which most frequent was epigenetic regulation (53%), followed by B-cell development (47%), RAS signaling (46%) and cell cycle (40%). A number of novel recurrent genetic lesions were also identified, including those in DOT1L and PHF6. DOT1L encode an H3K79 methyltransferase and was inactivated by frameshift/nonsense mutations and/or deletions in 19 cases. Although frequently found in T-ALL, mutations of PHF6 had not previously been reported in B-ALL but were detected in 14 cases in the current cohort and strongly associated with TCF3-PBX1 translocation. Significant positive correlations were also demonstrated for an additional 10 combinations of common genetic lesions, suggesting functional links between these combinations. Thus, ERG deletions were highly associated with IGH-DUX4 rearrangement, while mutations in KRAS, NRAS, and CREBBP were significantly enriched in hyperdiploid cases. ETV6-RUNX1 fusion also showed positive correlations with alterations in ETV6, CDKN1B, ATF7IP, VPREB1, BTG1, and WHSC1. Furthermore, mutually exclusive relationship between ETV6-RUNX1 translocationsand FLT3mutations were also identified. Finally, we analyzed the prognostic impact of driver mutations. In multivariate analysis of the entire cohort, 4 genetic alterations were significantly associated with poor prognosis (HR [95%CI]): IKZF1 mutations/deletions (2.6 [1.5−4.8]), EBF1 deletions (3.0 [1.4−6.5]), KDM6A mutations/deletions (2.8 [1.2−6.5]), and TP53 mutations (2.7 [1.2−5.9]). Additional factors (q < 0.1) were identified in subgroup analyses, including alterations in ETV6 (5.4 [1.2−24]), CDKN1B (7.4 [1.6−33]) and CDKN2A (4.2 [1.4−12]) in ETV6-RUNX1 ALL, KMT2D (5.9 [1.3−26]) in TCF3-PBX1 ALLand TP53 (38 [4.1−364]) in IGH-DUX4ALL. Conclusions We revealed the landscape of genetic lesions in pediatric B-ALL including novel targets of recurrent mutations with clinical relevance of common genetic lesions. Our results should help in the better stratification of patients. Disclosures Ogawa: Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.
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Alecsa, Mirabela-Smaranda, Mihaela Moscalu, Laura-Mihaela Trandafir, Anca-Viorica Ivanov, Cristina Rusu, and Ingrith-Crenguta Miron. "Outcomes in Pediatric Acute Lymphoblastic Leukemia—A Single-Center Romanian Experience." Journal of Clinical Medicine 9, no. 12 (December 15, 2020): 4052. http://dx.doi.org/10.3390/jcm9124052.
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Background: This study evaluates the main (para)clinical aspects and outcomes in a group of Romanian children diagnosed with acute lymphoblastic leukemia (ALL), under the conditions of antileukemic treatment according to an adapted ALL IC Berlin–Frankfurt–Munster (BFM) 2002 protocol. Methods: We performed a retrospective single-center study of 125 children diagnosed with ALL between 2010 and 2016. Standard forms were used for data collection of variate clinical and paraclinical parameters. Results: The children were predominantly male (64.8%) and their median age at diagnosis was 5 years. A total of 107 patients were diagnosed with precursor B-cell acute lymphoblastic leukemia (BCP)-ALL and 18 with T-cell acute lymphoblastic leukemia T-ALL. Multiplex reverse transcription polymerase chain reaction RT-PCR assay for ETV6-RUNX1, BCR-ABL, E2A-PBX1, KMT2A-AFF1, and STIL-TAL1 fusion genes was performed in 111 patients. ETV6-RUNX1 translocation was detected in 18.9% of patients, while BCR-ABL1 and E2A-PBX1 rearrangements were seen in 2.7% and 3.6%, respectively. Complete remission at the end of induction phase was obtained in 89.6% of patients. The overall relapse rate was 11.2%, with 11 early and 3 late relapses. The 5-year overall survival rate in BCP-ALL was 81.6% and in T-ALL 71.4%. Conclusions: The 5-year overall and event-free survival rates in our study were slightly lower than those reported in developed countries, so the patients’ outcomes are encouraging.
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Baldus, Claudia D., Michael D. Radmacher, Clara D. Bloomfield, Peter Martus, Stefan Schwartz, Nicola Goekbuget, Dieter Hoelzer, Eckhard Thiel, and Wolf K. Hofmann. "The Identification of ERG Related Pathways Pinpoints to Mechanisms Implicated in the Inferior Outcome of Acute T-Lymphoblastic Leukemia (T-ALL) with High ERG Expression." Blood 108, no. 11 (November 16, 2006): 1828. http://dx.doi.org/10.1182/blood.v108.11.1828.1828.
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Abstract The ETS transcription factor ERG is expressed during early, normal T-cell development and shut off once T-cell commitment is complete. In addition, ERG is involved in various chromosomal translocations and implicated in oncogenic pathways in Ewing sarcoma, prostate cancer, and acute myeloid leukemia. Due to the specific regulation during T-cell development and its oncogenic potential we investigated the prognostic impact of ERG expression in T-lymphoblastic leukemia (T-ALL). We have shown in a cohort of 105 adult patients (pts) with newly diagnosed T-ALL that high ERG expression is an independent risk factor predicting inferior relapse-free survival (RFS, P=0.003; 5-year RFS: high ERG 34% vs. low ERG 72%; Baldus et al., JCO. Vol. 24 10/2006). Gene expression profiling (Affymetrix U133 plus 2.0) was performed on diagnostic bone marrow samples of 31 adult T-ALL pts to define an ERG related gene expression signature indicative of its pathogenic role in T-ALL. ERG mRNA expression levels were determined by quantitative real-time RT-PCR and pts were dichotomized at ERG′s median expression level into low ERG (n=16) and high ERG expressers (n=15). After a filtering step that reduced the number of probe sets to just over 10,000, we identified 39 probe sets (representing 35 known genes) differentially expressed (P<0.001) between low ERG and high ERG expressers. Compared to the low ERG group, 23 probe sets had significantly higher expression levels in the high ERG group, and 16 probe sets had significantly lower expression levels in the high ERG group. This signature contained three probe sets for ERG, each one showing about a 2-fold higher expression in the high ERG group as compared to the low ERG group; supporting our RT-PCR results. Of the overexpressed genes in the high ERG group, 5 genes were involved in DNA binding (MYO18A, DNAJC1) or transcription factor activity (ERG, ETV6, DIP). ERG and ETV6 are both ETS transcription factors and are frequently rearranged resulting in oncogenic fusion genes involved in leukemia. Moreover, ETV6 has been shown to be essential for the establishment of early hematopoiesis. In addition, genes with ATPase and ATP binding activity (ATP2B4, ATP2C1, MYO18A), potentially involved in signal transduction pathways were related to high ERG expression. Up-regulation of AKR1C1, a member of the aldo-keto reductase superfamily, was demonstrated in the high ERG group. Interestingly, overexpression of AKR1C1 has been found in various types of cancers and was correlated with tumor progression and resistance to chemotherapy. In conclusion, an ERG associated gene expression signature was identified in T-ALL and provides insights into ERG co-regulated genes. The delineation of these ERG related pathways may pinpoint mechanisms responsible for the more malignant phenotype resulting in the inferior outcome of T-ALL with high ERG expression.
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Bastos, André Uchimura, Ana Carolina de Jesus, and Janete Maria Cerutti. "ETV6-NTRK3 and STRN-ALK kinase fusions are recurrent events in papillary thyroid cancer of adult population." European Journal of Endocrinology 178, no. 1 (January 2018): 83–91. http://dx.doi.org/10.1530/eje-17-0499.
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Objective PTC-specific analysis identified novel fusions involving RET, BRAF, NTRK1, NTRK3, AGK and ALK genes in adults and pediatric PTCs. Although many novel fusions are PTC-specific events and, therefore, are ideal for diagnosis purposes, validation across additional and larger patient cohorts is essential for introducing these potential diagnostic or prognostic biomarkers into the clinical practice. As most of the BRAF, NTRK3 and ALK fusions were initially found in pediatric PTC or in more aggressive thyroid carcinomas, and there is a great disparity across population, in this study, we screened a large set of adult-sporadic PTC cases for the most prevalent kinase fusion lately described in the TCGA. Design and methods The prevalence of the fusions was determined by RT-PCR in 71 classical PTC, 45 follicular variants of PTC (FVPTC), 19 follicular thyroid adenomas (FTAs) and 22 follicular thyroid carcinomas (FTCs). Results ETV6–NTRK3 was exclusively found in FVPTC, in both encapsulated and infiltrative variants, but was not found in FTAs and FTCs. STRN-ALK was found in both classical PTC and FVPTC. No AGK-BRAF fusion was identified in this series, endorsing that AGK–BRAF is a genetic event mainly associated with pediatric PTCs. Conclusions The identification of kinase fusions in thyroid carcinomas helps to expand our knowledge about the landscape of oncogenic alterations in PTC. As ETV6–NTRK3 and STRN–ALK are recurrent and not identified in benign lesions, they can certainly help with diagnosis of thyroid nodules. Further analysis is needed to define if they can also be useful for prognosis and guiding therapy.
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Palanichamy, Jayanth Kumar, Gunjan Sharma, Thakur Nidhi, Anita Chopra, and Sameer Bakhshi. "Role of IGF2BP1 and Target Genes in ETV6-RUNX1 Positive B-Acute Lymphoblastic Leukemia." Blood 134, Supplement_1 (November 13, 2019): 3818. http://dx.doi.org/10.1182/blood-2019-128019.
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Acute Lymphoblastic Leukemia is the most prevalent hematological neoplasm in Indian children and around 85% are of B-cell origin (B-Acute Lymphoblastic Leukemia (B-ALL)). ETV6-RUNX1 (E/R) is one of the common translocations seen in B-ALL. We have identified Insulin like Growth Factor 2 mRNA Binding Protein 1 (IGF2BP1), an oncofetal protein, to be specifically overexpressed in bone marrow samples of E/R positive B-ALL patients. We have tried to characterize the role of this protein in B-ALL. We collected 193 new paediatric pre B-ALL bone marrow samples. Most of the patients were under 5 years of age (73%) and only 3% of patients were more than 10 years. 66% of patients had no known translocation. Among the ones with translocation, ETV6-RUNX1 and BCR-ABL translocation were the commonest (11% each). 10% were E2A-PBX positive and only 3% had an MLL translocation. The translocation profile was very different from that of previously published reports. E/R translocation generally portends a good prognosis. There was a significantly lower initial WBC count in E/R positive group when compared to the other groups. However, there was no significant difference in other blood parameters including initial Hemoglobin levels and platelet counts. Interestingly, the proportion of patients with a high number of blasts in peripheral blood on day 8 post chemotherapy was actually significantly higher in the E/R +ve group indicating that there might be a proportion of patients in the E/R +ve group with a worse prognosis. There was no difference in minimal residual disease between the E/R translocation positive and negative groups. Real time RT-qPCR analysis of IGF2BP1 and ETV6 mRNA expression in bone marrow samples revealed significantly higher expression of IGF2BP1 in E/R positive B-ALL compared to E/R negative B-ALL samples. ETV6 mRNA relative expression on the other hand was found to be significantly lower in the E/R positive group. This corresponds to existing data that there is usually loss of the other wild type ETV6 allele in these patients. We used data from multiple studies to narrow down on true targets of IGF2BP1 in B-ALL. We used a published eCLIP dataset of IGF2BP1 to identify cognate RNA targets. Over expression of these targets in E/R +ve samples was confirmed from the other previously published high throughput gene expression studies on B-ALL (MILE (microarray innovations in leukemia) study (n = 3242) and Palanichamy et al, 2016, J Clin Invest). We identified 13 such genes which were bound by IGF2BP1 and regulated by it in E/R +ve B-ALL. One of these targets, EGFL7, has previously been shown to play an important role in haematopoiesis and involved in Acute Myeloid Leukemia pathogenesis. c-MYC is a known target of IGF2BP1 and. being a proto oncogene has an important role in B-cell proliferation. However, the biological and clinical role of cMYC and EGFL7 genes had not been evaluated in ETV6-RUNX1 positive Pre B-ALL. Real time analysis of EGFL7 and cMYC expression in the patient bone marrow samples showed significantly altered expression in both groups. As expected, EGFL7 showed a significantly higher expression in E/R +ve samples. Unexpectedly, cMYC showed lower expression in E/R positive group. The expression of EGFL7 also showed a significant positive correlation with initial hemoglobin levels and a negative correlation with initial WBC counts. To study the effect of IGF2BP1 binding to the EGFL7 or c-MYC UTRs, they were cloned downstream of firefly luciferase and coexpressed with IGF2BP1 in HeLa cells. Interestingly, the dual luciferase assay revealed no stabilization of either of these UTRs by IGF2BP1. This reveals a much more complex regulation by IGF2BP1 of its targets which might involve RNA methylation. To utilize the expression of IGF2BP1 and its target genes as a diagnostic marker for the presence of the E/R translocation, ROC curves using FISH/PCR as the gold standard test were prepared. The AUC (Area under curve) for ROC of the expression of IGF2BP1, c-MYC and EGFL7 were 0.917, 0.7 and 0.78 respectively. We also analyzed ROC curves using various combinations of these genes. Gene expression of IGF2BP1 alone was the best indicator of the presence of the ETV6-RUNX1 translocation with the highest sensitivity and specificity. In conclusion, relative expression of IGF2BP1 can serve as an excellent diagnostic marker for the E/R translocation with more than 90% sensitivity and 85% specificity. Disclosures No relevant conflicts of interest to declare.
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Brito-Babapulle, Finella MC, and Barbara Czepulkowski. "Defining The morphological Appearance Of ETV6-Abl Diseases. Evaluation Of a Case." Blood 122, no. 21 (November 15, 2013): 5271. http://dx.doi.org/10.1182/blood.v122.21.5271.5271.
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Abstract A 30 year old female presented@30 weeks pregnancy with a few circulating myeloid blasts in her blood but a normal full blood count. She had previously used an epipen for acute allergic reactions and had bone pain at presentation. There was splenomegaly on examination. Bone marrow aspirate was extremely difficult to obtain but trephine roll showed eosinophils, basophils, occasional mast cells and occasional blasts A population of 23% hypergranulated basophilic cells were seen on aspirate whose aetiology is not clear and are assigned to the basophil/mast cell series. For want of a better term these cells are called Finella’s cells. Histological examination of trephine biopsy showed extreme hypercellularity, Grade III/IV reticulin fibrosis large numbers of eosinophils and basophils with mast cells confirmed by CD 117 staining. The large hypergranulated basophilic cells (Finella’s cells) were not visible on trephine staining. An isodicentric X on cytogenetic analysis and a bcr-abl probe showed a split abl with 2 signals. An ETV6-ABL1 gene rearrangement was present. The c-kit D816V mutation was not identified nor was FIP1L1-PDGFRA present. Subsequently the morphologic features described above allowed the suspicion in a further case. Various appearances of the bone marrow and blood allow the suspicion of a particular disease which then leads to the performance of the diagnostic test to confirm the suspected genetic abnormality. This is epitomised in the diagnosis of chronic myeloid leukaemia based on a high white cell count, myelocyte peak and excess of myeloid cells with an increase in cells such as eosinophils and basophils. The diagnosis is then made by demonstrating the presence of the Philadelphia chromosome or bcr-abl gene rearrangement. In contrast in cases with an ETV6-abl, the patient has a white cell count that is not markedly increased with a leucoerythroblastic blood film and a bone marrow examination that shows a superficial resemblance to a chronic myeloproliferative disease with marked myelofibrosis and large numbers of eosinophils and basophils some of which are morphologically abnormal. Megakaryocytes when seen show discrete nuclear fragments and hypolobation and eosinophils and basophils are increased leading to the case being mistakenly diagnosed as an eosinophilic disorder rather than a specific disease entity i.e ETV6-abl. It cannot be classified with the mastocytic leukaemia’s as the number of masts cells is not greater than 5% and they are not clustered but loosely distributed. The aetiology of Finella’s cells are unclear. Mast cells in blood smears are easily identifiable by their large granules which disfigure the nuclear outline and a central area of pallor or by their “slipper” shape and granule content. In this case the cells are larger than mast cells circular, have no central area of pallor,the excessive granularity masking the nuclear outline and leading one to question whether a PML-RARA fusion is present which it is not. The presence of eosinophils, basophils and these cells should make the viewer suspect an ETV6-abl disorder which is normally a cryptic fusion which will not be identified without use of the appropriate FISH/ RT-PCR test. The disease appears to undergo blast transformation as seen with CML and early transplantation is recommended as happened successfully with our case. Although several other cases have been reported in the literature by various authors and called bcr-abl negative CML, myeloproliferative neoplasms, myeloproliferative disorders with eosinophilia etc peripheral blood eosinophilia is not present and FIP1L1-PDGFRA is not present. The morphologic features that lead one to suspect the presence of theETV6-abl have not been previously delineated. THE ETV6-ABL fusion protein has tyrosine kinase activity and responds to imatinib or second generation TKI’s and therefore demonstrating its presence when faced by the classic morphologic appearance is of clinical importance. Disclosures: No relevant conflicts of interest to declare.
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Miller, Christopher B., Charles G. Mullighan, and James R. Downing. "Mutations in the B-Cell Transcription Factor PAX5 Found in B-Progenitor Acute Lymphoblastic Leukemia Impair Normal PAX5 Activity." Blood 108, no. 11 (November 16, 2006): 613. http://dx.doi.org/10.1182/blood.v108.11.613.613.
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Abstract Using genome-wide profiling of DNA copy number abnormalities using high-resolution single nucleotide polymorphism arrays, we recently identified a high frequency of genomic aberrations involving the PAX5 gene in pediatric B-progenitor ALL. PAX5 is a critical transcriptional regulator of B lymphocyte commitment and differentiation. Mutations, including partial tandem duplication, complete and focal deletions, point mutations in the DNA-binding or transactivation domain, and three translocations that encode PAX5 fusion proteins were observed in 31.7% of B-ALL. The PAX5 deletions were mono-allelic and resulted in either loss of the entire gene, or the deletion of only a subset of the exons leading to the production of PAX5 proteins that lacked the DNA-binding paired domain (exons 2–4) and/or the transcriptional activation domain (exons 7–10). In murine systems, the complete absence of PAX5 results in the arrest of B-cell development at the pro-B-cell stage prior to immunoglobulin heavy chain rearrangement, whereas haploinsufficiency leads to a partial block in B-cell development. Importantly, in the primary leukemia samples, the mono-allelic loss of PAX5 was associated with reduced expression of PAX5 by flow cytometry and quantitative RT-PCR, suggesting that haploinsufficiency contributes to the block in differentiation characteristic of B-progenitor ALL. To determine if the other identified PAX5 mutations result in hypomorphic alleles, we analyzed the DNA-binding and transcriptional activity of the encoded proteins. DNA-binding activity was assessed by electrophoretic mobility gel-shift assays using a labeled oligonucleotide probes from the promoters of the PAX5 target genes CD19 and CD79A (mb-1), and transcriptional activity was assessed by a luciferase-based reporter assays using the PAX5-dependent reporter plasmid, luc-CD19. Analysis was performed on the paired-domain mutants P80R and P34Q, the focal deletions Δe2-5, Δe2-6, Δe2-7, Δe2-8, and Δe6-8, and the PAX5-ETV6 and PAX5-FOXP1 translocation-encoded fusion proteins. As expected, DNA-binding was abrogated in deletion mutants that lacked the paired domain (Δe2-5, Δe2-6, Δe2-7, Δe2-8). In contrast, the PAX5 Δe6-8, which retains the paired DNA binding domain but lacks a significant portion of the transcriptional regulatory domain, had normal DNA binding activity. Importantly, the paired domain point mutants impaired DNA-binding in a promoter specific manner, with P80R having a marked reduction in binding to both the CD19 and mb-1 promoters, whereas P34Q showed reduced binding only to the mb-1 promoter. Surprisingly, the PAX5-ETV6 and the PAX5-FOXP1 translocations had markedly reduced DNA-binding activity despite retention of the PAX5 paired domain. As expected each of the mutants with impaired or absent DNA-binding activity were found to have markedly reduced transcriptional activity when compared to wild type PAX5. Similarly, those mutants with altered or deleted transcriptional activation domains had reduced transcriptional activity, as did the two PAX5 translocation-encoded fusion proteins (PAX5-ETV6 and PAX5-FOXP1). Moreover, transfection of increasing amounts of PAX5-ETV6 or PAX5-FOXP1 together with a fixed amount of wild type PAX5 revealed that the fusion proteins competitively inhibit the transcriptional activation of wild type PAX5. Taken together, these data indicate that the identified PAX5 mutations impair DNA-binding and/or transcriptional activity. This loss of normal PAX5 function in turn would contribute to the observed arrest in B-cell development seen in ALL.
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Iqbal, Zafar, Sabir Noreen, Aleem Aamer, Awan Tashfeen, Tahir Naeem, Asad Sultan, Ammara H. Tahir, et al. "Characterization of Common Fusion Oncogenes As Prognostic Molecular Identities in Adult Acute Lymphoblastic Leukemia Identifies the Need for Genetic Testing At Presentation, Molecular Prognostication and Differential Treatment." Blood 120, no. 21 (November 16, 2012): 5115. http://dx.doi.org/10.1182/blood.v120.21.5115.5115.
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Abstract Abstract 5115 Introduction: Acute lymphoblastic leukemia (ALL) is a heterogeneous lymphoid disorder with many genetic abnormalities of which fusion oncogenes (FGs) are very common with a known role in leukemogenesis (Harrison & Foroni, 2002). Although prognostic significance of FGs is well characterized in pediatric ALL, the role of FGs in adult ALL is not well established (Moorman et al., 2007). Methods: We studied the frequency and association of five most common FGs namely BCR-ABL, MLL-AF4, ETV6-RUNX11, E2A-PBX1 and SIL-TAL1 with disease biology and treatment outcome in adult ALL. FGs were studied at diagnosis in 104 adult ALL patients using RT-PCR (Van-Dongen et al, 1999) and Interphase FISH. Results: FGs were found in 78. 8% (82/104) subjects (Table 1). Overall survival (OS) and relapse free survival (RFS) were 26. 17 and 11. 147 months, respectively (Figures 1–2). Patients with MLL-AF4 (12. 19%) showed an elevated total leukocyte count (TLC), prominent organomegaly, frequent central nervous system (CNS) involvement, and a poor clinical outcome (OS=8. 8 months). SIL-TAL1 (35. 36%) was associated with lymphadenopathy, frequent organomegaly, low platelets count and poor survival. Patients with BCR-ABL (20. 3%) had high TLC (p-value <0. 001), splenomegaly (p-value <0. 001), low platelets count (p-value <0. 001), poor outcome (OS=9. 3 & RFS=6. 3 months) and 10% less chances of CR as compared to BCR-ABL negative cases. ETV6-RUNX1 (4. 8 %) was mostly associated with low TLC, less common organomegaly, high CR rates and higher OS (30. 2 months), but their long term survival negatively affected by late relapses. Patients with TCF3-PBX1 (16. 3%) were associated with younger age (10/17, 59%), lower TLC (14/17, 82%), platelet count higher than 50×109/l (12/17, 71%), less common hepatomegaly (2/17, 12%), less common splenomegaly (3/17, 18%), early CR (11/17, 65%) but high relapse rate (13/17, 76. 1%) and shorter OS (11. 6 months). Conclusions & Discussion: High relapse rates and shorter OS despite favorable prognosis manifested by clinical features and high early CR rates in TCF3-PBX1 highlights the need for their identification at presentation and intensified treatment protocols to manage high relapse rate (Foa et al., 2003). Association of SIL-TAL1 with lymphadenopathy can help in better identification of this adult ALL subgroup at low resource centers. We found much higher frequency of TCF3-PBX1 and lower frequency of ETV6-RUNX1 than previously reported (Van Dongen et al, 1999). Although MLL-AF4 positive ALL is rarely observed in adult ALL, its frequency was 9. 7% in our adult ALL patients, which added to overall poor outcome of adult ALL in our population. Characterization of TCF3-PBX1 as poor prognostic molecular entity in our adult ALL population, low frequency of favorably prognostic ETV6-RUNX1, high frequency of poor prognostic MLL-AF4 and TCF3-PBX1 reflects ethnic and geographic differences in the biology and treatment of adult ALL (Burmeister et al., 2010). Therefore, it identifies the need for molecular testing in routine clinical settings at diagnosis and its implication in molecular prognostication and differential treatment. It also indicates the need for in-depth molecular analysis using advanced techniques and their implication in understanding the leukemogenesis and clinical management of adult ALL. Disclosures: No relevant conflicts of interest to declare.
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Zaliova, Marketa, Claus Meyer, Gunnar Cario, Martina Vaskova, Rolf Marschalek, Jan Stary, Jan Zuna, and Jan Trka. "The Central Region of TEL (ETV6) Is Dispensable for the TEL/AML1 (ETV6/RUNX1) Leukemogenesis." Blood 114, no. 22 (November 20, 2009): 1601. http://dx.doi.org/10.1182/blood.v114.22.1601.1601.
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Abstract Abstract 1601 Poster Board I-627 TEL/AML1 (ETV6/RUNX1) fusion gene, the most frequent genetic abnormality of childhood ALL, usually results from genomic breakpoint in TEL intron 5 and AML1 introns 1 or 2 (“classical” TEL/AML1). At the protein level the helix-loop-helix domain and exon 5 coded corepressor domain of TEL are typically fused to almost entire AML1 including transactivation and DNA binding domains. TEL/AML1 fusion as a single event does not lead to leukemia development but induces a preleukemic state characterized by expansion of B-cell precursors with enhanced self-renewal and impaired differentiation to more mature B-cell stages. It was demonstrated that TEL/AML1 preleukemic activity requires three functional domains: HLH domain, corepressor domain and DNA binding domain. One of the proposed mechanisms of TEL/AML1 function is that TEL/AML1 acts as an aberrant transcription factor and deregulates AML1 targets. TEL/AML1 induced transcriptional repression is mediated through interaction with corepressors and histone deacetylases. Both HLH and corepressor domains are able to interact with corepressors and are essential for the TEL-induced transcriptional repression. Parallel screening of TEL/AML1 fusion in 305 consecutive patients treated on BFM protocol by routine FISH and RT-PCR revealed TEL/AML1-positivity in 69 patients by both methods and RT-PCR false negativity in 2 out of 71 FISH-positive cases. In these two patients, we performed further transcriptomic and also genomic fusion site analysis of both TEL/AML1 and reciprocal AML1/TEL fusions and found an unusual genomic breakpoint within the TEL intron 4 resulting in a “variant” TEL/AML1 fusion lacking TEL exon 5-coded corepressor domain in both patients. Previous studies on animal models showed that TEL exon 5-coded region modulates the disease phenotype induced by TEL/ABL1 and TEL/TRKC fusion genes. We aimed to investigate the possible impact of the exon 5 loss in the “variant” TEL/AML1 fusion on disease behavior and TEL/AML1 function. We compared clinical features (age, white blood cell count, proportion of blasts in bone marrow and peripheral blood, detailed immunophenotype including TEL/AML1-specific combination of CD27 and CD44 antigens) and treatment outcome of all the 71 TEL/AML-positive patients from the same treatment protocol and we did not find any difference between the 2 “variant” and 69 “classical” cases. Moreover, we performed genome-wide gene expression profiling (GEP) of the two “variant” TEL/AML1 cases and the results show that the “variant” patients cluster within the “classical” TEL/AML1 cases and thus they are characterized by identical pattern of gene expression. We cloned the “variant” TEL/AML1 fusion into expression vector and analyzed its potential to repress transcription from AML1 target (granzyme B) promoter using luciferase assay. While the wild type AML1 substantially activates the promoter (350% of basal activity), the “variant” and the “classical” TEL/AML1 exhibited identical level of transcriptional repression (63% and 66%, respectively). Moreover, the repressive effect of both “variant” and “classical” TEL/AML1 is dependent on the presence of the AML1 consensus binding sites within the promoter region - mutation of both binding motifs results in elimination of the repressive effect (99% and 112% of the basal activity, respectively). Taken together, our data show that loss of the corepressor region of TEL does not significantly influence the TEL/AML1 function (or at least its ability to repress transcription of AML1 targets) and that the “variant” TEL/AML1 fusion is associated with leukemias that are undistinguishable from the “classical” TEL/AML1 cases. In contrast to recent studies, performed on artificial mouse or in-vitro models, we conclude - based on the data acquired from the genuine, naturally occurring childhood leukemias - that the corepressor region of TEL is dispensable for the TEL/AML1 leukemogenesis. This work was supported by grants MSM0021620813 and MZO00064203. Disclosures No relevant conflicts of interest to declare.
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Lainey, Elodie, Aurelie Caye-Eude, Lucile Broseus, Emmanuelle Clappier, Wendy Cuccuini, Cristina Leschi, Odile Fenneteau, et al. "Immunophenotypic Profiling of Childhood B-Cell Precursor Acute Lymphoblastic Helps Identifying Genetic Subtypes, Including Recently Identified Ones." Blood 128, no. 22 (December 2, 2016): 2910. http://dx.doi.org/10.1182/blood.v128.22.2910.2910.
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Abstract Background B-cell precursor acute lymphoblastic leukemia (B-ALL) comprises distinct entities characterized by recurring genetic alterations. These classifying alterations are associated with gene expression profiles, reflecting the specific biology of each genetic subgroup. In patients with none of these classifying alterations, referred as "B Other", RNAseq recently led to the identification of a huge number of in frame fusions which are likely to define novel genetic subgroups. Leukemic blasts often show aberrant antigen expression, giving rise to leukemia-associated immunophenotypic profiles (LAIP) distinct from their normal immature B-lineage counterparts. These variations in protein expression are likely to reflect the specific biology of B-ALL. We evaluated how LAIP were associated with the various classifying genetic lesions, including recently identified ones. Materials and Methods A cohort of 299 childhood B-ALL was studied prospectively. Genetic typing by cytogenetics, MLPA and RT-PCR identified high hyperdiploidy (Heh, n=77), ETV6-RUNX1 (n=81), TCF3-PBX1 (n=21), TCF3-HLF (n=3), MLL rearrangement (n=25), BCR-ABL/Phie-like fusions (n=10), ERGdel (n=8), TSLPR (n=8), iAMP21 (n=6), Hypodiploidy (n=6). The remaining patients were considered "B-Other" (n=54). Immunophenotyping was performed by 6-8-colors flow cytometry with a panel of 29 antibodies targeting surface proteins routinely used for ALL diagnosis and MRD assessment. The log of Ratio Fluorescence Intensity (RFI) of B-ALL/normal B precursor cells was calculated for each marker. RNAseq was performed in 18 B-Other and 8 BCR-ABL/Phie-like. Fusions were detected using TopHat2. Samples were classified using hierarchical clustering and/or principal component analyses (R software). Results Unsupervised clustering identified 6 groups with shared pattern of LAIP. Four groups were associated with known classifying lesions: ETV6-RUNX1 (of which 87% co-clustered), Heh (82%), TCF3-PBX1 (100%) and MLL (96%). ALL that did not cluster according to their genetic group mostly clustered (23/26; 89%) in two groups, hereafter named "B Other-1" and "B Other-2", which mainly contained ALL lacking any previously known cytogenetic lesion. Interestingly, 3 of 5 ETV6-RUNX1 with IKZF1del clustered in the "B Other 2", suggesting that misclassifications may not be due to poor LAIP specificity but rather reveal distinct biological features in some ALL. Although comprising too few cases to define specific groups, 8/10 B-ALL with BCR-ABL/Phie-like, 3/3 TCF3-HLF, and 8/8 TSLPR co-clustered in "B Other 2", and 7/8 ERGdel in "B Other 1". No clear clustering was observed for Hypodiploid and iAMP21. B-Other ALL were allocated either to oncogenic groups, e.g. TCF3-PBX1 (n=7) or MLL (n=7), or to the "B Other" groups (n=35). Only one B Other, harboring an IGH-IL3 fusion, co-clustered with BCR-ABL/Phie-like ALL. RNAseq of 18 B-Other identified no additional Phie-like fusion and none of these B-Other co-clustered with a group of 8 controls BCR-ABL/Phie-like. This makes unlikely that some Phie-like cases remained unidentified. However, other fusions involving MEF2D (n=3), PAX5 (n=5) or ZNF384 (n=2) were evidenced in B Other ALL. Cases with MEF2D or ZNF384 clustered both at the transcriptome and protein level. Using LAIP clustering, MEF2D ALL layed close to those with TCF3 fusions. ZNF384 cases clustered in the MLL group, consistent with a lack of CD10 expression combined to myeloid antigen expression. PAX5 ALL were allocated to the "B Other 2" group with no clear clustering and, more surprisingly, no consistent effect on CD19 expression. Secondary aberrations such as deletions in IKFZ1 or CDKN2A/B genes did not significantly affect clustering. Conclusion Unsupervised clustering based on surface protein expression allowed to classify most childhood ALL genetic groups, including Phie-like, MEF2D or ZNF384, which are challenging to identify due to the diversity of the underlying gene fusions. LAIP can thus rapidly orientate diagnosis, in particular for Phie-like which are candidates to targeted therapies. Interestingly, B-Other that clustered in known genetic groups may present biologically equivalent alterations undetected in our routine workup. Further studies will allow to assess whether cases co-clustering with ETV6-RUNX1 (n=2) or ERGdel (n=6) correspond to the recently described ETV6-RUNX1-like or DUX4 group respectively. Disclosures Baruchel: Servier: Consultancy; Novartis: Consultancy; Jazz: Consultancy; Baxalta: Research Funding; Celgene: Consultancy.
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Popov, Alexander, Grigory Tsaur, Tatiana Verzhbitskaya, Tatyana Riger, Yulia Yakovleva, Anna Ivanova, Alexander Druy, Egor Shorikov, Leonid Saveliev, and Larisa Fechina. "Qualitative and Quantitative Concordance of Minimal Residual Disease Data Assessed by Multicolor Flow Cytometry and PCR of Fusion Gene Transcripts In Childhood B-Cell Precursor Acute Lymphoblastic Leukemia." Blood 116, no. 21 (November 19, 2010): 1720. http://dx.doi.org/10.1182/blood.v116.21.1720.1720.
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Abstract Abstract 1720 Minimal residual disease (MRD) monitoring by flow cytometry (FC) or polymerase chain reaction (PCR) is a strong tool for risk-adapted treatment in childhood acute lymphoblastic leukemia (ALL). Monitoring of IgH/TCR rearrangements by real-time quantitative PCR (RQ-PCR) is well-standardized but very laborious and costly approach. Hence other methods - FC and PCR of fusion gene transcripts (FGt) – can be used for long-term MRD monitoring. Aim. To evaluate qualitative and quantitative concordance between MRD data assessed by FC and PCR of FGt in children with B-cell precursor ALL (BCP-ALL) during treatment. Methods. Concurrent detection of MRD by multicolor FC, RQ-PCR and RT-PCR was performed in 184 follow-up bone marrow samples from 46 children with BCP-ALL. Among them 21 patients (pts) carried ETV6-RUNX1 FGt, 3 pts – TCF3-PBX, while in other 22 pts various types of MLL-rearrangements were detected. 18 pts had CD10(-) and 28 pts – CD10(+) BCP-ALL. 62 samples were obtained during remission induction while 122 – during consolidation/intensification. 6–8-color FC was used. FGt CN was measured by RQ-PCR according to recommendations with normalization to ABL. MRD value was calculated as previously described. Results. Sensitivity of FC MRD detection varied from 10-4 to 10-5. RQ-PCR sensitivity ranged from 5×10-5 to 1×10-5. 65 of 184 samples were MRD-negative by both methods, 13 (7.07%) - were negative by FC but positive by RT-PCR, while in one sample (0.54%) tumor cells were detected by FC but not by PCR. Remaining 105 samples were MRD-positive by both methods. High qualitative concordance (92.39%) between FC and RT-PCR data was found. Similar concordance was observed in MLL-rearranged (91.60%), ETV6-RUNX1-positive (95.45%) and TCF3-PBX-positive (88.89%) cases was observed (p=0.650). Samples with and without normal lymphoid regeneration were analyzed separately, because presence of normal B-cell precursors (BCP) in follow-up samples is known to be an obstacle for FC data analysis. Qualitative concordance in BCP-negative and BCP-positive samples was very similar (92.31% and 92.47% respectively, p=0.814). Concordance of data assessed by two methods in samples obtained during remission induction and during consolidation/intensification was also very close (90.32% and 92.62% respectively, p=0.799). Among remission induction samples high qualitative concordance was observed in both day 15 and day 36 (end of induction therapy) samples (91.30% and 89.19% respectively, p=0.859). These results are in contrast with the previously shown data that at the end of remission induction concordance between FC and molecular techniques is relatively low [G. Gaipa et al, ASH-2008]. As the flow cytometric data analysis in CD10(+) and CD10(-) BCP-ALL patients bases on different approaches, these types of leukemia were also analyzed separately. High qualitative concordance was found in samples from both CD10(+) and CD10(-) BCP-ALL (89.66% and 94.85% respectively, p=0.295). Despite high qualitative concordance between FC and RT-PCR data, low quantitative concordance between FC and RQ-PCR results was found (R2=0.291). Significant quantitative difference in FC and RQ-PCR data could be associated with variability of FG expression during treatment that does not correspond to the cells' number. Moreover, percentage of tumor blasts among all nucleated cells is calculated during FC MRD detection, while MRD value in RQ-PCR of FGt is corresponded to the initial FGt and control gene levels. FC appears to be better for the quantitative MRD assessment however FGt detection by RT-PCR is more appropriate for MRD qualitative detection due to higher sensitivity. Hence, FC is more applicable for MRD monitoring during early treatment phases, when the precise MRD value is essential. In contrast, PCR of FGt is more useful for later time-points where MRD-positivity corresponds to poor outcome. Conclusion. Tandem application of FC and PCR of FGt seems to be a useful tool for long-term MRD monitoring in childhood BCP-ALL. Disclosures: No relevant conflicts of interest to declare.
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Ruminy, Philippe, Nimrod Buchbinder, Thomas Larson, Vinciane Marchand, Bérangère Joly, Sylvain Mareschal, Mathieu Viennot, et al. "Highly Multiplexed Targeted Sequencing of Recurrent Fusion Genes in Acute Leukemia." Blood 124, no. 21 (December 6, 2014): 2335. http://dx.doi.org/10.1182/blood.v124.21.2335.2335.
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Abstract In acute leukemia, recurrent chromosomal translocations which result in the fusion of two genes are frequent. Some of these markers have a well established prognostic and therapeutic impact and are routinely screened at diagnosis by cytogenetics and RT-PCR. Yet, due to the limitations of these methods, only a few among the dozens of known rearrangements are systematically tested. Many abnormalities which could provide important clinical information thus remain ignored, mainly due to the impossibility of performing a cost effective multi-targeted screening. To overcome this obstacle, we have developed a simple and parsimonious assay which allows a reliable detection of dozens of fusion genes in only a few hours. Our assay was designed to screen simultaneously more than 50 translocations involving 70 genes, recurrent in acute myeloid (AML), acute lymphoid (ALL) and chronic myeloid (CML) leukemia. cDNA samples obtained from leukemic cells are first incubated with a mix of oligonucleotides probes which are complementary to the ends of the exons, at the abnormal junctions on the fusion mRNAs (Figure). For most genes, different probes were designed to detect different transcripts resulting from alternative genomic breakpoints (for example on exons 1, 13, 14 and 19 for BCR, and on exons 2 and 3 for ABL). The mix we created thus regroups more than 150 probes and targets more than 400 different fusion transcripts. All left probes have a common tail (T1) at their 5' ends, all right probes a common tail (T2) at their 3' ends. Additional probes were also included to detect the most frequent mutations of NPM1 (A, B, D). If one translocation is present in the sample, two probes hybridize side to side on the fusion cDNA. A DNA ligase is next used to create a covalent link between these probes, allowing their amplification by PCR with the T1 and T2 primers. If a PCR product is amplified, the two partners are identified by sequencing analysis. To test this method, we applied it to a retrospective series of 430 patients, (252 AML and 178 childhood ALL). In B-ALL (147 cases), all 33 ETV6-RUNX1, 6 BCR-ABL and 5 TCF3-PBX1 as well as 6 MLL rearrangements (3 AF4; 1 ENL, 1 AF9 and 1 AFF4) identified at diagnosis by conventional methods were detected, as well as 5 previously unnoticed P2RY8-CRLF2 junctions. We only failed to detect one MLL-AF4 fusion detected by cytogenetics but not by RT-PCR, probably due to the poor quality of the RNA. Further analysis of 10 MLL-AF4 positive adult ALL patients allowed the correct identification of this fusion in all cases. In T-ALL (31 cases), we detected 6 known (4 SIL-TAL, 2 CALM-AF10) and 5 previously undetected fusions (2 NUP214-ABL, 1 MLL-ENL, 1 ETV6-ABL, and a new PLZF-ABL junction). In AML, we detected 86 fusions: 23 PML-RARA, 2 PLZF-RARA, 18 CBFB-MYH11, 12 RUNX1-RUNX1T1, 4 NUP98-NSD1, 2 BCR-ABL, 1 DEK-NUP214, 1 CALM-AF10, 1 MOZ-CBP, 22 MLL rearrangements (13 PTD, 3 AF9, 2 AF6, 1 AF10, 1 ENL, 1 AF1Q and 1 MAPRE) and 44 NPM1 mutations. Two PML-RARA cases, one with a BCR2 breakpoint (not included in our design), one weakly positive by RT-PCR but negative by cytogenetics, two t(11;17) validated by FISH but negative by RT-PCR, and one NPM1J mutation (not included in our assay) were not detected. Importantly, 20 translocations in this series, including 14 MLL fusions and a cytogenetically cryptic t(8;21) had not been identified at diagnosis. Furthermore, all these new abnormalities (in AML and ALL) could be confirmed by conventional RT-PCR and sequencing analysis, demonstrating the specificity of the method. In the whole cohort of 430 patients, the three methods thus detected 157 fusions. 85 (54.1%) and 112 (71.3%) had been detected at diagnosis by cytogenetics and RT-PCR respectively, and 152 (96.8%) by our assay. Two were revealed only by cytogenetics, one only by RT-PCR and 30 only by our method. In conclusion, we have developed a simple multiplexed assay which can reveal a very large number of recurrent gene fusions in leukemia. Its short turn-around time (we repeatedly tested up to 40 patients in parallel and the results can be obtained in less than one day) and low cost (only one PCR module, one pyrosequencer and basic molecular biology reagents are needed) make it particularly suitable for a daily practice. Its capacity to detect many abnormalities which are almost never tested in daily practice could provide many important diagnosis and prognosis information, and enable the stratification of patients in prospective clinical trials. Figure 1 Figure 1. Disclosures Ruminy: Centre Henri Becquerel: Patents & Royalties. Marchand:Centre Henri Becquerel: Patents & Royalties.
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Lemelle, Lauriane, Delphine Guillemot, Hervé Brisse, Arnaud Gauthier, Matthieu Carton, Anne-Laure Hermann, Nadege Corradini, et al. "Tag-n-trak study: Preliminary analysis of an unselected biobank tumors with NTRK fusion transcript, the French SFCE society contribution." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 10540. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.10540.
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10540 Background: NTRK ( Neurotrophic receptor tyrosine kinase) fusion transcript was initially described in infantile fibrosarcoma (IFS) and is now known to be present in many other rare types of tumor with a lower incidence. The main objective of the study is to describe the presentation and outcome of unselected tumors with NTRK fusion transcript (NTRK-FTT) to better consider the role of TRK inhibitors (TRKi) in such entities. Methods: We selected patients (pts) from the Institut Curie biobank (adults and children) with NTRK-FTT and which were assessed by RT-qPCR then RNA sequencing in a prospective or retrospective process, between 2001 and 2019. Results: We identified 62 NTRK-FTT among 2119 screened tumors (2.2 cases/year with RT-qPCR and 6.9 cases/year with RNA seq after 2016), NTRK3 (44 cases) (including 41 NTRK3-ETV6), NTRK2 (9) or NTRK1 (9). Most of pts had under 2 years (y) (74%) and only 7 pts were adults. We report preliminary analysis of clinical observation for the first 27 pts. Median age was 0.4 y [0-60.2]. Pathologic diagnosis was IFS (12 cases), various CNS tumors (4), atypical teratoid rhabdoid tumor (1), myofibroblastic inflammatory tumor (2), benign tumors (3), cellular congenital mesoblastic nephroma (2), lipofibromatosis like neural tumor, myxoïd liposarcoma, unclassifiable sarcoma (1 each). Two pts had metastatic tumors. Among all, 22 had surgery (1 mutilating), 15 chemotherapy, 4 radiotherapy, and 5 received TRKi, as second line treatment (2 pts) or ≥ third line (3 pts). After a median follow-up of 50 months [range, 1-155], 20/27 pts remained in complete remission, 3 had stable residue, and 4 had progressive disease (associated to 1 disease-related and 1 toxic deaths). Five-year OS is 88% [95%CI, 73.5-100] and 5y-EFS 59.5% [95%CI, 41.7-84.9]. Conclusions: This descriptive study showed that NTRK-FTT is rare (2.9%), encompasses a variety of different histotypes (n = 14). Systematic RNA sequencing allowed to depict 3.5 times more NTRK-FTT than targeted RT-qPCR. Overall outcome is favorable despite frequent tumor events. According to tumor type and the uncertainties regarding the long-term side effects of TRKi, the benefit/risk ratio must be carefully evaluated before using these drugs in first line.