Fusion of SFQP to ABL in a Patient with a T(1;9)(p34;q34) and Acute Lymphocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4444-4444
Author(s):  
Nicholas C.P. Cross ◽  
Andrew J. Chase ◽  
Milton Drachenberg ◽  
W. Mark Roberts ◽  
Jerry Z. Finklestein ◽  
...  
Keyword(s):  
Complete Remission ◽  
Fusion Gene ◽  
Chimeric Protein ◽  
Sh2 Domain ◽  
Lymphocytic Leukemia ◽  
Tyrosine Kinase Domain ◽  
Fish Analysis ◽  
Fusion Partner ◽  
Rt Pcr ◽  
Polypyrimidine Tract Binding Protein

Abstract We have investigated a child who presented with pre-B ALL and an acquired t(1;9)(p34;q34). BCR-ABL was not detected by RT-PCR or FISH analysis, however FISH did indicate that the ABL gene at 9q34 was disrupted. To identify the putative partner locus in this case, a modified 5′RACE strategy was employed that selected against normal ABL transcripts. Several clones were recovered in which ABL was fused to SFPQ (also known as PSF), a gene mapping to 1p34 that encodes a polypyrimidine tract-binding protein-associated splicing previously identified as a fusion partner of the helix-loop-helix transcription factor TFE3 in papillary renal cell carcinomas. Both SFPQ-ABL and reciprocal ABL-SFPQ transcripts were detectable by RT-PCR, and disruption of these two genes was further confirmed by amplification and sequencing of the forward genomic breakpoint. SFPQ-ABL, the likely oncogenic product, is predicted to encode a protein that retains the coiled coil domain of SFPQ and the entire tyrosine kinase domain and C-terminal sequences of ABL. The breakpoint in ABL was downstream of that seen for other ABL fusion genes and the chimeric protein is predicted to lack the ABL-encoded SH3 domain and part of the SH2 domain. The patient was treated according to the Children’s Cancer Group Protocol 1961 and subsequently received augmented BFM therapy with doxorubicin and double delayed intensification. He achieved complete remission but suffered extramedullary testicular relapse at 4.5 years. Following orchiectomy and intensive chemotherapy he remains in complete remission more than 6 years after diagnosis. We conclude that SFPQ-ABL is a novel fusion gene associated with ALL. Although the patient here responded to conventional chemotherapy, SFPQ-ABL is likely to be sensitive to imatinib and use of this agent might be considered in further cases.

A Novel FOXP1-PDGFRA Fusion Gene in A Case of Chronic Eosinophilic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2830-2830
Author(s):  
Yuka Sugimoto ◽  
Akiko Sada ◽  
Fumihiko Monma ◽  
Kohshi Ohishi ◽  
Masahiro Masuya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Genomic Dna ◽  
Low Dose ◽  
Fusion Gene ◽  
Fish Analysis ◽  
Fusion Partner ◽  
Rt Pcr ◽  
Total Rna

Abstract Abstract 2830 Introduction Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 is a new major category in the 2008 WHO classification of myeloid malignancies. FIP1L1-PDGFRA fusion gene is currently the most common abnormality in this category, but there are some other fusion genes incorporating part of PDGFRA. In a case of myeloproliferative neoplasms (MPN) with eosinophilia and hepatosplenomegaly, karyotype by G-banding and fluorescence in situ hybridization (FISH) for 4q12 rearrangements indicated a PDGFRA rearrangement other than FIP1L1-PDGFRA, and a novel FOXP1-PDGFRA fusion gene was identified. Case presentation A 44-year-old male visited a clinic because of wet cough for one year. His peripheral blood showed leukocytosis of 43.15 × 109 /L with eosinophilia up to 57.5%, mild erythrocytosis (Hb 17.3 g/dL), and thrombocytopenia of 86 × 109 /L. CT scan of the abdomen revealed hepatosplenomegaly. He was referred to our hospital and received oral PSL (1 mg/kg) first, because pulmonary eosinophilic infiltration was suspected by follow-up CT findings. Pulmonary infiltration and his cough disappeared rapidly in a week, but his leukocytosis with eosinophilia was exacerbated again with PSL tapering. His bone marrow at the time of admission disclosed hypercellular marrow with myeloid hyperplasia and eosinophilia, of which karyotype was 46, XY, t(3:4)(p13;q12), inv(9)(p12q13) in all of 20 metaphases. FISH analysis with tricolor 4q12 rearrangement probe set indicated that PDGFRA was disrupted in 97.3% of his peripheral blood cells. These cytogenetic abnormalities of his bone marrow cells suggested involvement of PDGFRA fusion gene except for FIP1L1-PDGFRA and did not disappear after steroid administration for 2 weeks. After low-dose of imatinib (100 mg/day) was started, he achieved a hematological response within 5 days, and PSL could be gradually tapered off. 3 months after therapy, he obtained complete cytogenetic response (CCyR). He has been in CCyR and free of symptoms for more than 6 months with only low-dose imatinib. Methods and Results Genomic DNA and total RNA were isolated from white blood cells in his peripheral blood at diagnosis. Complementary DNA was synthesized from total RNA. FIP1L1-PDGFRA fusion transcript was proved to be negative by RT-PCR. Molecular cloning with 5′-RACE-PCR revealed a novel mRNA in-frame fusion between exon 23 of FOXP1 and a truncated PDGFRA exon12. Reciprocal PDGFRA-FOXP1 transcripts were confirmed by RT-PCR analysis and FOXP1-PDGFRA genomic DNA sequence was determined with genomic PCR. As in the case with FIP1L1-PDGFRA, the breakpoint of PDGFRA in FOXP1-PDGFRA was located between the two tryptophan (W) residues of the putative WW-domain. Meanwhile, the other breakpoint was near inverted repeat in intron 23 of FOXP1, which is presumed to be very fragile site. By FISH analysis after magnetic cell sorting with MicroBeads, the 4q12 abnormality attributed to FOXP1-PDGFRA fusion gene was detected in granulocytes, but not in CD19-positive B or CD3-positive T cells. Discussion In a case with chronic eosinophilia harboring 46, XY, t(3:4)(p13;q12), inv(9)(p12q13), novel FOXP1-PDGFRA fusion gene was identified. Similar karyotypic abnormality harboring t(3:4)(p13;q12) was reported in a case of MPN with chronic eosinophilia, but responsible fusion gene was not identified (Myint H, et al. Br J Haematol. 1995). FOXP1 is a transcription factor which is implicated in a variety of cellular processes and has a role in immune regulation and carcinogenesis (Wlodarska I, et al. Leukemia. 2005). As a fusion partner of FOXP1, PAX5 and ABL1 are reported in cases with acute lymphoblastic leukemia. Thus, this is a first report showing that FOXP1-PDGFRA fusion gene is involved in hematologic malignancy. It is likely that FOXP1-PDGFRA is constitutively activated tyrosine kinase, which does not depend on dimerization but on the disruption of an autoinhibitory juxtamembrane domain encoded by exon 12 of PDGFRA from its structure. Eosinophilia responded well to low dose of imatinib as observed in CEL with FIP1L1-PDGFRA. Conclusion FOXP1-PDGFRA was identified in CEL for the first time. This is the eighth reported fusion gene associated with PDGFRA in CEL so far. Our patient with FOXP1-PDGFRA promptly responded to low-dose of imatinib as same as other cases with PDGFRA abnormalities. Further investigation is still in progress. Disclosures: No relevant conflicts of interest to declare.

FLT3 Is Fused to ETV6 in a Myeloproliferative Disorder with a t(12;13)(p13;q12) Translocation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2901-2901
Author(s):  
Hoang Anh Vu ◽  
Phan Thi Xinh ◽  
Seiko Shimizu ◽  
Michihiko Masuda ◽  
Toshiko Motoji ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Blot Analysis ◽  
Fusion Proteins ◽  
Fusion Gene ◽  
Hematologic Malignancies ◽  
Myeloproliferative Disorder ◽  
Tyrosine Kinase Domain ◽  
Common Mutation ◽  
Fish Analysis ◽  
Fusion Partner

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.

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 ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1439-1439 ◽  
Author(s):  
Julie M. Gastier-Foster ◽  
Andrew J. Carroll ◽  
Denise Ell ◽  
Richard Harvey ◽  
I-Ming Chen ◽  
...  
Keyword(s):  
B Cell ◽  
Gene Rearrangement ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Cytogenetic Abnormality ◽  
Fish Analysis ◽  
Oncology Group ◽  
Rt Pcr ◽  
Cell Precursor ◽  
Pediatric All

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.

Transforming and Tumorogenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosin-Kinase.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4683-4683
Author(s):  
Álvaro Cuesta-Domínguez ◽  
Mara Ortega ◽  
Cristina Ormazabal ◽  
Matilde Santos-Roncero ◽  
Marta Galán-Díez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Nude Mice ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Chimeric Protein ◽  
Tyrosine Kinase Domain ◽  
Molecular Response

Abstract Abstract 4683 Chromosomal translocations in human tumors frequently produce fusion genes whose chimeric protein products play an essential role in oncogenesis. Recent reports have found a BCR-JAK2 fusion gene in cases of chronic or acute myeloid leukemia, but the protein had not been characterized. We describe a BCR-JAK2 fusion gene by fluorescence in situ hybridization and RT-PCR amplification from bone marrow at diagnosis of a patient with acute lymphoblastic leukemia. After induction therapy, real time PCR showed persistent molecular response correlating with hematological remission maintained up to present. BCR-JAK2 is a 110 KDa chimeric protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis showed that BCR-JAK2 was constitutively phosphorylated and was located to the cytoplasm. BCR-JAK2 transformed the IL-3-dependent murine hematopoietic cell line Ba/F3 into IL-3 independent growth and induced STAT5b phosphorylation and translocation into the cell nuclei. The treatment with a JAK2 inhibitor abrogated BCR-JAK2 and STAT5b phosphorylation, leading to apoptosis of transformed Ba/F3 cells. To test whether BCR-JAK2 has tumorogenic ability in vivo, we performed experiments with nude mice, in which we injected subcutaneously cells transduced with the control vector and cells expressing BCR-JAK2. Notably, we only obtained tumors in the flank injected with BCR-JAK2 expressing cells, thus confirming the tumorogenic activity of the BCR-JAK2 fusion protein. We conclude that BCR-JAK2 is a new tyrosine-kinase that induces proliferation and cell survival, which can be abrogated by JAK2 inhibitors. In vitro studies demonstrate that BCR-JAK2 displays transforming activity. Moreover, the nude mice model reveals its ability to cause tumors. Disclosures: No relevant conflicts of interest to declare.

Characterization of 14 NUP214-ABL1 Fusions in T-Cell Acute Lymphoblastic Leukaemia (T-ALL) Exhibits a Genomic Heterogeneity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2075-2075
Author(s):  
Carlos Graux ◽  
Marina Lafage ◽  
Nicole Dastugue ◽  
Francine Mugneret ◽  
Roland Berger ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Cultured Cells ◽  
Outcome Data ◽  
Tyrosine Kinase Domain ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Genomic Amplification ◽  
Chromosomal Alterations ◽  
Male Predominance

Abstract The recently described episomal amplification of the NUP214-ABL1 fusion in 6% of T-ALL leads to the expression of a constitutively activated chimeric tyrosine kinase sensitive to imatinib. We collected additional cases in order to better characterize this new entity with respect to genetic presentations and clinical course. We collected 14 new NUP214-ABL1 positive cases by FISH (LSI-BCR/ABL1 ES, Vysis and ABL1 break-apart home-made probes) or RT-PCR (fusion transcript) screening. FISH analysis detected episomal amplification of NUP214-ABL1 in 11 patients with a highly variable number of nuclei with amplification (<1% to 90%). Interestingly, one case showed a higher percentage of nuclei with amplification when using frozen non cultured cells rather than cultured fixed cells (10% vs 1%) suggesting loss of episomes during culture. FISH showed also intrachromosomal amplification of NUP214-ABL1 in two cases: one presented a HSR at the original 9q34 site without detectable episomes, the other associated HSR (probably on a chromosome 10), episomes (<1% of nuclei) and 9q34 chromosomal insertions including NUP214 and ABL3′ that encode the tyrosine kinase domain but not ABL5′, on variable chromosomes including 14p (33%). One NUP214-ABL1 RT-PCR positive case did not show any FISH aberration. Median age: 16 y (3–45) with a male predominance (10:4). There were no T-cell lymphoblastic lymphoma. Immunophenotype (EGIL): mature (n=2), cortical (n=6) or pre-T (n=4). Karyotype: structural chromosomal alterations in 8 patients (including 4 with 10q24/HOX11 rearrangements), only numerical chromosomal alterations in 4 (including 2 with + 8), normal in 1, failed in 1. All samples with available information (10/14) showed a HOX11 or HOX11L2 abnormality. Among the 13 cases with available outcome data, we observed 5 early relapses, including both patients with NUP214-ABL1 HSR, and 1 refractory ALL. These observations emphasize the interest of combining both (quantitative) RT-PCR and FISH for the screening and characterisation of NUP214-ABL1 fusion and amplification, demonstrate the coexistence of different NUP214-ABL1 genomic presentations in one patient (episomal amplification, 9q34 insertions, HSR) compatible with the model in which genomic amplification start with episome formation in order to create the NUP214-ABL1 fusion followed by their amplification and optional secondary reintegration, confirm occurrence of NUP214-ABL1 in T-ALL with HOX11 and HOX11L2 involvement, raise the question of the rather worse prognosis for cases with intrachromosomal amplification as previously suggested, raise the signification of minor NUP214-ABL1 clones and variable genomic presentations in the leukemogenesis of this subgroup of T-ALL that could potentially benefit from imatimib. ° on behalf of the GFCH (Groupe Francophone de Cytogénétique Hématologique) and the BCGHO (Belgian Cytogenetic Group for Hematology and Oncology).

Molecular Profiling of Cancer Testis Antigens in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4701-4701
Author(s):  
Jason A Dubovsky ◽  
Emmanuel Berchmans ◽  
John J. Powers ◽  
Lin Hui-Yi ◽  
Yang Gao ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Chi Square ◽  
Clinical Criteria ◽  
Cancer Testis Antigens ◽  
Mutational Status ◽  
Cancer Testis

Abstract Abstract 4701 Background Chronic Lymphocytic Leukemia (CLL) is characterized by the progressive accretion of long-lived mature B-lymphocytes. Although the classical Rai and Binet staging is still commonly used, a new molecular understanding has identified specific signatures which could help predict disease progression and survival. Given the recent success of immunotherapeutic strategies and immunomodulatory drugs in the treatment of CLL we sought to identify potentially immunologically relevant targets and their relation to well known disease criterion. Methods Our study characterized the mRNA expression of 29 known cancer-testis antigens (CTAs) in 66 patients with CLL at varying stages of disease using a RT-PCR based expression panel. Relevant clinical criterion such as RAI stage, B2m, ZAP-70, IGVH mutational status, CD38, cytogenetics by FISH analysis, WBC count, age, gender, and treatment among others were then taken into account. The binary RNA expression data associated with the clinical and demographic factors were evaluated using chi-square or Wilcoxon rank sum analysis. Results Of the cancer-testis antigens tested, the MAGE family of CTAs revealed statistically significant correlations with multiple clinical criteria. Our analysis reveals a correlation between previous chemo-immunotherapy treatment and MAGE-A1, B2, E1, MAD-CT-2, SPA-17, and PAGE-5 expression. Beyond treatment, total white blood cell count was shown to have a significant association with MAGE family members A1, A3, and B2 expression. In addition, MAD-CT-2 and MAGE-B2 were significantly correlated with the expression of FMC-7 and SSX-4 and LAGE-1 correlated with the presence of B-cell symptomatology. Conclusions Preliminary RT-PCR based CTA phenotyping has unveiled interesting correlations to clinical criteria, opening multiple avenues for future immunotherapeutic interventions as well as possible prognostic value in CLL. Further investigation to better understand the biological value of this information in warranted. Disclosures: Pinilla: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

Overexpression of MEL1 as a Novel Fusion Partner of AML1 in the Blastic Phase of Chronic Myeloid Leukemia with the Recurrent Cryptic Translocation t(1;21)(p36.3;q22).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4332-4332
Author(s):  
Sawcene Hazourli ◽  
Pierre Chagnon ◽  
Raouf Fetni ◽  
Lambert Busque ◽  
Josee Hebert
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Zinc Finger Protein ◽  
Fish Analysis ◽  
Fusion Partner ◽  
Rt Pcr ◽  
Enhancer Element ◽  
Intron 1 ◽  
Cryptic Translocation ◽  
Pr Domain

Abstract Located at 1p36.3, MEL1 is a member of the MDS1/EVI1 gene family and encodes a zinc finger protein with a N-terminal PR-domain. It has been proposed that the overexpression of a modified version of MEL1 lacking the PR domain is oncogenic, whereas MEL1 retaining the PR domain is anti-tumorigenic. MEL1 is known to be overexpressed in some myeloid malignancies with reciprocal translocation t(1;3)(p36.3;q21) characterized by trilineage dysplasia and poor prognosis. It is suggested that the translocation of RPN1 gene at 3q21 in the vicinity of MEL1 gene might activate MEL1 expression through an enhancer element. Here we characterized a recurrent cryptic translocation t(1;21)(p36.3;q22) that fuses MEL1 to AML1 gene in a blastic transformation of chronic myeloid leukemia (CML). Fluorescence in situ hybridization (FISH) analysis with BAC/PAC clones revealed that the breakpoints are in intron 1 of MEL1 and between intron 1 and exon 8 of AML1. RT-PCR analysis showed that AML1-MEL1, but not the reciprocal MEL1-AML1 was expressed. Many splicing variants are present, and all fusions splice the 5′ end of AML1 that contains the RUNT domain with almost the entire MEL1. Furthermore two fusion transcripts contained open reading frames making possible the translation of two forms of AML1-MEL1 fusion proteins. To investigate if AML1-MEL1 leads to an inappropriate expression of MEL1 we performed a quantitative RT-PCR with primers outside and within the fused MEL1 allowing the detection of the normal and the rearranged allele respectively. Interestingly, our data show that while no normal MEL1 transcript was detected, there was an overexpression of the fused MEL1. These results suggest that similarly to AML1-EVI1 gene, overexpression of MEL1 could be regulated by the AML1 promoter in leukemic cells with the AML1-MEL1 fusion gene and might also play an important role in the progression of CML. Moreover, in contrast to previous studies showing an antioncogenic role for the PR domain, our findings indicate that in some leukemias, the overexpression of MEL1 is not restricted to the MEL1 PR-lacking form. This suggests that the mechanism by which the PR domain has his effect, is more complex than previously thought.

High Incidence of Primary Resistance to Imatinib in the Group of Ph-Negative BCR-ABL-positive CML Patients,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3772-3772
Author(s):  
Grigory Tsaur ◽  
Olga Plekhanova ◽  
Alexander Popov ◽  
Tatyana Riger ◽  
Anna Ivanova ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Direct Sequencing ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Tyrosine Kinase Domain ◽  
Molecular Response ◽  
Rt Pcr ◽  
Primary Resistance

Abstract Abstract 3772 Background. Around 1–2% of the CML patients do not have t(9;22)(q34;q11) detectable by conventional cytogenetics, but carry BCR-ABL fusion revealed by fluorescence in situ hybridization (FISH) and/or reverse-transcriptase PCR (RT-PCR). Outcome of Imatinib (IM) treatment in this group of patients remains unclear. Aim. To evaluate IM treatment efficacy in Ph-negative BCR-ABL- positive CML patients in comparison with Ph-positive ones. Methods. Initial diagnostics including chromosome banding analysis (CBA) and RT-PCR was performed in 251 CML patients who received 400 or 600 mg IM once a day. This group consisted of 181 patients in early chronic phase (CP), 65 patients in late CP and 4 patients in accelerated phase (AP). CBA was done after 24 hours culture. G-banding was performed by a trypsin-Giemsa method. Karyotypes were described according to ISCN (2005). At least 20 metaphases for each sample were analyzed. FISH assay using Dual-Colour Dual-Fusion BCR-ABL Translocation Probe (Abbott, USA) was applied on at least 200 interphase nuclei (I-FISH) and on all available metaphases. CBA and FISH were performed at the time of diagnostics and every 6 months of IM treatment. Cytogenetic response (CyR), partial cytogenetic response (PCyR) and complete cytogenetic response (CCyR) in Ph-positive CML patients were evaluated by CBA in contrast to Ph-negative patients those were assessed by I-FISH only. In Ph-negative CML patients CCyR were assumed as less than 1% of BCR-ABL -positive nuclei (N. Testoni et al, Blood, 2009). Quantitative measurement of BCR-ABL/ABL transcripts ratio by real-time quantitative PCR (RQ-PCR) was done every 3–6 months. Detection of point mutations in the BCR-ABL tyrosine kinase domain (TKD) was performed by direct sequencing of RT-PCR products. According to European LeukemiaNet recommendations (M. Baccarani et al, JCO, 2009), failures were defined as no complete hematological response (CHR) at 3 months, no CyR at 6 months, less that PCyR at 12 mo, less than CCyR at 18 months, loss of CHR, CCyR or major molecular response (MMR) at any time during treatment, newly acquired BCR-ABL mutation poorly sensitive to IM (G250E, E255V/K, T315I), or progression to the AP and/or blast crisis (BC). In Ph-negative patients 6 months and 12 months time-points, when CyR and PCyR have to be estimated, were excluded from the analysis due to lack of direct correlation between percentage of BCR-ABL -positive nuclei and CBA data (N. Testoni et al, Blood, 2009). Failure-free survival (FFS) and overall survival (OS) were calculated separately for Ph-positive and Ph-negative patients' groups. Results. In respect to t(9;22)(q34;q11) detection by CBA at the time of initial diagnosis all CML patients were divided into 2 groups: Ph-positive (n=244) and Ph-negative (n=7). In spite of presence of normal karyotype, all patients in the second group harboured BCR-ABL fusion gene revealed by FISH and RT-PCR. Groups were not significantly different in age at diagnosis, sex, Sokal risk group distribution, stage of disease at the time of IM treatment beginning, types of BCR-ABL fusion transcripts. Median time of follow-up was 43 months (range 26–66) in Ph-negative group and 36 months (range 18–105) in Ph-positive. Treatment results are shown in table. Although 6 of 7 Ph-negative CML patients achieved CHR by 3 months, only 1 of them was in CCyR by 18 mo, that was significantly lower than in Ph-positive group (p<0.001). Moreover, Ph-negative patients had high percentage of BCR-ABL -positive nuclei in BM both at 12 months (median 35% (range 1–60%)) and at 18 months of IM therapy (median 49% (range 0–92%)). Two Ph-negative CML patients progressed to AP and died subsequently. None of Ph-negative patients had BCR-ABL mutations, duplication or amplification. FFS in Ph-negative CML patients treated by IM was significantly lower than in 244 Ph-positive ones 0.14±0.13 vs 0.62±0.03 (p=0.007), while OS was comparable: 0.70±0.15 vs 0.85±0.02, (p=0.47), respectively. Conclusions. In our series treatment outcomes in Ph-negative CML patients who received IM at a dose of 400 or 600 mg once daily were significantly worse in comparison with Ph-positive ones. However, dose escalation or switching to second-generation TKI prevented further disease progression or CML-related deaths. Resistance in the observed group seems to have BCR-ABL-independent mechanisms. Disclosures: No relevant conflicts of interest to declare.

Recurrent Fusion of PCM1 to JAK2 in Atypical Chronic Myeloid Leukemia and Acute Leukemia Associated with the t(8;9)(p21-22;p23-24).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2963-2963
Author(s):  
Andreas Reiter ◽  
Christoph Walz ◽  
Ann Watmore ◽  
Claudia Schoch ◽  
Ilona Blau ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Hematological Malignancies ◽  
Coiled Coil ◽  
Janus Kinase ◽  
Tyrosine Kinase Domain ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Idiopathic Myelofibrosis ◽  
Secondary Aml ◽  
Atypical Cml

Abstract We have identified a novel, recurrent t(8;9)(p21–22;p23–24) in six patients with diverse hematological malignancies: atypical CML (n=4), secondary AML following idiopathic myelofibrosis (n=1) and pre B-ALL (n=1). Because of the involvement of several different tyrosine kinases in atypical CML, we focused our analysis on this class of gene. Initial FISH studies of one patient indicated that the janus kinase 2 gene (JAK2), located at 9p24, was disrupted. RACE-PCR was then used to identify the 8p21 partner gene as PCM1, a large centrosomal protein that contains multiple coiled-coil domains. RT-PCR and FISH analysis confirmed the fusion in this case, and also identified PCM1-JAK2 in the five other t(8;9) patients (RT-PCR and FISH, n=4; RT-PCR only, n=1; FISH only, n=1). Four different types of in-frame mRNA junction were identified, but in all cases the chimeric mRNA is predicted to encode a protein that retains several of the predicted coiled-coil domains from PCM1 and the entire tyrosine kinase domain of JAK2. Reciprocal JAK2-PCM1 mRNA could not be amplified in any patient. Clinically, 4 patients displayed CML-like hyperplasia with variable degrees of myelofibrosis and eosinophilia. Similar to typical CML, the clinical course of these patients was variable: one is alive 11 months after allogeneic stem cell transplantation, one transformed to acute leukemia 5 years after diagnosis, one died 4 days after presentation and one achieved a major cytogenetic response with interferon but died due to neurodegenerative disease and pneumonia at 7.5 years. Of the two remaining patients, one presented with secondary AML following idiopathic myelofibrosis and remains in remission 15 years after diagnosis following intensive chemotherapy and maintenance with interferon. The final patient died shortly after induction therapy for pre B-ALL. In conclusion, PCM1-JAK2 is a novel recurrent fusion gene in hematological malignancies that is likely to deregulate hemopoiesis in a manner similar to BCR-ABL. Patients with PCM1-JAK2 disease are attractive targets for signal transduction therapy.

Study on Clinical and Biological Characteristics of Childhood Acute Leukemia with MLL Gene Rearrangements.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4473-4473
Author(s):  
Jun He ◽  
Zi-xing Chen ◽  
Yong-quan Xue ◽  
Jin-lan Pan ◽  
Hai-long He ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Fusion Gene ◽  
Molecular Genetic ◽  
Chromosome Translocation ◽  
Fish Analysis ◽  
Gene Rearrangements ◽  
Older Children ◽  
Chromosome 11 ◽  
Rt Pcr ◽  
Mll Gene

Abstract The rearrangement of MLL gene is reported in 70%~80% of infant and in 5%~10% of older children (under the age of 15) with acute leukemia (AL). The biological features associated with alterations in MLL gene are hyperleukocytosis, CD10−/CD19+ phenotype and very poor prognosis. To explore the MLL rearrangement in details in our AL children patients and obtain more information on the relationship between the MLL gene abnormality and clinical outcomes. The following study has been conducted. A total of 298 patients with AL attended The Affiliated Children’s Hospital of Soochow University, including 16 cases with MLL rearrangements, were recruited in this study. Of the cohort, 11 were diagnosed as ALL, 5 were AML. 9 of 16 patients were in infant age (up to 2 year) and the rest were between the age of 2 to 13 years. Fluorescence in situ hybridization (FISH) analysis using LSI MLL dual color probe. Multiplex reverse transcriptase- polymerase chain reaction (multiplex RT-PCR) were used to discriminate 13 different fusion transcripts. These results were analyzed together with R banding karyotyping and immunolphenotyping determined by flow cytometry. We have found MLL rearrangements in 16 cases of childhood AL which were accounted for 5.4% of 298 AL patients, and 56.3% of infant AL. Among 106 cases analyzed by multiplex RT-PCR, MLL gene rearrangement was found in 11 cases, including MLL/AF4 fusion gene in 2, MLL/AF6 fusion gene in 1, MLL/AF6, MLL/ELL combined with MLL/AFX or HOX11 in one of each, MLL/AF9 in 2, MLL/AF10 in 1, MLL/ELL in 2. MLL partial tandem duplication in 1. In addition an activated HOX11 gene was found in 1 case.. In 27 cases assayed by FISH, MLL gene rearrangements have been detected in 9 cases (36.0%). In 16 patients with MLL gene rearrangements, 14 (87.5%) exhibited clonal chromosome abnormalities involved chromosome 11 in 11 cases, presenting as t(4;11) in 2, t(6;11), t(8;11), t(7;8;11), and t(9;11) in one of each, respectively, trisomy 11 in 2 and 11q- in 3 cases. Among these 16 patients, 11 were B-ALL, including Pro-B and Pre-B ALL; 5 of AML-M5, 3 of these 5 M5 patients were CD7+ and CD2+. Of these 16 patients 8 received chemotherapy and 7 of them achieved complete remission, while the other 8 patients eventually gave up treatment. Our results demonstrated that multiplex RT-PCR combined with FISH provided a more accurate and sensitive method for detection of MLL gene rearrangements, including chromosome translocation, deletion and duplication. Our findings lead to the detection of novel rearrangements at molecular genetic level. These findings regarding the MLL rearrangement provide most important information in guiding therapy and predicting prognosis in childhood AL. Besides our results also provide evidence in support of the value of 11q23/MLL in WHO classification categories.

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