Detection of Common Chimeric Fusion Transcripts in Acute Lymphoblastic Leukemia (ALL) Patients Using Multiplex Reverse Transcriptase Polymerase Chain Reaction Assay: A North Indian Tertiary Care Centre Experience

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4234-4234 ◽  
Author(s):  
Neelam Varma ◽  
Prateek Bhatia ◽  
Jogeshwar Binota ◽  
Ram Kumar Marwaha ◽  
Subhash Varma ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Tertiary Care ◽  
Rna Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts ◽  
Pediatric All ◽  
B Lineage

Abstract Abstract 4234 Introduction: Chromosomal abnormalities constitute an important parameter to identify prognostically relevant subgroups in acute lymphoblastic leukemia (ALL). Many western studies report the incidence of common chimeric fusion transcripts as 30–35%, however the data regarding the Indian patients is very scarce. Aims and Objectives: The aim of the present study was to detect presence of common chimeric fusion transcripts of t(12;21), t(9;22), t(1;19) and t(4;11) in adult and pediatric ALL cases using the Multiplex RT-PCR assay. Materials and Methods: This prospective study included 100 consecutive ALL cases diagnosed during last one year; 54 patients were <12 years of age. Diagnosis of ALL was made on the basis of bone marrow (BM) examination and flowcytometric immunophenotypic (FCM-IP) analysis. Approximately 2 ml of peripheral blood (PB) sample or 0.5 ml of BM sample was collected in EDTA. RNA extraction and cDNA synthesis was performed using commercial kits, according to standard protocols. RT-PCR assay was carried out using primers specific to the fusion transcripts of TEL-AML1 t(12;21); BCR-ABL t(9;22); E2A-PBX t(1;19) and MLL-AF4 t(4;11). The results of RT-PCR and FCM-IP were blinded and compared later. Results: Out of the 100 cases enrolled in the study, 54% were pediatric and 46% adult cases. Among the pediatric ALL cases, 49 were diagnosed as B-lineage ALL, 3 T-lineage ALL and 2 Biphenotypic ALL. Whereas among the adult ALL cases, 30 were diagnosed as B-lineage ALL, 12 T-lineage ALL and 4 Biphenotypic ALL. A total of 26% showed positivity for various fusion transcripts: 13% each pediatric and adult cases being positive. Of the 12 positive pediatric B-lineage ALL cases, 8 were positive for TEL-AML1 transcripts, 1 for BCR-ABL transcripts, 1 for E2A-PBX transcripts and 2 for MLL-AF4 transcripts. Out of the 11 positive adult cases, 7 were positive for BCR-ABL transcripts, 2 were positive for TEL-AML1 transcripts and 1 each for E2A-PBX transcripts and MLL-AF4 transcripts. BCR-ABL transcripts were detected in 1 out of 2 pediatric biphenotypic ALL and 2 out of 4 adult biphenotypic ALL cases. None of the pediatric cases was positive for the MLL-AF4 transcripts. None of the 4 transcripts were detected in any T-lineage ALL. Conclusion: Results of our study are comparable to those of other western and Asian studies, with TEL-AML1 transcripts being the most common fusion transcripts seen in pediatric B-ALL cases and BCR-ABL in adult cases. However two Indian studies had reported much lower incidence of TEL-AML1 and MLL-AF4 transcript in B-ALL cases. Identification of good prognostic groups and rationalization of therapy assumes great importance in our set ups as many patients are economically under-privileged. Relationship of these transcripts to the prognosis of our ALL patients will be evaluated in future. Disclosures: No relevant conflicts of interest to declare.

Multiplex RT-PCR assay for the detection of major fusion transcripts in Taiwanese children with B-lineage acute lymphoblastic leukemia

2002 ◽  
Vol 39 (1) ◽  
pp. 12-17 ◽  
Author(s):  
Der-Cherng Liang ◽  
Lee-Yung Shih ◽  
Chao-Ping Yang ◽  
Iou-Jih Hung ◽  
Shu-Huey Chen ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts ◽  
B Lineage

scholarly journals Multiplex RT-PCR Assay for Detection of Common Fusion Transcripts in Acute Lymphoblastic Leukemia and Chronic Myeloid Leukemia Cases

2016 ◽  
Vol 17 (2) ◽  
pp. 677-684 ◽  
Author(s):  
Nittaya Limsuwanachot ◽  
Teerapong Siriboonpiputtana ◽  
Kanlaya Karntisawiwat ◽  
Takol Chareonsirisuthigul ◽  
Suporn Chuncharunee ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts

CD22 Expression in Pediatric B-Lineage Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4119-4119
Author(s):  
Armin G. Jegalian ◽  
Alan S. Wayne ◽  
Robert J. Kreitman ◽  
Francis J. Mussai ◽  
Ira Pastan ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Surface Glycoprotein ◽  
B Cell Signaling ◽  
Limited Variation ◽  
Before And After ◽  
Pediatric All ◽  
The Mean ◽  
B Lineage

Abstract Abstract 4119 While the majority of pediatric patients with newly diagnosed B-lineage acute lymphoblastic leukemia (ALL) are cured with standard chemotherapy regimens, treatment is associated with multiple toxicities, and ALL remains the most frequent cause of cancer mortality in childhood. CD22, a B-lineage surface glycoprotein involved in B cell signaling and adhesion, is expressed in most cases of B-lineage ALL. We are conducting clinical trials of anti-CD22 immunotoxins [RFB4(dsFv)-PE38] for pediatric ALL. To assess eligibility for such targeted therapy, CD22 expression by ALL cells was studied in peripheral blood and/or bone marrow aspirate samples from 50 patients with relapsed ALL. The level of CD22 expression by ALL cells was quantitated by measuring mean anti-CD22 antibody binding per ALL cell (ABC) under saturating conditions using flow cytometry and the BD Biosciences QuantiBRITE system for fluorescence quantitation. Patients ranged in age from 3 to 22 years (median 10 years) and included 27 males and 23 females. CD22 expression was detected in all samples, and the vast majority of cases demonstrated expression of CD22 in 100% of leukemic blasts. CD22 antigen density in ALL cells varied widely among patients at baseline (range 451 - 14,519; mean 4276; median 3824; standard deviation 2976; see graph). CD22-directed immunotoxin therapy was initiated in 29 of the 50 patients, 19 of whom had samples quantitated for CD22 expression levels both before and after immunotoxin therapy. Most patients exhibited limited variation in the mean number of anti-CD22 molecules bound per ALL cell when comparing multiple specimens. In conclusion, CD22 expression varies widely in pediatric B-lineage ALL and persists despite repeated exposure to CD22-directed therapy. (MedImmune, LLC, sponsored the clinical studies of anti-CD22 immunotoxin CAT-8015.) Disclosures: No relevant conflicts of interest to declare.

The Heterogeneous Fusion Gene Landscape in Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4081-4081
Author(s):  
Yanara Marincevic-Zuniga ◽  
Johan Dahlberg ◽  
Sara Nilsson ◽  
Amanda Raine ◽  
Jonas Abrahamsson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Acute Lymphoblastic Leukemia ◽  
Gene Fusion ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Fusion Genes ◽  
Fusion Transcripts ◽  
Pediatric All

Abstract Background: Next generation sequencing allows for the detection of expressed fusion transcripts across the transcriptome and has spurred the discovery of many novel chimeric transcripts in various cancers. Structural chromosomal rearrangements that lead to fusion transcripts are a hallmark of acute lymphoblastic leukemia (ALL) and serve as markers for diagnosis and stratification of pediatric ALL patients into prognostically relevant subgroups. Improved delineation of structural alterations in ALL could provide additional information for prognosis in ALL and for improved stratification of patients into treatment groups. Methods: To identify novel fusion transcripts in primary pediatric ALL cells we performed whole transcriptome sequencing of 134 BCP and T-ALL patient samples collected at diagnosis. Our study include samples from patients with the well-known ALL subtypes t(12;21)ETV6-RUNX1, high hyperdiploid (51-67 chromosomes), t(9;22)BCR-ABL1, 11q23/MLL and dic(9;20), in addition to patients with undefined karyotype or non-recurrent cytogenetic aberrations ("undefined" and "other") (n=58). FusionCatcher was used for the detection of somatic fusion genes, followed by a stringent filtering pipeline including gene fusion validation by Sanger sequencing in order to reduce the number of false positives. Principal component analysis (PCA) of patients with fusion genes was performed using genome wide gene expression levels and DNA methylation levels (Infinium HumanMethylation450 bead array). Results: We identified and validated 60 unique fusion events in almost half of the analyzed patients (n=69). Of the identified fusion genes, 60% have not previously been reported in ALL or other forms of cancer. The majority of the fusion genes were found in a single patient, but 23% were recurrent, including known ALL fusion genes (n=10) and novel fusion genes (n=7). We found that BCP-ALL samples displayed a higher number of validated fusion genes (54%) compared to the T-ALL samples (28%) moreover in BCP-ALL patients with "other" and "undefined" karyotypes, we detected fusion genes in 71% and 61% of the samples, respectively. High hyperdiploid patients had the lowest rate of validated fusion genes (24%) compared to the other well-known subtypes, where we detected subtype-associated fusion genes in 97% of cases. We also identified promiscuous fusion gene partners, such as ETV6, RUNX1, PAX5 and ZNF384 that fused with up to five different genes. Interestingly, PCA revealed molecularly distinct gene expression and DNA methylation signatures associated with these fusion partners. Conclusion: RNA-sequencing of pediatric ALL cells revealed a detailed view of the heterogeneous fusion gene landscape, identifying both known and novel fusion genes. By grouping samples based on recurrent gene fusion partners we are able to find shared gene expression and DNA methylation patterns compared to other subtypes of ALL, suggesting a shared molecular etiology within these distinct subgroups, offering novel insights into the delineation of fusion genes in ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Quantitative analysis of TEL/AML1 fusion transcripts by real-time RT-PCR assay in childhood acute lymphoblastic leukemia

Leukemia ◽  
2000 ◽  
Vol 14 (8) ◽  
pp. 1526-1528 ◽  
Author(s):  
P Ballerini ◽  
J Landman Parker ◽  
I Laurendeau ◽  
M Olivi ◽  
M Vidaud ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Acute Lymphoblastic Leukemia ◽  
Real Time ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts

Outcome of Relapse After Allogeneic Transplantation for Childhood Acute Lymphoblastic Leukemia In Complete Remission. A Study of the Pediatric Diseases and Acute Leukemia Working Parties of the EBMT Group

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1291-1291
Author(s):  
Adriana Balduzzi ◽  
Myriam Labopin ◽  
Vanderson Rocha ◽  
Nabila Elarouci ◽  
Giorgio Dini ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Hematopoietic Cell Transplantation ◽  
Conflicts Of Interest ◽  
Pediatric Population ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Prognostic Impact ◽  
Dismal Prognosis ◽  
B Lineage ◽  
Donor Lymphocyte Infusions

Abstract Abstract 1291 Introduction. Childhood acute lymphoblastic leukemia (ALL) relapse occurring after hematopoietic cell transplantation (HCT) has a very dismal prognosis. Its treatment is still controversial and ranges from palliative treatment or chemotherapy to donor lymphocyte infusions, second transplant or experimental approaches. Objectives. The aim of this study is to assess the actual outcome in a pediatric population. The primary endpoint of this study is the 2-year probability of survival of children with ALL relapsing after allogeneic HCT; the secondary endpoint is the relationship between outcome and time of relapse after transplant, for which the following categories were considered: <3, 3–6, 6–12, > 12 months. Patients. Patients younger than 18 years of age undergoing first HCT from any allogeneic donor for ALL in first (CR1) or second (CR2) remission between January 1st 1998 and December 31st 2007 reported to the EBMT were eligible for the study. Results. Out of 3628 transplanted children with ALL reported to the EBMT, 836 (median age 9 years, male 66%) relapsed at a median of 6 months (range 1–67; 25th, 75th 4, 12 months) after HCT. The HCT was performed in CR1 (60%) or CR2 (40%) for a B-lineage (60%) or T- (13%) or unknown (27%) immunophenotype ALL, from an HLA-matched related (44%), unrelated (59%) or mismatched related (7%) donor, with marrow (61%), peripheral (28%) or umbilical (11%) stem cells. Out of 836, 81% died at a median of 2 months (25th,75th centiles:1,7) and 19% were reported as alive at last follow-up at a median of 22 months after relapse (range: 1–130). The 3-year probability of overall survival (3y-OS) was 14% (SE 1). As to immunophenotype, disease phase and donor type, 3y-OS was 15% (SE 2) in B-lineage and 8% (SE 3) in T-ALL, 18% (SE 2) in patients transplanted in CR1 and 11% (SE 6) in CR2 and 17% (SE 2) in patients transplanted from an HLA-identical sibling and 12% (SE 2) from any other donor. According to time of relapse after transplant, 3-year OS was 6% (SE 2), 10% (SE 2), 15% (SE 2) and 27% (SE 4) in those who relapsed in the first quarter, second quarter, second semester or after the first year, respectively. Donor lymphocyte infusions were reported for 7% and a second HCT for 16% of the 836 relapsed children. The probability of undergoing a second HCT within 1 year after relapse was 17% (SE 1); this probability was 6% for relapses occurring <6 months and 25% for later relapses. 3y-OS of those who underwent a second HCT was 32% (SE 5). Conclusions. The multivariate analyses confirmed the prognostic role of disease phase and immunophenotype, but not of the type of donor, assessed the strong prognostic impact of the time elapsed in CR after HCT before relapse, being earlier relapses at worse outcome compared with later relapses, possibly due to the chance of undergoing a second HCT, which role per se was not statistically significant. Disclosures: No relevant conflicts of interest to declare.

Triptolide Induces Apoptosis In Acute Lymphoblastic Leukemia Cells by Inhibition of the Oncoprotein MDM2

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4934-4934
Author(s):  
Mei Huang ◽  
Lubing Gu ◽  
Muxiang Zhou
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Biological Action ◽  
Leukemic Cells ◽  
Transcriptional Level ◽  
Antitumor Effects ◽  
Mdm2 Expression ◽  
Pediatric All ◽  
Chinese Plant

Abstract Abstract 4934 Triptolide, a nature product derived from the Chinese plant Tripterygium wilfordii, is reported to exhibit antitumor effects in a broad range of cancers. Recent studies indicate that the antitumor activity of triptolide is associated with its biological action to inhibit expression of many oncoproteins and anti-apoptotic or survival factors that were expressed in the cancer cells. Herein, we demonstrate that triptolide induces apoptosis in a subgroup of acute lymphoblastic leukemia (ALL) cells that overexpress MDM2 oncoprotein by inhibiting the MDM2 expression. In pediatric ALL, overexpression of MDM2 by leukemic cells is typically associated with a wild-type (wt) p53 phenotype and resistance to conventional chemotherapeutic drugs such as doxorubicin. In the present study, we evaluated the role of triptolide in regulating MDM2 and in inducing apoptosis, as compared to doxorubicin, using ALL lines and primary ALL samples. In contrast to doxorubicin, which induced p53 activation and a subsequent upregulation of MDM2, triptolide strongly induced persistent inhibition of MDM2 followed by a steady-state activation of p53, which resulted in potent apoptosis of the MDM2-overexpressing ALL cells tested, even if they were doxorubicin-resistant. We discovered that triptolide's inhibition of MDM2 in ALL cells occurred at the post-transcriptional level through inhibition of mRNA synthesis. Because p53 function is inhibited by MDM2 in chemoresistant/MDM2-overexpressing ALL cells, potent killing of these cells by triptolide suggests that this naturally-derived agent may be a novel therapeutic for refractory ALL. Disclosures: No relevant conflicts of interest to declare.

ERG Intragenic Deletion Characterizes a Distinct Oncogenic Subtype of B-Cell Precursor Acute Lymphoblastic Leukemia with a Favourable Outcome Despite Frequent IKZF1 Deletions

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 121-121
Author(s):  
Emmanuelle Clappier ◽  
André Baruchel ◽  
Jérôme Rapion ◽  
Aurélie Caye ◽  
Ahlème Khemiri ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Favourable Outcome ◽  
Fish Analysis ◽  
Cell Precursor ◽  
Pcr Assay ◽  
Genetic Lesion ◽  
Intragenic Deletion

Abstract Abstract 121 The genetic landscape of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in children above 10 years and adolescents remains poorly defined. Specifically, more than half of these patients have none of the cytogenetic abnormalities that define oncogenic subtypes and underlie risk stratification. To uncover new genetic abnormalities in these unassigned cases, we studied 85 BCP-ALL from patients aged 10 to 17 diagnosed at St-Louis hospital (Paris, France), for which the main classifying genetic lesions were assessed (i.e. high hyperdiploidy, t(12;21)/ETV6-RUNX1, t(1;19)/TCF3-PBX1, t(9;22)/BCR-ABL1, iAMP21, MLL translocations, low hypodiploidy, and near haploidy). Fifty of these BCP-ALL presented no classifying genetic lesions. Paired leukemic and remission samples could be analysed by high density array-CGH (Agilent 1M arrays) in 17 of these unassigned cases. We focused on acquired, focal, and recurrent copy-number abnormalities. A mono-allelic intragenic deletion of the ETS-related Gene (ERG) was found in 3 cases. ERG belongs to the ETS family of transcription factors and is implicated in chromosomal translocations associated with several cancer types including acute myeloid leukemia. The possibility of a cryptic unbalanced translocation was ruled out in the 3 cases by FISH analysis. The deletions encompassed exons 3 to 7, or 3 to 9, and the breakpoints were tightly clustered. Based on the breakpoint sequences we designed a PCR assay that allowed us to screen ERG intragenic deletions in the whole cohort. ERG deletion was identified in 9 additional cases, none of them having any of the known classifying genetic lesions, bringing up to 25% (12 out of 50) the frequency of ERG deletion in unassigned BCP-ALL of children older than 10. These results suggested that ERG deletion characterized a novel oncogenic subtype of BCP-ALL. Of note, these results were consistent with independent data of Harvey et al. (2010) that reported ERG deletions in a distinct gene-expression cluster. To confirm and extend these findings in the whole population of paediatric BCP-ALL, we used our breakpoint-specific PCR assay to screen ERG deletions in an independent cohort of 822 unselected patients aged 1 to 17, enrolled in the EORTC 58951 trial. ERG deletion was identified in 31/822 (3.7%) patients. Again, none of them had another known classifying genetic lesion, confirming that ERG deletion characterizes a distinct oncogenic subtype. Patients with ERG deletion were significantly older compared to other patients (median 7.0 vs 4.0, p=0.002), but they had similar white blood counts at diagnosis. They had a favourable outcome, with a 8-year event free survival (EFS) of 82.4% and overall survival (OS) of 96.0%, which is similar to EFS of 83.4% and OS of 91.6% obtained for patients having no very high risk initial features (i.e. no t(9;22)/BCR-ABL1, MLL rearrangement or haploidy/low hypodiploidy). IKZF1 deletion is a cooperative genetic lesion that has been recently shown to be associated with a poor outcome in BCP-ALL. Remarkably, the incidence of IKZF1 deletions in patients with ERG deletion was significantly higher than in other BCR-ABL1-negative patients, especially when considering the IKZF1 intragenic deletion Δ4-7 (10/31, 32.3% vs 34/744, 4.6%, P<0.001), and this regardless of age. Surprisingly, IKZF1 deletion had no impact on the prognosis of ERG deleted patients. Indeed, patients combining ERG and IKZF1 Δ4-7 deletions had a better outcome than other BCR-ABL1-negative patients with IKZF1 deletions (8-year EFS 83.3% vs 53.0%, hazard ratio (HR) 0.19, 95% CI 0.02–1.41; p=0.069). Altogether, we have identified a novel oncogenic subtype of BCP-ALL characterized by ERG deletion. This subtype is frequently associated with IKZF1 deletions, suggesting a preferred oncogenic cooperation. Importantly, despite having older age and frequent IKZF1 deletions, which are factors usually predictive of a poor prognosis, patients with ERG deletion have a favourable outcome. Therefore, this genetic abnormality may be systematically assessed as part of the diagnostic work-up of BCP-ALL and taken into account when considering treatment stratification. Disclosures: No relevant conflicts of interest to declare.

Novel Chromosomal Rearrangements and Sequence Mutations in High-Risk Ph-Like Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 67-67
Author(s):  
Kathryn G. Roberts ◽  
Ryan D Morin ◽  
Jinghui Zhang ◽  
Martin Hirst ◽  
Richard C. Harvey ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Cytokine Receptor ◽  
Negative Regulator ◽  
Rna Seq ◽  
Kinase Signaling ◽  
Rt Pcr ◽  
B Lineage

Abstract Abstract 67 Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, and relapsed B-lineage ALL remains a leading cause of cancer death in young people. Recent genomic analyses by our group and others identified a unique subtype of BCR-ABL-negative, high-risk B-ALL, with deletion or mutation of IKZF1 and a gene expression profile similar to BCR-ABL1-positive ALL (Ph-like ALL). Up to 50% of Ph-like patients harbor rearrangements of the cytokine receptor gene, CRLF2, with concomitant JAK mutations detected in ∼30%. However, the nature of genetic alterations activating kinase signaling in the remaining cases is unknown. To identify novel genetic alterations in Ph-like ALL, we performed transcriptome sequencing (RNA-seq) on 11 cases of Ph-like B-ALL (10 from the P9906 Children's Oncology Group trial and 1 from the St Jude Total XV study), and whole genome sequencing (WGS) on two of these. Using multiple complementary analysis pipelines including deFuse, Mosaik, CREST and CONSERTING, we identified novel rearrangements, structural variations and sequence mutations dysregulating cytokine receptor and kinase signaling in 10 cases. Putative rearrangements and sequence mutations were validated using RT-PCR, genomic PCR and Sanger sequencing. The spectrum of alterations included 3 cases with known IGH@CRLF2 rearrangement, 2 cases with the NUP214-ABL1 rearrangement, 1 case each with the in-frame fusions EBF1-PDGFRB, BCR-JAK2 or STRN3-JAK2, and 1 case with a cryptic IGH@-EPOR rearrangement. Detailed analysis of RNA-seq data revealed a 7.5 kb insertion of EPOR downstream of the enhancer domain in the IGH@ locus, which was not detected by fluorescence in situ hybridization. WGS identified an in-frame activating insertion in the transmembrane domain of IL7R (L242>FPGVC) in 1 index case, and recurrence screening identified similar IL7R sequence mutations in 8 cases from the P9906 cohort (N=188). This patient also harbored a focal homozygous deletion removing the first two exons of SH2B3 that was not evident by SNP array analysis. SH2B3 encodes LNK, a negative regulator of JAK2 signaling. Notably, all patients harbor genetic lesions affecting B-lymphoid development (e.g IKZF1), suggesting these events cooperate to drive B-lineage ALL. To determine the frequency of each fusion, candidate RT-PCR was performed on 231 cases from the COG AALL0232 trial of high-risk B-ALL, 40 (17%) of which were identified as Ph-like using Predictor Analysis of Microarrays (PAM). The EBF1-PDGFRB fusion was detected in 3 additional patients, each containing an intact PDGFRB kinase domain. No additional cases of NUP214-ABL1, BCR-JAK2, or STRN3-JAK2 were identified. Phosphoflow analysis on 3 primary ALL samples demonstrated increased CKRL phosphorylation in the NUP214-ABL1 case and tyrosine phosphorylation in the cases with BCR-JAK2 and STRN3-JAK2 fusions. Importantly, this activation was reduced with the tyrosine kinase inhibitors (TKI) imatinib, dasatinib and the T315I inhibitor XL228 in cells harboring the ABL1 fusion, and the JAK2 inhibitor, XL019, in the JAK2-rearranged samples. Furthermore, the novel EBF1-PDGFRB fusion transformed Ba/F3 cells to growth factor independence, induced constitutive activation of pSTAT5, pAkt, pERK1/2, and responded with low IC50 values to imatinib, dasatinib and the specific PDGFRB/FGFR inhibitor, dovitinib. Using complementary genomic approaches we show that rearrangements, sequence mutations and DNA copy number alterations dysregulating cytokine receptor and kinase signaling are a hallmark of Ph-like ALL. These data support the screening of patients at diagnosis to identify those with Ph-like ALL, characterize the genomic lesions driving this phenotype, and to determine those that may benefit from TKI treatment. Disclosures: Hunger: Bristol-Myers Squibb: Author's children own stock in BMS, Membership on an entity's Board of Directors or advisory committees.

MLLT10 Gene Promiscuity Unravels Involvement of RNA Processing Genes in Pediatric T-Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1431-1431
Author(s):  
Lucia Brandimarte ◽  
Valentina Pierini ◽  
Danika Di Giacomo ◽  
Paolo Gorello ◽  
Caterina Matteucci ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Rna Processing ◽  
Lymphoblastic Leukemia ◽  
Fusion Partner ◽  
Whole Genome ◽  
Rt Pcr ◽  
Specific Primers ◽  
Fusion Transcripts ◽  
Partner Gene

Abstract Abstract 1431 Background T-cell Acute Lymphoblastic Leukemia (T-ALL) affects about 15% of children with ALL. MLLT10 at 10p12 encodes for a transcription factor and is involved in leukemogenic fusions with PICALM or MLL in about 10% of childhood T-ALL with overexpression of HOXA cluster genes. In case of gene fusion MLLT10 retains the “octapeptide motif-leucine zipper” (OM-LZ) domain that appeared to be essential for leukemic transformation in mouse models (Deshpande AJ et al. Leukemia 2011) as it interacted with critical components of the chromatin modifying machinery, such as H3K79 methyltransferase hDOT1L (Okada Y et al. Cell 2005). Recently, in a case of early T-cell precursor ALL, NAP1L1 at 12q21 was identified by whole-genome sequencing as a new MLLT10 fusion partner gene (Zhang J et al. Nature 2012). We identified two new fusion transcripts involving MLLT10 in 2 cases of pediatric T-ALL suggesting MLLT10 is a promiscuous fusion partner gene in T-ALL. Aim Characterization of new MLLT10 fusion transcripts in 2 pediatric cases of T-ALL. Methods Total RNAs were extracted from cryopreserved bone marrow cells and retrotranscribed with MLLT10 specific reverse primer using 5'-RACE kit (Invitrogen). cDNAs were amplified in nested PCR using AAP/AUAP (Abridged Anchor Primer, Abridged Universal Amplification Primer, Invitrogen) as forward and MLLT10 specific primers as reverse. To confirm fusion transcripts we performed RT-PCR experiments using Thermoscript RT-PCR System (Invitrogen). cDNAs were amplified in nested PCR using HNRNPH1 and DDX3X specific primers as forward in patient 1 and 2 respectively and MLLT10 specific primers as reverse in both samples. PCR products were subcloned into pGEM-T easy vector (Promega) and sequenced. Whole genome analysis was performed applying Combined-Interphase Fluorescence In Situ Hybridization (CI-FISH) for 32 candidate genes as previously described (Gorello P et al. Haematologica 2010); Single Nucleotide Polymorphisms (SNPs) were performed following manifacturer's instructions (Affymetrix); Gene Expression Profiling (GEP) was applied to investigate the hypothesis that both new MLLT10 fusion genes shared leukemogenic properties with other MLLT10 fusions, particularly PICALM-MLLT10. Results In patient 1 the karyotype was 46,XX,inv(10)(p12q?)/46,XX. CI-FISH on bone marrow nuclei showed MLLT10 rearrangements (55%) and IKZF1 deletions (10%). The 10q disruption was located at 10q25.3 in a region of about 12 kb flanked by fosmid G248P87999G12, retained on inv(10), and RP11–411P18 translocated to chromosome 5. SNPs analysis was normal for copy number and LOH profile. In patient 2 cytogenetics failed. CI-FISH revealed a 9p deletion, with loss of PAX5 (9p13), CDKN2A/B (9p21) and JAK2 (9p24) in about 75% of interphase nuclei. MLLT10 break apart was abnormal in about 60% of nuclei, with break within RP11–418C1 spanning MLLT10 exons 1–3. SNPs analysis showed a 47,5Mb loss at 9p24.3-p11.2 and a 33,51Mb gain at 17q21.32-q25.3. 5'-RACE-PCR showed HNRNPH1 in patient 1 and DDX3X in patient 2 as two new MLLT10 partner genes in T-ALL. RT-PCR, cloning and sequencing confirmed these results. We found 2 in frame splicing variants in both cases: HNRNPH1 exon 11 (nt.1324)-MLLT10 exon 15 (nt. 2097) and HNRNPH1 intron 10 (nt. 6701)-MLLT10 exon 15 (nt. 2097) in patient 1; DDX3X exon 2 (nt. 958)-MLLT10 exon 3 (nt. 510) and DDX3X exon 1 (nt. 900)-MLLT10 exon 4 (nt. 590) in patient 2 (nucleotide numbers refer to GenBank accessions NM_005520.2 and NC_000005.9 for HNRNPH1, NM_001356.3 for DDX3X and NM_004641.3 for MLLT10). GEP showed that the new MLLT10 fusions were similar to 4 cases with PICALM-MLLT10 fusion but different from 5 other T-ALL, i.e. 2 with MLL translocations, 2 with inv(7)(p15;q34)/TCRB-HOXA and 1 with SET-NUP214 fusion, with a HOXA signature. Conclusions These two cases add new insights in multiple genomic recombinations affecting MLLT10 in pediatric T-ALL. Both new partner genes, i.e. HNRNPH1 and DDX3X, are involved in RNA processing and have not been reported to be involved in any genomic specific translocations, so far. The presence of the MLLT10 OM-LZ domain in both new fusions as well as the GEP of leukemic cells suggest that these MLLT10 recombinations activate the same leukemogenic pathways as identified for PICALM-MLLT10 and suggest promiscuity of MLLT10 in recombinations with leukemogenic genes. Disclosures: No relevant conflicts of interest to declare.

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