scholarly journals Fusion gene detection by RNA sequencing complements diagnostics of acute myeloid leukemia and identifies recurring NRIP1-MIR99AHG rearrangements

Haematologica ◽  
2021 ◽  
Author(s):  
Paul Kerbs ◽  
Sebastian Vosberg ◽  
Stefan Krebs ◽  
Alexander Graf ◽  
Helmut Blum ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Rna Sequencing ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Mirna Cluster ◽  
Fusion Genes ◽  
Rna Seq ◽  
Gene Detection ◽  
Clinical Routine ◽  
Acute Myeloid

Identification of fusion genes in clinical routine is mostly based on cytogenetics and targeted molecular genetics, such as metaphase karyotyping, FISH and RT-PCR. However, sequencing technologies are becoming more important in clinical routine as processing-time and costs per sample decrease. To evaluate the performance of fusion gene detection by RNA sequencing (RNAseq) compared to standard diagnostic techniques, we analyzed 806 RNA-seq samples from acute myeloid leukemia (AML) patients using two state-of-the-art software tools, namely Arriba and FusionCatcher. RNA-seq detected 90% of fusion events that were reported by routine with high evidence, while samples in which RNA-seq failed to detect fusion genes had overall lower and inhomogeneous sequence coverage. Based on properties of known and unknown fusion events, we developed a workflow with integrated filtering strategies for the identification of robust fusion gene candidates by RNA-seq. Thereby, we detected known recurrent fusion events in 26 cases that were not reported by routine and found discrepancies in evidence for known fusion events between routine and RNA-seq in three cases. Moreover, we identified 157 fusion genes as novel robust candidates and comparison to entries from ChimerDB or Mitelman Database showed novel recurrence of fusion genes in 14 cases. Finally, we detected the novel recurrent fusion gene NRIP1-MIR99AHG resulting from inv(21)(q11.2;q21.1) in nine patients (1.1%) and LTN1-MX1 resulting from inv(21)(q21.3;q22.3) in two patients (0.25%). We demonstrated that NRIP1-MIR99AHG results in overexpression of the 3' region of MIR99AHG and the disruption of the tricistronic miRNA cluster miR-99a/let-7c/miR-125b-2. Interestingly, upregulation of MIR99AHG and deregulation of the miRNA cluster, residing in the MIR99AHG locus, are known mechanism of leukemogenesis in acute megakaryoblastic leukemia. Our findings demonstrate that RNA-seq has a strong potential to improve the systematic detection of fusion genes in clinical applications and provides a valuable tool for fusion discovery.

RNA-Sequencing Unveils Cryptic Fusions in Patients with Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1278-1278
Author(s):  
Fabiana Ostronoff ◽  
Matthew Fitzgibbon ◽  
Martin McIntosh ◽  
Rhonda E. Ries ◽  
Alan S. Gamis ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Rna Sequencing ◽  
Myeloid Leukemia ◽  
Prognostic Markers ◽  
Normal Karyotype ◽  
Fusion Transcript ◽  
Rna Seq ◽  
Illumina Hiseq ◽  
Normal Controls ◽  
Acute Myeloid

Abstract Abstract 1278 Introduction: Acute myeloid leukemia (AML) represents a heterogeneous group of malignancies with great variability in response to therapy. In recent years, an increasing list of molecular markers with prognostic significance in AML has been identified; nonetheless, new prognostic markers and therapeutic targets are still needed. The aim of this study was to identify and verify fusion transcripts using RNA-Sequencing (RNA-Seq) that would be otherwise undetectable by conventional karyotyping. Methods: Transcriptome Sequence data is generated by high-throughput short-read RNA-Seq performed for each AML sample on the Illumina HiSeq. Poly(A) RNA is captured with poly(T) magnetic beads, fragmented, copied to cDNA libraries with reverse transcriptase and random primers. Each library is subjected to 50-cycle paired-end sequencing on the Illumina HiSeq at Hudson Alpha. Filtered Fastq files are processed with TopHat-Fusion [Kim2011,Trapnell2009] alignment software to discover cryptic fusions in RNA-Seq data without relying on known, annotated models. This process yielded an average of 20 million alignable reads per sample. Cord blood blast cell transcripts are also processed and serve as normal controls. A series of filtering steps eliminate junctions commonly found to be in error. Filtered junctions found in at least 3 AML samples and no normal controls are retained as AML-associated candidate junctions. Visual curation of candidates is performed using Integrative Genomics Viewer. Candidate fusions were verified by RT-PCR amplification of the AML-associated fusions in the index cases. Fusion transcript product, as well as the break point junction was verified by Sanger sequencing Results: Diagnostic specimens from 70 patients with de novo AML that included patients with normal karyotype (NK, N=31), core-binding factor (CBF) AML (N=33) and other (N=6) were sequenced. Age at diagnosis varied from 10 months to 69 years (Median 12 years). White blood cell count (WBC) and blast percentage were 49×109/L (range, 2.4 to 496×109/L) and 78% (40% to 100%), respectively. Bioinformatic evaluation of the RNA-Seq data revealed 67 high-value novel fusions that were not detected by conventional karyotyping: 54 (80.6%) were intra- and 13 (19.4%) inter-chromosomal junctions. The number of novel translocations varied in different cytogenetic groups, with 22 novel fusions detected in those with NK (16 intra and 6 inter-chromosomal junctions), 37 CBF (32 intra and 5 inter-chromosomal junctions) and 8 in “other” (6 intra and 2 inter-chromosomal junctions). Thirteen novel fusions (19.4%) were found in at least 2 or more screened-patients: two (15.4%) inter- and 11 (84.6%) intra-chromosomal junctions. Median number of fusions identified per patient was 2 (range, 1 to 6). Novel fusions involving PDGFR-β gene were identified in two patients, each with a different translocation partner (G3BP1 and ETV6, which was an intra and inter-chromosomal fusions, respectively). Sequencing of the fusion transcript junctions verified the fusion junctions and demonstrated in frame fusions of G3BP1 and ETV6 to the kinase domain coding region of PDGFR-β, identical junction to that seen in cases of imatinib sensitive idiopathic hypereosinophilic syndrome (IHES). Frequency validation in 100 adult and 100 pediatric cases identified one additional patient with G3BP1-PDGFR-β. Cryptic NUP98/NSD1 was identified and verified in two patients with normal karyotype as well as NUP98/HOXD13 translocation in one patient. Frequency determination of NUP98/NSD1 demonstrated prevalence of 7.8% in patients with NK, and that of 13% in patients with FLT3/ITD. Patients who harbored both NUP98/NSD1 fusion and FLT3/ITD had a dismal remission induction rate (CR rate in FLT3/ITD with and without NUP98/NSD1 was 28% vs. 73%; p=0.002). Conclusion: Our data show the applicability of RNA-Seq as a tool to discover cryptic fusion transcripts in AML. These novel fusions may define new independent prognostic markers and potential therapeutic targets for patients with this highly treatment-resistant disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Novel ZEB2-BCL11B Fusion Gene Identified by RNA-Sequencing in Acute Myeloid Leukemia with t(2;14)(q22;q32)

PLoS ONE ◽  
2015 ◽  
Vol 10 (7) ◽  
pp. e0132736 ◽  
Author(s):  
Synne Torkildsen ◽  
Ludmila Gorunova ◽  
Klaus Beiske ◽  
Geir E. Tjønnfjord ◽  
Sverre Heim ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Rna Sequencing ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Acute Myeloid

scholarly journals RNA Sequencing Reveals Novel and Rare Fusion Transcripts in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3627-3627 ◽  
Author(s):  
Antonella Padella ◽  
Giorgia Simonetti ◽  
Giulia Paciello ◽  
Anna Ferrari ◽  
Elisa Zago ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Rna Sequencing ◽  
Cytogenetic Analysis ◽  
Myeloid Leukemia ◽  
Fusion Transcript ◽  
Gene Fusions ◽  
Fusion Genes ◽  
Exon 2 ◽  
Frame Fusion ◽  
Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is a highly heterogeneous disease and a complex network of events contribute to its pathogenesis. Chromosomal rearrangements and fusion genes have a crucial diagnostic, prognostic and therapeutic role in AML. A recent RNA sequencing (RNAseq) study on 179 AML revealed that fusion events occur in 45% of patients. However, the leukemogenic potential of these fusions and their prognostic role are still unknown. To identify novel rare gene fusions having a causative role in leukemogenesis and to identify potential targets for personalized therapies, transcriptome profiling was performed on AML cases with rare and poorly described chromosomal translocations. Bone marrow samples were collected from 5 AML patients (#59810, #20 and #84 at diagnosis and #21 and #32 at relapse). RNAseq was performed using the Illumina Hiseq2000 platform. The presence of gene fusions was assessed with deFuse and Chimerascan. Putative fusion genes were prioritized using Pegasus and Oncofuse, in order to select biologically relevant fusions. Chimeras not supported by split reads, occurring in reactive samples, involving not annotated or conjoined genes were removed. The remaining fusions were prioritized according to mapping of partner genes to chromosomes involved in the translocation or to Chimerascan and deFuse concordance. The CBFβ-MYH11 chimera was identified in sample #84, carrying inv(16) aberration, thus confirming the reliability of our analysis. Sample #59810 carried the fusion transcript ZEB2-BCL11B (Driver Score, DS=0.7), which is an in-frame fusion and a rare event in AML associated with t(2;14)(q21;q32). The breakpoint of the fusion mapped in exon 2 of ZEB2 (ENST00000558170) and exon 2 of BCL11B (ENST00000357195). Differently from previous data, this fusion transcript showed 3 splicing isoforms. Type 1 isoform is the full-length chimera and it retains all exons of both genes involved in the translocation. Type 2 isoform was characterized by the junction of exon 2 of ZEB2 and exon 3 of BCL11B. In type 3 isoform, exon 2 and 3 of BCL11B were removed, resulting in an mRNA composed by exon 2 of ZEB2 and exon 4 of BCL11B. Gene expression profiling showed an upregulation of ZEB2 and BCL11B transcripts in the patient's blasts, compared to 53 AML samples with no chromosomal aberrations in the 14q32 region. The same samples showed the WT1-CNOT2 chimera, which is a novel out-of-frame fusion (DS= 0.008) related to t(11;12) translocation, identified by cytogenetic analysis. Two new in-frame fusion genes were identified in sample #20: CPD-PXT1 (DS=0.07), which appeared as the reciprocal fusion product of t(6;17) translocation, and SAV1-GYPB, which remained cryptic at cytogenetic analysis (DS=0.8, alternative splicing events are being investigated). SAV1 was downregulated in sample #20 compared to our AML cohort, suggesting the putative loss of a tumour-suppressor gene. Sample #21 carried a t(3;12) translocation and RNAseq identified a novel fusion event between chromosomes 19 and 7, involving the genes OAZ and MAFK (DS=0.9). Finally, no chimeras were confirmed in sample #32 having a t(12;18) translocation. Our data suggest that fusion events are frequent in AML and a number of them cannot be detected by current cytogenetic analyses. Gene fusions cooperate to AML pathogenesis and heterogeneity and we are further investigating the oncogenic potential of the identified translocations. Moreover, the results firmly indicate that different approaches, including G-banding, molecular biology, bioinformatics and statistics, need to be integrated in order to better understand AML pathogenesis and improve patients' stratification, High-resolution sequencing analysis currently represent the most informative strategy to tailor personalized therapies. Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:BMS: Speakers Bureau; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

scholarly journals Comprehensive diagnostics of acute myeloid leukemia by whole transcriptome RNA sequencing

Leukemia ◽  
2020 ◽  
Vol 35 (1) ◽  
pp. 47-61 ◽  
Author(s):  
Wibowo Arindrarto ◽  
Daniel M. Borràs ◽  
Ruben A. L. de Groen ◽  
Redmar R. van den Berg ◽  
Irene J. Locher ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Targeted Therapy ◽  
Rna Sequencing ◽  
Myeloid Leukemia ◽  
Simultaneous Detection ◽  
Fusion Genes ◽  
Genetic Aberrations ◽  
Whole Transcriptome ◽  
Tandem Duplications ◽  
Acute Myeloid

AbstractAcute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide risk-adapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1 irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3 and KMT2A were detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform.

scholarly journals Novel Fusion Genes Involving Hnrnpc and Rarg in Acute Myeloid Leukemia Mimicking Acute Promyelocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5274-5274
Author(s):  
Zhan Su ◽  
Xin Liu ◽  
Yan Xu ◽  
Weiyu Hu ◽  
Chunting Zhao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Myeloid Leukemia ◽  
Sanger Sequencing ◽  
Fusion Gene ◽  
Promyelocytic Leukemia ◽  
Fusion Genes ◽  
Hnrnp C ◽  
Acute Myeloid

Abstract The roles of Heterogeneous nuclear ribonucleoproteins(hnRNPs) in regulating tumor development and progression, either as oncogenes or as tumor suppressors, were well documented. HnRNP C is one of the members of hnRNPs,and differential expression of hnRNP C has been found in series of tumor cells. However, the role of hnRNP C in leukemia has not been reported to date. Here, we report the first novel gene fusion event between HNRNPC and retinoic acid receptor gamma (RARG) in acute myeloid leukemia mimicking acute promyelocytic leukemia. This translocation produced the HNRNPC-RARG fusion gene and its reciprocal, RARG-HNRNPC. A 43-year-old man was referred to our hospital with fever and a sore throat.Laboratory investigations revealed the following patient characteristics: (1) white blood cell count 12 × 109/L (blasts 1% and abnormal promyelocytes 86%). (2) Morphologic analysis of the bone marrow aspirate showed 86.5% microgranular atypical promyelocytes (Figure 1a, 1b). (3) Analysis from flow cytometry showed that the blasts were positive for CD33, CD13, CD45, and cMPO and negative for CD14, CD34, CD16, CD56, HLA-DR, B- or T-cell markers. Thus, the patient started all-trans retinoic acid (ATRA) treatment immediately. Afterwards, chromosomal analysis revealed 47 metaphases, and most of them were involved in t(14;17). Fluorescence in situ hybridization and RT-PCR assays did not identify the PML/RARA, NPM-RARA, PLZF-RARArearrangement. ATRA therapy lasted for 3 weeks, but no response was observed. Next, the patient received 2 cycles of induction chemotherapy until a complete response. Afterwards, he received 6 cycles of chemotherapy. Unfortunately, the leukemia relapsed 1 year later, and all treatments (including ATRA and arsenious acid) failed to produce any effects. The patient died from sepsis. To identify molecular alterations, transcriptome sequencing analysis was performed. A 213-bp RARG-HNRNPC fusion product was specifically amplified from the patient's cDNA, as predicted (Figure 1c). Sanger sequencing showed that RARG exon 9 was fused in-frame to HNRNPC exon 3(Figure 1d). The RARG 5'-region encoding the ligand-binding domain was fused to the HNRNPC3'-region, where a cluster of phosphorylation sites is located(Figure 1e). We also found a reciprocal chimeric transcript. The amplicon size of HNRNPC-RARG fusion was 186-bp (Figure 2a). Sanger sequencing demonstrated that HNRNPC exon 3 was fused in-frame to RARG exon 5 (Figures 2b). The HNRNPC 5'-region encodes an RNA recognition motif (RRM), and the segment from RARG encodes a DNA binding domain (DBD, Figure 2c). HnRNP C ubiquitously expressed RNA-binding protein (RBP) which are believed to influence pre-mRNA metabolism such as splicing, polyadenylation, stability, transport, andtranslation mediated by internal ribosome entry site. HnRNP C also plays an essential role in cell progression and the regulation of several DNA repair proteins. Retinoic acid receptors (RARs) are transcription factors that belong to the nuclear hormone receptor family.RARA, RARB, and RARG are three RARs subtypes which share highly similar sequences and functions. A study showed RARG seems to act as a major regulator maintaining the balance between HSC self-renewal and differentiation. Acute myeloid leukemias mimicking acute promyelocytic leukemia, or acute promyelocytic-like leukemias (APLL), share the same morphology and immunocytochemistry features with typical acute promyelocytic leukemia (APL) except the RARA rearrangements, and little is known about the molecular mechanisms of APLL. The sequences and function of the RARG and RARA are highly alike, and therefore can logically explain the similarity of biological characteristics between the two entities. Three other fusion genes harboring RARG ( including NUP98-RARG , PML-RARG and CPSF6-RARG) have been found in APLL. Unfortunately they showed resistance to treatment with ATRA or ATRA plus arsenic. Moreover, poor prognosis was observed likewise. All the above confirm that RARG rearrangements are not random but recurrent genetic abnormalities. In conclusion, we present a novel HNRNPC-RARG fusion gene and its reciprocal in APLL, and suggest that at least a portion of APLLs have RARG gene rearrangements. We propose that RARG-rearranged APLL may be a novel candidate subtype of acute myelocytic leukemia, or even of APL. Disclosures No relevant conflicts of interest to declare.

Genomic structure and nucleotide sequence of AML1-ETO fusion genes in children with acute myeloid leukemia

2004 ◽  
Vol 216 (03) ◽  
Author(s):  
C Gall ◽  
T Langer ◽  
M Metzler ◽  
S Viehmann ◽  
J Harbott ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nucleotide Sequence ◽  
Myeloid Leukemia ◽  
Genomic Structure ◽  
Fusion Genes ◽  
Acute Myeloid

scholarly journals Combined use of interferon alpha-1b, interleukin-2, and thalidomide to reverse the AML1-ETO fusion gene in acute myeloid leukemia

2021 ◽  
Author(s):  
Ruihua Mi ◽  
Lin Chen ◽  
Haiping Yang ◽  
Yan Zhang ◽  
Jia Liu ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Interferon Alpha ◽  
Myeloid Leukemia ◽  
Clinical Medicine ◽  
Fusion Gene ◽  
Interleukin 2 ◽  
Effective Rate ◽  
Dose Administration ◽  
Combined Use ◽  
Acute Myeloid

AbstractThis study aims to explore the effect of the ITI (interferon alpha-1b, thalidomide, and interleukin-2) regimen on the AML1-ETO fusion gene in patients with t(8;21) acute myeloid leukemia (AML) who were in hematologic remission but positive for the AML1-ETO fusion gene. From September 2014 to November 2020; 20 patients with AML (15 from The Affiliated Cancer Hospital of Zhengzhou University, 4 from The First Affiliated Hospital; and College of Clinical Medicine of Henan University of Science and Technology, and 1 from Anyang District Hospital) with hematological remission but AML1-ETO fusion gene positivity were treated with different doses of the ITI regimen to monitor changes in AML1-ETO fusion gene levels. Twenty patients were treated with a routine dose of the ITI regimen, including 13 males and 7 females. The median patient age was 38 (14–70 years). The fusion gene was negative in 10 patients after 1 (0.5 ~ 8.6) month, significantly decreased in 4 patients after 2.8 (1 ~ 6) months, increased in 4 patients, and unchanged in 2 patients. The 4 patients with elevated levels of the fusion gene were treated with an increased dose of the ITI regimen, and all four patients became negative, for a total effective rate of 90%. The ITI regimen reduces AML1-ETO fusion gene levels in patients with AML who are in hematologic remission but are fusion gene–positive. Improvement was observed in patients’ response to a higher dose administration, and patients tolerated the treatment well.

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