Multiplexed targeted sequencing of recurrent fusion genes in acute leukaemia

Abstract

2 Dozens of recurrent chromosomal rearrangements have been described in acute leukaemia, where they often result in the fusion of two genes and in the expression of hybrid proteins.These genetic markers often delineate distinct clinical entities, and have been incorporated into the World Health Organisation classification. Today, their evaluation at diagnosis thus provides crucial information for risk stratification, treatment decisions and residual disease evaluation 1,2 .Conventional cytogenetics is usually regarded as the gold standard method for the detection of these rearrangements. It is often considered mandatory at diagnosis but it requires a high level of expertise, is time consuming, and is not suitable for the detection of many cryptic rearrangements 3,4 . Complementary approaches such as fluorescent in situ hybridisation (FISH) and RT-PCR are thus often needed 5 , but the cost of a comprehensive analysis can rapidly become prohibitive. Many markers that may provide important clinical information are therefore never tested, lowering the chances for the patients to benefit from the optimal treatments. To address this issue, we have developed a rapid and parsimonious ligationdependant RT-PCR amplification assay (LD-RTPCR) 6 that allows for the detection of dozens of gene rearrangements (Figure 1).We created a mix of 153 LD-RTPCR probes to target more than 50 recurrent fusion genes and three NPM1 mutations ( Figure 1B and C). The procedure is detailed in supplemental methods, and representative results are provided in supplemental figure 1. As shown in supplemental figure 2A, the results inform on the identity of the two partner genes, but also on the localisation of the breakpoint, guiding the choice of an appropriate assay for residual disease evaluation. The analysis of serial dilutions showed that approximately 3.10 3 copies of transcripts are sufficient for the identification of the two partners (Supplemental figure 2B).To validate this method, we applied it to a retrospective cohort of 540 patients treated in our institutions ( Figure 2 and supplemental table 2). Sixty eight rearrangements were detected in the 180 childhood ALL cases we tested. Fifty two (76.5%) had been identified using RT-PCR at diagnosis and 20 (29.4%) using cytogenetics. Sixty seven of these rearrangements (98.5%) were detected using LD-RTPCR. As expected in this cohort, the most common were the ETV6-RUNX1 fusion (33 cases), rearrangements of the MLL gene (8 cases, four with AFF1, two with MLLT1, one with AFF4 and one with MLLT3) 7 , and the BCR-ABL1 (six cases), TCF3-PBX1 (five cases) and STIL-TAL1 fusions (four cases) 2 . The LD-RTPCR assay only failed to detect one t(4;11)(q21;q23), which was identified at diagnosis using cytogenetics but which could not be confirmed using RT-PCR. The breakpoint of this rearrangement may thus be unusual, or involve genes other than MLL and AFF1. It also validated four junctions which © 2015 Macmillan Publishers Limited. All rights reserved.3 were detected using cytogenetics, but which had not...

NUP214-ABL1 fusion defines a rare subtype of B-cell precursor acute lymphoblastic leukemia that could benefit from tyrosine kinase inhibitors

“…The blood count showed hemoglobin at 9.2 g/dL, platelet count at 58 000/mm 3 and hyperleukocytosis at 260 000/mm 3 with 95% of blast cells. The bone marrow aspirate was of high cellularity with 93% of peroxidase negative staining blast cells.…”

A rare case of acute myeloid leukemia with ARHGEF12 (LARG, 11q23.3) and MAPRE1 (EB1, 20q11.21) fusion gene in an elderly patient

“…The t(11;v)(q23;v) is an independent poor risk factor and the eligible patients will benefit from allogeneic stem cell transplantation in first remission (17). KMT2A gene mutations have different prognostic value depending on the fusion partner and testing for these anomalies is incorporated in some protocols (11). In our case, the patient outcome was favorable, with at least partial response.…”

“…Salsa MLPA P377 Hematological malignancies mix and P335 ALL-IKZF1 probemixes were used to analyze chromosomal regions that are currently known to have significant importance on AML risk stratification, diagnostic or prognostic. Ligation-dependent reverse transcription polymerase chain reaction (LD-RT-PCR) analysis technique was used in order to detect specific gene fusion (more than 50) for acute leukemias as previously reported (11). We adapted the protocol used by Ruminy P et al (11) and we performed the sequencing reaction by capillary electrophoresis.…”

“…Ligation-dependent reverse transcription polymerase chain reaction (LD-RT-PCR) analysis technique was used in order to detect specific gene fusion (more than 50) for acute leukemias as previously reported (11). We adapted the protocol used by Ruminy P et al (11) and we performed the sequencing reaction by capillary electrophoresis. Also, we removed the primers for NPM1 (type A-D mutations) from this protocol.…”

NARASIMHA: Novel Assay based on Targeted RNA Sequencing to Identify ChiMeric Gene Fusions in Hematological Malignancies

“…Researchers have therefore developed focussed target enrichment strategies (or gene-panels) for detection of CGF. Typically, these panels utilize capture probes 1,2 or multiplexed PCR approaches 3,4 to enrich targets of interest and detect the CGF, if present. These assays too require prior knowledge of the exact sequence of both partners involved in the formation of CGF thus failing to overcome a principal hurdle of being unable to detect a CGF involving a promiscuous gene where recombination with several partners is known to occur (for e.g., KMT2A-rearranged leukemia) 5 .…”