Identification of Multiple, Patient-Specific MLL Fusion Transcript Isoforms in Childhood Leukemia Using Anchored Multiplex PCR-Based Enrichment (AMP-E)

Abstract

Chromosomal translocations involving 11q23, resulting in rearrangements of the mixed lineage leukemia gene (MLL, re-named KMT2A) are frequent events in childhood leukemia. MLL is highly promiscuous, with approximately 80 fusions now characterized. Although fluorescence in situ hybridization (FISH) has high specificity for detecting MLL-rearrangements (MLL-r), sensitivity is limited and the translocation partner gene (TPG) cannot always be identified. In contrast, long-distance inverse-PCR (LDI-PCR) permits sequence-specific characterization of MLL breakpoints and the resultant fusion gene, which can then be used for monitoring minimal residual disease (MRD). A limitation of LDI-PCR is the relatively large input of DNA (≈ 1μg) required, with a blast cell percentage of > 20-30% to achieve sufficient sensitivity. Next-generation sequencing (NGS) approaches such as RNAseq and whole-genome sequencing (WGS) have the potential to identify multiple gene fusions, however their ability to detect the full spectrum of MLL fusions is limited by coverage, read depth and thereby cost. Such limitations can potentially be overcome with targeted sequencing panels, although their performance against "gold standard" assays, such as LDI-PCR, is unknown. We therefore aimed to assess the ability of a novel, targeted NGS approach for characterizing patient-specific MLLgene rearrangements from low inputs of RNA.The Archer™ FusionPlex™ Heme and Myeloid panels utilize anchored multiplex PCR-based enrichment (AMP-E) to rapidly enrich a number of targets, including MLL, creating libraries for NGS. The NGS libraries are generated using rapid workflows and are compatible with nucleic acid inputs of ≈ 20-200ng. Briefly, double stranded cDNA is generated from patient RNA and subjected to end repair, adenylation and ligation with unique, half-functional adaptors. Following two rounds of nested PCR with primers attached to common sequencing adaptors, the resulting target amplicons become functional and ready for clonal amplification and sequencing. Using AMP-E, we tested 23 paediatric MLL-r samples (15 ALL, 8 AML) that had previously been analyzed by LDI-PCR and were known to harbor 8 different MLL fusions, including MLL-AFF1 (n = 8), -MLLT3 (5), -MLLT10 (3), -ELL (2), -DCP1A (1), -MLLT1 (1), - AFF3 (1), and -TNRC18 (1). A patient sample known to express BCR-ABL1 was used as a positive control and a cytogenetically normal AML sample in remission was used as a negative control in each panel. The median blast count for samples analyzed was 86.1% (range 25%-97%). On average, 100ng of RNA was used per sample, with RIN values ranging from 2.7 to 9.1. Libraries generated using either the Archer™ FusionPlex™ Heme or Myeloid kit were sequenced to sufficient read depths by Illumina MiSeq® and NextSeq®, respectively. Bioinformatic analyses were performed with the Archer™ Analysis 4.1 software. Results were then compared with fusions identified by LDI-PCR.There was high concordance between AMP-E and LDI-PCR, with all MLL fusion genes identified by LDI-PCR also detected by AMP-E. Of note, an ALL sample with t(11;19), unable to be characterized by LDI-PCR, was identified by AMP-E to express MLL-MLLT1. The control BCR-ABL1 fusion was identified in every run and there were no false-negative results. Furthermore, AMP-E identified multiple MLL-fusion transcripts in 56.5% of patients. Analysis of paired diagnosis-relapse samples from an AML patient with MLL-MLLT3demonstrated that the two discrete transcripts present at diagnosis persisted at relapse, with emergence of a third transcript.In summary, detection of MLL gene fusions in acute leukemia using AMP-E is both sensitive and specific. The low RNA requirement, rapid workflow, compatibility with Illumina MiSeq® and cloud-based proprietary analysis software, together with the array of additional fusions and mutations detected by the Archer™ panels, show promise for translation into clinical diagnostic settings. The persistence of discrete transcript isoforms at relapse also highlights the potential for AMP-E to identify multiple, patient-specific MLL fusion transcripts which may have utility in refining prognostication, MRD monitoring and informing future functional studies of MLL-driven leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.