Rapid Capture NGS Identifies Genomic Rearrangements in BCP-ALL for MRD Diagnostics

Background

Immunoglobin heavy chain (IGH), T-cell receptor δ (TRδ) rearrangements and translocations have frequently been described in patients with B-cell precursor (BCP) ALL. In order to detect the various types of fusions we used a high-throughput sequencing (HTS) assay based on direct capturing and sequencing of enriched genomic DNA fragments. Our approach focused on enrichment and direct paired-end sequencing of IG/TR rearrangements to analyze clonality and to detect kinase- and/or cytokine-receptor fusion breakpoints. We selected the corresponding chromosomal regions spanning exonic as well as intronic genomic areas from IGH-JH, TRδ and from several "Ph-like"-associated genes (ABL1, ABL2, CSF1R, CRLF2, EPOR, JAK2, PDGFRß). For the identification of clonality and fusion breakpoints we used Vidjil (www.Vidjil.org) and "segemehl" bioinformatics algorithms, respectively. Clonal rearrangements and genomic breakpoints were subsequently compared for their use in detection of minimal residual disease (MRD).

Patients and methods

For combined clonality and breakpoint analysis, 162 BCP ALL patients (MLL^-; TEL-AML1^-, BCR-ABL1^-) were selected due to the MRD status at the end of induction (EOI) therapy, which include a substantial proportion of ALL samples (n = 68) with a high MRD burden (≥ 1 x 10^-2). To test the feasibility of the analytical approach, additional BCR-ABL1+ ALLsamples (n=19) were included for genomic breakpoint analysis. Captured genomic DNA from eighty ALL samples was sequenced on a HiSeq2500 Illumina device, the remaining samples were subjected to several MiSeq (Illumina) runs multiplexing up to 12 samples using MiSeq Reagent Kit v3 (600 cycle) chemistry.

Results

For TRδ, a total of 267 clonal rearrangements were characterized in 142/162 samples (88%). Beside clonal Vδ2-Dδ3, Dδ2-Dδ3, Vδ2-Jα, we identified a novel group of recurrent Dδ-Jα rearrangements in 46 samples (28%), comprising Dδ2 or Dδ3 segments fused predominantly to Jα29. For IGH-JH, 304 clonal rearrangements were identified in 152/162 samples, including several novel VDJ joinings usually not detectable by standard PCR-based approaches. In BCR-ABL1⁺ ALL, genomic breakpoints were identified in 17/19 cases. In BCP-ALL lacking a classical rearrangement, 50 gene fusions were detected, between IGH-JH and either DUX4, EPOR or CRLF2 in 21 of 162 cases. In addition, EBF1-PDGFRß or PAR1 deletions were detected in 5 and 10 cases, respectively. Prolonged MRD was observed in ALL carrying CRLF2^rearr, EPOR^rearr -, PDGFR-ß^rearr and as an unanticipated finding in 8/10 DUX4^rearr patients who mainly underwent hematopoietic stem cell transplantation in first remission (n=5) or had a relapse (n=2). MLPA revealed a non-deleted ERG status in these 8 DUX4^rearr ALL samples. A distinct subgroup of MRD high-risk patients were characterized by a complete lack of IGH-VDJ rearrangements, but they exhibited frequent IKZF1^del (7/10) and a variety of other genomic rearrangements (n=3 EPOR^rearr, n=3 DUX4^rearr, n=1 CRLF2^rearr, n=1 EBF1-PDGFRß or n=1 FOXP1-ABL1). Among gene-fusion positive ALLs, breakpoint- specific real-time quantitative MRD assays at EOI were established, which demonstrated equivalent or superior sensitivity and specificity compared to conventional TR/IG targeted MRD measurements.

Conclusions

The HTS approach presented here yields classical IGH/TRδ-related clonal MRD targets and genomic breakpoints from gene fusions at a time, the latter of which can subsequently serve as robust markers for follow-up monitoring. The MRD high-risk group (≥ 1 x 10^-2 at EOI) revealed an enrichment of aberrant gene fusions/rearrangements in 31 of 68 (46%) patients compared to 19 of 94 (20%) in the moderate-risk MRD group (>1 x 10^-4 < 1 x 10^-2). Rapid capture sequencing in a clinical diagnostics setting allows for specific ABL or JAK-STAT pathway directed early intervention in the majority of gene-fusion positive patients.