A Novel Multiplex NGS-Based Digital MLPA Assay for Copy Number Detection of 700 Target Sequences in Childhood Acute Lymphoblastic Leukemia

Background: Recurrent and clonal genetic alterations are characteristic of different subtypes of pediatric acute lymphoblastic leukemia (ALL) and several of them are strong independent predictors of patient outcome. Multiplex Ligation-dependent Probe Amplification (MLPA) has become one of the standard methods for detection of common copy number alterations (CNAs), including IKZF1 deletions, which are associated with a poor outcome. However, standard MLPA assays only accommodate a maximum of 60 MLPA probes. In order to increase the number of target sequences in one assay, a next generation sequencing (NGS)-based MLPA variant has been developed. This digitalMLPA assay is based on the well-known MLPA procedure but can include up to 1000 probes in a single reaction, uses minute amounts of DNA (≥20ng) and can be analysed on all Illumina NGS platforms.

Methods: A digitalMLPA assay was designed and optimised to detect CNAs of 55 key target genes and regions in childhood ALL, including deletions of B-cell differentiation and cell cycle control genes (PAX5, IKZF1/2/3, EBF1, RB1, CDKN2A/B, ETV6, and BTG1), iAMP21 and rearrangements of the PAR1 region, T-cell ALL associated aberrations (STIL-TAL1, LEF1, CASP8AP2, MYB, EZH2, MLLT3, NUP214-ABL1, PTEN, LMO1/2, NF1, SUZ12, PTPN2, PHF6), ERG and TP53 deletions. Several genetic alterations of potential prognostic and/or therapeutic relevance in ALL have also been included (NOTCH1, CD200/BTLA, VPREB1, TBL1XR1, PDGFRB-EBF1, IGHM, NR3C1/2, CREBBP, CTCF, ADD3, EPHA1, FHIT, SPRED1 and TOX). All target genes are covered by at least 3 digitalMLPA probes (~450 probes in total). Moreover, a set of 200 probes was designed for genome-wide detection of gross ploidy changes (high hyper- or hypodiploidy) and to determine the extent of CNAs, while also acting as reference probes for data normalization. Performance of all probes has been extensively tested on genomic DNA from healthy individuals and positive cell lines. Only probes with a standard deviation <0.12 were included. All probes were also tested under various experimental conditions, such as different salt, probe and polymerase concentrations. A series of 76 pediatric ALL patient samples (including both B-ALL and T-ALL samples), previously characterized for specific genetic aberrations by array CGH (aCGH) and/or SNP array, has been analysed single-blinded using the digitalMLPA assay. Results were compared to those obtained from standard MLPA assays (P335 ALL-IKZF1 and P327 iAMP21-ERG), which contain probes with different ligation sites for the respective genes. A dilution series of three B-ALL patient samples was analysed to determine the detection limit for subclonal aberrations.

Results: All aberrations previously identified by other methods in 67 pediatric ALL samples, were also identified using the digitalMLPA assay. These included whole chromosome gains and losses, whole gene deletions or gains, iAMP21, fusion genes (STIL-TAL1, NUP214-ABL1 and PDGFRB-EBF1) and intragenic gene deletions (IKZF1, ERG, CDKN2A/B, ETV6, PAX5 RUNX1, RB1, LEF1, NR3C1, RAG2, VPREB1, MLLT3, BTG1 and PTEN). Of interest was a case of high hyperdiploidy, which was correctly identified by digitalMLPA, while by aCGH analysis this case was misinterpreted as having multiple deletions. Among several other intragenic deletions, a heterozygous deletion of ERG exons 5-9 was observed in one patient. In addition, several cases of homozygous single-exon deletions were observed (e.g. IKZF1 exon 8, LEF1exon 3) and confirmed by aCGH. These findings should be further investigated with respect to their clinical impact. Results from the dilution series indicated that subclonal aberrations can be reliably detected by this digitalMLPA assay if present in at least 20- 30% of tumour cells.

Conclusions: This study demonstrates that digitalMLPA is a reliable technique that can be used to genetically characterize clinical samples of ALL patients. Experiments can be performed on small amounts of DNA in a high-throughput and cost-effective fashion. Because of the targeted approach, data analysis will be much easier as compared to array or other sequencing platforms. These results merit further consideration of (digital)MLPA as a valuable alternative for genetic work-up of newly diagnosed ALL patients.