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M. Zaliova et al.
fying lesions. Two parallel European studies demonstrated that ERGdel frequently co-occurs with deletion of the IKZF1 gene (IKZF1del) and attenuates its negative prog- nostic impact.1,3 Subsequent study further investigating the prognostic impact of IKZF1del in a context of addi- tional gene copy number changes identified a genetic pat- tern associated with poor outcome in children treated according to the AIEOP-BFM ALL 2000 protocol.4 This “IKZF1plus” genetic pattern is defined by co-occurrence of the IKZF1del with deletion of CDKN2A, CDKN2B, PAX5 and/or PAR1 region in the absence of ERGdel and will be used to refine risk stratification in the upcoming AIEOP- BFM ALL 2017 trial. The analysis of ERGdel thus becomes a part of the routine diagnostic algorithm in this large international clinical trial.
Several methods with different coverage and sensitivity can be used to detect ERGdel. Single nucleotide polymor- phism (SNP) array can reveal any type of deletion (of min- imal length varying with SNP array density) present in a major clone, representing more than 20-30% of the ana- lyzed sample. Multiplex ligation-dependent probe ampli- fication (MLPA) has similar sensitivity while the coverage depends on the design of the probe in the kits used. Genomic PCR has at least ten times higher sensitivity compared to SNP array/MLPA, while it only detects the most common deletion types which were considered in the PCR primer design. Importantly, the PCR approach was used in all the three aforementioned studies defining the prognostic impact of ERGdel in the context of IKZF1 del1,3 and IKZF1plus.4 Since ERGdel frequently occurs at sub- clonal levels that SNP arrays and MLPA fail to detect due to lower sensitivity,1 the genomic PCR should be used for diagnostics in accordance with these studies and the established definition of IKZF1plus. However, the genomic PCR-based ERGdel detection method used in those stud- ies1,4,5 suffers from at least two disadvantages: 1) due to variable length of amplified region and uneven level of ERGdel-positive clones, the PCR products must be sequence-verified; and 2) strict precautions must be adopt- ed when using Sanger sequencing in order to avoid carry-over contamination of samples, and sequence analy- sis can be complicated in samples with more distinct PCR products.
Based on the frequent subclonality of ERGdel and its instability between diagnosis and relapse, the ERGdel is considered a passenger genetic lesion.1,3 The early ALL gene expression profiling studies showed that it is specific to a subset of B-other ALL with a unique gene expression signature, likely representing a novel biological subtype.6,7 This novel ALL subtype was confirmed and further char- acterized by several recent studies, which also revealed its common genetic background, i.e. rearrangements of the DUX4 gene (DUX4r).7-9 In DUX4r-ALL, the expression of DUX4 (physiologically silent in somatic tissues) is activat- ed by juxtaposition under the control of ectopic regulatory element, most frequently the IGH gene enhancer. The ERG gene was identified among direct DUX4 targets in DUX4r-ALL. It has been demonstrated that DUX4 dereg- ulates ERG gene transcription in a complex manner; it induces expression of non-canonical ERG transcripts, including ERGalt which inhibits wild-type ERG and pro- motes leukemogenesis in mice. It is possible that DUX4 also renders the ERG gene prone to deletions via inducing (epi)chromatin changes.9 The DUX4r-ALL subtype has only recently been defined and for the moment there are
no simple tools for DUX4r detection and DUX4r-ALL clas- sification; thus, there are no data on its prognostic impact. A single American study reported favorable outcome of DUX4r-ALL suggesting that the favorable prognostic impact of ERGdel observed earlier is indeed inherent to this ALL subtype.9 However, ERGdel can only be found in a subset of DUX4r-ALL and its prognostic impact within this subgroup has not been explicitly addressed.
Here we analyzed the presence of ERGdel by SNP array, genomic PCR followed by Sanger sequencing, and by a newly-designed deep amplicon sequencing proce- dure (AmpliSeq). We aimed to determine to what extent the different sensitivity of methods impacts ERGdel detection and classification into IKZF1plus category. Next, we wanted to assess whether ERGdel can be detected in a significantly higher proportion of DUX4r-positive patients using a potentially more sensitive method and to what extent positivity could serve as a surrogate marker for the DUX4r-ALL classification. Moreover, we wanted to elucidate whether ERGdel-positive DUX4r- ALL differs from ERGdel-negative DUX4r-ALL, and whether the possible differences depend on the method used to detect ERGdel. Finally, use of AmpliSeq enabled us to study in detail also the repertoire and clonality of ERGdel in order to better understand its origin during leukemia clone evolution.
Methods
Patients and samples
The study analyzed diagnostic and remission bone marrow or peripheral blood samples from 118 children (aged 1-18 years) diag- nosed with B-other ALL (negative for ETV6/RUNX1, TCF3/PBX1, BCR/ABL1, KMT2A-rearrangements, hyperdiploidy, hypodiploidy) and treated in the Czech Republic between August 1998 and July 2017 according to the BFM ALL protocols. Patients were selected according to the availability of the biological mate- rial and/or of already existing genomic data. A retrospective part of the cohort (treated August 1998-July 2010; n=30) was enriched for patients presenting with immunophenotypic features shown to be associated with ERGdel (CD2-positivity, immunophenotyp- ic switch).10 The remaining 88 patients represent 84% of all con- secutively diagnosed and prospectively analyzed B-other patients treated according to the AIEOP BFM ALL 2009 protocol (consecu- tive sub-cohort; August 2010 – July 2017). The study was approved by the Institutional Review Board of the University Hospital Motol and informed consent was obtained in accordance with the Declaration of Helsinki.
Genomic polymerase chain reaction to detect ERG (ETS transcription factor) deletions (ERGdel)
The presence of ERGdel was analyzed by multiplex PCR, as described previously.3 Two more primers corresponding to addi- tional centromeric breakpoint sites1 were added: 5’-GCGGC- TACTTGTTGGTCCAAGAA-3’ and 5’-CTATCCTGAA- CATTGCTGCCAG-3’. PCR products were analyzed on agarose gel; positive samples were sequenced by Sanger method.
Single nucleotide polymorphism array
Copy number aberrations (CNA) and regions of uniparental dis- omy (UPD) were analyzed in 104 patients using HumanOmni Express BeadChip (Illumina, San Diego, CA, USA) or CytoScan HD arrays (Affymetrix, Santa Clara, CA, USA). For six patients, the results from the analysis on GeneChip Mapping 250K Nsp and
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haematologica | 2019; 104(7)