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ERG deletion in BCP-ALL
lytical tools for these data can be expected in the near future, which would further facilitate the diagnostic use of ERGdel AmpliSeq.
Although we have sequenced a proportion of patients with very high coverage to be able to analyze the ERGdel repertoire in depth, the coverage used for diagnostic pur- poses can be substantially reduced without a significant increase in false negativity. Even with the reduced cover- age, our data show slightly higher sensitivity of AmpliSeq over the PCR. However, the discordance between PCR and AmpliSeq is significantly lower compared to the dis- cordance between SNP array and PCR, and, importantly, our data suggest that biological and clinical characteristics of the PCR-defined and AmpliSeq-defined ERGdel groups are very similar.
Previous studies have shown the rarity of ERGdel in non-B-other BCP-ALL. In the present study we did not find any ERGdel-positive patient within non-DUX4r B-other ALL; thus, ERGdel was 100% specific for the DUX4r-ALL subtype. However, even with the use of AmpliSeq at high coverage, approximately 1 in 5 to 1 in 4 of DUX4r-ALL remained ERGdel negative, confirming that ERGdel cannot serve as a marker for DUX4r-ALL clas- sification. Similarly, we confirmed that nor do ERGalt transcripts represent a reliable surrogate marker for DUX4r ALL; they can be present in non-DUX4r ALL and, moreover, they are absent or expressed at low levels in a proportion of DUX4r ALL, and are undistinguishable from non-DUX4r ALL. Interestingly, higher levels of ERGalt were associated with the presence of PCR-defined ERGdel within DUX4r ALL in the present study. Since the ERGdel- negative DUX4r ALL cases in particular had zero or low levels of ERGalt, even simultaneous detection of ERGdel and ERGalt does not allow the DUX4 ALL subtype to be reliably identified.
Our study revealed additional biological and clinical dif- ferences between ERGdel positive and negative patients within the DUX4r-ALL. We found significantly more CNA in ERGdel-positive patients. Although we did not find any difference in expression of DUX4 and RAG1/2 at mRNA level, we believe that a higher CNA number may still reflect a higher rate of illegitimate V-(D)-J recombina- tion in ERGdel-positive patients. Moreover, our data show significantly better outcome of ERGdel-positive compared to ERGdel-negative DUX4r-ALL patients defined by PCR- based techniques. Although this finding needs to be vali- dated by further studies, it suggests that ERGdel might have additional value for outcome prediction than has been described so far.
A potential driving role for ERGdel in leukemogenesis
has remained controversial until recently. Originally, it
was thought that the ERGdel resulted in the expression of
aberrant ERG protein with a potential dominant negative
impact over wild-type ERG.6 However, our previous study
demonstrated that this protein is not expressed from the
leukemic samples has only rarely been reported so far. Moreover, as the aberrant ERG protein encoded by the intact ERG allele has a driver role in DUX4r-ALL,9 the bial- lelic ERGdel would be biologically disadvantageous. This is strongly supported by findings from in vitro study where silencing of ERG in a DUX4r-ALL NALM-6 cell line with
Figure 6. ERGdel patterns. Three different patterns of ERGdel are depicted. (Sub)clones with distinct ERGdel are represented by different colors; cells with- out ERGdel are shown in gray. Gain of ERGdel in leukemia-founding cell resulting in clonal pattern is probably extremely rare, if present at all. In acute lymphoblas- tic leukemia with a pseudoclonal pattern, only a single ERGdel gained by prog- eny of leukemia-founding cell is detected. However, an additional distinct sub- clonal ERGdel may co-exist at the level below sensitivity of the detection meth- ods. Our data suggest that the most frequent ERGdel pattern is polyclonal, where multiple distinct ERGdel subclones co-exist, and based on the total pro- portion of ERGdel-positive cells, the ERGdel can be detected by single nucleotide polymorphism array/polymerase chain reaction (PCR) or by PCR only. Patterns were visualized using fishplot package for R.
ERGdel allele15 and Zhang et al. have shown recently that 9
it is indeed encoded by ERGalt transcript. These findings further strengthen the likely passenger role of ERGdel. Our current study shows that ERGdel is independently acquired in multiple members of leukemic cell popula- tions and could just represent collateral DNA damage resulting from continuous DUX4-induced exposure of the ERG gene locus. Our interpretation of ERGdel clonality presumes that this deletion is predominantly monoclonal at the single cell level. Biallelic ERG deletion in bulk
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