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CD371 cell surface expression: a unique feature of DUX4-rearranged acute lymphoblastic leukemia

B-cell precursor acute lymphoblastic leukemia (B-ALL), the most common childhood malignancy, comprises genetically, biologically and clinically heterogeneous disease entities. Given the concepts of risk-adapted or targeted therapies, their precise delineation is becoming increasingly important. The recently discovered IGHDUX4 or, less commonly, ERG-DUX4 rearranged subtype of B-ALL accounts for 5-7% of the cases and is characterized by the expression of C-terminally truncated DUX4 isoforms, a highly distinctive gene expression signature and a profound deregulation of ERG. Somatic ERG deletions, which occur in roughly half of DUX4 patients, are indicative of a favorable outcome and attenuate the negative prognostic effect of adverse factors such as IKZF1 deletions alone or in combination with other deletion events. Given that ERG deletions are secondary events driven by the overexpression of DUX4, they delineate only a subset of DUX4 cases and it remains to be determined whether only those patients with ERG deletions or the entire cohort of DUX4 patients has a superior outcome. However, a reliable identification of DUX4 leukemia currently requires gene expression profiling or next-generation sequencing approaches,which are not yet feasible either for large-scale screening studies or in a diagnostic setting for many study centers. Herein we show that expression of the cell surface antigen CD371 (CLL-1), encoded by CLEC12A and easily detectable by flow cytometry, is a unique feature of DUX4-rearranged B-ALL and identifies virtually all DUX4 cases. Our evaluation of the immunophenotype of DUX4 leukemia provides solid evidence that CD371 cell surface expression is a highly specific surrogate marker to identify this otherwise difficult to ascertain genetic subgroup. This notion is based on the finding that of 46 DUX4 cases, 42 showed strong and three weak CD371 antigen expression (Table 1), while all other genetic subtypes were basically negative (Online Supplementary Tables S13). In order to detect DUX4 cases we performed RNAsequencing of two independent cohorts of patients with childhood or young adolescent leukemia (Online Supplementary Methods; Online Supplementary Tables S1 and S2). The Austrian cohort (cohort 1) consisted of 101 bone marrow samples from 92 patients (n=80 diagnostic, n=3 relapse samples, n=9 diagnosis/relapse matched pairs). It comprised mostly B-other cases (n=65) lacking sentinel genetic alterations as well as representative cases of all major genetic subtypes (Online Supplementary Table S1). Initially, most samples were subjected to RNAsequencing without prior knowledge of their immunophenotypic details, but later on nine more CD371 samples with unknown genetic subtype and available material were sequenced. A second cohort (cohort 2) from the Czech Republic consisted of 55 primarily B-other cases, analyzed in parallel for CD371 expression (Online Supplementary Table S2). DUX4 samples were identified by the analysis of DUX4 expression levels (Figure 1A), the presence of DUX4 fusion transcripts (Online Supplementary Tables S1 and S2) and their distinctive gene expression profile (Online Supplementary Methods; Online Supplementary Figure S1). Of 48 cases classified as DUX4, 44 harbored IGH-DUX4 and one a DUX4-ZNF384 fusion, while three lacked any DUX4 fusion (Online Supplementary Tables S1 and S2); but all displayed the gene expression signature typical of DUX4 leukemia (Online Supplementary Figure S1). As determined by single nucleotide polymorphism array analysis (Online Supplementary Methods) and genomic multiplex polymerase chain reaction, 32% (15/47) and 65% (13/20) of the patients, respectively, showed ERG deletions (Online Supplementary Tables S1 and S2; Online Supplementary Figure S2), confirming that with either method they are detectable only in a subset of DUX4 cases. About one-third of DUX4 cases displayed IKZF1 deletions (Online Supplementary Methods; Online Supplementary Tables S1 and S2; Online Supplementary Figure S2), which is comparable to the frequency detected in ERG-deleted leukemia. While in the ALL-BFM 2000 clinical trial ERG-deleted patients were more frequently allocated to the intermediate-risk group and had a favorable outcome, in the ALLBFM 2009 study, as a consequence of more refined minimal residual disease stratification, DUX4 patients were more commonly treated in the high-risk arm (Online Supplementary Tables S1 and S2). However, to date, the follow-up of the latter cohort of patients is still too short to draw any conclusions on whether a poor initial treatment response in DUX4 leukemia eventually results in a