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M. Zaliova et al.
IKZF1plus-positive patients in the present study were MRD-positive at day 33 and thus predicted to have a high- er risk of relapse.
The frequency and distribution of kinase aberrations is shown in Figure 4. Kinase aberrations that can be grouped into JAK/STAT-class were found in 23 patients who har- bored lesions of CRLF2 (n=21), JAK2 (n=7), JAK1 (n=6), IL7R (n=2), and SH2B3 (n=1). The most common aberra- tions were CRLF2 gene rearrangements which had previ- ously been shown to frequently occur at subclonal level.42 Accordingly, in addition to 12 P2RY8-CRLF2 and four IGH- CRLF2 fusions affecting a major leukemic clone (CRLF2r, n=16), P2RY8-CRLF2 fusions affecting a minor leukemic clone (P2RY8-CRLF2-low) were detected by RT-PCR in ten patients. The CRLF2r always resulted in sur- face CRLF2 positivity assessed by flow cytometry, was identified by RNA-sequencing, in the case of P2RY8-CRLF2 evidenced by PAR1 deletion on SNPa and was frequently accompanied by the activating CRLF2 mutation F232C (5 out of 16 cases) or by JAK1/2 mutation (6 out of 16 cases). On the other hand, all patients with P2RY8-CRLF2-low were negative for fusion transcript and PAR1 region dele- tion by RNA-sequencing and SNPa, respectively, and all but one were negative for surface CRLF2 by flow cytometry (a small CRLF2-positive subclone was reported in one patient). qPCR showed a clear separation (>1 log distance) of the two groups of P2RY8-CLRF2-positive patients (Online Supplementary Figure S8). While CRLF2r (and JAK/STAT-class aberrations in general) were significantly associated with a BCR-ABL1-like subtype, P2RY8-CRLF2- low occurred exclusively in the B-rest ALL subset (Table 2).
In the total B-other cohort, we found only one ABL-class aberration. The ETV6-ABL1 fusion was found in a BCR- ABL1-like ALL patient and was accompanied by another two in-frame fusions resulting from a single chromosomal rearrangement that we recently characterized in a separate report.43
Aberrations activating RAS/RAF-MAPK signaling (aber- rations of NRAS, KRAS, FLT3, BRAF, PTPN11, NF1) which may not be considered as suitable therapeutic targets because of their frequent subclonality, were found in 47 patients spread across all ALL subtypes.
Importantly, 50 out of 51 kinase aberrations detected by WES were also detected by RNA-sequencing.
Discussion
Although several large genomic studies have been pub- lished over the last few years, these were mostly based on non-consecutive cohorts of patients.4,6,7,21 Here, we present the first European population-based genomic study of pediatric B-other ALL based on a consecutive cohort of uniformly treated patients with a 100% inclusion rate. The only other consecutive study, the Swedish study by Lilljebjörn et al.,2 based on 54 B-other ALLs (considering the B-other definition used herein) with an inclusion rate of 69%, showed a similarly high proportion of the “B-rest” ALL (40-45%) and similarly infrequent ZNF384r and MEF2Dr ALL subgroups. Although the frequency of the remaining three subtypes differs between both studies (ETV6-RUNX1-like ALL 5% vs. 11%, DUX4r ALL 27% vs. 15%, BCR-ABL1-like ALL 15% vs. 28%), these differ- ences are not statistically significant. Moreover, in the case of BCR-ABL1-like ALL, the difference might be influenced
by heterogeneity in this subtype definition, which repre- sents a broader phenomenon12 and reflects the low speci- ficity of a BCR-ABL1-positive/-like gene expression signa- ture.
Despite the limitations of our analysis of clinical param- eters imposed by the small size of the individual subtypes, we do present two important observations. First, we show a strong association of ZNF384r with mixed, B-myeloid immunophenotype. Although this association had already been established,5,44-46 here we demonstrate that ZNF384r ALL represents the majority of all consecu- tively diagnosed non-infant pediatric B-other B-myeloid MPALs (4 out of 7; 57%). Second, we show a significant association of DUX4r ALL with worse early response to therapy. This ALL subtype is now often considered equiv- alent to ERGdel-positive ALL. However, an important pro- portion of DUX4r ALL lacks the ERGdel. We have shown previously that despite the association of ERGdel with worse early therapy response, this did not translate into worse outcome in the MRD-based BFM ALL 2000 proto- col.31 Although two recent studies reported a favorable outcome of DUX4r ALL,2,4 its potential heterogeneity with respect to ERGdel has still not been studied. Thus, the out- come of DUX4r ALL in BFM studies, both in general and stratified by ERGdel, still remains to be addressed.
Previous studies demonstrated the frequent occurrence and remarkable diversity of PAX5 aberrations in child- hood BCP ALL.9,47-49 We focused here on three types of PAX5 aberrations that, in our study, were mutually exclu- sive with established ALL subtypes and altogether repre- sented a large proportion of B-other ALL (20%). Moreover, we suggest here that one of them, the PAX5 P80R mutation, likely represents a primary lesion that defines a novel biological ALL subtype, whose existence and character, however, remain to be confirmed and defined in detail by future studies. Among PAX5 fusions, we identified four novel PAX5 fusion partners further broadening an already sizeable PAX5 recombinome.50 Although the PAX5 fusions in our study lacked a specific gene expression signature, which questions their potential to define a unique biological subset, with respect to their frequency (which exceeds that of some already estab- lished subtypes) future efforts to evaluate their clinical impact are to be expected. Interestingly, the patients with PAX5 fusions included in our study had a significantly bet- ter early treatment response compared to the other patients; none was stratified as high risk (Online Supplementary Table S10).
IKZF1plus genotype has been recently described to confer poor outcome (30-40% 5-year event-free survival) in non- standard risk patients on the ALL BFM 2000 study.17 We have identified 14 IKZF1plus-positive patients, representing 14% of B-other ALLs. Because BCR-ABL1-like ALL is enriched for IKZF1del and PAR1 deletion (included in the IKZF1plus definition), it has been assumed that IKZF1plus and BCR-ABL1-like cases may largely overlap, although this has still not been demonstrated. In our study, 57% of patients with the IKZF1plus genotype were classified as BCR-ABL1-like. Thus, although IKZF1plus was significantly enriched in BCR-ABL1-like ALL, a large proportion of IKZF1plus-positive patients were still not classified into this subtype. Only 2 out of 14 IKZF1plus-positive ALLs were stratified into standard risk, both belonging to the B-rest subset. Larger cohorts will be needed to determine whether a refined ALL subtype classification could help to
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haematologica | 2019; 104(7)