Ph-like and IKZF1plus Features in Children with Down Syndrome and B Cell Precursor Acute Lymphoblastic Leukemia

Children with Down Syndrome have an augmented risk for B-cell acute lymphoblastic leukemia (DS-ALL), which is associated with an inferior outcome compared with non-DS ALL due to increased chemotherapy-related toxicity and excess of relapses, thus demanding tailored therapeutic strategies. DS-ALL exhibits distinct somatic features from non-DS ALL: approximately 60% of DS-ALL patients have aberrant expression of CRLF2, frequently associated with JAK2 mutations, resulting in a higher incidence of the Philadelphia-like (Ph-like) transcription signature, and approximately 30% carry IKZF1 deletions. In non-DS patients treated in the AIEOP-BFM ALL 2000 trial, the feature "IKZF1plus", defined as the co-occurrence of IKZF1 deletion with deletions in CDKN2A, CDKN2B, PAX5 or PAR1 in the absence of ERG deletion, has been described as a Minimal Residual Disease (MRD)-dependent very-poor prognostic profile in childhood BCP-ALL. IKZF1plus status has been incorporated in the new stratification strategy and experimental treatment approaches in the ongoing AIEOP-BFM ALL 2017 trial, while patients carrying ABL-class fusion genes, characteristic of the Ph-like signature, are eligible to the current amended EsPhALL 2017 protocol. However, no study has yet addressed the prognostic relevance of these two features in DS-ALL patients. Aim of this study was to evaluate the incidence and the prognostic value of the Ph-like profile with its recurrent fusion events, and of the IKZF1plus feature in a large cohort of children with DS-ALL treated in Italy or Germany in AIEOP-BFM ALL protocols. Seventy DS-ALL patients treated in Italian centres from 2000 to 2014 were evaluated for their cytogenetic status, including the Ph-like ALL profile, while the IKZF1plus feature was investigated in a larger cohort of 134 patients treated in Italian and German centres from 2000 to 2011. We confirmed that DS-ALL is a heterogeneous disease, characterized by many genetic profiles, similar to those observed in non-DS ALL, but with a different subtype distribution. Forty-six out of 70 (65.7%) AIEOP DS-ALL patients displayed the Ph-like ALL gene expression signature, mostly characterized by CRLF2 and IKZF1 alterations. Importantly, Cumulative Incidence of Relapse (CIR) analyses showed that the Ph-like feature had a negative prognostic impact also in DS-ALL (27.7%±6.8 vs. 13%±7; p=0.04). It is also noteworthy that most of the Ph-like cases were not at high risk based on conventional stratification criteria (82.6%). Analyzing patients without CRLF2 overexpression, we identified fusion genes involving PAX5 or kinases (ABL1 and NTRK3) in two out of 18 Ph-like patients and in two out of 8 patients for whom no GEP data were available. Notably, these patients remain in continuous complete remission; however, due to the small number, the prognostic value cannot be assessed. In the Italian and German joint cohort, we observed 35.6% patients positive for P2RY8::CRLF2 fusion, 24.8% for IKZF1 deletion and 18% for IKZF1plus feature. In accordance with previous reports, we observed that while P2RY8::CRLF2 was not significantly associated to a different outcome, IKZF1 deletion was associated with an inferior event-free survival (EFS, 42.9%±8.9 vs. 72.7%±4.6; p<0.001) and with a higher CIR (35.2%±8.6 vs. 17%±3.9; p=0.007). Importantly, this study suggests that IKZF1plus is the feature associated with the most negative prognosis, especially when determined by the combination of IKZF1 deletion and P2RY8::CRLF2 fusion (13/15 had an adverse event: relapse or treatment-related death). Performing an ex-vivo drug screening with 174 drugs in early to late clinical trials on blasts of 3 IKZF1plus DS-ALL patients and on 14 controls (5 B-cell lymphoblastoid cell lines, 3 PBMCs, 3 T-cells and 3 CD34+ cells, all derived from healthy donors) we observed the efficacy of drugs known to be effective in Ph-like patients such as Birinapant, a SMAC mimetic, and HDAC inhibitors. In conclusion, we reported here in a large cohort of DS-ALL that only few Ph-like cases were positive for gene fusions involving PAX5 or kinases. The IKZF1plus feature in DS-ALL was 3 times more frequent than in non-DS ALL. Ph-like signature and IKZF1 deletions were associated with poor outcome, with the risk of relapse further increased for IKZF1plus patients. These alterations characterize subgroups of DS-ALL patients who need tailored therapeutic strategies.