Recurrent Genetic Fusions Redefine MLL-Germline Acute Lymphoblastic Leukemia in Infants

Introduction. B-cell-precursor Acute Lymphoblastic Leukemia (BCP-ALL) occurring in children < 1 year old is a rare and very aggressive disease, associated with the presence of MLL/KMT2A rearrangements in 80% of cases. The MLL rearrangement (MLL-R) originates prenatally in utero and plays a role in transcription factor dysregulation. Infant MLL-R ALL is typically associated with dismal outcome. On the contrary, infant MLL germline (MLL-G) ALL, treated with current intensive therapies, may have a relatively favorable prognosis (6-year Event Free Survival (EFS) of 73.9 % in the Interfant-06 study). Recent studies showed that NUTM1 gene is frequently involved in fusions with different partner genes in infant MLL-G ALL conferring a favorable prognosis. The present study aims at characterizing the presence and prognostic role of fusion genes in infants with MLL-G BCP-ALL, treated in Italy with Interfant or AIEOP-BFM ALL protocols.

Methods. We applied a custom RNA targeted NGS panel, named Ovation Fusion Custom Panel Target Enrichment System (Nugen/Tecan), with probes recognizing 95 leukemia-related genes to detect fusion transcripts, starting from a low amount of total RNA (ranging from 10 to 200 ng). In a subset of cases, whole transcriptome analysis was performed. The fusion genes were identified by both the STAR-Fusion and an in-house bioinformatics pipeline. MLL rearrangements were assessed by FISH with a MLL split signal and/or multiplex RTPCR for the most recurrent t(4;11), t(9;11) and t(11;19) fusion transcripts.

Results. Among 37 consecutive infants with MLL-G BCP-ALL, 30 had material available and were successfully screened; 29 were enrolled in the Interfant protocols and 1 in the current AIEOP-BFM ALL 2017 protocol. Strikingly, 22/30 (73%) carried a fusion gene, whereas 8 resulted negative by both RNA targeted panel and whole transcriptome RNA-seq. A NUTM1 fusion was identified in 9/30 cases (30%), with Acin1 (n=5), Cux1 (n=2), ZNF618 (n=1) or BRD9 (n=1) as fusion partner. Remarkably, 6/30 cases (20%) had a PAX5-rearrangement with several partner genes, such as DNAJA1 (n=3), FBRSL1 (n=1), MBNL1 (n=1) and GRHPR (n=1). Moreover, we identified other fusion genes in 7 patients: TCF3/PBX1 (n=2), TCF3/ZNF384 (n=1), ETV6/ABL1 (n=1), P2RY8/CRLF2 (n=1), and a new KDM2B/GATAD2B fusion in a pair of monozygotic Infant twins.

MLL-G ALL patients presented with favorable prognostic features: 24/30 were older than 6 months, 29/30 had WBC count < 300 x 109/L at onset and 28/30 had CD10 positive immunophenotype. In addition, 26/30 cases were prednisone good responders, and all achieved complete remission at the end of the induction phase. In 21 patients with available data, at the end of induction, MRD was negative in 7, <5x10-4 in 11, and ≥ 5x10-4 in only 3 patients (2 with a PAX5 fusion and one carrying TCF3/ZNF384). In 19 patients with available data, at the end of Consolidation Phase IB, MRD was high (≥ 5x10-4) only in one patient, carrying a PAX5 rearrangement.

Outcome analysis revealed that in 9 patients with NUTM1 fusions (mean age at diagnosis 6.6 months) no events occurred (3-year EFS was 100%), in keeping with data from an ongoing collaborative IBFM project (J. Boer, personal communication). Conversely in the 6 patients with PAX5-rearrangements (mean age at diagnosis 11.0 months), 4 events occurred (3-year EFS 25.0% ±20.4). The 3-year EFS of the remaining patients was 57.1% ±18.7 for those with other fusion genes (N=7) and 83.3% ±15.2 for those with no fusions (N=8).

Conclusion. This study shows that infant MLL-G BCP-ALL is associated with a marked genetic heterogeneity. For the first time, an unexpectedly high rate of fusion genes was detected, with specific response and outcome. We found that PAX5 fusions are recurrent and associated with a worse outcome compared to the NUTM1-rearranged class. Of note, we previously demonstrated with in vitro and in vivo experiments using cells from older children that specific treatments (i.e. Nintedanib) could be of benefit for PAX5 fusion-positive cases.

If confirmed in a larger cohort, these data would support the redefinition of the infant ‘MLL-germline’ ALL subgroup, where specific genetic features might have a prominent biological and prognostic role, with a rationale for risk stratification and potential benefit from genetically-driven treatments.