The Molecular Landscape of KMT2A-Rearranged Leukemia from Infancy to Adulthood Reveals Age and Leukemia-Specific Mutational Patterns
Authors: Pilheden Mattias , Falqués Ton , Ahlgren Louise , Sturesson Helena , Walsh Michael P , Song Guangchun , Castor Anders , Pronk Cornelis , Marquart Hanne Vibeke , Lausen Birgitte , Barbany Gisela , Pokrovskaja Tamm Katja , Norén-Nyström Ullrika , Fogelstrand Linda , Lohi Olli , Hyrenius Wittsten Axel , Zhang Jinghui , Downing James R. , Ma Jing , Hagstroem-Andersson Anna 
Published: November 2021

Abstract
Genetic rearrangements involving the KMT2A gene (KMT2A-R) are seen in around 10% of acute leukemia overall. KMT2A-R occurs in all ages and usually correlates with high-risk clinical features, in particular in infants aged 0-12 months of age with acute lymphoblastic leukemia (ALL). To uncover age- and leukemia-subtype specific molecular patterns in KMT2A-R ALL and acute myeloid leukemia (AML), we performed whole genome (WGS), whole exome (WES), and RNA-sequencing on a well-annotated Nordic KMT2A-R cohort of 104 patients, including infant ALL (n=33), childhood ALL (n=18), adult ALL (n=15), and pediatric AML (n=38) patients. For 77 patients, we performed WGS (40x) at diagnosis and remission as well as WES (140x) on the diagnostic sample, and remaining patients underwent WES only (n=27). RNA-sequencing was performed on 58 cases with available RNA.
Twenty-two genes were recurrently altered and remarkably, NRAS, KRAS, FLT3, PAX5, TP53, CDKN2A/B and IKZF1 accounted for 70% of mutations. The landscape of mutations suggested the presence of leukemia and age-specific associations with MYST4, PTPN11, and SETD2 uniquely altered in AML and PIK3CD, DNAH11, NOTCH1, CSMD3 and CDKN2A/B in ALL. Some genes were mutated in both KMT2A-R ALL and AML, but were more common in one disease, such as FLT3 and KRAS in AML and PAX5, TP53 and IKZF1 in ALL. Moreover, age-associated patterns were seen in ALL with NRAS more frequently mutated than KRAS in infant ALL (26% vs 15%), and KRAS more frequently mutated than NRAS in childhood ALL (24% vs 18%), with adult ALL having fewer such mutations (NRAS 13%; KRAS 7%). Alterations of CDKN2A/B and TP53 were absent in infant ALL, detected in childhood and adult ALL only. PAX5 alterations were primarily detected in childhood ALL (22%, 9% infant ALL, 7% adult ALL), with all three PAX5-altered infant cases having the KMT2A-MLLT3 fusion gene. Finally, KMT2A-R pediatric AML had the highest fraction of FLT3 mutations (24%, 9% infant ALL, 11% childhood ALL, 0% adult ALL) and all but one mutation occurred in KMT2A-MLLT3 rearranged cases and most were kinase domain point mutations.
We next expanded our analysis to include non-recurrent alterations. PI3K/RAS pathway alterations were detected across ages and subtypes with the highest fraction in pediatric AML (63%) and the lowest in adult ALL (27%, 43% infant ALL, 41% childhood ALL). Further, cell cycle related genes were primarily mutated in childhood (39%) and adult ALL cases (33%) and rarer in infant ALL (12%) and pediatric AML (16%) and genes within the B-cell pathway were more commonly altered in childhood ALL (29%) than in infant ALL (9%). Finally, in line with our previous study (Andersson et al, Nat Genet 2015) epigenetic mutations were absent in infant ALL, but present in 20-35% of the other patients.
RNA-sequencing identified the KMT2A-fusion in 56/58 cases, with low exonic coverage preventing detection of the fusion in two cases. The reciprocal KMT2A fusion was only expressed in 13/39 cases where it was predicted to be expressed based on karyotype or whole genome sequencing data with 11/13 cases having the KMT2A-AFF1 fusion gene. In addition, RNA-sequencing identified 6 in-frame and 12 out-of-frame fusion genes that had formed either as part of the KMT2A-R itself or that were independent genetic events. Further, a novel in-frame KMT2A-ACIN1 fusion was identified in a child aged 1 year with B-precursor ALL. ACIN1 encodes Apoptotic Chromatin Condensation Inducer 1 and the fusion was formed through an insertion of 14q11 into 11q23. ACIN1 is also rearranged as part of the ACIN1-NUTM1 that we identified in an infant with ins(15;14)(q22;q11.2q32.1) (Andersson et al Nat Genet 2015). To study the ability of KMT2A-ACIN1 to induce leukemia in mice, we injected retrovirally transduced mouse bone marrow cells containing the fusion into syngeneic mice and KMT2A-MLLT3 was used as control. All mice succumbed to disease at an average of 112 days for KMT2A-ACIN1 (n=12) and 63 days for KMT2A-MLLT3 (n=5) and mice displayed splenomegaly and leukocytosis with an immunophenotype indicative of AML. Primary leukemia cells isolated from moribund mice gave rise to leukemia in sublethally irradiated recipients with reduced disease latency. In conclusion, these results highlight the differential molecular patterns in KMT2A-R leukemia across infancy to adulthood thereby providing novel pathogenetic insight.

Disclosures
No relevant conflicts of interest to declare.