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The KMT2A gene encodes a lysine-specific methyltransferase which is essential in epigenetic regulation during early development and hematopoiesis. KMT2A, also known as MLL (mixed-lineage leukemia),...

Introduction: Although survival dependence on Bcl2 is a well-known aspect of the pathophysiology of chronic lymphocytic leukemia (CLL), the mechanisms of Bcl-2 dysregulation are incompletely understood. Recurrent translocations involving BCL2 and immunoglobulin genes, including t(14;18)(q32;q21) and variants such as t(2;18) or t(18;22), are classically observed in follicular lymphoma or germinal center diffuse large B-cell lymphoma (GC DLBCL), but are uncommon ( Methods: Clinically annotated primary samples from BCL2-R CLL patients identified by karyotype were obtained from the French Innovative Leukemia Organization network and Dana-Farber Cancer Institute. Primary samples from CLL without BCL2 rearrangement were used as a control (ctrl CLL). Next generation sequencing (NGS) was performed using a custom-designed panel of 29 genes, including among others: BIRC3, NOTCH1, FBXW7, MLL2, RAS pathway, SF3B1 and TP53. The mean coverage obtained was 2000X (limit of detection (LOD): 1%). Digital droplet PCR (ddPCR) was used to quantify NOTCH1 c.7544_7545delCT (LOD: 0.025%). Protein expression (Bcl2, Mcl1, Bim) was assessed by Western blot. Baseline BH3 profiling was performed as per Ryan et al., Bio Chem 2016. To mimic the lymph node microenvironment, viability assays were performed in co-culture with the stromal cell line NK.tert. Viability was assessed by AnnexinV/Hoechst staining. Ex vivo drug treatments included: BCL2i (inhibitor): venetoclax; MCL-1i: AZD5991, S63845 and BCLXLi: A133. Statistical analyses were by unpaired and paired t-test with a two-tailed nominal p ≤ 0.05 considered as significant. Results: In our cohort of 110 patients, the median age was 70 years old, and 79% were male. BCL2-R were t(14;18) in 77.2%, t(18;22) in 16.3% and t(2;18) in 6.3% of patients. The translocation involving BCL2 gene was isolated in 23.6% of cases, and was associated with trisomy 12 in 45.4% of patients. The most frequently mutated genes in this cohort were in the NOTCH pathway (NOTCH1 mutation: 43.6 %, mostly subclonal (mean of variant allelic frequency: 6.1%) and FBXW7: 4.5%)) and RAS pathway (KRAS, NRAS, BRAF: 9.1%). BCL2 mutations were observed in 22.8% of the 57 examined cases. No mutation previously described in venetoclax resistant CLL, such as F104L or G101V variant, were observed. Furthermore, MLL2 mutations were observed in 14.5% cases and were significantly associated with complex karyotype (p=0.01) and trisomy 12 (p=0.04). Others mutated genes were: BIRC3 (5.4%), TP53 (3.6%), SF3B1 (1%) and MYD88 L265P(1%). No mutations in EZH2, CREBBP or EP300 were found. In 15 CLL representative samples from each group (BCL2-R and ctrl), Bcl2 protein expression was significantly higher in BCL2-R CLL (ratio Bcl2/actin 0.94 vs 0.74, p=0.009) as was expression of the pro-apoptotic protein Bim (ratio Bim/actin: 2.059 vs 1.524, p=0.007). BH3 profiling demonstrated that BCL2-R CLL and ctrl CLL samples (n=23 in each group) had comparable overall priming (cyto-C release 66.1% vs 63.3%, ns) and Bcl-2 dependence (cyto-C release 75.4% vs 76.3%, ns). Both also had low dependence on Bcl-xL (cyto-C release 8.2% vs 8.8%, ns). In contrast, Mcl-1 dependence was found to be significantly lower in BCL2-R CLL (cyto-C release 15.6% vs 37.4%, p Conclusion: The genomic landscape of BCL2-R CLL is characterized by a high frequency of trisomy 12, subclonal NOTCH and RAS pathway mutations, as well as BCL2 and MLL2 mutations. Protein expression, BH3 profiling and viability assays data are consistent with nearly exclusive dependence on Bcl-2. Our data suggest that Bcl-2 inhibition should be favored over Mcl-1 inhibition in BCL2-R CLL. Disclosures Herbaux: Roche: Consultancy, Honoraria, Research Funding. Laribi:abbvie: Honoraria, Research Funding; amgen: Research Funding; novartis: Honoraria, Research Funding; takeda: Research Funding. Ysebaert:Roche: Consultancy; Janssen: Consultancy; AbbVie: Consultancy. Morel:Janssen: Honoraria. Guieze:abbvie: Honoraria, Other: advisory board, travel funds; janssen cilag: Honoraria, Other: advisory board, travel funds; roche: Other: travle funds; gilead: Honoraria, Other: travel funds; astrazanecka: Honoraria, Other: advisory board. Brown:Sun: Research Funding; Acerta: Consultancy; Pharmacyclics: Consultancy; Genentech: Consultancy; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other; Invectys: Membership on an entity's Board of Directors or advisory committees, Other: DSMC; Gilead: Consultancy, Research Funding; BeiGene: Consultancy; Catapult: Consultancy; Dynamo Therapeutics: Consultancy; Eli Lilly and Company: Consultancy; Juno/Celgene: Consultancy; Kite: Consultancy; MEI Pharma: Consultancy; Nextcea: Consultancy; Novartis: Consultancy; Octapharma: Consultancy; Pfizer: Consultancy; Rigel Pharmaceuticals: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Loxo: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Janssen: Honoraria; AbbVie: Consultancy.

Mutations in receptor tyrosine kinase/RAS signaling pathway genes are frequent in core-binding factor (CBF) acute myeloid leukemias (AMLs), but their prognostic relevance is debated. A subset of CBF AML patients harbors several signaling gene mutations. Genotyping of colonies and of relapse samples indicates that these arise in independent clones, thus defining a process of clonal interference (or parallel evolution). Clonal interference is pervasive in cancers, but the mechanisms underlying this process remain unclear, and its prognostic impact remains unknown. We analyzed a cohort of 445 adult and pediatric patients with CBF AML treated with intensive chemotherapy and with deep sequencing of 6 signaling genes (KIT, NRAS, KRAS, FLT3, JAK2, CBL). A total of 152 (34%), 167 (38%), and 126 (28%) patients harbored no, a single, and multiple signaling clones (clonal interference), respectively. Clonal interference of signaling mutations was associated with older age (P = .004) and inv(16) subtype (P = .025) but not with white blood cell count or mutations in chromatin or cohesin genes. The median allele frequency of signaling mutations was 31% in patients with a single clone or clonal interference (P = .14). The repertoire of KIT, FLT3, and NRAS/KRAS variants differed between groups. Clonal interference did not affect complete remission rate or minimal residual disease after 1-2 courses, but it did convey inferior event-free survival (P < 10-4), whereas the presence of a single signaling clone did not (P = .44). This inferior outcome was independent of clinical parameters and of the presence of specific signaling clones. Our results suggest that specific clonal architectures can herald distinct prognoses in AML.

Key Clinical Message Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with poor prognosis that should be investigated in routine practice. Single-nucleotide polymorphism (SNP)-array provides a useful method to detect such cases showing a highly characteristic profile.