High Incidence of RAS Signalling Pathway Mutations in MLL–rearranged Acute Myeloid Leukemia

Chromosomal translocations of the MLL gene on chromosome 11q23 are associated with a unique subset of acute lymphoblastic or acute myeloid leukemias (AML). In adults, MLL rearrangements are detected in 3% of de novo AML and in 10% of therapy-related AML (t-AML) cases and are associated with poor prognosis. In addition to disease defining mutations recent high-throughput sequencing studies had shown that almost all myeloid malignancies accumulate a large number of cooperating gene mutations.

Determination of somatic mutations occurring in cases harboring MLL rearrangements and investigation of the prognostic impact of molecular and additional chromosomal aberrations.

We investigated a cohort of 110 adult AML (80 de novo, 30 t-AML) cases harboring an 11q23 translocation. The cohort was composed of 66 females and 44 males; median age: 55.8 years. MLL translocation partners were as follows: MLLT3 (n=46), MLLT4 (n=15), ELL (n=15); MLLT10 (n=8), others (n=26). Chromosome banding analysis data was available in all cases and survival data in 78 cases (median overall survival (OS) was 10.1 months). Patients were screened for mutations in ASXL1 (n=98), CBL (n=62), CEBPA (n=61), FLT3-ITD (n=103), FLT3-TKD (n=95), IDH1 (n=96), IDH2 (n=84), KRAS (n=107), NPM1 (n=101), NRAS (n=106), PTPN11 (n=99), RUNX1 (n=110), and TP53 (n=110) using amplicon deep-sequencing (454 Roche Life Sciences, Branford, CT), direct Sanger sequencing or melting curve analysis.

Overall, mutations were detected in 59/110 (53.6%) cases. We discovered that 42/110 (38.2%) MLL-translocated AML cases harbored mutations within the RAS signalling pathway (KRAS mut: 23/107; 21.5%; NRAS mut: 22/106; 20.8%; PTPN11 mut: 3/99, 3.0%) or alterations in the RAS regulating FLT3 gene (FLT3-ITD: 4/103, 3.9%, and FLT3-TKD: 10/95, 10.5%). Additional mutations were detected in the tumor suppressor gene TP53 (8/110; 7.3%), ASXL1 (6/98; 6.1%), RUNX1 (4/110; 3.6%), and IDH1 (1/96). No mutation was detected in IDH2, CBL, CEBPA, and NPM1. Most cases showed only one mutation (n=39, 66.1%), whereas 17 cases (28.8%) showed two and 3 cases (5.1%) three mutations in different genes. No difference of mutation distribution was seen between de novo and t-AML. In this cohort, no associations amongst gene mutations were observed, however, FLT3-ITD was associated with MLL-ELL (3/14 vs 1/89, P=0.008) and PTPN11 mutations with MLLT10-MLL (2/8 vs 1/91, P=0.017) alterations. In addition, KRAS mut and NRAS mut correlated with high WBC count (KRAS mut: 103.0±79 vs 59.2±67 x10⁹/L, P=0.016; NRAS mut: 94.7±57 vs 60.4±72 x10⁹/L, P=0.080). Further, we were interested in the prognostic impact of single gene mutations. NRAS mut and TP53 mut showed both a non-significant inferior impact on OS, i.e. OS after 2 years: 19.1% vs 46.4%, P=0.62; 0% vs 41.3%, P=0.114. Further, TP53 mutations were correlated with shorter event-free survival (EFS) (EFS after 2 years: 0% vs 20.0%, P=0.029). No associations with prognosis were observed for the remaining genes and translocation partners.

In contrast, age was associated with OS and EFS (<60 years, n=59 vs ≥60 years, n=51: OS after 2 years: 51.4% vs 26.3%, P=0.003, EFS after 2 years: 28.0% vs 7.7%, P=0.004). Within the cohort of cases ≥60 years, TP53 mutations (n=5) were associated with worse EFS and OS in comparison to TP53 wild-type cases (n=45) (EFS after 2 years: 8.4% vs 0%, P= 0.006; OS after 2 years: 28.5% vs 0%, P=0.045). Of note, no correlations between mutation frequency and age were observed. We next focused on whether the number of mutations showed any impact on survival. This analysis revealed that cases with more than one mutation (n=20) showed shorter EFS (EFS after 2 years: 10.0% vs 27.3%, P=0.020).

Finally, we concentrated on AML with t(9;11)(p22;q23)/MLLT3-MLL, recognized as a distinct WHO-entity. We neither detected an association of MLLT3-MLL (n=46) with OS (P=0.445) or EFS (P=0.644) in comparison to the remaining translocation partners nor a distinct gene mutation profile. However, NRAS mutations correlated with shorter OS and EFS in cases with MLLT3-MLL (after 2 years OS: 17.8% vs 48.3%, P=0.045; after 2 years EFS: 17.8% vs 35.2%, P=0.056).

In patients with MLL-translocations a high number of secondary alterations (53.6%), predominantly in RAS pathway components (38.2%), were detected. This may have implication on novel therapeutic options in this unfavorable AML subset.

Grossmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.