Landscape of Secondary Genetic Lesions in Acute Myeloid Leukemia with Inv(16)/CBFB-MYH11

Introduction: Acute myeloid leukemia (AML) with inv(16)(p13q22) or t(16;16)(p13;q22) accounts for 5-7% of adult AML and overall is associated with a favorable outcome. However, secondary genetic lesions have been shown to negatively impact on outcome.

Aims: To assess the frequency and clinical impact of additional mutations and chromosomal aberrations in AML with inv(16)/CBFB-MYH11.

Patients: We analyzed 138 patients (pts) who were referred to our laboratory for diagnosis of de novo AML between 2005 and 2014 (54 females; 84 males; median age 54 years, range: 20-88 years). All patients were proven to have inv(16)(p13q22) or t(16;16)(p13;q22) /CBFB-MYH11 by a combination of chromosome banding analysis, fluorescence in situ hybridization and RT-PCR. All 138 samples were analyzed by next generation sequencing using a 22-gene panel targeting ASXL1, CBL, DNMT3A, ETV6, EZH2, FLT3-TKD, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, RAD21, RUNX1, SF3B1, SMC1A, SMC3, SRSF2, TET2, TP53, U2AF1, and WT1.

Results: In total, 127 pts showed an inv(16)(p13q22), 10 pts a t(16;16)(p13;q22). One pt showed a normal karyotype with a cytogenetically cryptic CBFB–MYH11 rearrangement confirmed by RT-PCR.

Using standard chromosome banding analysis, additional cytogenetic aberrations (ACA) were observed in 52 pts (38%). The most frequent secondary chromosome aberrations were +8 (15/52; 29%), +22 (15/52; 29%) and +21 (5/52; 10%). With regard to blood counts, cases with sole inv(16) had significantly elevated white blood cell counts compared to patients with inv(16) and ACA (78x10⁹/L vs 20x10⁹/L; p<0.001).

112/138 (81%) pts had at least one mutation in addition to CBFB-MYH11, 47/112 (42%) had at least two additional mutations (maximum: four).  Most common were mutations in NRAS (35%), KIT (32%), FLT3-ITD and FLT3-TKD (20%) and KRAS (17%). Mutations in other genes (ASXL1, CBL, DNMT3A, RUNX1, SRSF2, TET2 and WT1) were found in less than 10% of cases. Comparing AML with CBFB-MYH11 with the other core binding factor AML entity, i.e. AML with RUNX1-RUNX1T1 (Krauth et al., Leukemia 2014), the former showed a higher incidence of additional mutations  (81% vs 50%), however, the landscape of mutated genes was comparable. Solely, the frequency of ASXL1 mutations was higher in RUNX1-RUNX1T1 positive AML compared to CBFB-MYH11 positive AML (12% vs <1%). We additionally analyzed concomitant mutations in CBFB-MYH11 positive AML according to functional pathways. Mutations resulting in activated signaling (FLT3-ITD and FLT3-TKD, KRAS, NRAS, KIT) were identified in the majority of cases (n=107/138; 78%), while mutations of tumor suppressors (CBL, TP53, WT1) were detected in 18/138 cases only (13%). Mutations of myeloid transcription factors (CEBPA, RUNX1, ETV6), mutations of genes that modify the epigenetic status (ASXL1, EZH2, TET2, DNMT3A, IDH1/2 and MLL mutations), mutations of cohesin complex genes (SMC1A, SMC3 and RAD21) and spliceosome genes (SF3B1, U2AF1, SRSF2 and ZRSR2) were identified in less than 10% of cases.

There was no difference in frequency and types of additional mutations between patients with inv(16) sole and those with inv(16) and ACA with the exception of WT1 mutations, which were more frequent in patients with inv(16) and ACA (8/51; 16% vs 2/84; 2%; p=0.006).

Data regarding the prognostic impact of the concurrent genetic lesions, trisomy 22 and KIT mutations, in CBFB-MYH11 AML are controversial. In our cohort, survival analysis revealed no impact of trisomy 22 or concomitant KIT mutations on prognosis of CBFB-MYH11 AML. However, within patients with inv(16) sole those with concomitant KRAS mutations had a significantly worse overall survival (OS) compared to KRAS wild-type patients (2 year OS: 43% vs 23%; p<0.001).  

 Conclusions:

1. Secondary genetic lesions are detected in 91% of inv(16)/CBFB-MYH11 positive AML patients.
2. NRAS mutations were the most frequent secondary lesions followed by KIT mutations, FLT3-ITD and FLT3-TKD.
3. inv(16)/CBFB-MYH11 positive AML show high frequency of mutations resulting in activated signaling.
4. Considering controversial studies, trisomy 22 and concomitant KIT mutations had no prognostic impact in our cohort of 132 inv(16)/CBFB-MYH11 AML cases. The only additional genetic marker with a significant adverse prognostic impact on OS was KRAS mutation.