Gene Mutations Identified in Chemorefractory Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is a genetically heterogeneous disease. Recently, multiple recurrently mutated genes have been identified in AML and implicated in various mechanisms of leukemogenesis. However, knowledge regarding the association of gene mutations with primary or secondary resistance to chemotherapy is incomplete.

Methods: We analyzed a discovery cohort of 45 patients with chemorefractory AML that were enrolled in a phase 2 clinical trial of a novel conditioning regimen prior to allogeneic stem cell transplant for patients with non-remission AML. DNA was extracted from FACS-purified leukemic cells procured from patients after failure to achieve complete remission (CR) after ≥2 cycles of induction chemotherapy after initial diagnosis ("primary refractory", N=22) or after rapid relapse (<6 months) or failure to achieve CR after ≥1 cycle of induction chemotherapy after relapse ("secondary refractory", N=23). Since TP53 mutations have been previously associated with refractory disease, we selected 29 TP53 wild-type cases from the discovery cohort and performed whole exome sequencing (WES) with a mean read depth of 72X (range 30-140). All somatically acquired gene variants identified by WES in protein-coding genes were verified by Sanger sequencing. In addition, we performed Sanger re-sequencing of 11 recurrently mutated genes in AML (TP53, RUNX1, DNMT3A, TET2, FLT3, NPM1, IDH1, IDH2, ASXL1, NRAS and KRAS) in all 45 cases. Given lack of published WES data in refractory AML, we then compared these mutation frequencies to a cohort of 151 AML patients enrolled consecutively at one center (the "university cohort") with known responses to chemotherapy or The Cancer Genome Atlas (TCGA) data which comprises de novo AML only.

Results: WES of 29 TP53 wild-type refractory AML cases revealed a total of 351 confirmed somatic mutations with a median of 13 protein-coding mutations per case (range 5-22). Genes mutated in 7% (2 of 29 cases), and excluding the 11 known recurrently mutated genes listed above, that were not previously described in AML (COSMIC review) included ADAM23, CPNE7, and SIX5. We also identified mutations in NOMO3 and OAS2 in 7% (2 of 29 cases), which have been previously described in AML but at lower frequencies (1.7% [6 of 347; COSMIC] and 0.2% [1 of 347] respectively) based on review of the literature. The genes SRSF2, NOMO3 and OAS2, which were all identified in 7% (2 of 29 cases) in our discovery cohort, had no reported mutations found in the TCGA (p=0.02). Additional genes, which were found in our discovery cohort in 7% (2 of 29 cases) respectively, and were also found in the TCGA, include BCOR, FOXP1, FRYL, PHF6, STAG2, PTPN11 and SETD2.

Mutational profiling of the 11 recurrently mutated genes in AML revealed a striking paucity of NPM1 mutations in primary refractory AML (range 0% [discovery cohort] - 3% [university cohort]) compared with chemosensitive AML (31% [university cohort]; p<0.001). TP53 mutations, however, were enriched in primary refractory AML (range 23% [discovery cohort] - 38% [university cohort]) compared with chemosensitive AML (4% [university cohort]; p<0.001). Of note, while FLT3 -ITD mutations were infrequently observed in primary refractory AML (range 0% [discovery cohort] - 14% [university cohort]) compared to chemosensitive AML (27% [university cohort]; p<0.01), they were highly enriched in secondary refractory AML (61% [discovery cohort] - 30% [university cohort]; p=0.03).

Conclusions: 1) Whole exome sequencing of 29 TP53 wild-type refractory AML revealed recurrent mutations in ADAM23, CPNE7, NOMO3, OAS2 and SIX5. The function and prevalence of these gene mutations are not well-characterized in AML, including refractory AML and should be determined in a larger cohort of patients; 2) TP53 mutations were significantly enriched in primary refractory disease; 3) Conversely, FLT3 -ITD mutations were significantly enriched in secondary but not primary refractory disease, suggesting the frequent emergence of a chemorefractory FLT3-ITD mutated clone following treatment with conventional chemotherapy; and, 4) NPM1 mutations were significantly under-represented in primary refractory AML. While larger sequencing studies of refractory AML cases are needed, these data do not support gene mutations other than in TP53 as frequent causes of primary refractoriness to chemotherapy in AML.