Recurrent Genetic Variants of Long Non-Coding RNAs (lncRNAs) Independently Associate with Clinical Outcome of Younger Adults with Cytogenetically Normal Acute Myeloid Leukemia

BACKGROUND: Aberrant expression of lncRNAs has been shown to independently associate with clinical outcome of younger adult patients (pts) [aged <60 years (y); Papaioannou et al. Haematologica 2017;102:1391-1400] and older pts (aged ≥60 y; Garzon et al. PNAS 2014;111:18679-84) with cytogenetically normal acute myeloid leukemia (CN-AML). However, the prognostic significance of genetic variants (single nucleotide polymorphisms or somatic mutations) in lncRNAs and their association with established prognostic gene mutations have not been studied.

METHODS: We performed whole transcriptome profiling (RNA-seq) in 377 younger adults with de novo CN-AML and investigated the associations of recurrent genetic variants located within expressed lncRNAs with clinical outcome and gene mutations. All pts were treated on frontline Cancer and Leukemia Group B (CALGB)/Alliance for Clinical Trials in Oncology protocols. Analysis of 11 gene mutations, reported to be prognostic in AML, was performed by targeted amplicon sequencing (ASXL1, DNMT3A, FLT3 -TKD, IDH2, IDH1, NPM1, RUNX1, TET2, WT1), Sanger sequencing (double CEBPA) and fragment analysis (FLT3 -ITD).

RESULTS: We first generated a comprehensive list of the genetic variants that are located in lncRNAs in transcriptomes of younger CN-AML pts. To analyze unequivocally non-coding genetic variants and to avoid ambiguity in their genomic location, all variants that overlapped exons of protein coding genes and those that mapped to segmental duplications or repeat masked regions of the genome were excluded from further analyses. We focused on 207 variants whose frequency differed between our AML cohort and the healthy population, and on 88 variants that were novel. Of the variants tested, 8 showed significant association with at least 1 outcome endpoint [disease-free (DFS), overall (OS) or event-free (EFS) survival], and 3 of them significantly correlated with DFS, OS and EFS. There was no association between complete remission rates and lncRNA variants. The 3 variants which associated with all survival endpoints were: a thymidine (T)-to-cytosine (C) variant in lncRNA RP5-1074L1.4, an adenosine (A)-to-C variant in lncRNA HCG4, and a C-to-T variant in lncRNA HLA-V . The C variant of RP5-1074L1.4 was detected in 182 (64%) of the 284 pts who expressed the lncRNA and associated with longer DFS (P <.001; 5-y DFS, 41% v 27%), OS (P=.04; 5-y OS, 40% v 35%) and EFS (P <.001; 5-y EFS, 36% v 22%). The C variant of HCG4 was found in 41 (18%) of the 234 pts with detectable expression of the lncRNA. Pts harboring the C variant had longer DFS (P=.01; 5-y DFS, 61% v 35%), OS (P=.02; 5-y OS, 58% v 39%) and EFS (P=.04; 5-year EFS, 49% v 29%). Finally, the T variant of HLA-V, which was detected in 58 (26%) of the 227 pts who expressed the transcript, associated with longer DFS (P=.005; 5-y DFS, 53% v 30%), OS (P=.01; 5-y OS, 55% v 33%) and EFS (P=.007; 5-y EFS, 45% v 24%).

With regard to associations with prognostic gene mutations, pts with the C variant of RP5-1074L1.4 were more likely to harbor FLT3 -TKD than pts with the T variant (P=.02). Pts with the T variant in HLA-V were less likely to harbor RUNX1 mutations than pts with the C variant (P=.02). The C variant of HCG4 did not associate with differences in the frequencies of prognostic mutations.

In multivariable analyses, the variables considered in the models (other than the prognostic lncRNA variants) were: white blood cell and platelet counts, hemoglobin, extramedullary involvement, the presence of FLT3 -TKD, FLT3 -ITD and CEBPA, DNMT3A, NPM1, RUNX1 and WT1 mutations, and expression levels of MN1 and miR-155. The C variant of RP5-1074L1.4 was an independent marker for longer DFS (HR: 0.53, P <.001), OS (HR: 0.64, P=.007) and EFS (HR: 0.55, P <.001). The C variant of HCG4 independently associated with longer DFS (HR: 0.52, P=.03) and OS (HR: 0.61, P=.05), whereas the T variant of HLA-V independently associated with longer DFS (HR: 0.46, P=.003), OS (HR: 0.38, P <.001) and EFS (HR: 0.48, P=.001).

PCR assays and targeted Sanger sequencing experiments in leukemic blasts and paired remission bone marrow samples are currently being conducted to examine whether the prognostic variants are acquired genetic events or germline variants.

CONCLUSIONS: We conclude that a small subset of genetic variants that are located within lncRNAs are clinically relevant and provide independent prognostic information in younger adults with CN-AML.