Cryptic Genomic Lesions in Adverse-Risk Acute Myeloid Leukemia Identified by Integrated Whole Genome and Transcriptome Sequencing

Abstract

Acute myeloid leukemia (AML) is a heterogeneous group of hematologic malignancies characterized by the proliferation of myeloid cells blocked in their ability to differentiate. Evaluation by G-banding and fluorescence in situ hybridization is an essential aspect in the initial disease characterization and even now is fundamental in identifying cytogenetic abnormalities that can inform disease diagnosis, prognosis, and treatment decision [ 1 , 2 ]. For instance, the identification of t(8;21)(q22;q22) or t(15;17)(q22;q12), which generate RUNX1-RUNX1T1 or PML-RARA gene fusions, respectively, confers favorable prognosis when treated accordingly [ 1 ]. In recent years, advancements in next-generation sequencing and efforts by large genomics studies have led to a classification of 11 AML subgroups based on cytogenetic abnormalities as well as mutations in genes, such as NPM1 or CEBPA [ 1 ]. AML with a complex karyotype, defined by the presence of three or more unrelated chromosomal aberrations and the absence of favorable cytogenetic rearrangements, is associated with TP53 mutations and strikingly poor outcome [ 1 , 3 ]. While conventional cytogenetics is still a powerful technique, complex chromosomal aberrations test the limits of cytogenetic resolution. Moreover, detection of cryptic genomic lesions, especially gene fusions, by orthogonal methods can lead to adjustment of treatment regimens and/or identification of biomarkers [ 4 ]. The frequency at which cryptic gene fusions are present within complex karyotypes is currently unknown [ 5 ]. In this study, we investigated whether whole-genome sequencing (WGS) and whole-transcriptome sequencing (RNA-seq) can resolve these complex aberrations in a series of patients where conventional cytogenetics could not resolve the driver event. Sequencing was performed on nine patients with adverse-risk AML; seven cases had complex karyotypes and the others had unusual translocation events (Table 1 and Supplementary Table 1 ). The study was approved by the Research Ethics Board of the University Health Network (REB# 01–0573) and written informed consent was obtained from all patients. Table 1 Comparison of cytogenetic and genomic findings from nine patients with adverse-risk AML Full size table WGS and RNA-seq libraries were generated using NEBNext DNA Library Prep (New England Biolabs) and TruSeq Stranded Total RNA Sample Prep (Illumina), respectively, and were sequenced on the Illumina HiSeq 2000 platform. Bioinformatic tools used here are listed in Supplementary Table 2 . Briefly, we used BWA-MEM for WGS alignment, CREST for structural variant (SV) calling, and HMMcopy for copy number variation (CNV) detection. In parallel, we used STAR for RNA-seq alignment, STAR-Fusion for fusion transcript detection, and GATK for SNV/indel calling from RNA-seq data. By performing low-coverage WGS (mean genome coverage of 11.3×; Supplementary Table 1 ) of leukemia DNA without matched germline DNA, we aimed to detect SVs and CNVs in a manner comparable to conventional cytogenetics. Furthermore, we compared gene fusions and rearrangements detected by CREST with fusion transcripts detected by STAR-Fusion to cross validate. DNA breakpoints and fusion transcripts were validated by PCR and RT-PCR, respectively, followed by Sanger sequencing. Analysis of the leukemia genomes concordantly identified 39 out of 74 (53%) cytogenetic abnormalities annotated by visual inspection (Table 1 and Supplementary Table 3 ). The concordance rates for translocations/inversions, chromosomal gains/losses, and subchromosomal gains/losses were 74%, 54%, and 37%, respectively (Supplementary Table 1 ). For five cases with composite karyotypes, which were used to capture the karyotypic heterogeneity, their average concordance rate was lower than that of the other four cases (59% vs. 100%; Wilcoxon rank-sum test, p = 0.015). This is likely due to the challenge of detecting subclonal CNVs and SVs with low-coverage WGS. Nonetheless, many cryptic genomic lesions, including submicroscopic deletions of BCOR , TP53 , and FOXO3/LACE1 , that affect known leukemia-causing/modifying genes were detected. For instance, t(X;8)(p21.2;q24.1) that was reported following conventional cytogenetics in case 2 was revealed by genomic investigation to be linked to two other SVs that resulted in the deletion of BCOR (Supplementary Fig. 1 ). Complex and unbalanced rearrangement patterns, which can be more difficult to discern by G-banded karyotyping, were detected in five cases and could be linked to cytogenetic abnormalities resembling the actual events. Furthermore, in accordance with known association between TP53 mutation and complex karyotype [ 3 ], each of the three cases with the most cytogenetic abnormalities—cases 3, 7, and 8—had a point mutation and a copy number loss of TP53 (Table 1 ). Notably, four out of nine leukemias in our cohort harbored gene fusion events that were not identified by cytogenetics: ETV6-MECOM (case 3), NUP9