Recurrent Copy Number Variants Are Highly Prevalent in Acute Myeloid Leukemia

Introduction: Large chromosomal copy number variants (CNVs) detected by conventional karyotyping are common in AML; however, small segmental CNVs or copy-neutral loss of heterozygosity (CN-LOH) are not detected by conventional techniques and require sequence-based approaches. Specifically, acquired CN-LOH occurs through duplication of genetic material and subsequent loss of the reciprocal chromosome resulting in a hemizygous state. CN-LOH has been demonstrated in AML, mostly occurring in regions of known recurrent mutations (ex: FLT3 , JAK2 , TET2 ), but its overall prevalence and involvement in AML pathogenesis has yet to be determined. Initial studies regarding the significance of CNVs suggest possible prognostic impact. We aimed to explore the prevalence of segmental CNVs and CN-LOH in a cohort of pediatric and adult AML specimens that underwent clinical comprehensive genomic profiling (CGP) (FoundationOne Heme Sequencing).

Methods: CGP for targeted DNA and RNA sequencing was performed on 932 patients with AML (n=179 pediatric age ≤ 18 years, n=753 adult age ≥ 19 years). Specimen sites included bone marrow, peripheral blood, and biopsy of extramedullary disease. Following strict quality control (QC) measures to exclude any samples with transplant history, contamination, low purity, and lack of aneuploidy, all level CNV and LOH events were reported using a proprietary algorithm. A total of 323 samples met QC standards for LOH events, with all level copy number analyses available for 236 (73%) patients. We analyzed the loci of 91 genes to determine the type and prevalence of CNV events for amplifications (CNA; CN>2), deletions (CND; CN<2), CN-LOH, copy-loss LOH (CL-LOH) events at the genomic or chromosome level excluding sex chromosomes.

Results: Among the 323 samples (n=59 pediatric, n=264 adult), the prevalence of LOH events was 91% (294). Identified LOH events included single gene LOH or chromosomal LOH, including arm or whole chromosome. Chromosomal events occurred in 172 (53%) of patients, and most frequently involved the following arms: 7q (n=59), 7p (n=57), 13q (n=25), 18q (n=23), 18p (n=22), 5q (n=19), and 17p (n=19). Among patients with chromosomal LOH events, 131 (76%) had events in the 7 chromosomal arms with the highest frequency (Fig 1A). Genomic LOH events were detected in 287 (89%) patients, and were detected in all 91 of the genes analyzed, with a frequency of 1-58. Further, CN-LOH events were detected in 162 (50%) patients occurring in 89 (98%) of genes analyzed (Fig 1B), with a frequency of 1-17. Among the 20 genes with highest frequency of LOH, 11 of them occurred at the 7q and 13q regions, which is consistent with previous findings that these are areas of frequent genomic losses. LOH, including CN-LOH, events overall commonly occurred in genes located in the 5q, 7p, 13q, 16q, and 17p regions. Recurrent LOH events in 3 of the most commonly affect genes occurred in approximately 31% of patients ( EZH2 , n=103, BRAF , n=102, and KMT2C , n=99), all located on 7q. CN-LOH events were detected in oncogenes such as CDK6 , MET , FLT3 , JAK2 , ABL1 .

Among the 236 samples evaluable for CNA assessment, 217 (92%) had at least one CNA or CND detected. CNAs were detected in 163 patients (69%) and occurred in all 91 of the genes analyzed (Fig 1C). Among samples with CNAs detected, the median number of somatically acquired CNAs was 5 (range 1-91). Recurring CNAs were detected across all chromosomes, but were most common in genes located on chromosome 1q, 8, 11q, 13q, 17q, 19, and 21q. CNDs were detected in 165 patients (70%) and occurred in 83 of genes (91%) analyzed. Among samples with CNDs detected, the median number of CNDs was 6 (range 1-58).

Conclusion: CNVs and LOH events were commonly detected in this sample of 323 AML specimens. We show that LOH is highly prevalent, with genomic events detected in 89% of cases analyzed and CN-LOH events specifically detected in 50%. We found highly recurrent genes located at a few distinct chromosomal loci, suggesting some regions may be more prone to chromosomal instability. Future research in AML is needed to understand the mechanisms behind genomic and large-scale chromosomal disruptions, including CN-LOH. Identification of segmental CNVs and CN-LOH may identify cryptic variants that might aid in more appropriate diagnosis, management, disease monitoring, as well aid in the interpretation of mutation data as these events might alter the variant allele fraction of sequence variants.

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