Detection of Chromosomal Aberrations in Acute Myeloid Leukemia By Copy Number Alteration Analysis of Exome Sequencing Data

Exome sequencing is widely used and established to detect tumor-specific sequence variants such as point mutations and small insertions/deletions. Beyond single nucleotide resolution, sequencing data can also be used to identify changes in sequence coverage between samples enabling the detection of copy number alterations (CNAs). Somatic CNAs represent gain or loss of genomic material in tumor cells like aneuploidies (e.g. monosomies and trisomies), duplications, or deletions. In order to test the feasibility of somatic CNA detection from exome data, we analyzed 13 acute myeloid leukemia (AML) patients with known cytogenetic alterations detected at diagnosis (n=8) and/or at relapse (n=11). Corresponding remission exomes from all patients were available as germline controls resulting in 19 comparisons of paired leukemia and remission exome data sets.

Exome sequencing was performed on a HiSeq 2500 instrument (Illumina) with mean target coverage of >100x. Exons with divergent coverage were detected using a linear regression model on mean exon coverage, and CNAs were called by an exact segmentation algorithm (Rigaill et al. 2012, Bioinformatics). For all samples, cytogenetic information was available either form routine chromosomal analysis or fluorescent in situ hybridization (FISH). Blast count were known for all but one AML sample (n=19). Copy number-neutral cytogenetic alterations such as balanced translocations were excluded from the comparative analysis.

By CNA-analysis of exomes we were able to detect chromosomal aberrations consistent with routine cytogenetics in 18 out of 19 (95%) AML samples. In particular, we confirmed 2 out of 2 monosomies (both -7), and 9 out of 10 trisomies (+4, n=1; +8, n=8; +21, n=1), e.g. trisomy 8 in figure 1A. Partial amplifications or deletions of chromosomes were confirmed in 10 out of 10 AML samples (dup(1q), n=3; dup(8q), n=1; del(5q), n=3; del(17p), n=1; del(20q), n=2), e.g. del(5q) in figure 1B. In the one case with inconsistent findings of chromosomal aberrations between exome and cytogenetic data there was a small subclone harboring the alteration described in only 4 out of 21 metaphases (19%).

To assess the specificity of our CNA approach, we analyzed the exomes of 44 cytogenetically normal (CN) AML samples. Here we did not detect any CNAs larger than 5 Mb in the vast majority of these samples (43/44, 98%), only one large CNA was detected indicating a trisomy 8.

Estimates of the clone size were highly correlated between CNA-analysis of exomes and the parameters from cytogenetics and cytomorphology (p=0.0076, Fisher's exact test, Figure 1C). In CNA-analysis of exomes, we defined the clone size based on the coverage ratio: . Clone size estimation by cytogenetics and cytomorphology was performed by calculating the mean of blast count and abnormal metaphase/interphase count. Of note, clones estimated by CNA-analysis of exomes tended to be slightly larger. This may result from purification by Ficoll gradient centrifugation prior to DNA extraction for sequencing and/or the fact that the fraction of cells analyzed by cytogenetics does not represent the true size of the malignant clone accurately because of differences in the mitotic index between normal and malignant cells.

Overall, there was a high correlation between our CNA analysis of exome sequencing data and routine cytogenetics including limitations in the detection of small subclones. Our results confirm that high throughput sequencing is a versatile, valuable, and robust method to detect chromosomal changes resulting in copy number alterations in AML with high specificity and sensitivity (98% and 95%, respectively).

Figure 1: (A) Detection of trisomy 8 with an estimated clone size of 100%

(B) Detection of deletion on chromosome 5q with an estimated clone size of 90%

(C) Correlation of clone size estimation by routine diagnostics and exome sequencing (p=0.0076)