Genomic-Wide Analysis By High Resolution SNP Array Identifies Novel Genomic Alteration in Acute Myeloid Leukemia

Introduction: Novel array-based technique-single-nucleotide polymorphism (SNP) microarray can detect cytogenetic lesions mostly involving structural alterations with losses or gains of chromosomic material. These abnormalities are predictive of response and can help define therapeutic strategies. SNP microarray can also detect copy-neutral loss of heterozygosity (CN-LOH), which has a described role in Acute Myeloid Leukemia (AML) by inducing oncogene duplication, tumor suppressor inhibition and epigenetic reprogramming.

Aim: To improve conventional cytogenetic analysis and identify new genes relevant to leukemogenesis by SNP array-based genotyping.

Materials and Methods: We analyzed 279 AML patients (pts) at diagnosis by SNP Array 6.0 or Cytoscan HD Array (Affymetrix). Thirty-four samples were also analyzed by Whole Exome Sequencing WES (HiSeq 2000,Illumina). SNP Array data were analyzed by Nexus Copy Number™ v7.5 (BioDiscovery) and R Development Core Team, while WES data were analyzed by GATK and MuTect.

Results: Copy Number Alterations (CNAs) were scattered across all chromosomes (chrs). All pts showed  CNA events: 44.4% of CN gain, 21% of CN loss and 34.6% of CN-LOH.

Single copy gains mainly affected chrs X, 1, 2, 4, 9 and 8. Duplications occurred at chrs 2, 3, 5 and 14. Heterozygous loss events were detected in chrs 3, 5 and 14, while regions of deletion were located in chrs 6, 7 and 22. The CN-LOH event was the most common event and involved chrs 1, 2, 3, 4, 5, 6 and 8. We studied the deletome profile in our cohort (Fig.1) pf pts in order to define the minimal common deletion region.

SNP array analysis showed that several genes were preferentially deleted, including ADAM5 (12,9%), PHF6 (12,2%), AGPS (10%), SOX6 (7,9%), WT1 (6,5%), CRLF2 (5,4%) and LRRK1 (4,3%); while the genes preferentially amplified were GPC3 (70,25%), FLT3 (44,8%), FGF13 (36,2%), KIT (31,54%), AFF2 (31,5%), ETS1 (26,52%), MITF (22,2%), CASK (16,5%), CDY1 (14,33%), MECP2 (14,33%), FOXP2 (14%) and SMAD4 (12,9%).

Single-copy losses and deletions were enriched (p<0,001) for genes mapping into the following pathways: cancer-related (87 genes), metabolism (154 genes), ErbB signaling (34 genes), MAPK signaling (55 genes), ubiquitin mediated proteolysis (33 genes). Thirty-eight loss and 3 deletion events were shared by at least 14 pts (5%). Ten genes were deleted in at least 10 patients (3,6%).

Concerning single copy gain and amplified genes, the functional pathways significantly represented in our cohort were: cancer-related (103 genes), regulation of actin cytoskeleton (60 genes), MAPK signaling (64 genes), metabolic (171 genes) and cell adhesion molecules pathways (CAMs, 5 genes). Regarding gain events, 24 genes were shared by at least 42 pts (15%), 184 genes were shared by at least 14 pts (5%) and 58 genes by at least 28 pts (10%); whereas 15 genes have duplication events (in homozygosis) shared by at least 14 pts (5%).

Significant CN-LOH events included genes mapping to metabolic pathways (249 genes), pathways in cancer (103 genes), regulation of actin cytoskeleton (67 genes), calcium signaling pathways (57 genes), WNT signaling pathways (51 genes) and protein digestion and absorption (34 genes). CN-LOH events in 119 genes were shared by at least 14 patients (5%).

Finally, we could distinguish 3 clusters of pts, each one characterized by a peculiar pattern of amplified or deleted genes. In order to define relevant pathogenic mechanisms in our cohort, we combined the deletome profile with WES data obtained  from 34 pts. Interestingly, we found deletion of genes which are also targeted by mutations (BRCA2, LRRK1). Moreover, some deleted genes, as CASK, CDK6 and MAPT, were involved in pathways affected by genomic mutations (CASK deletion and MPP6 mutation, CDK6 deletion and PPM1B mutation, MAPT deletion and SPAG5 mutation).

Conclusion: By SNP array we have identified CNAs involving novel potential leukemia-related genes. Our results suggest that the comparison between SNP and WES data could provide important findings on the prognosis of AML pts. Minimal deleted regions deserve further investigation in order to identify new candidate oncogenes which could be relevant AML biomarkers.

Acknowledgement: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12(L. Bolondi), FP7 NGS-PTL project.

MCF and VG equally contributed to this work.

Fig.1