Using Combined High Density SNP/CNV Arrays as a Clinical Karyotyping Tool in Myeloid Malignancies.
Authors: Huh Jungwon , Gondek Lukasz , O’Keefe Christine , Theil Karl S. , Maciejewski Jaroslaw P. 
Published: April 2020

We have evaluated various types of single nucleotide polymorphism arrays (SNP-A) as a karyotyping platform in over 600 cases of bone marrow failure and various myeloid disorders including MDS, MDS/MPD, and AML and in over 360 controls. We have shown that SNP-A not only reliably confirms chromosome gains and losses identified by metaphase cytogenetics (MC) but also allows for detection of previously cryptic chromosomal lesions. Moreover, through the ability of combining copy number measurement with genotyping, SNP-A also enables detection of copy-neutral loss of heterozygosity (LOH), a lesion that cannot be recognized using traditional MC. We have previously shown that this type of lesion occurs frequently in myeloid disorders. Recently, ultra-high density SNP-A were introduced (Affymetrix 6.0 array platform) with modified chemistry and a combination of SNP (&gt;906,600) and copy number variant probes (CNV &gt;946,000) to allow for precise delineation of the location and size of submicroscopic chromosomal defects, copy-neutral LOH and germline CNVs. This technology will likely supplant previous SNP-A platforms in performance and opens a new era in clinical cytogenetic diagnostics. In this pilot trial, we applied the Affymetrix SNP 6.0 array to samples obtained at diagnosis from 193 patients (59 AML, 115 MDS, 19 MDS/MPD). For 95 samples, both Affymetrix 250K and 6.0 arrays were applied; 6.0 SNP-A allowed for more identification of 33 regions of copy number aberrations and 11 copy-neutral LOH, suggesting a higher resolution of this technology over 250K arrays. By MC, 43% (71/167) of patients had chromosomal aberrations including a complex karyotype; 48% (81/167) had a normal karyotype and 9% (15/167) showed no growth. By 6.0 SNP-A, aberrations were detected in 78% (150/193) of patients. Significantly, among the samples with either a normal karyotype or non-informative results, aberrations were found by SNP-A in 57% and 73%, respectively. In an illustrative case with a balanced translocation by MC, copy number changes were found at the junctions, indicating that the translocation breakpoints contained small deletions. Comparison of 6.0 SNP-A results with MC showed concordance for detection of MC-defined lesions. However, 9% (15/167) of defects were only detected by MC and could not be detected by SNP 6.0 array in patients with a low proportion of abnormal metaphases. Gains and losses detected by SNP-A 6.0 required a sensitivity threshold of 20% clonal cells. We identified recurrent lesions not previously reported as being CNV. These comprised 238 regions of loss, 257 regions of gain, and 97 regions of copy-neutral LOH. When feasible (or if indicated to resolve a discordance with MC or to exclude CNV), DNA from sorted CD3+ cells was used as a reference. Recurrent losses were located in 37%(71/193) patients at 5q, 7p, 7q, 13q, 17p, 20q, 1p, 12p, 3q and 4q. Recurrent gains in 28%(55/193) patients included 13q, 11p, 11q, 21q, 3p, 8p, 8q, 9q, 19p and 5p. Somatic copy-neutral LOH was identified in 20%(39/193) patients at 13q, 1p, 5q, 6p, 7q, 11q, 17q, 3p, 4q, and 12q. Homozygous deletion was observed in one patient with a 6 Mb loss at 5q34 (not visible on MC) within a heterozygous 5q11.2–5q35.3 deletion Copy neutral LOH was found in 29% (56/193) patients overall. AML patients appeared to have more frequent copy neutral LOH(34%) than MDS (24%), but not significant (P=0.2). The 5%(9/193) of patients had more than 2 chromosomes with copy-neutral LOH. Among the samples showing a normal karyotype, 20% (16/81) of patients showed copy -neutral LOH as the sole aberration without additional copy number aberrations. To our knowledge, this is the first systematic application of SNP-A 6.0 in a large cohort of patients with myeloid malignancies. Our results demonstrate that this technology complements, but does not replace conventional MC for identification of abnormalities in myeloid neoplasia.