Chromosomal lesions and uniparental disomy detected by SNP arrays in MDS, MDS/MPD, and MDS-derived AML

Abstract

Using metaphase cytogenetics (MC), chromosomal abnormalities are found in only a proportion of patients with myelodysplastic syndrome (MDS). We hypothesized that with new precise methods more cryptic karyotypic lesions can be uncovered that may show important clinical implications. We have applied 250K single nucleotide polymorphisms (SNP) arrays (SNP-A) to study chromosomal lesions in samples from 174 patients (94 MDS, 33 secondary acute myeloid leukemia [sAML], and 47 myelodysplastic/myeloproliferative disease [MDS/MPD]) and 76 controls. Using SNP-A, aberrations were found in around three-fourths of MDS, MDS/MPD, and sAML (vs 59%, 37%, 53% by MC; in 8% of patients MC was unsuccessful). Previously unrecognized lesions were detected in patients with normal MC and in those with known lesions. Moreover, segmental uniparental disomy (UPD) was found in 20% of MDS, 23% of sAML, and 35% of MDS/MPD patients, a lesion resulting in copy-neutral loss of heterozygosity undetectable by MC. The potential clinical significance of abnormalities detected by SNP-A, but not seen on MC, was demonstrated by their impact on overall survival. UPD involving chromosomes frequently affected by deletions may have prognostic implications similar to the deletions visible by MC. SNP-A-based karyotyping shows superior resolution for chromosomal defects, including UPD. This technique further complements MC to improve clinical prognosis and targeted therapies.

Figure 1

Analysis approach and type of lesions detected by SNP-A. (A) Normal male (left portion) and abnormal female karyotype with multiple lesions, including del(5q) (right portion). Whole genome scan is shown (top panel); each color represents copy number of a different chromosome (chromosome Y is not included on the array). Chromosome 5 is presented (middle panel) for both patients. Red dots depict single SNP signal intensity, while blue lines present an average value of SNP signal intensity. Green vertical bars represent heterozygous SNP loci, while blue bars show areas of LOH. In comparison to the normal chromosome 5 (left portion), the deletion 5q can be easily observed as a reduction in copy number and area of homozygous SNP loci (right portion). Idiogram of chromosome X is shown (bottom panel) demonstrating the haploid copy number with homozygous SNP loci (male DNA, left portion) and diploid DNA copy number with characteristic distribution of heterozygous SNP loci (female DNA, right portion). MC-detected chromosomal abnormalities (karyograms included) confirmed by SNP-A (B). (C) SNP-A–detected microdeletion of chromosome 21 and microduplication of chromosome 3. Copy number confirmation using Taq-Man Real-Time PCR shown as blue bars and compared with normal T cells (CD3+) obtained from the same patient. Microsatellite ID marker is displayed above the bars. Y axis (2^−ΔΔCt) presents the relative quantification scale, where 1 equals diploid DNA copy number. Examples of deleted and duplicated genes are included. Error bars represent SD.

Figure 2

SNP-A karyotyping results obtained through simultaneous testing blood and marrow and serial bone marrow exams. (A) Comparison of SNP-A results for both marrow and blood. (B) Results of serial testing performed during the disease course.

Figure 3

Detection and comparison of different types of lesions, including UPD, that affect chromosome 7 and influence the survival. (A) Two types of lesions resulting in LOH. Left portion demonstrates a deletion spanning part of the long arm of chromosome 7, shown here as reduction of copy number detected by SNP-A, which is concordant with MC finding (black arrow). Right portion demonstrates copy number neutral LOH (UPD), shown here as normal copy number (SNP-A) and normal karyogram by MC. Pink and blue bars below the idiogram indicate areas of LOH, with the thicker the blue bar the higher the probability of LOH. LOH was confirmed by MS genotyping using CD3+ cells as a nonclonal control. MS marker ID is displayed. Arrow indicates the allelic discrepancies. Location and type of lesions (loss-gray, gain-orange, upd-blue) affecting chromosome 7 are shown (B). Lesions previously not found by MC are marked with a black star. Kaplan-Meier analysis of the survival of patients (irrespective of the treatment received) with −7/del(7q) by MC and with new lesions affecting chromosome (del(7q)/upd(7q)) compared with patients with normal SNP-A analysis (C).

Figure 4

Chromosomal distribution of the lesions detected by SNP-A. Location and the size of the lesions are indicated next to the chromosomes' idiograms.

Figure 5

Type, frequency, and number of lesions detected by MC and SNP-A. (A) The frequency of chromosomal aberrations and noninformative results as detected by both MC and SNP-A. The insert in the SNP-A pie chart shows the portion of acquired UPD, either as the sole change or in addition to other abnormalities. (B) The percentage of patients with 0, 1, 2-3, and more than 3 lesions, respectively, as detected by MC (■) and SNP-A (□). Pie charts demonstrate distribution of low versus advanced stage of MDS within noninformative and normal karyotypes detected by MC and SNP-A.

Figure 6

Types and prognostic impact of new lesions found in patients with noninformative or normal MC. (A) The percentage of patients with normal or noninformative MC in whom new chromosomal lesions were found by SNP-A (left). Type and location of abnormalities are displayed in Table S1 and Figure 4. Stars mark the patients with sAML and noninformative MC in whom IPSS risk category cannot be determined. (B) Kaplan-Meier analysis of survival of patients (irrespective of the treatment received) with normal MC in whom new defects were identified using SNP-A as compared with these with a normal karyotype as evidenced by both negative SNP-A and MC. (C) Survival curves of sAML patients with normal SNP-A result versus patients with abnormal MC and those with normal MC in whom new lesions were found by SNP-A (left). Survival of sAML patients grouped into those with and without additional defects identified by SNP-A (right). (D) Survival curves of patients with MDS/MPD subgrouped into those with normal SNP-A, normal MC, but additional lesions by SNP-A, and patients with abnormal MC results. (E) Survival curves of patients with MDS and IPSS int-1 subgrouped based on the presence or absence of new lesions found by SNP-A irrespective of the MC result.