Assessing karyotype precision by microarray-based comparative genomic hybridization in the myelodysplastic/myeloproliferative syndromes

Abstract

Background: Recent genome-wide microarray-based research investigations have revealed a high frequency of submicroscopic copy number alterations (CNAs) in the myelodysplastic syndromes (MDS), suggesting microarray-based comparative genomic hybridization (aCGH) has the potential to detect new clinically relevant genomic markers in a diagnostic laboratory.

Results: We performed an exploratory study on 30 cases of MDS, myeloproliferative neoplasia (MPN) or evolving acute myeloid leukemia (AML) (% bone marrow blasts ≤ 30%, range 0-30%, median, 8%) by aCGH, using a genome-wide bacterial artificial chromosome (BAC) microarray. The sample data were compared to corresponding cytogenetics, fluorescence in situ hybridization (FISH), and clinical-pathological findings. Previously unidentified imbalances, in particular those considered submicroscopic aberrations (< 10 Mb), were confirmed by FISH analysis. CNAs identified by aCGH were concordant with the cytogenetic/FISH results in 25/30 (83%) of the samples tested. aCGH revealed new CNAs in 14/30 (47%) patients, including 28 submicroscopic or hidden aberrations verified by FISH studies. Cryptic 344-kb RUNX1 deletions were found in three patients at time of AML transformation. Other hidden CNAs involved 3q26.2/EVI1, 5q22/APC, 5q32/TCERG1,12p13.1/EMP1, 12q21.3/KITLG, and 17q11.2/NF1. Gains of CCND2/12p13.32 were detected in two patients. aCGH failed to detect a balanced translocation (n = 1) and low-level clonality (n = 4) in five karyotypically aberrant samples, revealing clinically important assay limitations.

Conclusions: The detection of previously known and unknown genomic alterations suggests that aCGH has considerable promise for identification of both recurring microscopic and submicroscopic genomic imbalances that contribute to myeloid disease pathogenesis and progression. These findings suggest that development of higher-resolution microarray platforms could improve karyotyping in clinical practice.

Figure 1

FISH confirmation studies. A) Four different chromosome 12 abnormalities in patient #1 and the corresponding aCGH plot. Three duplication/deletion/duplication regions were confirmed by FISH: amplification of CCND2/12p13.32 (RP11-928N17) is denoted by a 7R/2G FISH pattern; deletion at 12p12.3 (1R/2G pattern) confirmed using the RP11-147E12 FISH probe which maps between PLCZ1 and PLEKHA5; and gain of SOX5 (RP11-34a16) at 12p12.1 with a 4~8R/2~ 4G patterns. B) Submicroscopic CNAs in a trisomy 8 patient #21. BAC aCGH plots for chromosomes 8, 1 and 21. Using the dye swap method, the top plot shows trisomy 8 (FISH confirmed in 82.5%), the middle plot shows a gain (duplication) of 1p21.3p12 confirmed by FISH (32%), and the bottom chromosome 21 plot shows a 344-kb RUNX1/21q22.12 deletion. The duplication was confirmed by FISH on metaphase cells using a 1p21 probe (RP11-96F24), which maps within the duplicated segment, labeled in red, and a control probe that maps to 1q24 (RP11-104L21) labeled in green. Arrow indicates tandem 1p duplication in the metaphase cell. The interphase cell shows three red signals and two green signals. Triple-color interphase FISH (lower right) confirms RUNX1 deletion in 10% of trisomy 8-positive cells. The chromosome 8 centromere probe is labeled in aqua (signals not arrowed), a control probe for distal 21q is labeled in green (white arrows), and the 180-kb RUN1 probe (dJ1107L6) is labeled in red. RUNX1 deletion was present in 10% of trisomy 8-posiitve cells.

Figure 2

Unexpected aCGH results. (A) Hidden unbalanced der(7)t(3;7) rearrangement in patient #22. Plots for chromosomes 3 and 7 reveal a 3q gain and 7q loss. Using a homebrew breakapart probe set for EVI1 (bottom right), metaphase FISH confirmed the presence of both EVI1/3q26.2 FISH signals on both chromosome 3 homologues and a single EVI1 (green) telomeric probe to the der(7) chromosome at band 7q21.3 (upper right). This patient also showed a "cryptic" RUNX1 deletion (not shown). (B) Non-contiguous CNAs of chromosome 7 resulted in four distinct deletions (one in 7p and three in 7q) in patient #15. The 7 centromere was detected by FISH but is not represented on the BAC array. Using a commercially available 7cen/7q31 FISH probe set, a normal FISH result was reported in this patient because the 7 centromere and 7q31 (arrow) were present in two copies. (C) Complex 12p aCGH plot in patient #15 with normal-appearing pair of chromosomes 12. The dup/del/dup 12p CNAs detected by aCGH were not visible by cytogenetics. I-FISH confirmed gain in 12p13.31 with RP11-433J6, deletion at 12p13.2 using RP11-36K5, and duplication of 12p12.1 using RP11-34A16.