A Comprehensive Genetic Analysis of MDS Patients From Peripheral Blood Combining FISH- and SNP-Array-Analysis

Chromosomal banding analysis (CBA) of bone marrow metaphases is the gold standard to identify chromosomal abnormalities in myelodysplastic syndromes (MDS). We aim to comprehensively detect and follow chromosomal abnormalities during the course of the disease without the need of repeated bone marrow biopsies. In ongoing studies we attempt to achieve this goal by performing serial fluorescence in situ hybridization (FISH) analysis on CD34+ peripheral blood cells (PBC). The aim of this pilot study was to establish SNP-array-analysis (SNP-A) on CD34+ PBC to complement genetic analysis on peripheral blood by identifying chromosomal abnormalities not detectable by FISH and/or CBA.

We immunomagnetically enriched CD34+ PBC of 20 patients (pts) with MDS (16 pts), suspected MDS (1 pts) and secondary acute myeloid leukemia (sAML, 3 pts). SNP-A was performed with arrays from Affymetrix (3x SNP 6. 0, 4x Cyto 2. 7, 13x CytoScanHD). Fresh or frozen CD34+ PBC of 10 pts and in methanol/acetic acid fixed CD34+ PBC of 9 patients were successfully processed. One whole genome amplified sample was included. CBA and FISH-A was done for all patients.

By CBA, 3 pts had no chromosomal abnormalities, 8 pts had one abnormality, 6 pts had 2–4 abnormalities and 3 pts had more than 6 abnormalities. By SNP-A on CD34+ PBC, additional abnormalities could be revealed in 13/20 pts. In two pts they were also confirmed by FISH-A. Most of them were micro-deletions not detectable by CBA. In addition, SNP-A revealed uniparental disomies (UPD) in 5/20 pts. Of the 3 pts with no detectable abnormalities in CBA, one had a micro-deletion in 4q24 (TET2). The other two had an insufficient number of metaphases. One of them showed a highly complex karyotype by FISH-A and SNP-A on CD34+ PBC. The other one had suspected MDS and did not show any abnormalities by SNP-A.

The 17 pts with ≤ 6 abnormalities in CBA showed 55 abnormalities by CBA, FISH-A and SNP-A altogether. 34/55 (62%) abnormalities could be detected by SNP-A and/or FISH-A, but not by CBA. 24/55 (44%) abnormalities could only be detected by SNP-A. 4/55 (7%) of abnormalities were structural abnormalities or small clones and were only detected by CBA.

Serial analysis indicated clonal evolution: A patient with 16 abnormalities detectable by CBA and additional three by FISH and SNP-A developed two further micro-deletions (del(2)(q31q32), del(4)(q24q26)) within four months. When a MDS patient with a known 20q-deletion (isolated by CBA and FISH) progressed to AML 25 months after first diagnosis we detected 3 micro-deletions by SNP-A of peripheral blood (0. 98 Mb on 4q, 1. 31 Mb on 12q, 2. 55 Mb on 12q) thus resulting in 4 cytogenetic alterations fulfilling the criteria of complex and prognostically unfavorable abnormalities.

Recently it was shown that abnormalities detectable by SNP-A, but not by CBA, could worsen prognosis of MDS patients. We succeeded in detecting these additional abnormalities without the need of bone marrow biopsies out of peripheral blood. Nevertheless, by parallel FISH and SNP-A of CD34+ PBC, most abnormalities detectable by CBA of bone marrow metaphases could be detected. Comprehensive genetic analysis at close intervals thus is possible without the need of bone marrow biopsies to study clonal evolution. The information gained could be used for therapy decisions, to improve prognostication and to unravel genetic evolutionary steps towards acute leukemia.

No relevant conflicts of interest to declare.