FISH analysis of circulating CD34+ cells as a new tool for genetic monitoring in MDS: Verification of the method and application to 27 MDS patients

Abstract

In myelodysplastic syndromes (MDS) chromosomal anomalies can be identified in 50–80% of patients. They have a diagnostic and prognostic impact and are increasingly considered for therapeutic decisions. Cytomorphology and cytogenetic analyses of bone marrow (bm) cells define the goldstandard to diagnose MDS patients and to document treatment response. We present a novel method using peripheral blood (pb) for frequent cytogenetic monitoring: after immunomagnetic cell separation circulating CD34+ cells were analysed by fluorescence in situ hybridization (FISH). We compared FISH analyses of enriched and non-enriched pb and bm cells with conventional chromosome banding analyses of bm metaphases: analysing circulating CD34+ cells by FISH is a sensitive, reliable method to measure the abnormal cell clones in pb. This method is practicable, non-invasive, representative for the clonal situation in the bm, and has a predictive value. Its feasibility was proven in a cohort of 27 MDS patients.

Introduction

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem cell disorders leading to an ineffective hematopoesis with peripheral cytopenias [1], [2]. Chromosomal aberrations play a major role in pathogenesis, prognosis, diagnosis and, most recently, treatment allocation for subgroups of MDS patients [3], [4], [5], [6], [7], [8], [9]. In de novo MDS cytogenetic abnormalities can be detected in approximately 50% of patients, whereas patients with treatment related MDS show chromosomal anomalies in up to 80% [2], [4], [5], [7], [9], [10], [11]. Usually, these chromosome aberrations are demonstrated in bone marrow cells by cytogenetic analysis of metaphases using conventional banding techniques, and most of these chromosomal anomalies can alternatively or additionally be diagnosed by fluorescence in situ hybridization (FISH) [11], [12].

In 2001 Vehmeyer et al. [13] were able to show that in MDS an increased number of CD34+ progenitor cells circulates in the peripheral blood. An association between the number of peripheral stem cells and an advanced stage of the disease was demonstrated. In previous studies we could prove that in MDS and acute myeloid leukemia (AML) CD34+/CD38− bone marrow cells constantly show clonal chromosomal aberrations [14], [15].

In the present study we report on a novel method to monitor the response to treatment with 5-Azacytidine (5-Aza, Vidaza™) in MDS patients using FISH analyses of circulating CD34+ cells. Based on these data we are able to show that sequential molecular-cytogenetic analyses of peripheral blood specimens enable a frequent monitoring of treatment response without the necessity for repeated bone marrow biopsies.