Analysing Circulating CD34+ Cells by FISH Is Representative for the Clone Size in Bone Marrow in MDS Patients Under Different Therapy Modalities

Introduction: In myelodysplastic syndromes (MDS) the knowledge about chromosomal aberrations is important for diagnosis, pathogenesis, prognosis and treatment of cytogenetic subgroups of MDS patients. Usually, chromosomal anomalies in MDS patients are detected in bone marrow (bm) cells by chromosome banding analyses of metaphases. Alternatively or additionally they can be diagnosed by Fluorescence-in-Situ-Hybridization (FISH). We here present a novel method for cytogenetic monitoring of MDS patients from peripheral blood (pb) which is representative for the clone size in bm cells.

Patients and methods: After initial diagnosis by chromosome banding analyses of bm cells, CD34+ cells from pb were enriched by immunomagnetic activated cell sorting (MACS®) and then analysed by FISH. Sixteen MDS patients (7 AML, 4 RAEB-2, 1 RAEB- 1, 3 RCMD, 1 RA) treated with 5-Azacytidine (5-Aza) and 3 patients (all 5q-syndromes) treated with Lenalidomide (Len) were included in this study. FISH analyses of circulating CD34+ cells were performed once a month in every patient during follow-up.

Results: Our data show that, irrespective of FAB-/WHO subtypes or peripheral blast counts, the successful collection of enough CD34+ cells for FISH analyses from pb is possible: in average 80 000 to 400 000 CD34+ cells per sample (20ml pb) were enriched. Morphologic features of CD34+ cells by microscopic analyses were the same in all patients and did not depend on WHO subtypes or on the percentage of peripheral blasts: homogeneous, pure populations of cells with an immature appearance. Additionally, we compared the results of FISH analyses of non-enriched pb, enriched circulating CD34+ cells, non-enriched bm cells and enriched CD34+ bm cells with the results of classical cytogenetic analyses of bm using conventional banding techniques: Constantly, the smallest clone sizes were measured in non-enriched pb, followed by non-enriched bm cells, enriched CD34+ bm cells and enriched circulating CD34+ cells. In all cases the clone sizes of enriched circulating CD34+ cells measured by FISH were close to the results of classical cytogenetic analyses of bm metaphases representing the gold standard of cytogenetic examinations. In our patient cohort 10/16 patients treated with 5-Aza and 3/3 patients treated with Len developed a cytogenetic remission (cyR) detected by FISH analyses of circulating CD34+ cells. CyR was defined according to modified IWG criteria. Because there are no standardized response criteria for FISH analyses available, especially not for FISH from pb, as yet, we definded cyR as a reduction of more than 50% of circulating CD34+cells showing the respective aberration. Remarkably, in all cases with cyR under 5-Aza the hematologic improvement (HI) followed 1–3 cycles later. Under therapy with Len, cyR and HI were measured simultaneously. During follow up HI was still onlasting while there was a cytogenetic relapse detectable by a step-wise increase of aberrant circulating CD34+ cells.

Conclusions: Analysing circulating CD34+ cells by FISH is a reliable method to survey the size of an aberrant clone in pb in high-risk as well as in low-risk MDS patients. It is representative for the clonal situation in the bm and allows a very frequent monitoring during therapy with obviously predictive value. It may reduce the need for repeated bm biopsies. In all cases where a bm biopsy is not possible or unsuccessful or metaphases for conventional banding analyses are not available, our method still allows the measurement of the clone size from pb.