FISH-Analyses of Cirulating CD34+ Cells in MDS-Patients - A Suitable Method To Measure and Predict Response to 5-Azacytidine.

Abstract

In this study we present a novel method to measure response to treatment (here 5-Azacytidine (5-Aza)) that is less invasive and can be more frequently performed than bone marrow biopsy (bmb). Usually chromosomal aberrations are proven in bone marrow cells by classical cytogenetics. Most of them are suitable for a detection by FISH-techniques. In our study we have followed chromosomal anomalies in circulating CD34+ cells from peripheral blood in patients (pts) with MDS treated with 5-Aza. We could observe that the size of an abnormal clone in general is larger in circulating CD34+ cells as compared to non-enriched bone marrow cells. The percentage of circulating CD34+ cells showing the respective chromosomal anomaly is decreasing in response to therapy and therefore can be used as an indicator for response. Fourteen pts receiving 5-Aza are included in our ongoing study as yet. Thirteen are male, 1 is female, the average age at the time of diagnosis is 67 years (49–81 years). Starting 5-Aza 7 pts were diagnosed as AML, 3 as RAEB-2, 1 as RAEB-1, 1 as RCMD, 1 as CMML, 1 as RA. In all pts initial karyotyping of bone marrow cells was performed. There was 1 pt with normal karyotype, 5 pts (35.7%) showed a complex aberrant karyotype. The other pts had 1–2 chromosomal anomalies (+8, −7/7q-, 20q-, 5q-). All pts with clonal anomalies (13/14) were informative for further FISH-analyses. In average pts received 4 cycles 5-Aza (range 2–8 cycles). Before every therapy cycle circulating CD34+ cells from peripheral blood were enriched by immunomagnetic cell sorting (MACS) and analysed by FISH. Modified IWG-criteria were used to evaluate hematologic response. At present 10 pts could be analysed completely. All of them showed hematologic and/or cytogenetic response to therapy: 4 pts achieved complete remission (CR), 2 of them after allogeneic bone marrow transplantation. Remarkably, 3 out of 4 evaluable pts with complex aberrant karyotypes responded to 5-Aza. One further pt reached partial remission (PR), one stayed in stable disease (SD). Two pts showed at least a marrow response (PR/CR) without developing other improvements. Two further pts achieved at least hematologic improvement (HI). Remarkably, 9 out of 10 pts showed chromosomal aberrations suitable for FISH. Seven (77.8%) developed partial cytogenetic remission (cyPR) measured in circulating CD34+ cells. Six out of 7 pts with cyPR reached HI and/or CR. One was lost for follw up. CyPR preceded hematologic response as well as progression by 4–8 weeks. We conclude that 5-Aza leads to good results even in high-risk MDS-pts with complex aberrant karyotypes. Our data thus imply that analysing circulating CD34+ cells from peripheral blood by FISH techniques is a suitable method to monitor and to predict response to therapy with 5-Aza and possibly other agents in MDS-pts. It might be the more sensitive method to measure the aberrant cell clone than analysing non-enriched bone marrow cells. It could preserve pts from repeated bmb and allows a very frequent monitoring of therapy response.