Abnormal karyotype in bone marrow (BM) cells is detected in 30–70% of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias with myelodysplasia-related changes (AML). The same cytogenetic abnormalities are found in isolated CD34+ hematopoetic progenitor cells (HPCs) of these patients. According to recent studies cytogenetic abnormalities are described in 10–70% MDS and AML patients in BM derived mesenchymal stromal cells (MSCs) [Flores-Figueroa et al. 2008, Blau et al. 2011]. These abnormalities can be clonal and nonclonal (spontaneous). The goal of the study was to characterize and compare the cytogenetic changes in BM derived MSCs and in CD34+ HPCs isolated from BM and peripheral blood (PB) in MDS and AML patients.
The data from 35 patients is presented: 6 pts with AML (1 pt with therapy-related AML), 29 pts with MDS (refractory anemia − 4 pts, refractory cytopenia with multilineage dysplasia − 13 pts, refractory anemia with ringed sideroblasts − 2 pts, refractory anemia with excess blasts − 7 pts, 5q-syndrome – 3 pts). Median age was 60 years (range 19 to 77). Patients' assignment to different groups was made according to the 2008 World Health Organization (WHO) classification. Cytogenetic analysis was performed using G-banding and FISH. MSCs were derived from bone marrow mononuclear cells then plated in 75 cm3flacks and harvested after 2–5 passages. CD34+ HPCs were collected from BM and PB by magnetic separation with anti-CD34 MicroBead Kit human (Miltenyi Biotec).
BM karyotype was normal in 17 pts, cytogenetic abnormalities were found in 18 pts − 4 AML and 14 MDS: in 9 pts (2 AML/7 MDS) – isolated del(5q), monosomy 7, i(14), inv(3) or trisomy 8; in 8 pts (2 AMl/6 MDS) – two or more abnormalities; and 1 pt displayed only constitutional abnormality (inv(9)(p13q21)). It is worth to note that routine cytogenetic analysis didn't present any mitosis in 3 MDS pts however FISH analysis revealed del(5) and del(7) simultaneously in one of these pts, trisomy 8 in one pt and normal karyotype in another one pt. Cytogenetic analysis of MSCs was carried out in 23 pts – 4 AML and 19 MDS pts. BM karyotype was normal in 10 pts and abnormal in 13 pts. Karyotype in MSCs was normal in all AML pts and in 16 MDS pts. There were cytogenetic changes in 3 MDS pts: in 1 pt - constitutional inversion (inv9(p13q21)), in 1 pt - nonclonal translocation and constitutional inversion (46XY,t(2;22)(p10;q11),inv(9)(p13q21)/46XY,inv(9)(p13q21)) and in 1 pt - clonal abnormalities (46XY,add(2q)/46XY). Patient with clonal cytogenetic abnormalities in MSCs had complex karyotype in BM (45–46XY,−5,−13,der(19),add(q13?or p13?),−20,+marx2,+mar del(13q21)?/45–46XY idem, +dmin/46XY), but these changes were different. Constitutional inversion was confirmed in both cases by cytogenetic analysis in PB lymphocytes. FISH analysis was performed in CD 34+ HPCs magnetically isolated from BM and PB in 24 pts. Fourteen of these pts had normal BM karyotype and we confirmed that using FISH with LSI (5q33-q34), LSI (7q31)/CEP 7, CEP 8 (Vysis). We used the same DNA probes for evaluation of CD34+ HPCs from BM and PB in pts with normal BM karyotype. We used FISH in 10 pts with ascertained cytogenetic abnormalities in BM to confirm these findings with DNA probes LSI (5q33-q34), LSI (7q31)/CEP 7, CEP 8, LSI AML1/ETO, CEP X/CEP Y, LSI (20q12) (Vysis). FISH analysis didn't reveal any aberration in BM and PB CD34+ HPCs in patients with normal BM karyotype. In pts with distinguished abnormalities in BM the same pattern was demonstrated by FISH in CD34+ HPCs. We counted the percent of abnormal nuclei per 200. The means of percentages by FISH of BM, BM CD 34+ HPCs and PB CD34+ HPCs didn't differ and constituted 65.8%, 73.1% and 74.8% respectively.
Cytogenetic analysis in MSCs revealed aberrations only in MDS patients (3 from 16). No cytogenetic abnormalities in MSCs in AML pts was found. Karyotype in MSCs didn't coincide with BM karyotype in the same patients. Hematopoetic progenitor cells from BM and PB of MDS and AML patients displayed the same cytogenetic abnormalities as the full population of bone marrow cells. The data shows that hematopoetic and mesenchymal precursors in MDS and AML are cytogenetically distinct.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.