In vitro growth in acute myeloblastic leukaemia: relationship with other clinico‐biological characteristics of the disease

Abstract

The in vitro growth characteristics of a large series of acute myeloid leukaemia (AML) patients and their relationship with other clinical and biological disease characteristics were analysed. Patients with AML were studied, 181 with de novo AML and 45 with secondary AML (24 myelodysplastic syndrome, sAML-MDS, 21 myeloproliferative disorder, sAML-MPD). Leukaemic colony forming units (L-CFU) were assayed by plating peripheral blood (PB) blast cells in methyl-cellulose and using LCM-PHA as stimulant. In each case parallel cultures were made with and without stimulating factors. Plating efficiency (PE) was defined as the number of clusters plus colonies/10(5) cells plated. Autonomous growth (AG) was the number of colonies plus clusters growing without stimulant. The autonomous proliferative index (API) was calculated as the number of clusters + colonies without stimulating factor divided by the number of clusters + colonies with stimulating factor. No significant differences in the PE between de novo and secondary AML were found. Autonomous growth was significantly higher in sAML-MPD. The FAB subtype M3 leukaemias displayed a significantly greater PE and a significantly lower API when compared with the other FAB subgroups (P=0.0002). Upon analysing the relationship with the immunophenotype, only CD33 expression showed a significant relationship with the in vitro growth pattern; CD33+ cases displayed a higher PE (P=0.0002) and AG (P=0.0003) than CD33- cases. When patients were grouped according to the level of rh123 efflux (MDR1) it was observed that cases with >30% elimination showed a higher AG and API than those with <30% (P=0.03). Finally we found that patients with higher API (>0.05) displayed a significantly shorter overall survival as compared with patients with API<0.05 (P=0.04). The in vitro study properties of clonogenic cells produces relevant clinical information of leukaemic cell biology in AML patients.

New Strategies in the Treatment of Acute Myelogenous Leukemia (AML): In Vitro Culture of AML Cells—The Present Use in Experimental Studies and the Possible Importance for Future Therapeutic Approaches

“…Apoptosis is detected for an increasing fraction of cells during in vitro culture of native AML blasts [18], and the proliferative in vitro response of the leukemic cells is then caused by another subset of cells [7][8][9][10][11][12]. The number of apoptotic cells is determined by several factors, for example, a decreased fraction is observed in AML populations that show autocrine proliferation, in the presence of exogenous growth factors, and when an optimal culture medium is used [33,42,[44][45].…”

“…Concise Review [7][8][9][10][11][12]. An understanding of these AML models is therefore essential for the interpretation of experimental data and for the understanding of possibilities and limitations of the new therapeutic approaches.…”

“…Both native AML blasts and AML cell lines can be cultured in medium with inactivated fetal calf or human serum [7][8][9][10][11][12][31][32][33], and the serum then represents a nonstandardized parameter with unidentified mediators that may have effects on AML blasts [34]. For example, cytarabine cannot induce differentiation of the AML cell line ML-1 when cultured in serum-free medium, but the drug induces differentiation of ML-1 cells cultured in serum-containing medium by rendering the cells responsive to differentiation-inducing cytokines in the serum [34].…”

“…Gradient separation (specific density 1.077 g/ml) is widely used for preparation of AML cells [7][8][9][10][11][12], and this method results in populations of >95% purity when samples are derived from selected patients with a high number of AML blasts relative to other low density cells [18]. Additional positive and/or negative selection is commonly used when gradient-separated blasts contain a relatively high percentage of contaminating cells.…”

“…Density gradient separation used alone [7][8][9][10][11][12]18] The procedure is simple, cheap, fast, and suitable Patient selection is required because a high purity is for handling large sample volumes, and it has only achieved only when the sample contains a low number a minor influence on AML blast characteristics.…”