Dieuwke R. Huismans

Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay

The methyl-thiazol-tetrazolium (MTT) assay is a drug resistance assay which cannot discriminate between malignant and non-malignant cells. We previously reported that samples with > or = 80% leukaemic cells at the start of culture give similar results in the MTT assay and the differential staining cytotoxicity assay, in which a discrimination between malignant and non-malignant cells can be made. However, the percentage of leukaemic cells may change during culture, which might affect the results of the MTT assay. We studied 106 untreated childhood acute lymphoblastic leukemia (ALL) samples with > or = 80% leukaemic cells at the start of culture. This percentage decreased below 80% in 28%, and below 70% in 13%, of the samples after 4 days of culture. A decrease below 70% occurred more often in case of 80-89% leukaemic cells (9/29) than in case of > or = 90% leukaemic cells at the start of culture (5/77, P = 0.0009). Samples with < 70% leukaemic cells after culture were significantly more resistant to 6 out of 13 drugs, and showed a trend towards being more resistant to two more drugs, than samples with > or = 80% leukaemic cells. No such differences were seen between samples with 70-79% and samples with > or = 80% leukaemic cells after culture. We next studied in another 30 ALL samples whether contaminating mononuclear cells could be removed by using immunoamagnetic beads. Using a beads to target cell ratio of 10:1, the percentage of leukaemic cells increased from mean 72% (s.d. 9.3%) to mean 87% (s.d. 6.7%), with an absolute increase of 2-35%. The recovery of leukaemic cells was mean 82.1% (range 56-100%, s.d. 14.0%). The procedure itself did not influence the results of the MTT assay in three samples containing only leukaemic cells. We conclude that it is important to determine the percentage of leukaemic cells at the start and at the end of the MTT assay and similar drug resistance assays. Contaminating mononuclear cells can be successfully removed from ALL samples using immunomagnetic beads. This approach may increase the number of leukaemic samples which can be evaluated for cellular drug resistance with the MTT assay or a similar cell culture drug resistance assay.

In vitro resistance to cytosine arabinoside, not to daunorubicin, is associated with the risk of relapse in de novo acute myeloid leukaemia.

The efficacy of chemotherapy in acute myeloid leukaemia (AML) is limited by clinical drug resistance. We determined in vitro resistance to cytosine arabinoside (ARA‐C), daunorubicin (DNR), mitoxantrone (MITOX), m‐amsacrine (AMSA) and etoposide (VP16) in 49 adults with de novo AML using the MTT assay. Results showed that non‐ responders to chemotherapy were, in vitro, 2.9‐fold more resistant to DNR, but not more resistant to ARA‐C, compared to complete responders. However, complete responders who were in vitro resistant to ARA‐C had a 4‐fold higher risk of relapse (95% CI 1.3–12.5‐fold) compared to complete responders in vitro sensitive to ARA‐C. With a mean follow‐up of 12 months the probability of continuous complete remission (CCR) for patients in vitro sensitive to ARA‐C was 61% at 34 months (95% CI 28–82%), whereas all patients in vitro resistant to ARA‐C relapsed within 18 months from diagnosis. This difference appeared to be independent of other clinical features such as sex, age, white blood cell count, FAB classification, and CD34 expression. In vitro resistance to DNR was not related to the probability of CCR. We conclude that in vitro drug resistance assessed with the MTT assay appears to be associated with short‐ and long‐term clinical outcome in AML. Confirmatory studies comprising a sufficient number of patients for multivariate analyses should prove whether in vitro resistance to ARA‐C will appear to be an independent risk factor.

Cytotoxic Effects of Vitamin A in Combination with Vincristine, Daunorubicin and 6‐Thioguanine upon Cells from Lymphoblastic Leukemic Patients

We studied whether isotretinoin potentiated the effects of vincristine (VCR), daunorubicin (DNR), and 6‐thioguanine (6‐TG) against cells obtained from 24 patients with acute lymphoblastic leukemia (ALL). Treatment with 5 μg/ml isotretinoin alone resulted in a leukemic cell survival of 82%± 28.1%. So isotretinoin is toxic to ALL cells. Dose‐response curves were obtained for VCR, DNR and 6‐TG in the presence and absence of isotretinoin Isotretinoin showed additive leukemic cell kills in combination with VCR and DNR, When corrected for cell kill by isotretinoin alone, it appeared that isotretinoin did not significantly enhance leukemic cell kills by VCR, DNR and 6‐TG. No differences were found between samples from patients at initial diagnosis and at relapse with respect to cell kill by isotretinoin alone and with respect to a possible synergistic effect of isotretinoin and the cytostatic drugs. It is concluded that isotretinoin has additive antileukemic effects in combination with VCR or DNR. However, isotretinoin does not potentiate the antileukemic effects of VCR, DNR and 6‐TG against leukemic cells obtained from patients with ALL.

Cytostatic Drug Resistance in Childhood Relapsed Acute Lymphoblastic Leukemia

The treatment of childhood acute lymphoblastic leukemia (ALL) has improved greatly in recent decades. At present, dependent on the risk group, 82% to 100% of the children with ALL will achieve a complete remission (CR) when treated with combination chemotherapy [1]. Overall, about two-thirds will remain in continuous complete remission (CCR). Despite of the current available effective cytostatic drugs, still one-third will get a relapse. Patients suffering from a relapse do have a poor prognosis in which drug resistance of leukemic cells is supposed to playa major role [2].

Motility of Leukemic Cells in Collagen Gel Related to Immunological Phenotype in Childhood Acute Lymphoblastic Leukemia

Motility of leukemic cells was measured in a three-dimensional collagen matrix assay. Leukemic cells from 16 children with acute lymphoblastic leukemia (ALL) and normal peripheral blood lymphocytes (NPBL) from 6 healthy volunteers, were allowed to migrate into this collagen matrix for 48 h at 37 degrees C. NPBL migrated much further (300-600 micron) than leukemic cells (0-200 micron). Among the leukemic cases, only common ALL and one case of null ALL showed some migration (0-200 micron). T-All cells did not migrate at all under the circumstances of this experiment.

Capping of Leukemic Cells with Monoclonal Anti-HLA-A, B, C Related to Prognosis in Childhood Acute Lymphoblastic Leukemia

Capping of leukemic cells with a monoclonal antibody against HLA A,B,C determinants was studied in 53 cases of childhood acute lymphoblastic leukemia (ALL). Determination of the percentages of capped cells after different times of incubation with anti-HLA A,B,C show that T ALL and common ALL do have quite different kinetics of HLA capping. In T ALL all cases reach levels of percentage of capped cells above 30%, in common ALL only 11 of 31 cases cap well. Dilution of the antiserum in 6 common ALL cases results in an increase of capped cells, but the original kinetics of the common ALL capping remain. ALL cases with capping curves above 30% have a worse prognosis (shorter continuous complete remission) than cases with capping curves below 30% in the total group as well as in the non-high-risk group.

Significance of number of HLA determinants in HLA capping in childhood acute lymphoblastic leukemia.

The density of HLA determinants on the cell surface was studied in relation to HLA capping in childhood acute lymphoblastic leukemia (ALL). Analysis was performed with flow cytometry (FACS) and a subjective labeling score. These two methods gave comparable results. In T-ALL high percentages of capped cells were related to low numbers of HLA determinants. In c-ALL we found the opposite. This study shows that capping capacity and number of HLA determinants in childhood ALL are inversely related.