A. H. Loonen

Different types of non-P-glycoprotein mediated multiple drug resistance in children with relapsed acute lymphoblastic leukaemia

Although cellular drug resistance is considered to be an important cause of the poor prognosis of children with relapsed acute lymphoblastic leukaemia (ALL), the knowledge of drug resistance in these patients is very limited. Different aspects of drug resistance were studied in 17 children with relapsed ALL. The in vitro sensitivity profile was determined using the MTT assay. Cells from relapsed children were significantly more resistant to 6-thioguanine, prednisolone, cytosine arabinoside, daunorubicin (DNR), mustine-HCl and mafosfamide but not to L-asparaginase and vincristine (VCR) than cells from 41 children with ALL at initial diagnosis. Some relapsed patients showed a general drug resistance while others were resistant to only 1-3 drugs. The relevance of the multidrug resistance (MDR) model was analysed: In all DNR- and VCR resistant cases a co-resistance to drugs not involved in the MDR model was found. P-glycoprotein was not detected in any of 28 untreated and 14 relapsed samples tested. VCR- and DNR accumulation in the most resistant cells were not lower than in sensitive cells. Resistance modifiers did not potentiate the cytotoxicity of VCR and DNR. We conclude that resistance to anthracyclines and vinca alkaloids in childhood relapsed ALL is not due to P-glycoprotein mediated MDR. Different types of drug resistance varying from a resistance to only one drug to a general chemoresistance, can be detected in children with relapsed ALL. VCR and L-asparaginase seemed to be only infrequently involved in drug resistance. Knowledge of drug resistance might lead to more effective and less toxic therapies for children with relapsed ALL.

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.

Incidence of additional genetic changes in the TEL and AML1 genes in DCOG and COALL-treated t(12;21)-positive pediatric ALL, and their relation with drug sensitivity and clinical outcome

Clinical heterogeneity within t(12;21) or TEL/AML1-positive ALL (25% of childhood common/preB ALL) indicates that additional genetic changes might contribute to outcome. We studied the relation between additional genetic changes in TEL(ETV6) and AML1(RUNX1) (FISH), drug sensitivity (MTT assay) and clinical outcome in 143 DCOG and COALL-treated t(12;21)-positive ALL patients. Additional genetic changes in TEL and AML1 were present in 83% of the patients, and consisted of (partial) deletion of the second TEL gene (70%), an extra AML1 gene (23%) or an extra der(21)t(12;21) (10%). More than one additional change was observed in 20%. Disease-free survival (pDFS) of DCOG patients without additional genetic changes (4 years pDFS±s.e. 53±17%) and of those with an extra der(21)t(12;21) (60±22%) is poorer than that of compared to patients with other additional genetic changes in TEL or AML1 (79±6%; P-trend=0.02). This was mainly due to the occurrence of early relapses within 2.5 years after the first diagnosis. Similar observations were found in the COALL cohort, albeit not significant owing to limited follow-up. Multivariate analysis including age, WBC and genetic abnormalities in TEL and/or AML1 showed that especially, in vitro resistance to prednisolone (hazard ratio 5.78, 95% CI 1.45–23.0; P=0.01) is an independent prognostic factor in DCOG- and COALL-treated t(12;21)-positive ALL.

Cell proliferation is related to in vitro drug resistance in childhood acute leukaemia.

Bone marrow and peripheral blood samples from 362 patients with acute lymphoblastic leukaemia (ALL) proliferating cell and 90 patients with acute myeloid leukaemia (AML) were analysed for S-phase fractions, Ki67 antigen, and proliferating cell nuclear antigen expression. The S-phase fractions were correlated with in vitro drug resistance to 15 different anticancer agents. Leukaemia cells isolated from bone marrow had higher S-phase fractions than leukaemia cells isolated from peripheral blood (in initial ALL, median values resp. 6.9 and 2.7%, in initial AML resp. 5.3 and 1.3%; both P<0.01). Relapse ALL samples derived from bone marrow showed increased S-phase fractions (median 9.9%) compared with initial ALL samples (median 6.9%; P<0.01). ALL samples obtained at initial diagnosis showed higher S-phase fractions (median 6.9%) and higher Ki67 expression (median 30%) than initial AML samples (median resp. 5.3 and 14%; both P<0.05). The S-phase fractions were not related to white blood cell count, age, or gender. Within initial ALL, the S-phase fraction correlated significantly but modestly strong (ρ=0.3–0.5; P<0.05) with sensitivity to antimetabolites (cytarabine, mercaptopurine, thioguanine), L-asparaginase, teniposide, and vincristine. Similar results were found within subgroups of initial ALL (nonhyperdiploid and common/precursor-B-lineage ALL). In relapsed ALL and AML such correlations were not found. In conclusion, cell proliferation differs between leukaemia subgroups and increased proliferation is associated with increased in vitro sensitivity to several anticancer agents in initial ALL.

In vitro drug resistance and prognostic impact of p16INK4A/P15INK4B deletions in childhood T‐cell acute lymphoblastic leukaemia

p16 gene deletions are present in about 70% of primary paediatric T‐cell acute lymphoblastic leukaemia (T‐ALL) and 20% of common/precursor B‐cell ALL cases. It is not clear what the impact of the frequent p16 deletions is within the subgroup of T‐lineage ALL. We studied the relationship between p16/p19ARF deletions, using fluorescence in situ hybridization, and in vitro drug resistance and prognosis in childhood T‐ALL at diagnosis. The cellular drug resistance was measured with the methyl thiazol tetrazoliumbromide assay using a panel of drugs and the thymidylate synthase inhibition assay for methotrexate. There was a complete overlap of individual LC50 values of p16 gene homozygously deleted and p16 germ‐line cases for most of the nine classes of drugs tested. The only difference was for dexamethasone: the p16‐deleted group was more sensitive than the germ‐line p16 group (P = 0·030). The homozygously deleted p16 T‐ALL patients (n = 34) treated with the modern multiagent chemotherapy schemes of the Dutch Childhood Leukaemia Study Group ALL‐VII/‐VIII or Co‐operative ALL‐92/‐97 protocols have a significantly lower 5‐year disease‐free survival (DFS) than germ‐line p16 T‐ALL (n = 25) (65·1 ± 9·1% vs. 95·5 ± 4·4%, Plog rank= 0·021). Hence, this study identifies a subpopulation of primary childhood T‐ALL that appears to have an extremely high DFS. However, the observed differences in outcome do not seem to be related to intrinsic resistance for the tested drugs.

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].

Cellular Drug Sensitivity of Immunophenotypic Subgroups of Childhood Acute Lymphoblastic Leukemia

The immunophenotype is one of the prognostic factors in children with acute lymphoblastic leukemia (ALL) (Crist et al. 1984, 1985, 1989; Sallan et al. 1980; Greaves et al. 1981; Pui et al. 1986). B-cell ALL (B-ALL) cases have the worst prognosis and patients with B-cell precursor ALL the most favorable prognosis. Precursor B-ALL can be subdivided into three groups: pro-B-ALL (CD10−, cytoplasmic μ chain− [cμ−]; common ALL (c-ALL) (CD10+/cμ−) and pre-B-ALL (CD10+ or −/cμ+). The small group of patients whose cells lack the common ALL (c-ALL) antigen characteristic of the earliest stage of B-cell differentiation (pro-B-ALL) show poorer responses by comparison with the c-ALL+ cases. In some studies the pre-B-ALL cases did worse than the cALL cases, but this is probably due to a small subset of patients with a specific chromosomal abnormality on their leukemic cells (Crist et al. 1990). Also, patients with T-cell ALL (T-ALL) have an unfavorable prognosis. Some of these findings are still controversial because in some studies the immunophenotype is related to other clinical and biological features like white blood cell (WBC) count and organomegaly. Also, when effective treatments are used, the prognostic value of immunophenotype could be diminished (Borowitz 1990; Poplack and Reaman 1988; Miller 1988).

Correlation of In Vitro Drug Resistance Assessed by the MTT Assay with Long Term Clinical Outcome in Childhood Acute Lymphoblastic Leukemia

Clinical forms and mechanisms of drug resistance in childhood acute lymphoblastic leukemia (ALL) are poorly characterized. No studies have been reported on the relationship between initial chemosensitivity and final clinical outcome in ALL patients, most probably because of the lack of suitable in vitro assays. We recently reviewed the use of different assays in leukemic patients [1]: Clonogenic assays are not suitable for testing samples from patients with ALL. The short-term dye exclusion or differential staining cytotoxicity (DiSC) assay showed favorable clinical correlations and can be used in ALL patients but is too laborious for large-scale patient studies. We and others adapted the efficient MTTassay to study chemosensitivity in samples of leukemic patients and showed that the results of MTT and DiSC assays are comparable [1]. The MTT assay was found to predict initial response to chemotherapy in AML patients and to detect acquired drug resistance in ALL and AML patients. In this report we used the MTT assay to study the relation between drug sensitivity at initial diagnosis and the long-term clinical outcome in children with ALL.