A. J. P. Veerman

Relation between age, immunophenotype and in vitro drug resistance in 395 children with acute lymphoblastic leukemia : Implications for treatment of infants

The prognosis of infant ALL, characterized by a high incidence of the immature CD10 negative B-lineage ALL (proB ALL) is poor. This study aimed to determine the resistance profile of infant ALL cells. In vitro drug resistance was determined by the MTT assay of 395 children with ALL at initial diagnosis: there were 21 infants <1.5 years of which nine <1 year, 284 children aged 1.5–10 years (intermediate age group) and 90 children >10 years. Immunophenotyping resulted in 310 cALL/preB ALL, 69 T-ALL, 15 proB ALL and one unknown cases. The following drugs were tested: daunorubicin, doxorubicin, mitoxantrone, idarubicin (Ida), prednisolone (Pred), dexamethasone (DXM), vincristine (VCR), Asparaginase (Asp), 6-MP, 6-TG, AraC, VM26 and 4-HOO-ifosfamide (Ifos). Infants <1.5 years were significantly more resistant to pred (>500-fold), Asp (11-fold) and VM26 (2.7-fold) but significantly more sensitive to Ara-C (2.3-fold) compared to the intermediate age group. When analyzing infants <1 year of age similar results were found. prob all cells (seven infants <1.5 years; eight children >1.5 years) were significantly more resistant to glucocorticoids, Asp, thiopurines, anthracyclines and Ifos compared to cALL/preB ALL but more sensitive to Ara-C. Cells from children >10 years were significantly more resistant to Pred, DXM, Asp, Ida and 6-MP. T-ALL cells showed a strong resistance to Pred, Asp and VCR and a mild but significant resistance to all other drugs except thiopurines and VM26. We conclude that the poor prognosis of infant ALL is associated with a resistance to glucocorticoids and Asp. However, ALL cells from infants show a relatively high sensitivity to Ara-C which suggests that infants with ALL might benefit from treatment schedules that incorporate more Ara-C than the current treatment protocols.

Relation of cellular drug resistance to long-term clinical outcome in childhood acute lymphoblastic leukaemia

The clinical relevance of cellular drug resistance in children with acute lymphoblastic leukaemia (ALL) is unknown. The relation between in-vitro sensitivity to chemotherapeutic drugs at initial diagnosis and long-term clinical outcome was investigated in 44 children with ALL. The short-term MTT assay was used to assess sensitivity to prednisolone, vincristine, colaspase (asparaginase), daunorubicin, and thioguanine (instead of mercaptopurine which is unstable in vitro). For vincristine and colaspase there was no difference in outcome (probability of continuous complete remission) between sensitive and resistant patients. However, the probability of continuous complete remission was significantly lower in patients with resistant cells than in those with sensitive cells for thioguanine (p less than 0.01), daunorubicin (p less than 0.02), and prednisolone (p less than 0.05). For prednisolone there was a significant worsening of the prognosis (p less than 0.05) from the extremely sensitive patients through an intermediate group to the most resistant group. The prognostic significance of cellular drug resistance was independent of white-blood-cell count, age, sex, and hepatosplenomegaly. Leukaemic cells from boys were more resistant to thioguanine than those from girls. Thus, the short-term highly efficient MTT assay can help to predict long-term response to chemotherapy in childhood ALL.

Patient Stratification Based on Prednisolone-Vincristine-Asparaginase Resistance Profiles in Children With Acute Lymphoblastic Leukemia

PURPOSE To confirm the prognostic value of a drug resistance profile combining prednisolone, vincristine, and l-asparaginase (PVA) cytotoxicity in an independent group of children with acute lymphoblastic leukemia (ALL) treated with a different protocol and analyzed at longer follow-up compared with our previous study of patients treated according to the Dutch Childhood Leukemia Study Group (DCLSG) ALL VII/VIII protocol. PATIENTS AND METHODS Drug resistance profiles were determined in 202 children (aged 1 to 18 years) with newly diagnosed ALL who were treated according to the German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL)-92 protocol. RESULTS At a median follow-up of 6.2 years (range, 4.1 to 9.3 years), the 5-year disease-free survival probability (pDFS) rate +/- SE was 69% +/- 7.0%, 83% +/- 4.4%, and 84% +/- 6.8% for patients with resistant (PVA score 7 to 9), intermediate-sensitive (PVA score 5 to 6), and sensitive (SPVA score 3 to 4) profiles, respectively (sensitive and intermediate-sensitive v resistant, P </=.05). Resistant patients were at increased risk of an early event (nonresponse or relapse within 2.5 years of diagnosis) compared with sensitive and intermediate-sensitive patients (P =.03). The profile did not identify patients at higher risk of late relapse, which was also observed for DCLSG ALL-VII/VIII patients now analyzed at a median of 7.5 years of follow-up (range, 4.4 to 10.8 years). Despite being nondiscriminative for late relapses, the resistant profile was still the strongest prognostic factor for COALL-92 patients in a multivariate analysis including known risk factors (P =.07). CONCLUSION Drug resistance profiles identify patients at higher risk of early treatment failures and may, therefore, be used to improve risk-group stratification of children with ALL.

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.

Effects of Chemotherapy on Neurocognitive Function in Children With Acute Lymphoblastic Leukemia: A Critical Review of the Literature

Chemotherapy‐only treatment has increasingly become the standard of treatment for childhood acute lymphoblastic leukemia (ALL). The objective of this review is to assess the present state of knowledge of the neurocognitive effects of central nervous system (CNS)‐directed chemotherapy in children with ALL, and to formulate directions for future research. We performed a review of studies published since 1997, that included an ALL group treated with chemotherapy only and a control group. Twenty‐one studies met our inclusion criteria. There is evidence of subtle long‐term neurocognitive deficits survivors of childhood ALL after treatment with chemotherapy only. These involve mainly processes of attention and of executive functioning, while global intellectual function is relatively preserved. Young age at diagnosis and female sex emerged as risk factors. Pediatr Blood Cancer 2009;52:447–454.

BFM-oriented treatment for children with acute lymphoblastic leukemia without cranial irradiation and treatment reduction for standard risk patients: results of DCLSG protocol ALL-8 (1991-1996).

Modern treatment strategies, consisting of intensive chemotherapy and cranial irradiation, have remarkably improved the prognosis for children with acute lymphoblastic leukemia. However, patients with a potential for cure are at risk of severe acute and late adverse effects of treatment. Furthermore, in 25–30% of patients treatment still fails. The objectives of the DCLSG study ALL 8 were to decrease the toxicity and to increase the effectivity of BFM-oriented treatment. Decrease of toxicity was aimed at by confirmation of the results of the previous DCLSG study ALL-7, showing that the majority (94%) of children with ALL can succesfully be treated with BFM-oriented therapy without cranial irradiation, and by reduction of treatment for standard risk (SRG) patients. To increase the cure rate in medium risk (MRG) patients the efficacy of high doses of intravenous 6-mercaptopurine (HD-6MP) during protocol M and in SRG patients the efficacy of high doses of L-asparaginase (HD-L-ASP) during maintenance treatment was studied in randomized studies. Patient stratification and treatment were identical to protocol ALL-BFM90, with the following differences: no prophylactic cranial irradiation, SRG patients received only phase 1 of protocol I. Four hundred and sixty-seven patients entered the protocol: 170 SRG, 241 MRG and 56 HRG patients. The 5 years event-free survival rate for all patients was 73% (s.e. 2%); for SRG, MRG and HRG patients 85% (s.e. 3%), 73% (s.e. 3%) and 39% (s.e. 7%), respectively. In patients >1 year of age at diagnosis unfavorable prognostic factors were male sex, >25% blasts in the bone marrow at day 15 and initial white blood cell count (WBC) >50 × 109/l. The cumulative risk of CNS relapse rate was 5% (s.e. 1%) at 5 years. These results confirm that the omission of cranial irradiation in BFM-oriented treatment does not jeopardize the overall good treatment results, nor does early reduction of chemotherapy in SRG patients. No benefit was observed from treatment intensification with HD-L-ASP in SRG patients, nor from HD-6MP in MRG patients.

Chemotherapy and attentional dysfunction in survivors of childhood acute lymphoblastic leukemia: Effect of treatment intensity

Central nervous system (CNS) directed chemotherapy is replacing prophylactic cranial irradiation in treatment protocols for childhood acute lymphoblastic leukemia (ALL), mainly to reduce long‐term neuropsychological sequelae. We evaluated the effects of chemotherapy on attentional function in survivors of ALL and examined whether possible deficits are related to treatment intensity.

In vitro drug sensitivity of normal peripheral blood lymphocytes and childhood leukaemic cells from bone marrow and peripheral blood.

In vitro drug sensitivity of leukaemic cells might be influenced by the contamination of such a sample with non-malignant cells and the sample source. To study this, sensitivity of normal peripheral blood (PB) lymphocytes to a number of cytostatic drugs was assessed with the MTT assay. We compared this sensitivity with the drug sensitivity of leukaemic cells of 38 children with acute lymphoblastic leukaemia. We also studied a possible differential sensitivity of leukaemic cells from bone marrow (BM) and PB. The following drugs were used: Prednisolone, dexamethasone, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, vincristine, vindesine, daunorubicin, doxorubicin, mafosfamide (Maf), 4-hydroperoxy-ifosfamide, teniposide, mitoxantrone, L-asparaginase, methotrexate and mustine. Normal PB lymphocytes were significantly more resistant to all drugs tested, except to Maf. Leukaemic BM and PB cells from 38 patients (unpaired samples) showed no significant differences in sensitivity to any of the drugs. Moreover, in 11 of 12 children with acute leukaemia of whom we investigated simultaneously obtained BM and PB (paired samples), their leukaemic BM and PB cells showed comparable drug sensitivity profiles. In one patient the BM cells were more sensitive to most drugs than those from the PB, but the actual differences in sensitivity were small. We conclude that the contamination of a leukaemic sample with normal PB lymphocytes will influence the results of the MTT assay. The source of the leukaemic sample, BM or PB, does not significantly influence the assay results.

Comparison of the rapid automated MTT-assay with a dye exclusion assay for chemosensitivity testing in childhood leukaemia

Comparison of the rapid automated MTT-assay with a dye exclusion assay for chemosensitivity testing in childhood leukaemia

Causes of death - other than progressive leukemia - in childhood acute lymphoblastic (ALL) and myeloid leukemia (AML): the Dutch Childhood Oncology Group experience

We analyzed causes of death, other than resistant disease or relapse, in 875 children with acute lymphoblastic leukemia (ALL) and 229 with acute myeloid leukemia (AML), treated on three different Dutch Childhood Oncology Group (DCOG) ALL and three AML protocols. Overall, 23 (2.6%) ALL and 44 (19.2%) AML patients died. Early death (ED, before remission was reached) occurred in nine ALL (1%) and thirty AML (13.1.%) patients, including three and ten deaths before treatment was initiated. Chemotherapy-related mortality in remission (CRM) occurred in nine ALL (1.1%) and eight AML (4.4%) patients. For ALL, both ED and CRM declined over time, although this was not statistically significant. For AML a decrease in ED was observed (from 26% to approximately 10%), but counter-balanced by an increase in CRM (from 3 to 8%), maybe related to the scheduling of intensification blocks in AML-92/94. Including transplant-related mortality, death in CR rates in AML increased from 3 to 15% in the last study. The main cause of ED was hemorrhage, often associated with hyperleucocytosis, and infection for CRM. We conclude that mortality dropped favorably in ALL, but not in AML. Especially for AML, effective but less toxic therapy and better supportive care guidelines need to be developed.