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Experiments were performed to establish optimal conditions for specific and non-specific lymphocyte transformation in vitro, in cultures containing 1–3 × 105 human lymphocytes. The stimulation as measured by 14C-thymidine incorporation was evaluated when using various methods of lymphocyte purification and different numbers of contaminating erythrocytes, different kinds of tubes, serum from different sources, and varying PHA concentrations. Furthermore, the dependence of the 14C-thymidine incorporation on the thymidine concentration in the medium and on the total number of lymphocytes present per culture was determined. Two methods of harvesting the cells to measure the 14C-thymidine incorporation were compared. On the basis of this analysis, conditions for maximal sensitivity and reproducibility could be defined in measuring lymphocyte reactivity to PHA, allogeneic lymphocytes and antigens in a miniaturized culture system.

FLT3 is a receptor tyrosine kinase involved in the proliferation and differentiation of hematopoietic stem cells. FLT3 internal tandem duplications (FLT3/ITDs) are reported in acute myeloid leukemia (AML) and predict poor clinical outcome. We found FLT3/ITDs in 11.5% of 234 children with de novo AML. FLT3/ITD-positive patients were significantly older and had higher percentages of normal cytogenetic findings or French-American-British (FAB) classification M1/M2 and lower percentages of 11q23 abnormalities or FAB MS. FLT3/ITD-positive patients had lower remission induction rates (70% vs 88%; P = .01) and lower 5-year probability rates of event-free survival (pEF) (29% vs 46%; P = .0046) and overall survival (32% vs 58%; P = .037). Patients with high ratios (higher than the median) between mutant and wild-type FLT3 had significantly worse 2-year EFS rates than FLT3/ITD-negative patients (pEFS 20% vs 61%, P = .037), whereas patients with ratios lower than the median did not (pEFS 44% vs 61%; P = .26). FLT3/ITD was the strongest independent predictor for pEFS, with an increase in relative risk for an event of 1.92 (P = .01). Using an MTT (methyl-thiazol-tetrazolium)-based assay, we studied cellular drug resistance on 15 FLT3/ITD-positive and 125 FLT3/ITD-negative AML samples, but we found no differences in cellular drug resistance that could explain the, poor outcomes in FLT3/ITD-positive patients, We conclude that FLT3/ITD is less common in pediatric than in adult AML. FLT3/ITD is a strong and independent adverse prognostic factor, and high ratios between mutant and WT-FLT3 further compromise prognosis. However, poor outcomes in FLT3/ITD-positive patients could not be attributed to increased in vitro cellular drug resistance.

Specific cytogenetic abnormalities predict prognosis in childhood acute myeloid leukemia (AML). However, it is unknown why they are predictive and whether this is related to drug resistance. We previously reported that Down syndrome (DS) AML was associated with favorable resistance profiles. Here, we successfully analyzed drug resistance and (cyto-) genetic abnormalities of 109 untreated childhood AML samples using the 4-day total cell-kill methyl-thiazol tetrazolium (MTT) assay. Patients were classified according to the genetic abnormalities in the leukemic cells: t(8;21), inv(16), t(15;17), t(9;11), other 11q23 translocations, abnormalities of chromosome 5/7, trisomy 8 alone, normal karyotype, single random, and multiple (defined as 2 or more) abnormalities. The DS AML samples were excluded from the subgroup analysis. Samples with chromosome 5/7 abnormalities were median 3.9-fold (P = .01) more resistant to cytarabine than other AML samples. The t(9;11) samples were more sensitive to cytarabine (median 2.9-fold, P= .002), etoposide (13.1-fold, P = .001), the anthracyclines (2.9- to 8.0-fold, P < .01), and 2-chlorodeoxyadenosine (10.0-fold, P= .002) than other AML samples. The trisomy 8 and t(15;17) groups were too small for meaningful analysis. All other genetic subgroups did not show specific resistance profiles. Overall, we found no differences in drug resistance in samples taken at diagnosis between patients remaining in continuous complete remission (CCR) versus the refractory/relapsed patients. Within several genetic subgroups, however, relapsed/refractory patients were more cytarabine resistant when compared with patients remaining in CCR, but numbers were small and the results were not significant. We conclude that some, but not all, cytogenetic subgroups in childhood AML display specific drug-resistance profiles.

The difference in the current cure rates between adult and childhood acute lymphoblastic leukaemia (ALL) may be caused by differences in drug resistance. Earlier studies showed that in vitro cellular drug resistance is a strong independent adverse risk factor in childhood ALL. Knowledge about cellular drug resistance in adult ALL is still limited. The present study compared the in vitro drug resistance profiles of 23 adult ALL patients with that of 395 childhood ALL patients. The lymphoblasts were tested by the MTT assay. The group of adult ALL samples was significantly more resistant to cytosine arabinoside, L-asparaginase, daunorubicin, dexamethasone and prednisolone. The resistance ratio (RR) was highest for prednisolone (31.7-fold) followed by dexamethasone (6.9-fold), L-asparaginase (6. 1-fold), cytosine arabinoside (2.9-fold), daunorubicin (2.5-fold) and vincristine (2.2-fold). Lymphoblasts from adult patients were not more resistant to mercaptopurine, thioguanine, 4-HOO-ifosfamide, mitoxantrone and teniposide. There were no significant differences in drug resistance between adult T-cell (T-) ALL (n = 11) and adult common/pre-B-cell (B-) ALL (n = 10). Additionally, adult T-ALL did not differ from childhood T-ALL (n = 69). There were significant differences between adult common/pre-B-ALL and childhood common/pre-B-ALL (n = 310) for prednisolone (RR = 302, P = 0.008), dexamethasone (RR = 20.9, P = 0.017) and daunorubicin (RR = 2.7, P = 0.009). Lymphoblasts from adults proved to be relatively resistant to drugs commonly used in therapy. This might contribute to the difference in outcome between children and adults with ALL.

The efficacy of chemotherapy in acute myeloid leukaemia (AML) is limited by clinical drug resistance. We determined in vitro resistance to cytosine arabinoside (ARAC), daunorubicin (DNR), mitoxantrone (MITOX), m-amsacrine (AMSA) and etoposide (VP16) in 49 adults with de novo AML using the MTT assay. Results showed that nonresponders 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.

Daunorubicin (DNR) is a major front-line drug in the treatment of childhood acute lymphoblastic leukaemia (ALL). Previously, we showed that in vitro resistance to DNR at diagnosis is related to a poor long-term clinical outcome in childhood ALL and that relapsed ALL samples are more resistant to DNR than untreated ALL samples. In cell line studies, idarubicin (IDR), aclarubicin (ACR) and mitoxantrone (MIT) showed a (partial) lack of cross-resistance to the conventional anthracyclines DNR and doxorubicin (DOX), but clinical studies in childhood ALL have been inconclusive about the suggested lack of cross-resistance. In the present study we determined the in vitro cross-resistance pattern between DNR, DOX, IDR, ACR and MIT in 48 untreated and 39 relapsed samples from children with ALL using the MTT assay. The relapsed ALL group was about twice as resistant to DNR, DOX, IDR, ACR and MTT as the untreated ALL group. Thus, resistance developed to all five drugs. We found a significant cross-resistance between DNR, DOX, IDR, ACR and MIT, although in some individual cases in vitro anthracycline cross-resistance was less pronounced. We conclude that IDR, ACR and MIT cannot circumvent in vitro resistance to DNR in childhood ALL. Clinical studies may still prove whether IDR, ACR or MIT has a more favourable toxicity profile than DNR.

Ecto-5'-nucleotidase (ecto-5'NT) catalyzes the extracellular dephosphorylation of nucleotides like IMP. Cytoplasmic 5'NT (cyto-5'NT) and non-specific (e.g. acid- and alkaline) phosphatases (AP) regulate the intracellular degradation of nucleotides. High NT and AP activities might cause a resistance to the thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We studied the relation between these enzymes and immunophenotype, drug resistance and prognosis in 77 children with acute lymphoblastic leukemia (ALL). Enzyme activities were assessed radiochemically; in vitro drug resistance was measured with the MTT assay. AP activities were higher in T-ALL and B-ALL than in precursor B-ALL. Cyto-5'NT activity was very low in all phenotypes and accounted for a significant proportion of total IMPase activity only in the very immature CD10- c mu- precursor B-ALL. CD10+ ALL cases with high ecto-5'NT activities showed a trend (p = 0.065) for a lower probability of continuous complete remission than those with a low activity. Ecto-5'NT activity was not related to in vitro drug resistance to 6-TG. A weak correlation was found between in vitro 6-TG resistance and cyto-5'NT and AP activities. We conclude that high ecto-5'NT activities do not cause a resistance to 6-thiopurines in childhood ALL. Some patients have high cyto-5'NT and AP activities associated with 6-thiopurine resistance.

Decreased activity of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT), responsible for the conversion of 6-mercaptopurine and 6-thioguanine (6-TG) to their cytotoxic nucleotides, may cause resistance to these thiopurines in experimental leukemic systems. The clinical significance of this mechanism is as yet unclear. In 83 children with untreated acute lymphoblastic leukemia (ALL), we determined the prognostic value of HGPRT activity and the relation between HGPRT activity and resistance to thiopurines. HGPRT activity was determined radiochemically; in vitro resistance to 6-TG with the MTT assay. HGPRT level was significantly lower in T-ALL than in B-lineage ALL; no differences were found between sequential differentiation stages of B-lineage ALL. HGPRT activity was inversely related to the white-blood-cell count (WBC). Among patients with cALL and pre-B-ALL with WBC less than 50 x 10(9)/l, cases with a low HGPRT had a significantly poorer prognosis than those with a high HGPRT. WBC, age, sex, organomegaly and differentiation stage were comparable in both patient groups. No correlation was found between HGPRT activity and in vitro 6-TG resistance in cALL and pre-B-ALL patients. T-ALL cases were not more 6-TG-resistant than cALL and pre-B-ALL cases. Cells from 6 relapsed ALL cases did not show decreased HGPRT activity. We conclude that: (a) HGPRT is lower in T- than in B-lineage ALL and is constant in sequential differentiation stages of B-lineage ALL; (b) HGPRT activity is inversely related to tumor load; (c) low HGPRT activities are correlated with a poorer prognosis in precursor B-ALL but this cannot be explained by thiopurine resistance because (d) there is no relation between HGPRT activity and in vitro 6-TG resistance.

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 micrograms/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.

Ecto-5′nucleotidase (5′NT; CD73) expression was studied with a monoclonal antibody (7G2) and a radiochemical assay and compared with the expression of other antigens in B-cell-lineage leukemias on cells from 100 leukemic patients and two cell lines. A B-cell origin was confirmed by the expression of CD19 and HLA-DR. Four stages of B-cell leukemias were defined: stage I (pro-B) as CD10-, cytoplasmic mu- (c mu- ), surface Ig- (sIg-); stage II (cALL) as CD10+/c mu-/sIg-; stage III (pre-B) as CD10+ or -/c mu+/sIg-; and stage IV (B) as CD10-/c mu-/sIg+. A linear correlation was found between immunohistochemical and radiochemical determination of 5′NT (r = .86). 5′NT expression was low in T-cell leukemias and stage I, high in stages II and III, and low again in stage IV of B-cell leukemias. 5′NT expression was not related to c mu, CD20, CD21, CD22, CD34, and terminal deoxynucleotidyl transferase (TdT) expression, but was significantly related to CD10 and inversely related to kappa/lambda expression. However, the 5′NT activity in CD10+ leukemias (stages II and III) shows a very wide range. Within the group of CD10+ leukemias no differences were detected between 5′NT+ and 5′NT- cells in their expression of other B-cell antigens. We conclude that the place of 5′NT in leukemias corresponding to early stages of B-cell development has been characterized. 5′NT is expressed in CD10+ stages and decreases before the expression of sIgs. Future studies should make clear whether a high expression of this enzyme in CD10+ stages is a normal maturation phenomenon or a malignant phenomenon.

The knowledge about drug resistance in childhood leukemias and acute lymphoblastic leukemia (ALL) in general is limited. This is because of the lack of a suitable in vitro drug sensitivity assay, which is in part due to low in vitro ALL cell survival. We recently adapted the highly efficient 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay to test cells from ALL patients and showed that its results were comparable with those of the DiSC assay, up to now the most valid but laborious assay. In this study, in vitro drug sensitivity was assessed in cells from 82 children with leukemia, 79 of whom had ALL, with the MTT assay. Dose response curves were obtained for 6-mercaptopurine, 6-thioguanine (6-TG), prednisolone (Pred), daunorubicin (DNR), vincristine (VCR), cytosine arabinoside (Ara-C), L- asparaginase (L-Asp), mafosfamide, and mustine. A cytotoxic effect of methotrexate could be detected in only a few cases. Large interindividual differences in drug sensitivity were detected. Compared with leukemia cells from newly diagnosed patients, leukemia cells from relapsed patients were significantly more in vitro resistant to 6-TG, Pred, Ara-C, mafosfamide and mustine but not to DNR, VCR, and L-Asp. Improvements of culture medium and methods to increase MTT reduction were studied. From 10 components tested, addition of insulin and bovine serum albumin to serum-containing medium improved ALL cell survival. Addition of succinate did not increase the amount of MTT reduction. We conclude that the in vitro MTT assay highly facilitates large-scale studies on drug resistance of ALL patients that can lead to rational improvements in existing treatment protocols.

The combination of fludarabine, cytarabine (ara-C) and G-CSF (FLAG) is routinely used in the treatment of acute myeloid leukemia (AML). In this study we characterized the interactions between fludarabine, ara-C and G-CSF in vitro using AML blasts. Exposure to G-CSF alone resulted in a higher leukemic cell survival (LCS), which might be indicative of increased proliferation. The LCS decreased significantly from 69.7 to 54.0% when blasts were exposed to G-CSF 21 h prior to incubation with ara-C (p=0.01). In contrast, LCS increased significantly (from 55.6 to 69.0%; p=0.04) after sequential exposure to G-CSF and fludarabine. Exposure to 4 combinations of fludarabine (4 h; 0.14 microM and 0.55 microM) and ara-C (96 h; 0.21 and 0.82 microM) (FLA) resulted in additive cytotoxicity. The triple combination (FLAG), 21 h 5 microM G-CSF followed by 4 h fludarabine (0.14 and 0.55 microM) and finally ara-C (0.21 and 0.82 microM) for 96 h also resulted in an additive cell kill. In conclusion, these data support the clinical use of G-CSF in combination with ara-C, and the combination of ara-C and FLA. Pre-exposure to G-CSF before FLA (FLAG) did not result in increased cytotoxicity in our experiments, indicative of similar anti-leukemic activity.

The prognostic influence of age in childhood acute myeloid leukemia (AML) is less clear as in childhood acute lymphoblastic leukemia (ALL), where infants carry a poor prognosis. Reports on outcome in infant AML have been contradictory, and also the age cut-off point is less well defined as in ALL. We studied differences in cellular drug resistance, using the MTT-assay, between 28 infants (age ≤24 months) versus 122 older children (aged 2–18 years) with de novo AML. We found no differences in drug resistance within the infant AML group, comparing infants

Gemtuzumab ozogamicin in pediatric CD33-positive acute lymphoblastic leukemia: first clinical experiences and relation with cellular sensitivity to single agent calicheamicin

The features of proliferation in brain tumours are related with clinical prognosis for several types of brain tumours, especially gliomas. For childhood central primitive neuro-ectodermal tumours (cPNET), including medulloblastoma, this relation has previously been unclear. The aim of this study is to investigate the relationship between proliferative features of cPNET and in vitro resistance for cytostatic drugs measured with the 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium-bromide (MTT) assay. Tumour material was obtained from 23 surgical specimens of cPNET. The expression of the proliferation markers Ki-67, proliferating cell nuclear antigen (PCNA) and cyclin D1 was determined with immunohistochemistry, while S-phase and DNA ploidy were analysed by flowcytometric analysis cell scan (FACS). The in vitro resistance for 10 cytostatic drugs was determined with the MTT assay. Drug resistance levels were available in 19 (83%) of the 23 samples with a complete profile of 10 cytostatic drugs tested in 14 samples. An excellent correlation in drug resistance scores was found between pharmacologically related drugs. The Ki-67 staining in 20 samples varied from 10 to 60% and from 30 to 100% for PCNA. Cyclin D1 staining was negative in 11 out of 18 samples. The S-phase in 16 samples ranged from 2 to 16%. Increased staining of Ki-67 was related with actinomycin D sensitivity (r −.603; P=0.022), while cells with a higher S-phase percentage were more resistant to ifosfamide (r .952; P

Children with Down syndrome (DS) have an increased risk for leukemia. The prognosis for DS acute myeloid leukemia (AML) is better than for non-DS AML, but the clinical outcome of DS acute lymphoblastic leukemia (ALL) is equal to that of non-DS ALL. Differences in prognosis may reflect differences in cellular drug resistance. In vitro drug resistance profiles were successfully investigated on leukemic cells from 13 patients with DS AML and 9 patients with DS ALL and were compared with reference data from 151 non-DS AML and 430 non-DS B-cell precursor (BCP) ALL. DS AML cells were significantly more sensitive to cytarabine (median, 12-fold), the anthracyclines (2-7-fold), mitoxantrone (9-fold), amsacrine (16-fold), etoposide (20-fold), 6-thioguanine (3-fold), busulfan (5-fold), vincristine (23-fold), and prednisolone (more than 1.1-fold), than non-DS AML cells. Compared with DS ALL, DS AML cells were significantly more sensitive to cytarabine only (21-fold). After short-term exposure to methotrexate, DS AML cells were 21-fold more resistant than non-DS AML cells, but no difference was observed after continuous exposure. DS ALL cells and non-DS BCP-ALL cells were equally sensitive to all drugs, including methotrexate. Normal peripheral blood mononuclear cells from DS and non-DS children without leukemia showed highly resistant drug profiles. It was concluded that the better prognosis of DS AML might, at least partially, be explained by a specific, relatively sensitive drug-resistance profile, reflecting the unique biology of this disease. (Blood. 2002;99:245-251)

Retinoblastoma is frequently treated with chemotherapy to facilitate intraocular therapy, as well as to diminish or delay radiotherapy in invasive disease. It is also used more extensively in patients with dissemination to the central nervous system and/or the bone marrow. Once the disease has spread, the prognosis is poor. Radiotherapy is effective in ocular retinoblastoma, but is associated with facial deformation and a higher chance for second primary tumors in the irradiation field. These sequelae emphasize the need to determine more effective chemotherapy schedules and local treatment. The aim of this study is to investigate the relation between in vitro drug resistance for ten cytostatic drugs and histopathologic features in primary retinoblastoma. MATERIALS AND METHODS. Forty-four fresh samples of primary retinoblastoma were tested for in vitro drug resistance using the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The histopathologic features for differentiation, invasion and intra-ocular extension, necrosis, mitosis, and apoptosis were scored.The differentiation of the tumors revealed 24 poorly differentiated, 14 intermediately differentiated, and 6 well differentiated tumors. Tumor infiltration showed 3 minimal and 3 massive choroideal invasions, as well as 21 prelaminary and 2 postlaminary optic nerve invasions. The tumor was unifocal in 16 eyes and multifocal in 28 eyes, with extensive retinal involvement in 10 eyes and tumor seeding in 21 eyes. The MTT assay was successful in 82% of the samples after enzymatic handling of the tumor cells was omitted. Undifferentiated tumors were more sensitive to carboplatin (p = 0.034) and doxorubicin (p = 0.025), thiotepa (p = 0.051) and ifosfamide (p = 0.075) in comparison to differentiated tumors. Type of retinal involvement, invasion, focality, and seeding did not show a relationship with drug resistance. Calcified tumors were more resistant to actinomycin D and ifosfamide and more sensitive to vincristine; conversely, apoptotic tumors were more sensitive to ifosfamide and more resistant to vincristine (p = 0.027). Necrotic tumors were more sensitive to actinomycin D (p = 0.004), and mitotic tumors were more sensitive to idarubicin (p = 0.026). In 90% of the tumors extreme drug resistance to cytarabin was present.In retinoblastoma many histopathologic features are related to in vitro drug resistance. Undifferentiated tumors are more sensitive to several cytostatic drugs. Calcification and apoptosis show an inverse relation with in vitro drug resistance to ifosfamide and vincristine. Extreme drug resistance to cytarabin is observed; this drug should not be used in retinoblastoma treatment.

Cellular drug resistance is thought to be an important cause of the poor prognosis for children with relapsed or refractory acute lymphoblastic leukemia (ALL), but it is unknown when, to which drugs, and to what extent resistance is present. We determined in vitro resistance to 13 drugs with the MTT assay. Compared with 141 children with initial ALL, cells from 137 children with relapsed ALL were significantly more resistant to glucocorticoids, L-asparaginase, anthracyclines, and thiopurines, but not to vinca-alkaloids, cytarabine, ifosfamide, and epipodophyllotoxins. Relapsed ALL cells expressed the highest level of resistance to glucocorticoids, with a median level 357- and >24-fold more resistant to prednisolone and dexamethasone, respectively, than initial ALL cells, whereas the resistance ratios for the other drugs differed from 0.8- to 1.9-fold, intraindividual comparisons between initial and relapsed samples from 16 children with ALL showed that both de novo and acquired drug resistance were involved. Specific in vitro drug-resistance profiles were associated with high-risk relapsed ALL groups. In vitro drug resistance was also related to the clinical response to chemotherapy in relapsed/refractory childhood ALL. We conclude that drug resistance may explain the poor prognosis for children with relapsed/refractory ALL. These day may be helpful to design alternative treatment regimens for relapsed childhood ALL.

Cellular drug resistance is supposed to play a major role in chemotherapy failures which frequently occur in childhood acute non-lymphoblastic leukemia (ANLL). Therefore, we determined in vitro chemosensitivity to daunorubicin, doxorubicin, mitoxantrone, 6-thioguanine, etoposide, and cytosine arabinoside (Ara-C) in childhood ANLL using the colorimetric MTT assay. The 4-day MTT assay was successfully performed in 62/73 samples obtained from 53 children with ANLL. We obtained comparable results from bone marrow or peripheral blood samples, and from fresh or cryopreserved samples. In vitro chemosensitivity was not related to clinical features such as sex, age, white blood cell count, or FAB-types. The group of poor responders to chemotherapy was median 3-fold more resistant to Ara-C than the group of good responders, but identification of a threshold for Ara-C sensitivity predictive for individual responses was limited due to the great overlap of in vitro chemosensitivities between both groups. Children with relapsed ANLL were in vitro median 3-fold more resistant to Ara-C than the initial ANLL group. No significant differences for the other drugs were observed with respect to clinical response or disease status. These results suggest that in vitro resistance to Ara-C plays an important role in chemotherapy failures in childhood ANLL, but larger studies are necessary to establish the predictive value of Ara-C sensitivity assessed with the MTT assay.

Retinoids, analogs of vitamin A, have been shown to inhibit growth and induce maturation in a large variety of tumor types. The results of many studies on this subject have been reviewed extensively by Lippman et al. [1,2]. In these reviews it was suggested that retinoids have synergistic activity in combination with cytostatic drugs. The data to support this suggestion are however scarce and all reports on these studies dealt with cell lines or animal models [3–7]. No studies using cells derived directly from patients have been reported as far as we know. We recently adapted the MTT assay to assess in vitro cytotoxic effects of chemotherapeutic drugs upon cells derived from leukemic patients [8,9]. With this assay combinations of resistance modifying agents with anticancer drugs were tested using cells from leukemic patients [10]. In the present study we evaluated whether vitamin A was capable of potentiating the cytotoxic effect of anticancer drugs against tumor cells obtained from patients with acute lymphoblastic leukemia (ALL).

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).

The immunophenotype is one of the prognostic factors in children with acute lymphoblastic leukemia (ALL) [1–6]. B-ALL cases have the worst prognosis and patients with B-cell precursor ALL the most favorable prognosis. Among the patients with precursor B-ALL, those whose lymphoblasts have cytoplasmic.t heavy chain (cµ; preB ALL) and the small group of patients whose cells lack the common ALL (cALL) antigen characteristic of the earliest stage of B-cell differentiation (proB ALL) show poorer responses by comparison with the cALL+/cµ− cases (cALL). Also patients with T-ALL have an unfavorable prognosis. Part of these findings are still controversial because in some studies the immunophenotype is related to other clinical and biological features like white blood cell count (WBC) and organomegaly. Also, when effective treatments are used, the prognostic value of immunophenotype could be diminished [7–9].

Nowadays the use of combination chemotherapy in children with acute lymphoblastic leukemia (ALL) results in a complete remission rate of more than 95%. With the best currently available treatment, about two-thirds of these children will remain in continuous complete remission and can therefore be considered cured. Patients suffering from a relapse, however, have a cure rate which is much lower. One of the main causes of this poor prognosis is probably a resistance of the leukemic cells to a number of drugs used for treatment. At present the knowledge of drug resistance in childhood ALL is very limited. It is unknown for instance for which drugs resistance occurs and which mechanisms are of clinical importance. Currently, much attention is given to the multidrug resistance (MDR) phenomenon: a resistance to vincra alkaloids and anthracyclines, mediated by the drug efflux pump p-glycoprotein (-gp), which can at least be partially overcome by so-called resistance modifiers. Recently, we and others adapted and improved assays to detect drug resistance of leukemic cells obtained from patients showing good correlations between in vitro results and clinical response to chemotherapy [1–8]. Because of the development of these short-term assays it has recently become possible to study drug resistance of patients with ALL. In the present study we assessed the resistance profiles of children with relapsed ALL and the clinical relevance of the MDR model in these patients.

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.

The reduction of the tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) to a blue-black formazan product by living but not by dead cells can be used to measure chemosensitivity of tumor cells. The main advantages of the MTT assay are its simplicity, rapidity, and the fact that the results are read automatically with a microplate spectrophotometer. Several reports on the use of the MTT assay in chemosensitivity testing have been published, but all these studies dealt with established cell lines and not with specimens obtained directly from patients. Here we present a study in which the MTT assay has been adapted to assess the effect of antineoplastic drugs on lymphoblasts of children with 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.

In 83 children with acute lymphoblastic leukaemia (ALL) the immunological phenotype of the lymphoblasts was determined using E rosetting, monoclonal anti-T cell sera, surface immunoglobulin staining and common ALL antiserum. The data were compared with acid alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP) cytochemical data. The vast majority of T-ALL cells proved to be ANAE-negative (30/31) and AP-positive (29/31). Null-ALL was always AP-negative (12/12), the ANAE reaction was sometimes positive (4/12). In common ALL, findings ranged from negative to positive for both enzymes. Two cases of B-ALL were ANAE-negative and AP-negative. Enzyme cytochemical determinations gave thus preferential patterns, especially for T-ALL and null-ALL. In common ALL, considerable heterogeneity was found, which may be a reflection of differences in maturation between different cases of common ALL.

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.

In 53 children with acute lymphoblastic leukaemia (ALL), initial handmirror cell (HMC) count among lymphoblasts was studied in relation to the occurrence of a relapse in the central nervous system (CNS), taking into account the white blood cell count (WBC) and the immunological phenotype. The children were followed for a period limited by (1) the first CNS relapse, (2) death or (3) the closing day of this study. The median follow-up period was 30 months, range 2-106 months. HMC counts were available in bone marrow smears of 41 children and in cytospins of 35 children. Cytospins proved to give more reliable and consistent results than bone marrow smears. In the 35 'cytospin' children no CNS relapses occurred in the group of 16 children with non-T-non-B-ALL and HMC counts above 10%. However, in the group of 10 children with non-T-non-B-ALL and HMC counts below 10%, and in the group of 9 children with T-ALL (HMC less than or equal to 11%), 6 and 5, respectively, got a CNS relapse. The CNS relapse-free period was not significantly different between these last two groups, whereas both groups did differ significantly from the group mentioned first (p less than 0.01). This was not found in bone marrow smears of 41 children, presumably because of the inaccurate counting results. A low initial HMC count in cytospins is associated with an increased risk for the occurrence of a CNS relapse in children with ALL. This prognostic factor seems to be independent of other prognostic signs such as immunological phenotype and high WBC.

Capping with concanavalin A (ConA) and monoclonal anti-HLA-ABC backbone was studied in childhood acute lymphoblastic leukemia (ALL). Capping with ConA and HLA gave quite different results, both in common ALL and T-ALL. With ConA most cases capped poorly, comparable to results described in chronic lymphatic leukemia and lymphoma, but in several cases capping was comparable to that of normal lymphocytes. In HLA capping T-ALL cells capped better than common ALL cells. HLA capping of T-ALL cells is comparable to that of normal lymphocytes. HLA capping results in handmirror cell formation giving support to the hypothesis that capping and motility are associated events.

In the last two decades the survival rate of children with acute lymphoblastic leukemia (ALL) has improved greatly. However, with a complete remission percentage close to 100%, about 35% of the children still get a relapse. In a previous study it was shown that in childhood common-ALL, 5′nucleotidase (5′NT) positive cases have a poorer prognosis than 5′NT negative cases (Veerman et al., 1985). This might be due to the breakdown of the cytotoxic nucleotides of 6-MP by high cytoplasmic 5′NT activities. Alternatively, ecto 5′NT can provide purine requirements of the purine salvage pathway and therefore rescue cells from a blockage of purine de novo synthesis by 6-MP and/or MTX (Thompson, 1985; Pieters et al., 1987a). Thirdly, 5′NT positive cases might be relatively resistant to cytostatic drugs in general.

We showed that in childhood common acute lymphoblastic leukemia (c-ALL), 51 nucleotidase (5′NT) positive cases have a poorer prognosis than 5′NT negative cases. This might be due to the breakdown of the cytotoxic nucleotides of 6-mercaptopurine (6-MP) by high cytoplasmic 5′NT activities. Alternatively, ccto 5′NT can provide purine requirements of the purine salvage pathway and therefore rescue cells from a blockage of purine de novo synthesis by 6-MP and/or methotrexate. To test these hypotheses we need to determine the relation between these enzymes and drug sensitivity. We have adapted the MTT assay, which has only been reported on in studies dealing with established cell lines, to assess the chemosensitivity of cells obtained directly from patients. The assay is based on the reduction of MTT to formazan by living but not by dead cells. Formazan production is quantitated automatically with a microplate spectrophotometer. Incubation of ALL cells with 6-MP (4-125 μg/ml) and 6-thioguanine (1.6-50 μg/ml) for 2-4 days resulted in dose-response curves covering the range from 0% to 100%; cell survival. Comparison of the MTT assay with a dye exclusion assay in 10 patients with ALL demonstrated an identical success rate and comparable dose-response curves for both assays. Because automated quantitation of the chemosensitivity of leukemic cell samples involving about 80 drug concentrations takes only a few minutes with the MTT assay, this assay is a rapid, efficient, and objective method of measuring sensitivity to purine antagonists in ALL patients.