Peanut agglutinin, a marker for T-cell acute lymphoblastic leukemia with a good prognosis.

Peanut agglutinin (PNA) binding was studied in cells from 74 children with acute lymphoblastic leukemia (ALL). PNA positivity occurred in 50% of T-ALL (12 of 24) and was rare in other types of ALL. There was no clear relationship between PNA and initial white blood cell count, French-American-British classification, stage of differentiation of leukemic cells, hand mirror cells, and organomegaly at diagnosis. Prognosis, however, was significantly better (continuous complete remission, death) in the PNA-positive T-ALLs. It seems that PNA is a useful marker for a subgroup of T-ALL with a better prognosis.

Effects and interaction of 7-hydroxy methotrexate and methotrexate in leukaemic cells ex vivo measured by the thymidylate synthase inhibition assay.

In high dose therapy with methotrexate (MTX) the main metabolite 7-hydroxy-methotrexate (7-OH MTX) exceeds the plasma concentration of MTX achieving about tenfold higher levels. To investigate the interaction between 7-OH MTX and MTX ex vivo, the thymidylate synthase inhibition assay was used to quantify antifolate effects in patient blast samples, measuring the inhibition of the key enzyme thymidylate synthase (TS). In 18 leukemic samples (7 ALL, 11 AML) no dose-dependent TS inhibition was observed for 7-OH MTX. However, a statistically significant increase of TS inhibition (p<0.05) was observed for a 1:1 mixture of MTX and 7-OH MTX as compared to the effect of MTX alone. The half-maximal inhibitory concentrations in the short-exposure assay were 0.857 microM for MTX alone versus 0.088 microM for the 1:1 mixture with 7-OH MTX, respectively (p< or =0.05). This interaction was not observed with an excess of 7-OH MTX. Similar results were obtained in long exposure experiments. We conclude that there is a dose-dependent interaction between 7-OHMTX and MTX, despite the lack of TS inhibitory effects of the metabolite alone.

Different cellular drug resistance profiles in childhood lymphoblastic and non-lymphoblastic leukemia: a preliminary report.

The better prognosis of acute lymphoblastic leukemia (ALL) than of acute non-lymphoblastic leukemia (ANLL) in children, and the often observed better prognosis of myeloid-antigen (MyAg) negative ALL than of MyAg-positive ALL, may be related to differences in cellular drug resistance. We therefore compared the resistance to 12 drugs of 125 ALL and 28 ANLL samples with the MTT assay. ALL samples were median > 75-fold more sensitive to the glucocorticoids prednisolone and dexamethasone (p < 0.00001), and 2-fold more sensitive to vincristine (p = 0.05) than ANLL samples. Differences for the other drugs were not significant. MyAg-negative ALL samples were more sensitive to glucocorticoids than MyAg-positive ALL-samples (p < or = 0.04). Prednisolone, and dexamethasone if tested, had a stimulatory effect on leukemic cell survival in 36% of ANLL, but in only 2% of ALL samples (p < 0.0001). Vincristine, and vindesine if tested, had a similar effect in 11% of ANLL, and in 4% of ALL samples (p = 0.11). We conclude that the more favorable response of ALL against ANLL to combination chemotherapy in children may be explained by the higher antileukemic activity of glucocorticoids and of vincristine in ALL, while none of the drugs was more active in ANLL. Similarly, the better prognosis of MyAg-negative ALL than of MyAg-positive ALL may be explained by a relative sensitivity to glucocorticoids. Glucocorticoids and vinca-alkaloids induced leukemia cell proliferation in part of the samples, most frequently in ANLL. The findings may be useful in the design of new chemotherapeutic regimens for ALL and ANLL.

Prognostic significance of peanut agglutinin binding in childhood acute lymphoblastic leukemia.

We previously reported the favorable prognosis associated with positive peanut agglutinin (PNA) binding in childhood T cell acute lymphoblastic leukemia (ALL), and hypothesized that this may be related to glucocorticoid sensitivity (Veerman et al. Cancer Res 1985, 45: 1890). The purposes of this prospective study involving 202 children with newly diagnosed ALL were to determine the relationship between PNA binding and (1) immunophenotype; (2) in vitro resistance to prednisolone (PRD) and dexamethasone and other drugs; (3) clinical response to a systemic PRD monotherapy (plus one intrathecal injection with methotrexate); and (4) multidrug chemotherapy. PNA positivity was more frequent in T cell ALL (65% of 43 cases) than in pro-B (0% of seven cases), common (17% of 106 cases) and pre-B (16% of 45 cases) ALL (P < 0.001). PNA binding was not associated with in vitro resistance to PRD or dexamethasone. However, in 38 evaluable T cell ALL patients, nine of 13 PNA-negative cases were clinically poor responders to PRD, while all 25 PNA-positive cases were good responders to PRD clinically (P < 0.0001). The four clinically poor PRD responders with B cell precursor (BCP)-ALL were also PNA negative. Within T cell ALL, PNA-positive patients had a 3.4-fold (95% Cl, 1.1-10.4, P = 0.03) lower relative risk of any event, than PNA-negative patients. Within BCP-ALL, PNA binding was not of prognostic significance. In conclusion, PNA positivity, especially frequent in T cell ALL, is a marker for a subgroup of childhood ALL patients who are very likely to respond well to systemic PRD 'monotherapy'. In addition, PNA positivity is a favorable prognostic factor in T cell ALL.

mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method.

Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of beta-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR x FPGH)/(RFC x FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.

Ex vivo activity of methotrexate versus novel antifolate inhibitors of dihydrofolate reductase and thymidylate synthase against childhood leukemia cells.

Leukemic cells of 27 children [14 patients with initial acute lymphoblastic leukemia (iALL), 8 patients with relapsed ALL (rALL), and 5 patients with acute nonlymphoblastic leukemia (ANLL)] were evaluated for their sensitivity to methotrexate (MTX) and five novel antifolate drugs, which have the potential to circumvent MTX resistance. The novel antifolates include a polyglutamatable [edatrexate (EDX)] and a lipophilic (trimetrexate) inhibitor of dihydrofolate reductase and two polyglutamatable inhibitors (ZD1694 and GW1843U89) and one lipophilic inhibitor (AG337) of thymidylate synthase (TS). Drug activity was assessed via the determination of in situ inhibition of TS activity after exposing leukemic cells to antifolate drugs for: (a) 3 h, followed by a 15-h drug-free period; and (b) 18 h of continuous exposure. For human CEM leukemia cell lines with well-defined mechanisms of resistance to MTX, in situ TS inhibition correlated with the growth-inhibitory effects of MTX and the novel antifolates (r = 0.86-0.93; P < 0.01). Although a wide interpatient variability in MTX sensitivity was observed within the three leukemia groups, the median drug concentration required to inhibit TS activity to 50% of untreated controls (TSI50) for a 3-h exposure to MTX was similar for iALL and rALL cells but was up to 9-fold higher in ANLL cells. After a 3-h exposure, EDX, ZD1694, and GW1843U89 displayed a markedly (10-150-fold) increased potency over MTX in all leukemia groups with comparable TSI50 values for ANLL and iALL cells. Compared with a 3-h MTX exposure, continuous exposure resulted in lower TSI50 values for iALL (14-fold), rALL (14-fold), and ANLL cells (85-fold). In comparison to MTX, the TSI50 values in these groups were also lower for EDX (1.6-3.5-fold), ZD1694 (2.1-4.3-fold), and GW1843U89 (15-35-fold). On short-term exposure, the lipophilic drugs trimetrexate and AG337 displayed markedly less potency as compared with that of long-term exposure. In conclusion, the efficacy of novel antifolates against childhood leukemia cells can be tested with the in situ TS inhibition assay. These novel antifolates displayed a greater efficacy than MTX against childhood leukemia cells and may have potential for the circumvention of MTX resistance in ANLL cells.

A possible role for methotrexate in the treatment of childhood acute myeloid leukaemia, in particular for acute monocytic leukaemia.

Acute myeloid leukaemia (AML) is thought to be methotrexate (MTX)-resistant. However, a small study suggested that acute monocytic leukemia (AML-M5) is sensitive to MTX. We measured MTX accumulation/polyglutamylation in 20 AML-nonM5, 37 AML-M5 and 83 common/preB-acute lymphoblastic leukaemia (c/preB-ALL) samples. Membrane transport was determined in 11 childhood AMLs (including 3 AML-M5) and in 25 c/preB-ALL samples. MTX sensitivity was determined in 23 AML-nonM5, 15 AML-M5 and 63 common/preB-ALL samples using the thymidylate synthase (TS) inhibition assay. MTX transport was higher in AML samples compared with c/preB-ALL precluding a transport defect in AML. Accumulation of long-chain polyglutamates MTX-Glu(4-6) was 3-fold lower for AML-nonM5 compared with c/preB-ALL cells (median 268 versus 889 pmol MTX-Glu(4-6)/10(9) cells; P < or = 0.001); for AML-M5 samples, median accumulation of MTX-Glu(4-6) was 0 pmol/10(9) cells (P < or = 0.001). After short-term MTX exposure, AML-nonM5 was 6-fold more resistant to MTX compared with c/preB-ALL cells (2.16 versus 0.39 microM; P < 0.001), while AML-M5 was 2-fold more resistant (P = 0.02). In both AML-nonM5 and AML-M5 cells, MTX resistance was circumvented by continuous MTX exposure (median TSI(50) values: 0.052 and 0.041 microM, respectively) compared with a c/preB-ALL value of 0.066 microM. In conclusion, AML-M5 is relatively sensitive to MTX compared with other AML-subtypes even though polyglutamylation of MTX is poor. Using continuous exposure, AML-nonM5 and AML-M5 cells were at least as sensitive to MTX as c/preB-ALL cells. This report suggests that MTX might be an overlooked drug in the treatment of childhood AML.

Prognostic value of 5'nucleotidase in acute lymphoblastic leukemia with the common-ALL phenotype.

5'-Nucleotidase (5'NT) is an enzyme found on the surface membrane of leukemic cells with the c-ALL phenotype. We studied 79 children with acute lymphoblastic leukemia (ALL). Thirty six of them had the c-ALL phenotype. Within this c-ALL group, ten had 5'-NT positive leukemic cells. Clinical data were available in 33 c-ALL cases. Sex, age and initial white blood cell count were comparable between 5'NT positive and 5'NT negative c-ALL cases. In the 5'NT positive group the probability of complete continuous remission was significantly lower than in the 5'NT negative group (p less than 0.05).

Clinical and cell biological features related to cellular drug resistance of childhood acute lymphoblastic leukemia cells.

Several clinical and cell biological features, such as sex, age, leukemic cell burden, morphologic FAB type, and immunophenotype, have prognostic value in childhood acute lymphoblastic leukemia (ALL). The explanation for their prognostic significance is unclear, but might be related to cellular drug resistance. We prospectively studied the relation between the above mentioned features with resistance to 13 drugs in 144 childhood ALL samples obtained at initial diagnosis. The MTT assay was used for drug resistance testing. The interindividual differences in drug resistance were very large and exceeded those between the several subgroups. There was generally no significant relation between sex, leukemic cell burden, and FAB type with drug resistance. However, subgroups with a worse prognosis as defined by age (< 18 months and > 120 months at diagnosis) or immunophenotype (pro-B ALL and T-ALL) did show relatively resistant drug resistance profiles as compared to the subgroups with a better prognosis (age 18-120 months, common and pre-B ALL). Within the group of common and pre-B ALL and compared to the intermediate age-group, samples of the younger children were significantly more resistant to daunorubicin, mitoxantrone and teniposide, and samples of the older children were significantly more resistant to prednisolone and mercaptopurine. Pro-B ALL samples were significantly more resistant to 1-asparaginase and thioguanine, and T-ALL samples were significantly more resistant to prednisolone, dexamethasone, 1-asparaginase, vincristine, vindesine, daunorubicin, doxorubicin, teniposide, and ifosfamide, than the group of common and pre-B ALL cases. We conclude that the prognostic significance of age and immunophenotype in particular may be explained, at least partly, by its relation with resistance to certain drugs. The results of this study may be useful for future rational improvements of chemotherapeutic regimens in childhood ALL.

BCL-2 expression in childhood leukemia versus spontaneous apoptosis, drug induced apoptosis, and in vitro drug resistance.

The antileukemic activity of cytotoxic drugs is increasingly thought to be the result of induction of apoptosis. Several proto-oncogenes have been related to the regulation of this process. In this study we evaluated the relation between bcl-2 expression, spontaneous and dexamethasone (DXM) induced apoptosis, and in vitro resistance to DXM, prednisolone (PRD) and cytarabine (ARA) determined using the total cell kill colorimetric methyl-thiazol-tetrazolium salt (MTT) assay, in childhood acute lymphoblastic leukemia (ALL). Drug resistance was expressed as the LC50 value, the drug concentration lethal to 50% of the cells. Fourty-six samples taken at initial diagnosis (iALL) and 31 samples taken at relapse (rALL) were incubated in culture medium, with and without DXM. Bcl-2 expression and apoptosis were measured flowcytometrically, the latter using DNA histogram analysis. Bcl-2 expression was 1.4 fold higher in rALL than in iALL (p = 0.008). Both spontaneous and DXM induced apoptosis increased significantly from 0 to 48 hours (in up to 71%, 81% of the cells respectively). Bcl-2 expression was inversely correlated with the extent of spontaneous apoptosis after 24 hours in iALL (r = -0.40, p = 0.05). Relapsed samples, but not samples obtained at presentation, expressing high levels of bcl-2 displayed increased resistance to drug induced apoptosis (r = -0.63, p = 0.02). In iALL high bcl-2 expression appeared to be related to low LC50 values of ARA. No correlations were found for DXM or PRD. In conclusion, DXM excerts its cytotoxic effect at least partly by means of induction of apoptosis. Bcl-2 inhibits drug induced apoptosis in rALL. However in iALL bcl-2 expression is not associated with increased in vitro drug resistance, nor with increased resistance to drug induced apoptosis.

Impaired bortezomib binding to mutant ß5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells.

Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the ß5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZ-resistant cells revealed upregulation of mutant ß5 subunit of the proteasome. These newly identified ß5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the ß5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant ß5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the α7 subunit of the proteasome can overcome this drug resistance modality.

Immunological phenotype related to acid alpha-naphthyl acetate esterase and acid phosphatase in childhood acute lymphoblastic leukaemia.

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.

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.

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.

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.

Differential methotrexate resistance in childhood T- versus common/preB-acute lymphoblastic leukemia can be measured by an in situ thymidylate synthase inhibition assay, but not by the MTT assay.

Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P =.46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P =.001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = -.58, P <.001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

Favorable prognosis of hyperdiploid common acute lymphoblastic leukemia may be explained by sensitivity to antimetabolites and other drugs: results of an in vitro study.

DNA hyperdiploidy is a favorable prognostic factor in childhood acute lymphoblastic leukemia (ALL). The explanation for this prognostic significance is largely unknown. We have studied whether DNA ploidy was related to cellular resistance to 12 drugs, assessed with the methyl-thiazol-tetrazolium assay, in samples of 74 children with common (CD10+ precursor B-cell) ALL. Sixteen patients had hyperdiploid ALL cells and 58 patients had nonhyperdiploid ALL cells. Hyperdiploid ALL cells were more sensitive to mercaptopurine (median, 9.0-fold; P = .000003), to thioguanine (1.4-fold; P = .023), to cytarabine (1.8-fold; P = .016), and to I-asparaginase (19.5-fold; P = .022) than were nonhyperdiploid ALL cells. In contrast, these two ploidy groups did not differ significantly in resistance to prednisolone, dexamethasone, vincristine, vindesine, daunorubicin, doxorubicin, mitoxantrone, and teniposide. The percentage of S-phase cells was higher (P = .05) in the hyperdiploid ALL samples (median, 8.5%) than in the nonhyperdiploid ALL samples (median, 5.7%). However, the percentage of cells in S-phase was not significantly related to in vitro drug resistance. We conclude that the favorable prognosis associated with DNA hyperdiploidy in childhood common ALL may be explained by a relative sensitivity of hyperdiploid common ALL cells to antimetabolites, especially to mercaptopurine and to I-asparaginase.