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.

Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia.

Activating mutations in RAS and receptor tyrosine kinases such as KIT and FLT3 are hypothesized to cooperate with chimeric transcription factors in the pathogenesis of acute myeloid leukemia (AML). To test this hypothesis, we genotyped 150 pediatric AML samples for mutations in KIT (exons 8, 17), NRAS and KRAS (exons 1, 2) and FLT3/ITD. This is the largest cohort of pediatric AML patients reported thus far screened for all four mutations. Of the children with AML, 40% had a mutation in KIT (11.3%), RAS (18%) or FLT3/ITD (11.1%), and 70% of cases of core-binding factor (CBF) leukemia were associated with a mutation of KIT or RAS. Mutations in RAS or FLT3/ITD were frequently found in association with a normal karyotype. Patients with a FLT3/ITD mutation had a significantly worse clinical outcome. However, the presence of a KIT or RAS mutation did not significantly influence clinical outcome. We demonstrate that KIT exon 8 mutations result in constitutive ligand-independent kinase activation that can be inhibited by clinically relevant concentrations of imatinib. Our results demonstrate that abnormalities of signal transduction pathways are frequent in pediatric AML. Future clinical studies are needed to determine whether selective targeting of these abnormalities will improve treatment results.

In vitro drug-resistance profile in infant acute lymphoblastic leukemia in relation to age, MLL rearrangements and immunophenotype.

Acute lymphoblastic leukemia (ALL) in infants under 1 year is strongly associated with translocations involving 11q23 (MLL gene), CD10-negative B-lineage (proB) immunophenotype, and poor outcome. The present study analyses the relationship between age, MLL rearrangements, proB-lineage, and in vitro drug resistance determined using the MTT assay. Compared to 425 children aged over 1 year with common/preB (c/preB) ALL, the 44 infants were highly resistant to steroids (for prednisolone (PRED) more than 580-fold, P=0.001) and L-asparaginase (L-ASP) (12-fold, P=0.001), but more sensitive to cytarabine (AraC) (1.9-fold, P=0.001) and 2-chlorodeoxyadenosine (2-CdA) (1.7-fold, P<0.001). No differences were found for vincristine, anthracyclines, thiopurines, epipodophyllotoxines, or 4-hydroperoxy (HOO)-ifosfamide. ProB ALL of all ages had a profile similar to infant ALL when compared with the group of c/preB ALL: relatively more resistant to L-ASP and PRED (and in addition thiopurines), and more sensitive to AraC and 2-CdA. Age was not related to cellular drug resistance within the proB ALL group (<1 year, n=32, vs >/=1 year, n=19), nor within the MLL-rearranged ALL (<1 year, n=34, vs >/=1 year, n=8). The translocation t(4;11)(q21;q23)-positive ALL cases were more resistant to PRED (>7.4-fold, P=0.033) and 4-HOO-ifosfamide (4.4-fold, P=0.006) than those with other 11q23 abnormalities. The expression of P-glycoprotein, multidrug-resistance protein, and lung-resistance protein (LRP) was not higher in infants compared to older c/preB ALL patients, but LRP was higher in proB ALL and MLL-rearranged ALL of all ages. In conclusion, infants with ALL appear to have a distinct in vitro resistance profile with the proB immunophenotype being of importance. The role of MLL cannot be excluded, with the t(4;11) being of special significance, while age appears to play a smaller role.

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 (rho=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.

Features of proliferation and in vitro drug resistance in central primitive neuro-ectodermal tumours.

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<0.0001). In vitro drug resistance testing of central primitive neuro-ectodermal tumours (PNET) is feasible with the MTT assay. Ifosfamide resistance was related with increased Ki-67 and S-phase percentage of the tumour cells, while increased Ki-67 was also related with actinomycin D sensitivity. These findings suggest a cell cycle dependent activity of cytostatic drugs in vitro.

In vitro drug resistance profile of Philadelphia positive acute lymphoblastic leukemia is heterogeneous and related to age: a report of the Dutch and German Leukemia Study Groups.

BACKGROUND: The t(9;22)(q34;q11) translocation leading to the Philadelphia (Ph) chromosome resulting in BCR-ABL gene fusion is associated with a poor prognosis in acute lymphoblastic leukemia (ALL). PROCEDURE: We studied the relation between t(9;22), determined by karyotype, fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR), and in vitro drug resistance, measured by the MTT assay, in precursor B-cell ALL at diagnosis. The findings in twenty-one Ph-positive (Ph+) childhood common/precursorB (c/preB) cases were compared with 254 Ph-negative (Ph-) ALL cases. RESULTS: A large range of LC(50) values was found within the Ph+ patients. Moreover, LC(50) values did not differ significantly between Ph+ and Ph- samples for prednisolone, dexamethasone, L-asparaginase, vincristine, anthracyclines, thiopurines, epipodophyllotoxins, and 4H00-ifosfamide, even after matching for important prognostic features (age, white blood cell count (WBC), and immunophenotype). Adult Ph+ (n = 12) ALL was more resistant to prednisolone (> 270-fold, P = 0.030), and displayed an overall tendency to resistance when compared to matched cases of Ph- (n = 15) adult precursor B-cell ALL. Within Ph+ ALL, in vitro prednisolone resistance increased significantly with age (P = 0.006). The expression of lung resistance protein (LRP), but not P-glycoprotein (P-gp) or multidrug resistance protein (MRP), was significantly higher in all Ph+ patients. CONCLUSIONS: Both childhood and adult Ph+ precursor B-cell ALL samples display a heterogeneous in vitro resistance profile, with relatively sensitive and resistant cases. The adult Ph+ samples, however, are generally more resistant compared to matched Ph- controls, reaching significance for prednisolone. The correlation of prednisolone resistance with age within the Ph+ cases might help explain the poorer prognosis of adult Ph+ ALL.

Histopathologic features of retinoblastoma and its relation with in vitro drug resistance measured by means of the MTT assay.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

TEL/AML1 gene fusion is related to in vitro drug sensitivity for L-asparaginase in childhood acute lymphoblastic leukemia.

The t(12;21) translocation resulting in TEL/AML1 gene fusion is present in approximately 25% of patients with precursor B-lineage pediatric acute lymphoblastic leukemia (ALL). Studies suggest an association with a good prognosis; however, relapse can occur. We studied the relation between t(12;21), determined by fluorescence in situ hybridization or polymerase chain reaction, and in vitro drug resistance, measured by the MTT assay, in childhood B-lineage ALL at diagnosis. A total of 180 ALL samples were tested, 51 (28%) of which were positive for t(12;21). The median LC(50) values did not differ significantly between TEL/AML1-positive and -negative samples for prednisolone, dexamethasone, daunorubicin, thiopurines, epipodophyllotoxins, and 4-HOO-ifosfamide. However, the TEL/AML1-positive patients were relatively more sensitive to L-asparaginase (ASP; 5.9-fold; P =.029) and slightly but significantly more resistant to vincristine (1.5-fold; P =.011) and cytarabine (1.5-fold; P =.014). After matching for unevenly distributed patient characteristics-that is, excluding patients younger than 12 months, patients with CD10-negative immature B-lineage ALL, patients with Philadelphia chromosome, and patients who were hyperdiploid (more than 50 chromosomes) from the TEL/AML1 negative group-the only remaining difference was a relative sensitivity for ASP in the TEL/AML1-positive samples (10.8-fold; P =. 012). In conclusion, the presence of TEL/AML1 gene fusion in childhood precursor B-lineage ALL does not seem to be associated with a high in vitro drug sensitivity, except for ASP, indicating that these patients could benefit from treatment schedules with significant use of this drug.

Do P-glycoprotein and major vault protein (MVP/LRP) expression correlate with in vitro daunorubicin resistance in acute myeloid leukemia?

Two proteins that have been correlated with the occurrence of multidrug resistance in acute myeloid leukemia (AML) are P-glycoprotein (Pgp) and the major vault protein (Mvp/LRP). With the purpose of further quantifying the potential contributions of Pgp-mediated drug efflux and Mvp/LRP to drug resistance in AML we have investigated whether the transport function of Pgp and the expression of Mvp/LRP correlated with the accumulation of daunorubicin (DNR) and the in vitro resistance to DNR cytotoxicity (LC50 by MTT assay) in AML cells. In de novo adult AML, the steady-state DNR accumulation (in pmol/10(6) cells) correlated with Pgp activity or expression, whereas the LC50 for DNR did not correlate with Pgp activity (measured as the modulation of rhodamine 123 or DNR accumulation by the Pgp inhibitor PSC833) or Pgp expression (measured by flow cytometry with the MRK-16 antibody). The contribution of MRP1 expression to a reduced DNR accumulation seems minor compared to Pgp. In addition, the modulation of the DNR LC50 by PSC833 did not correlate with Pgp protein or activity. The steady-state DNR accumulation showed no correlation with the DNR LC50. The Mvp/LRP expression (immunocytochemical staining) did neither correlate with DNR accumulation nor with the DNR LC50. A significant negative correlation was seen between the Mvp/LRP immunocytochemical staining and Pgp activity, indicating that both markers define (partially) different populations. In conclusion, it is shown that Pgp function, but not Mvp/LRP or MRP1 expression correlate with a low steady-state DNR accumulation in de novo AML. The Pgp activity does, however, not predict the DNR sensitivity in AML measured as in vitro DNR LC50 with an MTT-based assay. The reason for that seems to be that a low DNR accumulation may not be the most important factor in determining the LC50. While the clinical usefulness of these drug resistance tests remains to be proven they do not seem to provide as yet a straightforward explanation for the major cause(s) of clinical chemotherapy failure.

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 (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.

In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia.

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.

In vitro chemosensitivity assessed with the MTT assay in childhood acute non-lymphoblastic leukemia.

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.

In vitro anthracycline cross-resistance pattern in childhood acute lymphoblastic leukaemia.

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.

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.

Cellular drug resistance profiles that might explain the prognostic value of immunophenotype and age in childhood acute lymphoblastic leukemia.

Immunophenotype and age have prognostic value in childhood acute lymphoblastic leukemia (ALL) but how this operates is not understood. In 84 children with ALL at initial diagnosis we studied the correlation between these factors and the in vitro resistance to eight drugs, determined with the 3-(4,5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay. B-lineage ALL samples were classified into four differentiation stages: the CD10- proB ALL; cALL; preB ALL with cytoplasmic mu positive ALL cells; and B-ALL with surface immunoglobulin-positive (Ig+) cells. cALL and preB ALL cases have the best prognosis; proB and T-ALL cases show a worse prognosis and B-ALL the poorest prognosis. Patients aged < 18 months and > 10 years have a poor prognosis compared to patients in the intermediate age group. Our results show that cALL and preB ALL cells were the most drug-sensitive cells compared to the other phenotypes. No differences were found between cALL and preB ALL cases with the exception that preB cells were more sensitive to mustine and mafosfamide (Maf). Compared to cALL and preB ALL cases, T-ALL cases were significantly more resistant to prednisolone (Pred), daunorubicin (DNR), L-asparaginase (L-Asp), cytosine arabinoside (AraC), and Maf; proB ALL cases were more resistant to Pred, DNR, L-Asp, and 6-thioguanine. The three B-ALL cases were resistant to vincristine and DNR. Two out of three B-ALL were resistant to Pred. Compared to cells from patients aged 18 months to 10 years, cells from children < 18 months were more resistant to Pred and DNR; cells from children > 10 years were more resistant to Pred. We conclude that cellular drug-resistance patterns might at least partly explain the prognostic value of immunophenotype and age in childhood ALL.

Relation of 5'-nucleotidase and phosphatase activities with immunophenotype, drug resistance and clinical prognosis in childhood leukemia.

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.

Hypoxanthine-guanine phosphoribosyl-transferase in childhood leukemia: relation with immunophenotype, in vitro drug resistance and clinical prognosis.

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.