IN VITRO DRUG SENSITIVITY AND APOPTOSIS INDUCTION IN NEWLY DIAGNOSED PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA: Correlation with Overall Survival

PurposeTo evaluate the effect of obesity (defined as a body mass index > 95th percentile for age and sex at diagnosis) on outcome of pediatric acute lymphoblastic leukemia (ALL).Patients and MethodsWe retrospectively analyzed data from 4,260 patients with newly diagnosed ALL enrolled from 1988 to 1995 onto five concurrent Children's Cancer Group studies. Results were verified in a second cohort of 1,733 patients enrolled onto a sixth study from 1996 to 2002.ResultsThe 1988 to 1995 cohort included 343 obese and 3,971 nonobese patients. The 5-year event-free survival rate and risk of relapse in obese versus nonobese patients were 72% ± 2.4% v 77% ± 0.6% (P = .02) and 26 ± 2.4 v 20 ± 0.6 (P = .02), respectively. After adjusting for other prognostic variables, obesity's hazard ratios (HRs) of events and relapses were 1.36 (95% CI, 1.04 to 1.77; P = .021) and 1.29 (95% CI, 1.02 to 1.56; P = .04), respectively. The effect of obesity was prominent in the 1,003 patients ≥ 10 years old at diagnosis; in this subset, obesity's adjusted HRs of events and relapses were 1.5 (95% CI, 1.1 to 2.1; P = .009) and 1.5 (95% CI, 1.2 to 2.1; P = .013), respectively. In a second cohort of 1,160 patients ≥ 10 years old, obesity's adjusted HRs of events and relapses were 1.42 (95% CI, 1.03 to 1.96; P = .032) and 1.65 (95% CI, 1.13 to 2.41; P = .009), respectively. The effect of obesity on outcome was unrelated to changes in chemotherapy doses, length of intervals between chemotherapy cycles, or incidence and severity of therapy-related toxicity.ConclusionObesity at diagnosis independently predicts likelihood of relapse and cure in preteenagers and adolescents with ALL.

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Sensitivity of Childhood Acute Lymphoblastic Leukemia (ALL) to Idarubicin Is Significantly Higher Than to Daunorubicin Treatment Based on Ex Vivo Apoptosis Induction.

Blood ◽

10.1182/blood.v104.11.4495.4495 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4495-4495

Author(s):

Aram Prokop ◽

Banu Bagci ◽

Guenaelle Lingfeld ◽

Lucia Badiali ◽

Karin Garbrecht ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Response Rate ◽

De Novo ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Cell Populations ◽

Apoptosis Induction ◽

Good Tolerability ◽

Childhood All

Abstract Anthracyclines, especially daunorubicin, play a very important role in the treatment of acute lymphoblastic leukemia (ALL) and the relapsed ALL in childhood. In the present study, primary lymphoblasts isolated from 65 children with de novo ALL (median: 5.8 years; range: 1.9 – 16.9 years) and relapsed ALL (median: 12.7 years; range: 1.3 – 17.9 years) were treated with daunorubicin (10 mmol/l) or idarubicin (2 mmol/l) in vitro. We could show that both anthracylines induce apoptosis, as evidenced by measurement of genomic DNA fragmentation. Interestingly, daunorubicin only induced modest apoptosis, whereas idarubicin displayed a significantly stronger apoptosis inducing effect. Furthermore the treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in good response in most of the resistant cell populations. Out of the 65 patients analysed in this study 23 were female (13 de novo ALL, 10 relapsed ALL) and 42 were male (29 de novo ALL, 13 relapsed ALL). Primary lymphoblasts were obtained by bone marrow aspiration and separated by centrifugation over Ficoll. Within these cell populations following immunologic subgroups were found: 35 c-ALL, 10 pre-B-ALL, 7 pro-B-ALL, 10 T-ALL and 3 pre-T-ALL. Daunorubicin induced apoptosis in 33 out of 65 lymphoblast populations (response rate 50.8 %). Nevertheless, a far higher response rate was observed for idarubicin with 59/65 (90,8 %) (p < 0.008), if response is defined as apoptosis induction higher than 1 %. Daunorubicin-resistance was found in 32/65 (49,2 %), resistance to both was observed in 6/65 (9,2 %). Treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in significant apoptosis induction in 26 out of 32 cell populations (81,3 %). We clearly demonstrated here that the in vitro treatment of lymphoblasts from children with de novo or relapsed ALL with idarubicin induces significantly higher response rates than daunorubicin treatment. The ex vivo sensitivity of daunorubicin-resistant lymphoblasts of childhood ALL to idarubicin treatment reflects the better potency of idarubicin to induce apoptosis and to overcome daunorubicin resistance. These data prompted us to study the clinical relevance of idarubicin in ongoing clinical trials to improve existing therapeutic regiments. First clinical data point to a good tolerability of idarubicin in the treatment of relapsed ALL in childhood.

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Pharmacokinetic and Pharmacodynamic Effects of PEG-Asparaginase in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia: Results from a Single Agent Window Study.

Blood ◽

10.1182/blood.v110.11.860.860 ◽

2007 ◽

Vol 110 (11) ◽

pp. 860-860

Author(s):

Inge M. Appel ◽

Karin M. Kazemier ◽

Anjo J.P. Veerman ◽

Elisabeth van Wering ◽

Monique L. Den Boer ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Clinical Response ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Hazard Ratio ◽

Leukemic Cells ◽

Newly Diagnosed ◽

Pharmacodynamic Effects

Abstract L-Asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia. The effectiveness is generally thought to result from a rapid depletion of asparagine in serum and cells. Several studies have shown that in vitro resistance to this drug is an independent prognostic factor in ALL. We investigated the clinical response of one in vivo dose of 1000 IU/m2 PEG-Asparaginase and its pharmacokinetic and pharmacodynamic effects in children with newly diagnosed ALL before the start of combination chemotherapy. 57 children (36M / 21F) were enrolled in the study: 2 pro B-ALL, 38 common/ pre B-ALL and 17 T-ALL. Genotyping of precursor B-ALL revealed 11 hyperdiploid, 8 TELAML1 positive, 2 BCRABL positive, no MLL rearrangement, 8 normal, 11 others. The clinical response to PEG-Asparaginase on day 0 (5 days after the PEG-Asparaginase infusion) was defined as good when the number of leukemic cells of peripheral blood was < 1 × 109/L, as intermediate when leukemic cells were 1-10 × 109/L, and as poor when leukemic cells were > 10 × 109/L. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common / pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (p=0.01). Both BCRABL positive ALL cases showed a poor response (p=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-Asparaginase (p=0.02) and both in vitro as well as in vivo response were prognostic factors for long-term event-free survival (Hazard ratio 6.4; p=0.004, and Hazard ratio 3.7; p=0.01, respectively). The L-Asparaginase activity in the serum was >100 IU/L for at least 15 days. The asparagine levels remained below the detection limit of 0.2 mM for at least 26 days with a concomitant rise in serum aspartate and glutamate. These findings confirm that PEG-Asparaginase will yield its pharmacodynamic effects for 2-4 weeks. After administration of one in vivo dose of 1000 IU/m2 PEG-Asparaginase no changes in apoptotic parameters or changes in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. Otherwise it is possible that in vivo mesenchymal cells from the bone marrow supply leukemic blasts with asparagine in response to treatment with L-Asparaginase. Conclusion: The clinical response to one dose of 1000 IU/m2 PEG-Asparaginase intravenously is related to phenotype and genotype and predicts outcome. These results suggest that children with ALL with a poor clinical response to PEG-Asparaginase might benefit from a more intensive antileukemic therapy.

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In Vitro Drug Sensitivity as a Predicitive Tool of Early Clinical Response in Childhood Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v110.11.4232.4232 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4232-4232

Author(s):

Faith C. Galderisi ◽

Linda C. Stork ◽

Ju Li ◽

Motomi Mori ◽

Solange Mongoue-Tchokote ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Clinical Response ◽

Induction Therapy ◽

Drug Sensitivity ◽

Lymphoblastic Leukemia ◽

Mixed Effects ◽

Mixed Effects Model ◽

Drug Sensitivity Assay ◽

Pediatric All

Abstract Background: Residual disease or rapidity of response to induction therapy is among the most powerful predictors of outcome in pediatric Acute Lymphoblastic Leukemia (ALL). Various methods to determine response during induction have been in use in clinical investigation. We hypothesize that drug sensitivity at the cellular level predicts rapidity of response to induction therapy in ALL. We recently developed a high resolution flow cytometry based cytotoxicity assay for in vitro cellular drug response profiling for pediatric ALL. This method has a turnaround time of 48 hours and the ability to measure drug effect specific to leukemic cells regardless of number of admixed normal cells. We report preliminary data that correlate results of this drug sensitivity assay with rapidity of response to induction therapy among patients with ALL. Methods: We performed in vitro tests, applying a multiparameter flow cytometric drug cytotoxicity assay, on bone marrow (BM) samples of 23 patients with newly diagnosed standard (n = 10), high (n = 11), and very high (n = 2) risk ALL, as defined by the Children’s Oncology Group (COG) and NCI risk classification. Fourteen patients were rapid early responders (RER) and 9 were slow early responders (SER) by COG criteria at day 15 and 29. Cryopreserved cells from BM samples were thawed and determined to have adequate viability by trypan blue dye exclusion. Drugs were tested at three different concentrations, each in triplicate. Concentrations tested were based on an empirically derived cut-off concentration (EDCC) adopted from published in vitro studies, chosen to produce a large scatter of survival index values among samples. Leukemic blasts were specifically identified by surface markers, CD 45, CD 19 and CD 10 or CD 3, while cytotoxicity was measured with Annexin V based apoptosis. Leukemic cell survival index (LCSI = Average Replicate /Average Control x 100) was determined at 48 hours after in vitro exposure to individual standard induction agents for pediatric ALL: vincristine, asparaginase, dexamethasone, prednisone and daunomycin. LCSI differences between RER and SER were compared using Wilcoxon rank sum test for each drug and concentration. The mixed effects model was used to evaluate the overall difference of LCSI between RER and SER over the three concentrations (referred to as “averaged concentrations”). Results: For dexamethasone, a significantly lower LCSI was seen in the RER compared with the SER cohort at individual and averaged concentrations: RER mean LCSI = 40.2%, SER mean LCSI = 70.1% (p = 0.01, mixed effects model). A trend toward a lower mean LCSI in the RER compared with the SER group was noted for asparaginase and vincristine at individual and averaged concentrations (p < 0.1). Mean LCSI was not different between the RER and SER groups for daunomycin and prednisone at individual or averaged concentrations. Conclusions: This in vitro drug sensitivity assay provides a response profile for dexamethasone that correlates with in vivo response to induction therapy. Research is ongoing with more patient samples in order to achieve a greater statistical power to detect a smaller difference for all drugs tested. Further research will also correlate clinical response with LCSI using drug combinations in vitro. Results of these studies will determine the potential value of this assay for early risk stratification and modification of therapy in de novo or relapsed ALL.

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In-Vitro Efficacy of the Deoxyguanoside Analogs Forodesine (BCX-1777) and ARA-G in Pediatric Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v114.22.2038.2038 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2038-2038

Author(s):

Irene Homminga ◽

Michel C. Zwaan ◽

Amel Seghouani ◽

Chantal Y. Manz ◽

Shanta Bantia ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Nucleoside Transporter ◽

Nucleotide Synthesis ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Expression Levels ◽

Lc50 Value ◽

Pediatric All

Abstract Abstract 2038 Poster Board II-15 Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with elevated deoxyguanosine (dGuo) plasma levels. DGuo is converted into dGTP inducing apoptosis in T-cells and this provides the rationale for the development of deoxyguanosine analogues as a potential treatment option for T-cell malignancies. Forodesine (BCX-1777; BioCryst-Mundipharma) is an efficient blocker of PNP activity, thereby boosting the conversion of dGuo into dGTP and raising intracellular dGTP levels. AraG (9-b-D-arabinofuranosyl-guanine) is a compound that is resistant to PNP-mediated degradation that is efficiently converted into AraGTP. AraGTP becomes incorporated in the DNA, blocking DNA synthesis and promoting apoptosis. In a phase II clinical trial, the AraG prodrug Nelarabine enforced a complete remission rate of 55% for pediatric T-ALL patients at 1st relapse. (Berg, JCO 2005). Clinical data of Forodesine treatment in pediatric ALL patients are not yet available. As tested on primary pediatric acute lymphoblastic leukemia (ALL) patient samples (4 T-ALL, 2 BCP-ALL), 1μM of Forodesine is sufficient to completely block PNP and abolish rapid dGuo degradation resulting in a median 7.9 (range 0.5-378) fold raise of intracellular dGTP levels. Accumulation of dGTP is comparable for T-ALL (n=31) and BCP-ALL (n=11) patient samples. This reflects equal intrinsic ability of salvage nucleotide synthesis for both T-ALL and BCP-ALL cells. Cytotoxic effect of Forodesine was tested on primary leukemia cells from newly diagnosed pediatric ALL patients in-vitro by incubating cells with Forodesine (1μM) in the presence of increasing concentrations of dGuo (0.001-50μM). In accordance with selective T-cell toxicity, T-ALL cells were more sensitive to Forodesine/dGuo treatment (median T-ALL LC50 value: 1.1μM dGuo/1μM Forodesine, n=27, p=0.001) compared to BCP-ALL cells, which had a median LC50 value of 8.8μM dGuo/1μM Forodesine (n=30). All patients that responded demonstrated dGTP accumulation (1.5-222.1 fold), although the raise of dGTP levels did not correlate with Forodesine/dGuo toxicity (r2= 0.10, p=0.22). Studying in-vitro responsiveness to AraG, T-ALL cells were more sensitive compared to BCP-ALL cells (p=0.0002) with a median AraG LC50 value of 20.5μM for T-ALL samples (n=24) versus 48.3μM for BCP-ALL samples (n=20). Remarkably, TELAML1 positive BCP-ALL cases were insensitive to AraG treatment (median LC50 value >50μM, n=9). No correlation was identified between in-vitro Forodesine/dGuo and AraG cytotoxicities (r2=0.05, p=0.29). Most patient samples that displayed AraG resistance still responded to Forodesine/dGuo treatment. This may be explained by the fact that the uptake of both drugs may be facilitated by different transporters. Using RQ-PCR we could demonstrate that AraG toxicity, in contrast to Forodesine, was significantly associated with ENT1 (equilibrative nucleoside transporter 1) expression levels (p=0.008), which was previously identified as strong predictor for AraC cytotoxicity in pediatric ALL (Stam RW. et al., Blood 2003). AraG cytotoxicity strongly correlated with AraC cytotoxicity (r2=0.71, p<0.0001). We found no significant correlation between Forodesine sensitivity and the expression levels of other nucleoside transporters (CNT1, CNT2, CNT3, ENT2), kinases (dCK, dGK), nucleotidases (NT5C1A, NT5C2, PNI) or other enzymes that are involved in dGuo metabolism (PNP, RRM1, RRM2). In conclusion, T-ALL cells are more sensitive to Forodesine/dGuo treatment in-vitro than BCP-ALL cells that have nearly 8 fold higher dGuo LC50 values. Resistance to AraG treatment does not preclude responsiveness to Forodesine treatment and vice versa, indicating that Forodesine and AraG rely on different cellular mechanisms for cytotoxicity, possibly involving differences in dependence on the nucleoside transporter ENT1. Disclosures: No relevant conflicts of interest to declare.

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