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A total number of 817 children with acute lymphoblastic leukemia (ALL) and 181 with acute myeloblastic leukemia (AML) were assessed for individualized tumor response testing (ITRT) profile as a prognostic factor in long-term follow-up. For each patient, ITRT, initial response to therapy and long-term outcome were assessed. In initial ALL, an impact on long-term response was shown in ITRT for 13 drugs, while in initial AML only for cytarabine. For patients with ALL, a combined five-drug ITRT profile for prednisolone, l-asparaginase, vincristine, cytarabine and daunorubicin or doxorubicin had predictive value for probability of disease-free survival (pDFS) in univariate analysis, whereas in multivariate analysis, bone marrow response by day 33 was the only prognostic factor. For patients with AML, no factor had prognostic value for pDFS in univariate analysis, while ITRT to cytarabine almost reached significance. In conclusion, ITRT can possibly be regarded as a risk factor in childhood acute leukemias.

The proteasome inhibitor, bortezomib, is known to be effective in the therapy of various neoplasms. The objective of this study was the analysis of the ex vivo activity of bortezomib in paediatric acute lymphoblastic leukaemia (ALL), in comparison to paediatric acute myeloid leukaemia (AML). A total of 159 patients entered the study, including 106 ALL (including 86 precursor-B-cell ALL, and 20 T-cell ALL) and 53 AML children. The ex vivo sensitivity to bortezomib and 16 other drugs was studied by MTT assay. Paediatric AML samples were more resistant than paediatric ALL samples to most of the tested drugs, except for cytarabine and thioguanine. With respect to immunophenotype, ex vivo drug resistance in T-cell ALL (T-ALL) was higher for most of the drugs. No differences in drug resistance between T-ALL and common/pre-B-cell-ALL were found for daunorubicin, mitoxantrone and 6-thioguanine. Bortezomib was the only compound which was more active in T-ALL than in common/pre-B-ALL paediatric samples. In conclusion, bortezomib had good ex vivo activity in paediatric T-ALL samples.

Glufosfamide is a new agent for cancer chemotherapy. The objective of the study was the comparison of the in vitro drug resistance profile of glufosfamide with other oxazaphosphorines in 106 samples of childhood acute leukemia by means of the MTT assay. The following drugs were tested: glufosfamide, 4-HOO-ifosfamide, 4-HOO-cyclophosphamide, mafosfamide cyclohexylamine salt, prednisolone, vincristine, L-asparaginase, daunorubicin and cytarabine. In the group of initial Acute Lymphoblastic Leukemia (ALL) samples, equivalent cytotoxicity values for glufosfamide, 4-HOO-ifosfamide, 4-HOO-cyclophosphamide and mafosfamide were 5.95, 9.92, 4.60 and 3.90 microg/ml, respectively. In comparison to initial ALL samples, the relative resistance for glufosfamide and 4-HOO-ifosfamide in relapsed ALL samples were 1.9 (p=0.049) and 1.3 (ns), and in initial Acute Myeloblastic Leukemia (AML) samples, respectively, 31 (p0.001) and 5 (p=0.001). All oxazaphosphorines showed highly significant cross-resistance. In conclusion, in vitro activity of glufosfamide is comparable to ifosfamide. Glufosfamide shows high activity against lymphoblasts both on diagnosis and on relapse, however it cannot circumvent resistance to other oxazaphosphorines.

Cellular resistance in childhood acute leukemias might be related to profile and function of multidrug resistance proteins and apoptosis regulating proteins. The aims of the study were: (1) analysis of expression of MRP1, PGP1, LRP, BCL-2 and p53 proteins; (2) correlation with ex vivo drug resistance, and (3) analysis of their prognostic impact on clinical outcome in childhood acute lymphoblastic (ALL) and acute myeloid (AML) leukemia. Total number of 787 children diagnosed for initial ALL (n = 527), relapsed ALL (n = 104), initial AML (n = 133) and relapsed AML (n = 23) were included into the study. Mean follow-up period was 3.5 years. Drug resistance for up to 30 anticancer agents was performed by the MTT assay. Expression of all proteins was tested by flow cytometry. Both initial AML and relapsed ALL samples showed higher drug resistance than initial ALL samples. No significant differences were found in drug resistance between initial and relapsed AML samples. The presence of multidrug resistance and apoptosis proteins had no impact on pDFS in iALL and iAML, however strong trend towards adverse prognostic impact of MRP1, PGP and LRP on pDFS in rALL was observed. The same trend was observed for each of analyzed co-expressions of tested multidrug resistance proteins. The phenomenon of cellular drug resistance in childhood acute leukemias is multifactorial and plays an important role in response to therapy. Expression of MRP1, PGP and LRP proteins, as well as their co-expression play possible role in childhood relapsed ALL.

Combined ex vivo drug resistance profile to prednisolone, vincristine and L-asparaginase (PVA) has prognostic significance in childhood de novo ALL (Den Boer JCO 2003, Styczynski JCO 2004). So far, no data exist to support prognostic value of in vitro drug resistance profile in childhood acute myeloid leukemia (AML). The aim of this study was the analysis of prognostic value of ex vivo drug resistance profile in childhood AML. A total number of 90 children (46 male, 44 female) aged 0.1–17 yrs (median 10 yrs), with de novo AML were included into the study. All patients were recruited between 1999 and 2004 and were treated according to ANLL-98 protocol. Patients with early deaths were excluded from the study. Drug resistance profile was done by the MTT assay in one laboratory for 4–24 drugs. According to median cytotoxicity for each of tested drugs, all patients were scored as resistant or sensitive to this drug. Patients who received a HSC transplantation were censored at the time of transplantation. Drug resistance profile and other known prognostic parameters, were examined by Kaplan-Meier method and by multivariate analysis using the Cox regression proportional hazard model. Probability of overall survival was 0.57±0.05, p(LFS)=0.72±0.05, mean LFS 3.36 yrs (95%CI=2.95–3.77). Relapses occurred in 17/90 children; 40/90 children died during follow-up. Children who relapsed during follow-up showed median higher in vitro resistance of leukemic blasts to most of tested drugs, except for cytarabine, cladribine, vincristine, mercaptopurine and thioguanine. Better LFS was observed for patients with favourable cytogenetics (1.00 vs 0.74±0.09, p=0.055); early BM response by day 15 (0.76±0.07 vs 0.54±0.13, p=0.013); BM remission by day 28 of induction (0.74±0.07 vs 0.35±0.26, p=0.018); ex-vivo sensitive patients to cyclophosphamide (0.83±0.15 vs 0.65±0.09, p=0.12), doxorubicin (0.81±0.12 vs 0.64±0.09, p=0.18), epirubicin (0.72±0.13 vs 0.61±0.12, p=0.26), fludarabine (0.73±0.12 vs 0.62±0.11, p=0.22), mitoxantrone (0.77±0.12 vs 0.51±0.13, p=0.07), treosulphan (0.88±0.12 vs 0.62±0.11, p=0.29), etoposide (0.70±0.13 vs 0.63±0.09, p=0.4) and for patients with leukemic cells being sensitive to fludarabine, treosulphan and mitoxantrone ie. FTM score (0.73±0.12 vs 0.50±0.14, p=0.034). In multivariate analysis, two factors showed prognostic value: early BM response by day 15 (p=0.0021; HR=0.29, 95%CI=0.13–0.64) and combined ex vivo drug resistance profile to fludarabine, treosulphan and mitoxantrone (FTM score), p=0.0048; HR=0.38, 95%CI=0.19–0.77. Combined ex vivo drug resistance profile to fludarabine, treosulphan and mitoxantrone has prognostic significance in childhood acute myeloid leukaemia, however cytogenetics and early bone marrow response to therapy seems to have stronger prognostic value.

Introduction: In vitro drug resistance profile has so far provided information on chemosensitivity of leukemic cells in acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). Previous studies have shown that for most tested drugs, patients with relapse had higher IRTR than those with de novo ALL or AML. The objective of this study was the analysis and comparison of the individualized tumor response testing (ITRT) between first and second relapse in children with ALL or AML. Patients: A total of 186 pediatric leukemic samples (154 ALL and 32 AML) were tested for ex vivo chemosensitivity for up to 22 drugs. ALL samples included 113 samples obtained at first relapse, and 41 obtained at second relapse. AML samples included 22 samples obtained at first relapse, and 10 obtained at second leukemic relapse. The distribution of patients between first and second relapse groups was comparable. Methods: In vitro drug resistance was tested by the MTT assay. The drug concentration that was inhibitory to 50% of the cells (IC50) was calculated from the dose-response curve and was used as a measure of ex vivo drug resistance for each sample. The relative resistance (RR) between groups analyzed for each drug was calculated as the ratio of the mean values of the IC50 of the respective groups for this drug. Only patients who had a successful MTT assay at diagnosis were included in the study. The following drugs were used: prednisolone, dexamethasone, vincristine, idarubicin, daunorubicin, doxorubicin, mitoxantrone, L-asparaginase, cytarabine, fludarabine, cladribine, clofarabine, treosulfan, thiotepa, melphalan, 4-HOO-cyclophosphamide, 4-HOO-ifosfamide, bortezomib, busulfan, 6-mercaptopurine, and 6-thioguanine. For patients with ALL, combined drug resistance profile to prednisolone, vincristine and L-asparaginase (PVA score) was also analyzed. Results: ALL: In comparison to first relapse, second relapsed childhood ALL were more resistant to most of tested drugs. Median PVA score in multiple relapsed patients was 8 vs 6 for patients at first relapse (p=0.004). The median relative resistance value between patients with multiple relapse and those with first relapse for all tested drugs was 2.0, indicating higher drug resistance on second relapse. Multiple relapsed ALL samples were more drug resistant to: prednisolone (>1.9-fold), dexamethasone (>1.5-fold), vincristine (3.1-fold), L-asparaginase (5-fold), mitoxantrone (2.4-fold), cytarabine (4.3-fold), mercaptopurine (2.2-fold), thioguanine (4.8-fold), etoposide (2.6-fold) and melphalan (2.7-fold). On the other hand, lymphoblasts at second relapse were comparably resistant to: daunorubicin, doxorubicin, 4-HOO-cyclophosphamide, 4-HOO-ifosfamide, busulfan, treosulfan, fludarabine, clofarabine and bortezomib. No drug showed a trend towards better cellular sensitivity at first versus second relapse. AML: No significant differences between ITRT at first and second relapse of childhood AML were found. Of the all drugs analyzed, no drug was found for which significantly higher resistance of myeloblasts was observed at second relapse when compared to first relapse of AML. The median RR value between second and first relapse of all tested drugs was 1.0; for 10 drugs the RR was less than 1 (i.e. assumed better sensitivity on subsequent relapse) and for another 11 drugs, the RR value was greater than 1 (i.e. higher drug resistance on subsequent relapse). No drug showed a trend towards better cellular sensitivity at first versus subsequent relapse, as the differences were not significant for each tested drug. Conclusion: In comparison to first relapse, leukemic blasts of children at second relapse of ALL are more in vitro resistant to most of tested drugs. Contrary, myeloblasts of children at second relapse of AML show drug resistance comparable to first relapse. (This study was supported by NCN Grant DEC-2011/03/D/NZ5/05749.) Disclosures No relevant conflicts of interest to declare.

Abstract 1456 BACKGROUND: Individualized tumor response testing (ITRT) is a recently accepted term for ex vivo drug resistance profile testing in cancer patients. Previous studies have shown that ITRT results in children correspond with clinical response in initial acute lymphoblastic leukemia (iALL), but not in initial acute myeloid leukemia (iAML). ITRT combined profile to prednisolone, vincristine, and L-asparaginase (PVA score) showed a strong association with clinical outcome in iALL (Den Boer et al JCO; Hollemann et al, NEJM). OBJECTIVE: Polish Pediatric Leukemia Study Group carried out between 1999–2010 a multi-institutional, non-interventional study on correlation between ITRT profile and clinical response in long-term follow-up. The objective of the study was to assess the role of ITRT profile as prognostic factor in childhood iALL and iAML. MATERIALS AND METHODS: A total number of 998 children (median age 6.5 yrs, range 3 days - 21 yrs) were enrolled into the study, including 817 with initial ALL (iALL) and 181 children with initial AML (iAML). The median follow-up was 4.6 yrs (range 0–10,4). ITRT (ex vivo drug resistance profile) was determined by the MTT assay. Following drugs were tested: prednisolone (P), vincristine (V), L-asparaginase (A), daunorubicin (D), doxorubicin (Dox), idarubicin (I), mitoxantrone (M), cytarabine (C), busulfan (B) and etoposide (E). Apart from ITRT to single drugs, combined ex vivo drug resistance profiles PVA, PVADC, PVADoxC in iALL and CVIDM in iAML were also determined. Probability of DFS was estimated by the Kaplan-Meier method and compared by the log-rank test. Univariate and multivariate analyses were performed in Cox regression model. RESULTS: (1) For iALL patients, neither single drug nor 3-drugs PVA combined ITRT profile had prognostic value for pDFS. 5-drug PVADC and PVADoxC profiles had predictive values, with p-values 0.049 and 0.017, respectively. For 295 iALL patients with ITRT-sensitive PVADC profile, 5-year pDFS=0.892. No ITRT factor was discriminative between very early, early and late ALL relapses. In univariate analysis in Cox model, following factors had prognostic value for pDFS in iALL: steroid resistance by day 8 (p=0.002), bone marrow (BM) response by day 15 (p=0.005), BM response by day 33 (p=0.001) and PVADC ITRT profile (p=0.052). In multivariate analysis in Cox model, only BM response by day 33 (p=0.001) had prognostic value for pDFS in iALL, while bone marrow BM by day 15 had borderline significance (p=0.055). (2) For iAML patients, ITRT to cytarabine had an impact on pDFS (0.81 vs 0.63, p=0.047). For 55 iAML patients with ITRT-sensitive to cytarabine, 5-year pDFS=0.816. 5-drug CVIDM profile had also predictive values, with p-value 0.020. No ITRT factor was discriminative between very early, and late AML relapses. In univariate analysis in Cox model, no factor had prognostic value for pDFS in iAML, while ITRT to cytarabine almost reached significance (p=0.054). Multivariate analysis for iAML was not performed. CONCLUSION: Ex vivo drug resistance profile (ITRT) can be regarded as a risk factor in childhood acute leukemias. Combined ITRT profile to prednisolone, vincristine, L-asparaginase, daunorubicin and cytarabine (PVADC) has predictive value in pediatric iALL, while ITRT profile to cytarabine has prognostic value in pediatric iAML. ACKNOWLEDGEMENTS: Supported by grants: EFS-POKL.04.01.01-00-191/08-00 nr 1/2010; MNiSW Nr N N407 541339; MNiSW Nr N407 078 32/2964; UMK 09/2009; UMK 10/2009. Disclosures: No relevant conflicts of interest to declare.