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Purpose Neuroblastoma (NB) is the most frequent extracranial tumor in children. The detection of bone marrow (BM) involvement is crucial for correct staging and risk-adapted treatment. We compared three methods regarding the detection of NB involvement in BM. Methods Eighty-one patients with NB were included in this retrospective study. BM samples were obtained at designated time points at study entry and during treatment or follow-up. The diagnostic tools for BM analysis included cytomorphology (CM), flow cytometry (FCM) and automatic immunofluorescence plus fluorescence in situ hybridization (AIPF). Results We analyzed 369 aspirates in 81 patients in whom AIPF, CM, and FCM were simultaneously available. During the observation period, NB cells were detected in 86/369 (23.3%) cases, by CM in 32/369 (8.7%), by FCM in 52 (14.1%), and by AIPF in 72 (19.5%) samples. AIPF and/or FCM confirmed all positive results obtained in CM and detected 11 additional positive BM aspirates in 294 CM negative samples (p Conclusion The combination of AIPF/FCM yielded the highest detection rate of NB cells in BM. AIPF was the single, most sensitive method in detecting these cells. Although CM did not provide any additional positive results, it is still a useful, readily available and cost-effective tool. The prognostic significance of FCM and AIPF should be confirmed in a prospective study with a larger number of patients.

Measurement of minimal residual disease (MRD) with multicolor flow cytometry (MFC) has become an important tool in childhood acute lymphoblastic leukemia (ALL), mainly to identify rapid responders and reduce their therapy intensity. Protocols of the Moscow-Berlin (MB) group use a comparatively low (for standard risk; SR) or moderate (for intermediate risk; ImR) treatment intensity from the onset, based on initial patient characteristics. Recently, we reported that 90% of SR patients-50% B cell precursor (BCP-ALL)-MFC-MRD negative at end of induction (EOI)-had 95% event-free survival (EFS). METHODS: In the present study, we applied this method to children with initial ImR features. In study MB 2008, 1105 children-32% of BCP-ALL patients-were assigned to the ImR group. Of these, 227 were treated in clinics affiliated with MFC laboratories of the MB group network, and included in this MFC-MRD pilot study. A single-point MFC-MRD measurement at the EOI with the threshold of 0.01% identified 65% of patients-20% of all BCP-ALL patients-with EFS of 93.5%. Taking both studies together, the combination of clinical parameters and a one-point MRD measurement identifies 70% of BCP-ALL patients with an excellent outcome after low- or moderate-intensity therapy and avoids overtreatment of a significant proportion of patients.

Children with other extramedullary relapse of acute lymphoblastic leukemia are currently poorly characterized. We aim to assess the prevalence and the clinical, therapeutic and prognostic features of extramedullary localizations other than central nervous system or testis in children with relapse of acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) treated on a relapsed ALL protocol. Patients and Methods: Patients with relapse of ALL and LBL, treated according to the multicentric ALL-REZ BFM trials between 1983 and 2015, were analyzed for other extramedullary relapse (OEMR) of the disease regarding clinical features, treatment and outcome. Local treatment/irradiation has been recommended on an individual basis and performed only in a minority of patients. Results: A total of 132 out of 2323 (5.6%) patients with ALL relapse presented with an OEMR (combined bone marrow relapse n = 78, isolated extramedullary relapse n = 54). Compared to the non-OEMR group, patients with OEMR had a higher rate of T-immunophenotype (p &lt, 0.001), a higher rate of LBL (p &lt, 0.001) and a significantly different distribution of time to relapse, i.e., more very early and late relapses compared to the non-OEMR group (p = 0.01). Ten-year probabilities of event-free survival (pEFS) and overall survival (pOS) in non-OEMR vs. OEMR were 0.38 ± 0.01 and 0.32 ± 0.04 (p = 0.0204) vs. 0.45 ± 0.01 and 0.37 ± 0.04 (p = 0.0112), respectively. OEMRs have been classified into five subgroups according to the main affected compartment: lymphatic organs (n = 32, 10y-pEFS 0.50 ± 0.09), mediastinum (n = 35, 10y-pEFS 0.11 ± 0.05), bone (n = 12, 0.17 ± 0.11), skin and glands (n = 21, 0.32 ± 0.11) and other localizations (n = 32, 0.41 ± 0.09). Patients with OEMR and T-lineage ALL/LBL showed a significantly worse 10y-pEFS (0.15 ± 0.04) than those with B-Precursor-ALL (0.49 ± 0.06, p &lt, 0.001). Stratified into standard risk (SR) and high risk (HR) groups, pEFS and pOS of OEMR subgroups were in the expected range whereas the mediastinal subgroup had a significantly worse outcome. Subsequent relapses involved more frequently the bone marrow (58.4%) than isolated extramedullary compartments (41.7%). In multivariate Cox regression, OEMR confers an independent prognostic factor for inferior pEFS and pOS. Conclusion: OEMR is adversely related to prognosis. However, the established risk classification can be applied for all subgroups except mediastinal relapses requiring treatment intensification. Generally, isolated OEMR of T-cell-origin needs an intensified treatment including allogeneic stem cell transplantation (HSCT) as a curative approach independent from time to relapse. Local therapy such as surgery and irradiation may be of benefit in selected cases. The indication needs to be clarified in further investigations.

Aim: Outcomes of children with high-risk (HR) relapsed acute lymphoblastic leukaemia (ALL) (N = 393), recruited to ALLR3 and ALL-REZ BFM 2002 trials, were analysed. Minimal residual disease (MRD) was assessed after induction and at predetermined time points until haematopoietic stem cell transplantation (SCT). Methods: Genetic analyses included karyotype, copy-number alterations and mutation analyses. Ten-year survivals were analysed using Kaplan-Meier and Cox models for multivariable analyses. Results: Outcomes of patients were comparable in ALLR3 and ALL-REZ BFM 2002. The event-free survival of B-cell precursor (BCP) and T-cell ALL (T-ALL) was 22.6% and 26.2% (P = 0.94), respectively, and the overall survival (OS) was 32.6% and 28.2% (P = 0.11), respectively. Induction failures (38%) were associated with deletions of NR3C1 (P = 0.002) and BTG1 (P = 0.03) in BCP-ALL. The disease-free survival (DFS) and OS in patients with good vs poor MRD responses were 57.4% vs 22.6% (P < 0.0001) and 57.8% vs 32.0% (P = 0.0004), respectively. For BCP- and T-ALL, the post-SCT DFS and OS were 42.1% and 56.8% (P = 0.26) and 51.6% and 55.4% (P = 0.67), respectively. The cumulative incidences of post-SCT relapse for BCP- and T-ALL were 36.9% and 17.8% (P = 0.012) and of death were 10.7% and 25.5% (P = 0.013), respectively. Determinants of outcomes after SCT were acute graft versus host disease, pre-SCT MRD (≥10 −3), HR cytogenetics and TP53 alterations in BCP-ALL. Conclusion: Improvements in outcomes for HR ALL relapses require novel compounds in induction therapy to improve remission rates and immune targeted therapy after induction to maintain remission after SCT. Trial registration: ALLR3: NCT00967057; ALL REZ-BFM 2002: NCT00114348

Purpose Neuroblastoma (NB) is the most frequent solid malignancy in children outside the central nervous system. Detection of bone marrow (BM) involvement is crucial for correct staging and risk-adapted treatment. We compared three different methods regarding the detection of NB involvement in BM. Methods Eighty-one patients with NB were included in this retrospective study. BM samples were obtained at designated time points at study entry and during treatment or follow-up. The diagnostic tools for BM analysis included cytomorphology (CM), flow cytometry (FCM) and automatic immunofluorescence plus fluorescence in situ hybridization (AIPF). Results We analyzed 369 aspirates in 81 patients in whom AIPF, CM, and FCM were simultaneously available. During the observation period, NB cells were detected in 86/369 (23.3%) cases, by CM in 32/369 (8.7%) samples, by FCM in 52 (14.1%) samples, and by AIPF in 72 (19.5%) samples. AIPF and/or FCM confirmed all positive results obtained in CM and detected 11 additional BM aspirates with NB cells in 294 CM negative samples (p

Introduction: Acute lymphoblastic leukemia (ALL) is the most common cancer affecting children and its cure rates are close to 90%. Nevertheless, relapsed ALL is still a major cause of cancer-associated deaths in children. Therefore, the multicenter trial Acute Lymphoblastic Leukemia-Relapse Study of the Berlin-Frankfurt-Münster Group (ALL-REZ BFM) 2002 was designed to improve the prognosis of children with relapsed ALL by introducing new chemotherapy elements. Little is known though about the prognostic relevance of deviations from the ALL protocols. This study aimed to investigate the characteristics of such protocol deviations and to determine their prognostic relevance for the relapsed disease. Methods: We performed a retrospective analysis of 686 children and adolescents up to 18 years of age with first relapse of ALL who were enrolled between 2002 and 2012 in the ALL-REZ BFM 2002 Study. Deviations were identified in terms of time and type. In order to investigate the prognostic importance of the time delay, the 90th percentile of delays between defined treatment elements (i.e. 15.5 days) was used as a cut-off point. Furthermore, deviations were categorized as avoidable (logistics, family/PI decisions) and non-avoidable (clear medical indication). The protocol deviation occurred during the course of therapy was therefore considered as a time-dependent variable. Disease-free survival, (DFS, induction failures excluded) and OS were analyzed using Kaplan-Meier estimate, log-rank test, Mantel-Byar test and Cox/logistic regression (univariate and multivariate analyses). Results: A total of 587 patients (86%) received the protocol therapy, whereas 99 patients (14%) underwent deviations during their treatment. Baseline characteristics were compared in both groups (Table 1). Deviations were categorized as: change of chemotherapy in 48% of the patients (n=47/99), stop of chemotherapy in 10 % (n=10/99), non-protocol compliant reaction to MRD findings in 28% (n=28/99) and change of radiation treatment in 14% (n=14/99). The cumulative incidence of protocol deviation was 6% and 15.5% at 3 and 9 months, respectively (Figure 1). The remission rate of patients with protocol deviation during induction was slightly lower compared to patients without such deviation (24/29, 83% vs. 579/657, 88%; P=0.38). The estimated 5-year probabilities of DFS and OS between the patients treated according to protocol therapy and those with deviations were significantly different (pDFS, Protocol therapy: 0.61 ± 0.02 vs. Deviation: 0.38 ± 0.05, P < 0.001; pOS, Protocol: 0.70 ± 0.02 vs. Deviation: 0.57 ± 0.05, P < 0.001; Figure 2, 3). In multivariate analyses protocol deviation, time point of relapse and immunophenotype turned out to be independent prognostic factors of the DFS (Table2). The hazard ratio of the protocol deviation was significant only in patients with the late relapse, whereas in patients with early/very early relapse no significant prognostic effect could be found [late (n=40): HR=2.53, 95%CI 1.53-4.21, P Moreover, DFS probabilities were comparable for avoidable (n=63) and non-avoidable (n=31) deviations (0.43 ± 0.07 vs. 0.40 ± 0.09, P=0.9; Non avoidable: HR=1.19, 95%CI=0.68-2.10, P= 0.53). In terms of the time-related deviations, the extent of the delays between induction and beginning of consolidation therapy was not significantly associated with the DFS [≤90th percentile (n=319/603); pDFS: =0.60 ± 0.02 vs. >90th percentile (n=36/603); pDFS: 0.56 ± 0.08, P=0.3]. Treatment delays during other phases of the protocol were also not related to the outcome. Conclusion: While protocol deviations did not significantly affect remission rates in this study, they were significantly related to inferior DFS. Since a relevant part of the protocol deviations could be classified as avoidable, strict protocol compliance should lead to improved outcomes. The prognostic impact of protocol deviations was in particular relevant in patients with late relapse with generally rather chemosensitive diseases and better prognosis. In this analysis, treatment delays did not influence the outcome. Figure 1 Figure 1. Disclosures von Stackelberg: Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Miltenyi: Consultancy, Honoraria.

Usually, central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is diagnosed by cytomorphology (CM) of cerebrospinal fluid (CSF) on cytospin slides. Multicolor flow cytometry (MFC) provides the opportunity to detect low numbers of leukemia cells undetectable by CM. The present study aimed at evaluating the clinical significance of MFC for the diagnosis of CNS involvement at initial manifestation of childhood ALL. In 155 children with ALL, CSF samples were studied in parallel by CM and MFC. Patients were treated according to protocol ALL-MB-2008 for childhood ALL. The prognostic impact of the leukemia burden in CSF was determined categorizing the findings as positive/negative. In addition, the absolute blast cell count per 1 ml of CSF was studied as a continuous variable. CSF positivity was significantly more frequent using MFC compared with CM (35.3% vs. 15.3% of patients). The outcome of MFC-positive and MFC-negative patients was not different in clinically relevant patient risk groups—CNS1, standard and intermediate-risk groups. Using the quantitative approach, at the threshold level of 20 blasts per ml of CSF, patients could be divided into two groups with a significantly different outcome, irrespective of the clinical risk group, the type of CNS-directed therapy, and the CNS status determined by CM. Our data do not support the concept of re-stratification and modification of therapy based on qualitative CSF investigation by MFC. However, MFC is a highly sensitive technique of CSF investigation improving the definition of CNS involvement in childhood ALL, and quantitative measurement of blast cells in CSF, if well-organized, can be a useful additional tool for stratification of patients in clinical trials.

The prognostic significance of genetic lesions in T-cell ALL still needs to be elucidated. Karyotyping and FISH were performed in samples from 120 patients with T-cell ALL registered in the trial Moscow-Berlin 2008. Most frequent rearrangements were TLX3 (N = 29; 24%) and TAL1 (N = 18; 15%), followed by KMT2A (N = 6; 5%), TLX1 (N = 5; 4.2%), and 11p13-15 (N = 5; 4.2%). In 16.7% of patients, the karyotype was normal, and in 30.8% 'other' aberrations were seen. Patients with a normal karyotype, TAL1, or KMT2A rearrangements had the most favorable outcome (probability of event free survival (pEFS): 82% ± 6%), while prognosis for patients with TLX3 and TLX1 rearrangements and 'other' aberrations was less favorable (pEFS: 62% ± 6%). Worst outcome was observed for five patients with 11p rearrangements (pEFS: 20% ± 18%). In summary, three subgroups of patients with T-cell ALL with significantly different outcomes could be defined by cytogenetic profiling.

The Associazione Italiana di Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Acute Lymphoblastic Leukemia (AIEOP-BFM ALL 2000) study has for the first time introduced standardized quantitative assessment of minimal residual disease (MRD) based on immunoglobulin and T-cell receptor gene rearrangements as polymerase chain reaction targets (PCR-MRD), at 2 time points (TPs), to stratify patients in a large prospective study. Patients with precursor B (pB) ALL (n = 3184) were considered MRD standard risk (MRD-SR) if MRD was already negative at day 33 (analyzed by 2 markers, with a sensitivity of at least 10-4); MRD high risk (MRD-HR) if 10-3 or more at day 78 and MRD intermediate risk (MRD-IR): others. MRD-SR patients were 42% (1348): 5-year event-free survival (EFS, standard error) is 92.3% (0.9). Fifty-two percent (1647) were MRD-IR: EFS 77.6% (1.3). Six percent of patients (189) were MRD-HR: EFS 50.1% (4.1; P < .001). PCR-MRD discriminated prognosis even on top of white blood cell count, age, early response to prednisone, and genotype. MRD response detected by sensitive quantitative PCR at 2 predefined TPs is highly predictive for relapse in childhood pB-ALL. The study is registered at http://clinicaltrials.gov: NCT00430118 for BFM and NCT00613457 for AIEOP. (Blood. 2010;115(16):3206-3214) © 2010 by The American Society of Hematology.

Impaired tumor suppressor functions, such as deficient p53, are characteristic for glioblastoma multiforme (GBM) and can cause resistance to DNA-damaging agents like cisplatin. We have recently shown that the INhibitor of Growth 1 (ING1) tumor suppressor is down-regulated in malignant gliomas and that the decrease of ING1 expression correlates with histological grade of malignancy, suggesting a role for ING1 in the pathogenesis and progression of malignant gliomas. Based on this background, the purpose of our current study was to examine the potential impact of ING1 protein levels on DNA-damage response in GBM. Using LN229 GBM cells, which express ING1 proteins and harbor mutant TP53, we are the first to show that DNA damage by cisplatin or ionizing radiation differentially induced the two major ING1 splicing isoforms. The p47ING1a isoform, that promotes deacetylation of histones, thus formation of heterochromatic regions of DNA, which are less susceptible to DNA damage, was preferentially induced by >50-fold. This might represent a response to protect DNA from damage. Also, ING1 knockdown by siRNA accelerated transit of cells through G1 phase, consistent with ING1 serving a tumor suppressor function, and caused cells to enter apoptosis more rapidly in response to cisplatin. Our results indicate that malignant gliomas may down-regulate ING1 to allow more efficient tumor growth and progression. Also, ING1 down-regulation may sensitize GBM cells with deficient p53 to treatment with cisplatin.

In this study, we examined the gene expression of interleukin (IL)-8, CXCR3, and CXCR4 in leukemic cells from 100 children with relapsed B-cell progenitors (BCP) acute lymphoblastic leukemia (ALL), using quantitative real-time polymerase chain reaction (RT-PCR). IL-8, CXCR3, and CXCR4 were expressed in almost all bone marrow (BM) samples. The CXCR4 expression significantly correlated with known prognostic factors at relapse: time point and site of relapse. Patients who had a combined BM relapse (n=21) had lower IL-8 and CXCR4 expression than those who had an isolated BM relapse (n=79). The CXCR3 expression was higher in female patients (n=39) than in male patients (n=61). However, this did not reach prognostic relevance in relapsed ALL.

Purpose In the Non-Hodgkin's Lymphoma–Berlin-Frankfurt-Munster (NHL-BFM) 95 trial, we tested, against the historical control of the combined trials NHL-BFM90 and NHL-BFM86, whether prophylactic cranial radiotherapy (PCRT) can be omitted for CNS-negative patients with stage III or IV lymphoblastic lymphoma (LBL) with sufficient early response. Patients and Methods Apart from the removal of PCRT in NHL-BFM95, the chemotherapy of the three trials was identical except for the amount of l-asparaginase and daunorubicin during induction. The therapy in NHL-BFM95 was accepted to be noninferior when compared with trials NHL-BFM90/86 if the lower limit of the one-sided 95% CI for the difference in the 2-year probability of event-free-survival (pEFS) between target patients of NHL-BFM95 and the historical controls of NHL-BFM90/86 did not exceed −14%. The target patient group consisted of stage III and IV patients who were CNS negative and responded well to induction therapy. Results The number of target patients was 156 in NHL-BFM95 (median age, 8.6 years; range, 0.2 to 19.5 years) and 163 in NHL-BFM90/86 (median age, 8.4 years; range, 0.6 to 16.6 years). For the target group, the pEFS rates at 2 and 5 years were 86% ± 3% and 82% ± 3%, respectively, in NHL-BFM95 (median follow-up time, 5.1 years; range, 2.1 to 9.1 years) compared with 91% ± 2% and 88% ± 3%, respectively in NHL-BFM90/86 (median follow-up time, 10.7 years; range, 5 to 15.4 years). The lower limit of the one-sided 95% CI for the difference in pEFS was −11% at 2 years and −13% at 5 years. In NHL-BFM95, one isolated and two combined CNS relapses occurred compared with one combined CNS relapse in NHL-BFM90/86. Five-year disease-free-survival rate was 88% ± 3% in NHL-BFM95 compared with 91% ± 2% in NHL-BFM90/86. Conclusion For CNS-negative patients with stage III or IV LBL and sufficient response to induction therapy, treatment without PCRT may be noninferior to treatment including PCRT.

We report on the first pediatric patient with a localized primary peripheral T-cell lymphoma, not otherwise specified, of the central nervous system (CNS). The solid lesion that was enhanced in magnetic resonance images of the left precentral region was totally resected. The histopathology revealed a peripheral T-cell lymphoma, not otherwise specified. Staging procedures showed that the lesion was confined to the CNS. Without any further therapy, the patient still remains in complete remission 6 years after diagnosis. Thus, we conclude that a peripheral T-cell lymphoma, not otherwise specified, of the CNS can occur in children. In the case presented here, complete resection sufficed.

Purpose: Relapse of disease and subsequent resistance to established therapies remains a major challenge in the treatment of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). New therapeutic options, such as proteasome and histone deacetylase inhibitors (HDACi) with a toxicity profile differing from that of conventional cytotoxic agents, are needed for these extensively pretreated patients. Experimental Design: Antiproliferative and proapoptotic effects of combined HDACi/proteasome inhibitor treatments were analyzed using BCP-ALL monocultures, cocultures with primary mesenchymal stroma cells from patients with ALL, and xenograft mouse models. The underlying molecular mechanisms associated with combined treatment were determined by gene expression profiling and protein validation. Results: We identified the proteasome inhibitor bortezomib as a promising combination partner for HDACi due to the substantial synergistic antileukemic activity in BCP-ALL cells after concomitant application. This effect was maintained or even increased in the presence of chemotherapeutic agents. The synergistic effect of combined HDACi/BTZ treatment was associated with the regulation of genes involved in cell cycle, JUN/MAPK, PI3K/AKT, p53, ubiquitin/proteasome, and NF-κB pathways. We observed an activation of NF-κB after bortezomib treatment and the induction of apoptosis-related NF-κB target genes such as TNFαRs after concomitant treatment, indicating a possible involvement of NF-κB as proapoptotic mediator. In this context, significantly lower NF-κB subunits gene expression was detected in leukemia cells from patients who developed a relapse during frontline chemotherapy, compared with those who relapsed after cessation of frontline therapy. Conclusion: These results provide a rationale for the integration of HDACi/BTZ combinations into current childhood BCP-ALL treatment protocols. Clin Cancer Res; 19(6); 1445–57. ©2013 AACR.

Background/Aims: With rising cure rates of childhood cancer, side effects of treatment are attracting increasing interest. The present analysis evaluates the influence of tumor localization, radiotherapy and chemotherapy on the age of menarche. Methods: 4,689 former pediatric oncology patients, diagnosed 1980–2004, were contacted in collaboration with the German Childhood Cancer Registry. Results: 1,036 out of 1,461 female participants reported their age at menarche and had an oncological diagnosis before menarche. The median age at menarche was 13 years, compared to 12.8 years in the German general population. A significant delay of menarche was seen in patients with pituitary radiation doses of ≧30 Gy (mean 13.6 years, SD 2.2) compared to Conclusion: Overall, female childhood cancer survivors showed a normal menarcheal age. Pituitary radiation dosage of ≧30 Gy, spinal and pelvic radiotherapy were associated with a moderate delay in the occurrence of menarche.

Fertility can be impaired by radiation and chemotherapy among childhood cancer survivors. Therefore, timely and adequate patient counselling about the risk of infertility and preservation methods is needed. The primary study objective was to assess remembered counselling among childhood cancer survivors. As a second objective, the impact of lacking patient counselling on offspring-related attitudes and behaviour was examined. Counselling regarding the late effects of gonadotoxicity that could be recalled by patients was assessed using a questionnaire sent by the German Childhood Cancer Registry. The questionnaire was answered by 2754 adult childhood cancer survivors (53.1% female, mean = 25.7 years). The proportion of patients who could not remember patient counselling about the late effects of chemo-/radiotherapy on fertility decreased significantly over time. In 1980 to 1984 67%, in 2000 to 2004 50% of the patients reported no memories of counselling (p < .001). Counselled patients feared significantly less that their children may have an increased cancer risk (4.4% vs. 6.7%, p = .03). They were also more likely to undergo fertility testing than patients who could not recall counselling (odds ratio = 2.91, 95% confidence interval [2.12, 3.99]). Patients reported an increased memory of patient counselling over the past 25 years. Still, a 50% rate of recalled counselling shows an ongoing need for adequate and especially sustainable counselling of paediatric cancer patients about infertility and other long-term adverse treatment effects. Those who reported a lack of counselling had offspring-related fears more frequently, which stopped them from having children.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and accounts for 25% of cancers that occur before 15 years of age and 19% among persons less than 20 years of age (Ries et al. 1999). Based on the most recent estimates from the Surveillance, Epidemiology and End Results (SEER) Program of the National Cancer Institute (NCI), there are about 3,000 cases of ALL diagnosed in the United States each year among persons less than 20 years of age. The incidence peaks at 80–90 cases/million at 2–3 years of age, begins to drop abruptly at age 5–6 years reaching a rate of about 20 cases/million at 8–11 years of age, and then gradually decreases to an annual rate of about 10 cases/million by 20 years of age.

To the Editor,Ondansetron is a serotonin (5-hydroxitriptamine, 5-HT) receptor antagonist which is widely used as anti-emetic agent in the prophylactic treatment of chemotherapy-induced nausea and v...

Prognosis for patients with glioblastoma multiforme (GBM) is poor. Inhibitors of histone deacetylases (HDACi) like trichostatin A (TSA) are promising alternatives to conventional treatment. Deficient tumor suppressor functions, such as TP53 mutations and p14(ARF)/p16(INK4a) deletions, are characteristic for GBM and can cause resistance to DNA damaging agents such as cisplatin and to HDACi like TSA. The type II tumor suppressor Inhibitor of growth 1 (ING1) is involved in DNA damage response and histone modification. We have previously shown that ING1 is downregulated in GBM and involved in glioma-induced angiogenesis and in cisplatin-induced apoptosis in malignant glioma cells. Hence, the goal of our present study was to investigate whether TSA affects ING1 protein expression and also whether modulating ING1 levels affects TSA-induced apoptosis in malignant glioma cells that contain deficient p53 function and inactive pl4(ARF)/p16(INK4a) signaling. If so, we asked, which apoptotic pathway might be the major mediator beyond this interaction. To test whether ING1 proteins function in TSA-induced apoptosis in GBM, we analyzed TSA effects in LN229 GBM cells, which harbor TP53 mutations and INK4a deletion, following ING1 knockdown by siRNA. Expression of ING1, acetylated core histones H3 and H4, and the proapoptotic proteins caspase 3 and Fas-associated death domain (FADD) was determined by Western blotting. Percentages of apoptotic cells were obtained by flow cytometry. TSA induced the major ING1 isoform p33(ING1b) and increased levels of both histone acetylation and apoptosis in LN229 cells. ING1 knockdown cells revealed marked resistance to TSA-induced apoptosis, impairment of caspase 3 activation, and suppression of FADD. The data suggest that ING1 contributes to TSA-induced apoptosis in GBM cells with deficient p53 and p14(ARF)/p16(INK4a) functions, possibly by regulating FADD/caspase 3 signaling.

Despite the growing knowledge on molecular risk factors of the most common malignant brain tumor in childhood, medulloblastoma, its biology remains only partially understood. A previous study investigating the entire mitochondrial genome of medulloblastoma revealed a number of somatic mutations in tumor and corresponding cerebrospinal fluid samples. In our present study we sought to corroborate these results on somatic and germ line mutations by comparing the complete mitochondrial genome sequences of medulloblastoma tissue in a further cohort of patients. Analysis of the entire mitochondrial genome by temporal temperature gel electrophoresis and direct sequencing revealed 6 somatic mutations in 6 of 15 medulloblastoma. All changes were insertions, deletions, or substitutions restricted to the np 303 to 315 poly-C tract of the D-loop region. Three were changes from heteroplasmy to homoplasmy. Two were changes from heteroplasmy to heteroplasmy and one mutation represented a change from homoplasmy to heteroplasmy. In addition, 25 distinct germ line variations were identified. These results are in support of our previous findings on frequency of somatic mitochondrial mutations in medulloblastoma. Somatic alterations were found only in the hypervariable D-loop region, supporting the idea that these control regions contain hot spots for both, germ line variations and somatic alterations of the mitochondrial genome.

The inhibitor of growth 1 (ING1) homologue ING4 has previously been implicated as a negative regulator of angiogenesis in a murine glioma and a multiple myeloma model. An association between ING1 and angiogenesis has not been reported yet. Our previous studies using tumor samples from patients have shown that ING1 levels are downregulated in glioblastoma multiforme (GBM), one of the most highly vascularized malignancies. Based on this background, the goal of this study was to test the effects of the major ING1 splicing isoforms, p47ING1a and p33ING1b, on pathological angiogenesis induced by human GBM cells. We used a chorioallantoic membrane (CAM) assay to examine whether LN229 human GBM cells can induce angiogenesis and whether alterations in ING1 expression, such as ING1 knockdown by siRNA or ectopic ING1 overexpression using ING1a and ING1b expression constructs, can affect this process. Increased ING1 protein expression significantly suppressed LN229 cell-induced angiogenesis in the CAM assay. While no effects on the proangiogenic factors VEGF or IL-8 were noted, the expression of angiopoietins (Ang) 1 and 4 were increased by the p47ING1a, but not by the p33ING1b isoform. Levels of Ang-2 were not sensitive to altered ING1 levels. Our data are the first to suggest that ING1 proteins suppress neoangiogenesis in GBM. Moreover, our results may support the idea that ING1 proteins regulate the expression of proteins that are critical for angiogenesis in GBM such as the angiopoietins.

The most common brain tumors in childhood and adolescence are low grade pilocytic astrocytomas (PA). Given that an increasing number of mitochondrial defects have been related to brain tumors and cancer in general, we asked whether PAs harbor mutations of mitochondrial DNA (mtDNA). Sequencing analysis of the complete mitochondrial genome of tumor tissue and corresponding blood samples from 19 patients with PA was performed. Of the 19 PA tissue samples, 16 (84%) showed somatic mtDNA mutations, and a total of 34 different somatic mtDNA mutations were identified. Of the 34 mtDNA mutations, 17 (50%) were found in genomic regions involved in pathways of oxidative phosphorylation. Three of the missense mutations in protein coding regions involved change of one amino acid: M60 V in ATP synthase subunit 6, L236I in cytochrome b, and L112 M in cytochrome c oxidase subunit 1. We were able to demonstrate that mtDNA mutations occur in PA and that they are frequently located in protein coding regions. The PA tumors were found to have the highest percentage of mitochondrial mutations of any of the neuroectodermal tumor entities studied to date. To reveal the prognostic importance of these mutations in the tumor biology of PA, larger series need to be studied prospectively.

We are reporting on a 14-year-old boy with a very early relapse of pre-B acute lymphoblastic leukemia (ALL) and anaplastic astrocytoma WHO degrees III; the astrocytoma was subtotally resected and subsequently treated with irradiation and chemotherapy. The leukemic relapse was refractory to the administered salvage therapy. Therefore, treatment with the human anti-CD20 monoclonal antibody (MoAb) IDEC-C2B8 (rituximab) was initiated. After a small fraction of the standard dose (375 mg/m(2)) had been administered, the infusion had to be interrupted because of an acute attack of pain in the lumbar region. Two days later, after resumption of the therapy, he developed a fatal course of systemic inflammatory response syndrome (SIRS) and died, possibly due to uncontrollable cytokine release syndrome associated with sepsis. The fatal course will be discussed based on a review of the literature.

In this multicenter crossover study, our aim was to evaluate the efficacy and acceptance of acupuncture as a supportive antiemetic approach during highly emetogenic chemotherapy in pediatric oncology.Eleven children receiving several courses of highly emetogenic chemotherapy for treatment of solid tumors were included. Randomization allocated patients to start chemotherapy either with antiemetic medication plus acupuncture or antiemetic medication alone. During all study courses, patients continued to receive their programmed and additional antiemetic medication as needed. Acupuncture was given at day 1 of chemotherapy and at subsequent days on patient's demand. The amount of baseline and additional antiemetic medication during chemotherapy was documented. Patients maintained a daily diary of vomiting episodes and completed an evaluated nausea score at the end of every course. Their body weight was taken before and after a chemotherapy course.Twenty-two courses with or without acupuncture were compared. The benefits of acupuncture in adolescents with respect to the reduction of additional antiemetic medication were observed. Acupuncture enabled patients to experience higher levels of alertness during chemotherapy and reduced nausea and vomiting. Except for needle pain, no side effects were noted. Patient's acceptance of acupuncture was high.Our data indicate that acupuncture might reduce antiemetic medication and episodes of vomiting in pediatric oncology.

BACKGROUND Recent studies have shown that cytokines/cytokine receptors (C/CR) affect leukemic cell growth and survival. The goal of the current study was to investigate possible correlations between gene expression patterns of C/CR in leukemic cells, clinical features, and outcome in children with acute lymphoblastic leukemia (ALL) at first disease recurrence. METHODS Between January 1997 and December 2000, bone marrow (BM) samples were collected from 68 children with first ALL recurrence at diagnosis. These patients were enrolled in the ALL-REZ 95–96 disease recurrence trials of the Berlin-Frankurt-Munster study group. C/CR gene expression (interleukin [IL]-7, IL-10, IL-12p40, IL-15, IL-18, IL-7Rα, IL-10R1, IL-15Rα, interferon-gamma [IFN-γ], vascular epithelial growth factor [VEGF], Flt1, and transforming growth factor-beta) was quantified by real-time reverse transcriptase-polymerase chain reaction and correlated with protein expression by immunofluorescence. RESULTS In comparison with T-lineage ALL specimens, expression of IL-10, IFN-γ, IL-15Rα, and Flt1 was significantly higher in B-cell precursor (BCP) ALL specimens (P < 0.01). Among BCP ALL samples, gene expression of IL-7Rα and Flt1 was higher in pre-B than in common or pro-B leukemic cells. Moreover, expression levels of VEGF, IL-7Rα, IL-10R1, and IL-15Rα were lower in lymphoblasts of patients with a combined BM recurrence than in those with an isolated recurrence (P < 0.05). Children with IL-15Rα expression above the median level had a significantly better probability of event-free survival (0.65 vs. 0.34, P = 0.04) and survival (0.71 vs. 0.37, P = 0.02) at 5 years. CONCLUSIONS Expression of distinct C/CR in ALL cells was associated with lineage commitment and differentiation of leukemic cells, as well as with prognosis. It remains to be evaluated whether these prognostic and biologic findings of distinct C/CR expression in leukemic cells also have therapeutical implications for future antileukemic strategies. Cancer 2005. © 2005 American Cancer Society.

At relapse, T-cell acute lymphoblastic leukaemia (ALL) has a worse patient outcome than B-cell precursor (BCP-) ALL. To investigate this further, we compared in vitro cellular drug resistance profiles of T-cell and BCP-ALL samples obtained at relapse. We investigated 237 paediatric relapsed ALL cases, including 151 samples taken at first relapse, of which 30 were T-cell ALL. In vitro drug resistance was measured using the 4-day methyl-thiazol-tetrazolium (MTT) assay and cellular immunophenotype was determined at central reference laboratories. Similar results were found for first relapsed ALL samples and for the total group: T-cell ALL samples were more resistant to 4-HOO-ifosfamide (1.4-fold, P = 0.019) and cisplatin (3.7-fold, P = 0.005). The samples were more sensitive to thiopurines such as mercaptopurine (2.1-fold, P = 0.007) and thioguanine (1.7-fold, P = 0.003). Resistance/sensitivity to 16 other drugs did not differ significantly. These results do not explain the relatively poor prognosis of T-cell ALL at relapse, but do suggest that the more intensive use of thiopurines in relapsed T-cell ALL may be beneficial.

Cytokines play a key role in the differentiation, growth and survival of hematopoietic cells in the bone marrow (BM) stroma microenvironment. The mechanisms by which stromal derangements may contribute to the evolution of hematopoietic neoplasias are largely unknown. Here, we characterized BM stromal cells isolated from children with acute lymphoblastic leukemia and determined the effect of the interaction between stromal cells and lymphoblasts on cytokine expression as well as the effect of prednisolone using mono- and co-culture models. The analyses demonstrate that (1) stromal cells and lymphoblasts display different patterns of cytokine gene expression individually. (2) Stromal cells influence gene expression of cytokines in lymphoblasts and vice versa. (3) Glucocorticoid substitution inhibit cytokine gene expression in stromal cells. These findings indicate that stromal cells are important components involved in malignant hematopoiesis and also in response to therapy.

Post-transplant lymphoproliferative diseases (PTLD) are severe complications of immunosuppressive treatment after solid organ transplantation (SOT). Most pediatric cases are associated with Epstein-Barr virus (EBV). Despite morphologic features similar to de novo lymphomas PTLDs are sensitive to immune interventions alone. We conducted a non-randomized prospective multicenter study to test a response-adapted sequential treatment with rituximab +/- chemotherapy in pediatric patients with CD20+ PTLD (Ped-PTLD Pilot 2005). Pediatric patients ( 49 patients (mean age 9.8 [0.8-17.4] years) were included, among them 18 renal, 11 liver graft recipients, and 20 patients after heart or lung transplantation. Median time from SOT to PTLD was 4.7 [0.15 – 15] years (17 early, 32 late PTLDs). Tumor cells contained EBV products in 44 of 49 cases. Histology was polymorphic in 12 patients, diffuse large B cell lymphoma (DLBCL) in 24, Burkitt lymphoma in 7, and other high grade B cell lymphomas in 6 patients upon central review. Median follow-up is 4.5 years. 32 patients (64%) received rituximab only, of which 26 (81% of responders, 53% of total study population) remain alive and in continuous complete remission with a median follow up of 4.9 years. The remaining 6 rituximab responders experienced relapse (n=4), secondary malignancy (n=1), or death unrelated to PTLD (n=1). 15 patients had stable or progressive disease after initial rituximab treatment and proceeded to chemotherapy. Of these, 10 patients (66%) converted to complete remission while 4 patients (27%) progressed and went off study to receive intensive chemotherapy. 1 patient died during mCOMP treatment, which could not be distinguished between tumor progression and infection. 2/49 patients died of PTLD before rituximab response could be evaluated. 2-year OS was 86% +/- 5% with 67% +/- 7% surviving event-free. Estimated 5-year OS and EFS were 83% +/-5% and 67% +/-7%, respectively. 6/7 patients with Burkitt histology required mCOMP chemotherapy. Patients after thoracic organ transplantation had a significantly inferior outcome compared to liver or renal graft recipients (5-years EFS 79%+/-8% vs 44% +/-11%, p=0.01). There was no significant impact of tumor EBV-status, histology, stage or early/late PTLD development. Treatment-related mortality was attributed to bowel perforation (n=1) and infection (n=2). No episode of graft rejection occurred during treatment for PTLD. In conclusion, response-adapted immuno-chemotherapy is an effective and well tolerated treatment regimen for CD20-positive PTLD after SOT in children. Chemotherapy could be spared in half of the PTLD patients, but was necessary in almost all Burkitt PTLD patients. Prognosis was significantly worse in patients after heart or lung transplantation compared to renal or liver allograft recipients. Future studies are envisioned to evaluate whether the inclusion of cellular immunotherapy may contribute to avoid chemotherapy in the majority of patients. Disclosures No relevant conflicts of interest to declare.

Minimal residual disease (MRD) has been demonstrated to be of high and independent prognostic value prior to hematopoietic stem cell transplantation (HSCT) in childhood acute lymphoblastic leukemia (ALL) by several studies. Within the ALL-REZ BFM 2002 trial, MRD of 10-3 has been identified as the best prognostic cut-off before HSCT in children with relapsed ALL. In this study we have investigated, whether interventional treatment intensification in patients with persisting MRD at a level of =/>10-3 is capable of reducing MRD prior to HSCT and improving survival. In the trial ALL-REZ BFM 2002 (recruitment of patients between 01'2002 and 09'2012) online MRD-monitoring has been performed in patients with ALL relapse and indication for allogeneic HSCT since 2010. MRD results obtained during the consolidation treatment phase and before HSCT have been disclosed to the treating centers. Treatment courses consisting of Clofarabine/Cyclophosphamide/Etoposide, Daunoxome or Idarubicine/Fludarabine/Cytarabine, or in case of T-ALL Nelarabine alone or with Cyclophosphamide/Etoposide were offered as interventional elements and administered to patients at the discretion of the treating physician aiming at reducing MRD and improving survival. A total of 30 patients with first ALL relapse and persisting MRD at a level of =/>10-3 at the end of consolidation treatment received an interventional intensification before HSCT (Intervention Group, IVG). Sixty patients with first relapse and persisting MRD at a level of 10-3 at the end of consolidation treatment did not receive an interventional intensification before proceeding to HSCT (non-IVG). MRD reduction of at least one log step was achieved in 78% and of two log steps in 59% of patients after interventional intensification. Relevant clinical characteristics as time to relapse, site of relapse, immunophenotype, consolidation treatment arm and type of HSCT were equally distributed between the IVG and the non-IVG. However, the proportion of patients with an MRD levels In conclusion, persisting MRD during consolidation treatment can be efficiently reduced by interventional intensified chemotherapy. However, this does not translate into a significant improvement of survival. MRD persistence after intensive conventional chemotherapy seems to identify particularly aggressive leukemias which are not successfully treated by further reduction of the MRD load. Therefore, targeted therapies need to be investigated and prospective controlled studies should be given preference to individualized interventions. Figure 1 Figure 1. Disclosures von Stackelberg: Amgen: Consultancy, Honoraria.

To the Editor .— We read with great interest “First Report of Autologous Cord Blood Transplantation in the Treatment of a Child With Leukemia” by Hayani et al.1 The authors reported on the first successful autologous cord blood transplantation in a child with acute lymphoblastic leukemia of the B-precursor subtype with isolated central nervous system (CNS) relapse …

Background Primary chemotherapy is a new treatment approach in retinoblastoma, aiming to avoid radiogenic adverse effects, such as second tumor–associated mortality, as observed following external beam irradiation. Objective To describe the clinical and histopathologic regression pattern after primary chemotherapy in retinoblastoma. Methods Five patients with sporadic bilateral retinoblastoma underwent planned enucleation of their functionally blind eye after 2, 3 (in 2 patients), 4, and 6 courses of primary chemotherapy with carboplatin, etoposide, cyclophosphamide, and vincristine. The eyes were examined histopathologically, using light microscopy and immunohistochemical analysis with proliferation markers. Results One patient had a type 1 (cottage cheese) regression and 4 patients had either a type 2 (fish flesh) or a type 3 (combined) regression pattern. Histopathologic examination revealed a complete tumor necrosis in 1 patient with type 1 regression after 3 courses of chemotherapy and in 1 patient with type 3 regression after 4 courses of chemotherapy. The remaining 3 patients with type 2 or type 3 regression had histologically still active proliferative tumor cells after 2, 3, and 6 courses of chemotherapy. Conclusion This article correlates histopathologically the clinically described efficacy of primary chemotherapy in the treatment of retinoblastoma, underlining, however, the necessity of careful observation and the use of ancillary treatment whenever there is no complete tumor regression.

Abstract 669 Background. Children with Down syndrome (DS) have a higher risk for developing not only acute lymphoblastic leukemia (ALL) but also significant adverse effects of chemotherapy compared to the overall pediatric population. Currently, it is unknown how children with DS, who develop a relapse of ALL, respond to treatment protocols that were optimized in the pediatric population without DS. We hypothesized that a concomitant diagnosis of DS is an independent prognostic factor of survival after treatment for relapsed ALL and that decreased tolerance of therapy impairs the success of relapse treatment in children with DS. Patients and Methods. The probability of event-free (EFS) and overall survival (OS) and the causes of treatment failure were determined for 51 children with DS and a matched cohort of 102 children without DS among 2736 children and young adults (up to 22 years of age) who were treated for relapsed ALL on a series of clinical trials conducted by the ALL-REZ-BFM Study Group between 1983 and 2012. Results. Among children with DS, who were enrolled on clinical trials of the ALL-REZ-BFM Study Group, ALL relapse more frequently exhibited favorable prognostic characteristics compared to the unmatched population of 2579 patients without DS. High risk forms of relapse were less frequent among children with DS (risk group S4, 10 vs. 28%, p DS was an independent prognostic factor of outcome after relapse of ALL in multivariate analysis. Conclusion. A higher rate of induction deaths and treatment-related mortality was the main barrier to successful outcomes of relapse therapy in children with DS whereas relapse rates were not different from patients without DS. An increased representation of children with DS including those with high risk features in recent time suggests that access to clinical trials for relapsed ALL has been successfully broadened for children with DS. Specific optimization of treatment modifications and supportive care have improved survival in children with DS and are suggested to further decrease the number of fatal treatment-related events during relapse therapy in this group. Disclosures: No relevant conflicts of interest to declare.

Abstract 2612 Poster Board II-588 Minimal residual disease (MRD)-measurements have been integrated into treatment protocols for childhood acute lymphoblastic leukemia (ALL) using T-cell-receptor(TCR)/immunoglobulin(IG) gene rearrangements. Additionally TCR/IG gene rearrangements can be applied to describe clonal relations and identify cell populations which undergo clonal evolution and selection during and after treatment. Patterns of TCR/IG gene rearrangements might differ between initial and relapse diagnosis and are interesting and very informative in order to describe both the quantitative extent at diagnosis and the kinetics during treatment of main- and subpopulations. In order to minimize the chance of a false negative MRD-result knowledge about the stability of TCR/IG-markers is important. Therefore, stability of TCR/IG-markers between initial and relapse diagnsosis was assessed in previous studies. These studies recommended to use two markers besides considering gene-locus and clonality profile. However, it has never been validated prospectively whether the TCR/IG-markers actually chosen for MRD-quantification and risk-group stratification are sufficient for clinically and biologically reliable MRD-monitoring during frontline treatment. We have aimed to compare marker profiles between initial and relapse diagnosis by a systematic approach in order to evaluate markers acutally chosen for initial MRD-quantification. Further, MRD-response to induction of initial and relapse treatment have been compared. One main objective of our study was to gain new insights in clonal heterogeneity, resistance and clonal evolution. We have performed a prospective study including patients treated according to the frontline-trial ALL-BFM 2000 and who suffered a relapse. The interim analysis included finally 45 patients showing a median time to relapse of 1.5 years (range 0.6 – 3.8). Therefore, mainly (73%) very early (during first 18 months after initial diagnosis) and early (between 18 and 30 months) relapses were included in the study. In 33% of patients (15/45) all markers identified at initial diagnosis remained stable at relapse diagnosis and no additional marker was gained at relapse. The remaining 67% of patients showed at least one clonal change. In 38% (17/45) of patients at least one gain of a TCR/IG gene rearrangement was seen; in 53% (24/45) of patients loss at relapse and in 16% (7/45) V-V-replacement or Vd2-Ja ongoing rearrangement product was seen at relapse. Considering markers actually chosen for initial MRD-quantification, in 62% of patients both markers remained stable, in 27% at least one, in 11% no marker. Early on-therapy relapses showed the same proportion of patients with a marker-gain, than later on-/off-therapy relapses. A backtracking-analysis of markers gained revealed the presence of nearly all markers (17/19: 90%) at initial diagnosis at different quantitative levels independently of time point of relapse. Paired initial and relapse molecular response to induction varied remarkably. Only 5 of 24 patients included in this analysis showed similar response (1 poor, 4 intermediate, 3 good responders) which demonstrates the unexpected high heterogeneity. It is interesting to note that complete identical markers for MRD-monitoring during initial and relapse treatment were only chosen in half of the patients (12/24). These observations challenges the classical and still existing opinion that early relapses derive from resistant leukemia because resistant MRD was not eliminated by polychemotherapy. Based on our data and data from other studies comparing genetic markers in paired samples between initial and relapse diagnosis, the extrapolation of an algorithm for MRD-marker choice is not possible and would be too artificial at the moment. From the biological point of view, the practical restriction of using only one or two markers for MRD quantification at several time points during early and later phases of the treatment should not be recommended. Despite the higher proportion of very early/early relapses in this interim analysis we observed a high heterogeneity of marker-profiles between initial and relapsed diagnosis and molecular response to treatment at both disease stages. Disclosures: No relevant conflicts of interest to declare.

The ETV6/RUNX1 fusion, resulting from the cryptic translocation t(12;21)(p13;q22), is the most common genetic rearrangement in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) with a prevalence of approximately 20–25% at first presentation. Although generally associated with favorable risk features and advantageous long-term survival rates, similar frequencies of ETV6/RUNX1 positivity at relapse question its prognostic significance. At relapse, a second continuous complete remission can again be achieved in the majority of patients with ETV6/RUNX1-positive ALL, however, a substantial proportion (approximately 25%) exhibit a poor response to treatment and eventually experience a subsequent relapse. Currently, critical secondary genetic events are assumed to be pivotal for t(12;21) positive ALL leukemogenesis. At the chromosomal level, additional numeric aberrations may also contribute to differences both in treatment response and course of disease, and may provide additional prognostic information. Aiming at identifying the incidence and the clinical relevance of additional genetic aberrations at the time point of first ALL relapse diagnosis, we performed whole genome high resolution tiling-path bacterial artificial chromosome (BAC) array CGH of leukemic cell DNA from 53 patients with ETV6/RUNX1-positive first relapse enrolled in the relapse trials ALL-REZ BFM of the Berlin-Frankfurt-Münster study group. Additional genetic aberrations were detected in all of the ETV6/RUNX1-positive leukemic bone marrow samples with a mean number of seven aberrations per ALL. Chromosomal losses were observed approximately 2.5 times more frequently than gains. A high proportion of the identified copy number alterations occurred in recurrently affected chromosomal regions. Copy number alterations most commonly detected by high-resolution array CGH were losses of 12p13 (49%, 26/53), 6q21 (34%, 18/53), 15q15.1 (23%, 12/53), 9p21 (21%, 11/53), 3p21 (21%, 11/53), 5q (19%, 10/53), 19q13 (17%, 9/53), 10q (13%, 7/53), 3p14.2 (11%, 6/53) and gains of 21q22 (32%, 17/53) and of 12p (21%, 11/53). Loss of the whole X-chromosome, detected in 17% (9/53) of the analyzed samples, was observed exclusively in females. In contrast, gain of Xq, identified in 13% (7/53) of the samples, was detectable solely in males. Evaluation of the clinical parameters in relation to recurrent copy number alterations revealed that loss of the whole X-chromosome was associated with a molecular good response to treatment (P=0.03), as assessed by sensitive minimal residual disease (MRD) monitoring. In contrast, loss of 5q31.3, detected in 11% (6/53) of the analyzed samples, was associated with a dismal molecular response to therapy (P=0.019) and with a tendency to a worse outcome (P=0.054). Thus, this study demonstrates that relapsed ETV6/RUNX1-positive ALL is characterized by (a.) multiple additional genomic alterations, in particular by a high incidence of chromosomal losses, which occur predominantly in recurrently affected chromosomal regions, some of which (b.) are of significant prognostic relevance. These findings support the notion that several additional chromosomal changes are not only required for the process of malignant transformation in ETV6/RUNX1-positive ALL (leukemogenesis) but also influence therapeutic success. Perspectively, high resolution genomic profiling will provide valuable information enabling a more refined, individualized therapy and an improved risk stratification in relapsed childhood ALL.

Despite advances in chemotherapeutic treatment of hematological diseases in childhood, there is significant rate of relapse with high death rates, so that research in new therapeutic molecules is on demand to combat resistance. Search for low molecular weight inhibitors in plant material therefore offers a wide spectrum of new anticancer compounds. Tryptanthrin is found in several plants like Isatis tinctoria or Polygonum tinctorium. Our experiments revealed that NT1, the modified 8-Nitro-3-chloro-derivative of tryptanthrin, specifically induced apoptosis in BJAB, Nalm6, Reh, Sup-B15 and p388 cells, as evidenced by DNA fragmentation, Annexin/PI staining, dissipation of the mitochondrial membrane potential measured via flow cytometry and processing of caspase-3 by Western Blot analysis. These events occurred in the absence of cellular lysis, i. e. in the absence of release of lactate dehydrogenase from the cells, thereby excluding NT1-induced necrosis. A dose-dependent inhibition of proliferation up to 96% hereby precedes the DNA fragmentation and apoptosis. In contrast, healthy leucocytes were not affected by treatment with NT1. NT1-induced cell death was furthermore functionally characterized by the use of different cellular model systems. Investigating control vector-(pcDNA3-mock-transfected) and pcDNA3-FADDdn-transfected BJAB cells, which were stably transfected with a dominant-negative FADD mutant lacking the N-terminal death effector domain, revealed that NT1-induced apoptosis is mainly executed via the CD95-dependent cascade. These findings were confirmed by real-time PCR, which unveiled an up to 8-fold increase of caspase-8, 20-fold upregulation of the FAS ligand and the members 9, 11B and 25 of the TNF receptor superfamily during NT1-induced apoptosis. The strong inhibitor of apoptosis Bcl-2 often leads to severe drug resistance in treatment of malignancies. Melanoma cells transfected with the pIRES plasmid (MelHO/pIRES) and the Bcl-2 cDNA in pIRES (Mel-HO/Bcl-2), which in consequence 30-fold overexpress the Bcl-2 protein, were incubated with NT1 for 72h. Apoptosis showed to be independent on increased Bcl-2 levels, which is in line with an upregulation of the anti-Bcl-2 protein Harakiri during NT1 treatment measured by real-time PCR. Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in pediatric leukemia. Recent studies have demonstrated that members of the inhibitor of apoptosis (IAP) family proteins like survivin or XIAP are upregulated in pediatric ALL or AML. XIAP mRNA expression was found to be 160-fold down regulated in BJAB cells treated with NT1 as compared to untreated cells. Thus, NT1 is able to sensitize resistant tumor cells to cytostatic therapy in combined treatment with other drugs as shown in further experiments. Analyzing primary leukemia cells from 24 children with de novo ALL, 4 with relapsed ALL and 6 with newly diagnosed AML, we could demonstrate that NT1 overcomes drug resistance against daunorubicin (p

In the current trial ALL-REZ BFM 2002 for treatment of children with relapsed acute lymphoblastic leukemia (ALL) assessment of minimal residual disease (MRD) has been applied to quantify response to induction therapy at a submicroscopic level in children with intermediate-risk relapsed ALL aiming to stratify treatment, and improve survival. Time point of measurement of molecular response to therapy in bone marrow (BM) and the MRD cut-off used are based on a prospective blinded MRD-study performed during the previous trial ALL-REZ BFM 96. Further, in order to gain new experiences about a possible control of post-induction treatment, MRD reduction kinetics have been measured during treatment until stem cell transplantation (SCT) or end of maintenance therapy. MRD-quantification has been performed using quantitative real-time PCR with clone-specific T-cell receptor/Immunoglobulin gene rearrangements. Between 01/2002 and 08/2008, in 161 patients with intermediate risk (S2) including late isolated BM relapses and early or late combined BM relapses of B-cell precursor ALL, MRD after the second induction course (block F2) was used for post-induction treatment stratification. About half of intermediate risk patients with BM involvement showed a poor response to therapy (MRD ≥10−3 after F2) and were thereupon allocated to SCT. Probability of event-free survival of these patients is 48% (SE [standard error] ±8.5%) in the current ALL-REZ BFM 2002 trial compared to 18% (SE±6.5%) in the previous trial ALL-REZ BFM 96. MRD reduction kinetics until SCT were very heterogeneous in the poor response group. About 10% of this group presented as molecular non-responder with a persisting MRD-level of ≥10−2 after the 5th treatment block. In comparison to the previous trial, in the current trial the proportion of early relapses which includes only combined relapses among S2 patients was higher (20%) in the group with a molecular good response (MRD