ALBERT

Description: Key Points AML induction with liposomal daunorubicin (80 mg/m2 per day for 3 days) shows antileukemic activity comparable to idarubicin (12 mg/m2 per day for 3 days). Liposomal daunorubicin promises to be more active in the t(8;21) subgroup and causes less treatment-related toxicity.

Description: In trial ALL-BFM 2000, high-risk (HR) acute lymphoblastic leukemia (ALL) is defined by inadequate initial response to induction treatment [poor prednisone response on treatment day eight (PPR), non remission on treatment day 33, and/or a high load of minimal residual disease (MRD, ≥10E-3) after 12 weeks of treatment (TP2) and/or by positive cytogenetics for a t(4;11) or t(9;22)]. Recently, we presented data on the prospective evaluation of MRD at additional time-points under HR-treatment and showed that patients with a persistently high MRD load after the application of three intensive HR blocks were neither curable by chemotherapy alone nor by the addition of stem cell transplantation (very highly resistant leukemia, VHRL, Schrauder et al., Blood, 2007; 110: 585). Thus, VHRL patients are a target group for the implementation of experimental treatment approaches. Unfortunately, despite MRD analysis being an excellent tool for the identification of this group of patients, the late time point of identification does not allow an early experimental intervention. In addition, MRD measurements do not yield any information on potentially targetable molecular mechanisms of resistance. Therefore, we developed a strategy for early identification of VHRL patients and their subsequent rational treatment based on the exploration of treatment targets. In practice, we apply different genome-wide screening approaches for a comprehensive molecular characterization. Here, we present data on gene expression profiling of HR patients performed in parallel to the ongoing extensive MRD analysis. All HR-patients with sufficient spare initial diagnostic specimens and at least 80% of blasts in bone marrow or peripheral blood were included. In addition, non-HR patients were profiled for the purpose of comparison. Microarrays containing more than 39,000 distinct cDNA clones (SFGF, Stanford, CA) were used. Data were analyzed using the entire set of genes and separately focusing on genes involved in apoptosis. Several publicly available analysis tools were applied. At the time of analysis, gene expression data of 106 patients with B-precursor immunophenotype and negative for TEL-AML1, MLL-AF4 and BCR-ABL rearrangements were analyzed: 43 Non-HR patients; 28 HR patients with a PPR, but low or absent MRD at TP2 (isolated PPR); 17 patients with a high MRD load at TP2 but good MRD reduction under HR blocks (MRD-HR); and 18 patients with persistently high MRD levels after three HR blocks (VHRL). By unsupervised clustering two cases with an unfavorable E2A-HLF rearrangement were identified. Applying Significance Analysis of Microarrays (SAM, PNAS2001, 98, 5116–5121), only 15 genes (all low expressed in PPR) were identified to be differentially expressed comparing isolated PPR and Non-HR patients (1000 permutations, FDR 〈 5%, fold change 〉1.5). In contrast, comparing VHRL patients (excluding the E2A-HLF-positive cases) to the entire group of Non-HR, isolated PPR and MRD-HR patients, 135 genes (57 down-, 78 upregulated in VHRL) were identified. Of interest, in addition to their E2A-HLF-specific pattern, the two E2A-HLF-positive patients did show the same pattern of resistance-associated genes as did VHRL patients without known molecular background. Focusing on genes involved in apoptosis in each single VHRL patient, at least one potentially targetable anti-apoptotic gene with an expression of at least 2-fold the median was identified. Connectivity Map searches identified several substances with highly significant connections to VHRL and Non-VHRL signatures, respectively. In summary, we have seen clear differences in gene expression patterns dependent on the degree of resistance. VHRL patients seem to be characterized by a specific GEP independent of their heterogeneous molecular background. The present data are currently evaluated in a prospective study and will be complemented by those obtained through additional other genome-wide screening approaches (e.g., CGH analysis and epigenetic profiling). The resulting information will then be utilized to build up an integrated VHRL classifier and to discover new treatment options with the ultimate goal of individualized experimental treatment of VHRL patients based on their specific molecular characteristics.

Description: Osteonecroses (ON) are common complications associated with the treatment of acute lymphoblastic leukemia (ALL). For symptomatic ON, incidences of 1.6% to 9.3% have been published in the literature. The range of incidence rates may be related to treatment, data acquisition methods and patients’ age distribution. Between 09/99 and 06/06, 3,048 protocol patients with ALL (age 1–17 years) were enrolled into the multicenter trial ALL-BFM 2000 (Germany, Austria, Switzerland). Data on symptomatic ON were prospectively collected and were reported in 111 patients (3.6%, 95% confidence interval (CI) 3.0–4.4%). The median age of the patients with ON was 12.8 years (range 1.3–17.9 years), the median time from ALL diagnosis until diagnosis of ON was 15 months (range 2–73 months; data available from 92 patients). The incidence in adolescents, in particular adolescent girls, was significantly higher than in younger children (age

Description: Purpose Monitoring of minimal residual disease (MRD) after allogeneic stem cell transplantation (allo-SCT) by quantitative real-time PCR (qRT-PCR) of rearranged Ig- and TCR-genes may highlight patients with highest risk for relapse to whom pre-emptive treatment may be offered. Patients and Methods In the prospective phase 3 trial ALL-SCT-BFM-2003 (recruitment period 09/2003 to 09/2011; time point of analysis May 2013), MRD was assessed in bone marrow immediately on days +30, +60, +100, +200 and +365 post transplantation in 115 patients. Of these, 48 were male and 67 were female patients. All received a myeloablative conditioning regimen with TBI and VP 16. Patients received their transplant in CR2 (n=94), CR3 (n=21), CR4 (n=1) or NR (n=2). The transplantations were performed with bone marrow from matched sibling donors (MSD, n=23), matched unrelated donors (MUD, n=71) or with T-cell depleted stem cells from mismatched donors (MMD, n=21). Fifty-four patients were younger than 10 and 61 patients were older than 10 years at the time of transplant. Standardized quantification of MRD was performed according to the guidelines of the Euro-MRD-Group and MRD results were not released to the clinicians. Results The total group of patients showed a pEFS of 0.52±0.10; cumulative incidence of relapse (CIR) and cumulative incidence of treatment related mortality (CI TRM) was 0.41±0.11 and 0.19±0.09, respectively. In 76 patients, MRD could also be assessed prior to transplant: patients who were MRD negative (n=41) had a three year pEFS of 0.62±0.04, patients with a MRD load of 1E-3 survived their disease. MRD values post transplant were analyzed as time-dependent covariates. When analyzed either for the different time points or for the highest MRD value post transplant, MRD results had always significant influence on survival. Taken the highest MRD value post transplant, probabilities of pEFS and CIR were 0.65±0.11 and 0.23±0.09 for MRD negative patients (n=72), 0.36±0.18 resp. 0.75 ±0.18 for patients with MRD

Description: Abstract 756FN2 Bone marrow (BM) aspiration at the end of induction therapy plays a crucial role for the evaluation of remission and the minimal residual disease (MRD), both critical for treatment stratification in modern treatment protocols for paediatric acute lymphoblastic leukaemia (ALL). However, the aspiration is repeated in 15–20% of patients, either due to non-representative morphology or to insufficient material needed for MRD analysis. We prospectively analysed 320 paediatric ALL patients treated according to ALL-BFM 2000 (n=301) or ALL IC-BFM 2002 (n=19) protocols with repeated BM aspiration at the end of induction therapy, on treatment day 33. Fourteen patients had more than one re-puncture. The median follow-up was 69 months, 45 (14%) patients had an event (relapse/death). The cause for the repeated BM aspiration was non-representative morphology (32%), insufficient material for MRD analysis (33%) or both (35% cases). In order to evaluate prognostic significance of the re-punctures and to determine which of the repeated samples should be used for the final treatment stratification we analysed MRD levels and MRD stratification, morphology, leukocyte count (WBC) and the length of treatment delay caused by waiting for the repeated aspiration. MRD data were collected and interpreted according to the EuroMRD guidelines in one central reference laboratory per each participating country. Morphology was evaluated centrally using an own scoring system (with a max value of 26 points). Treatment delay between the original and the last aspiration was one-third longer in patients with subsequent event compared to patients remaining in complete remission (CR) (median 8 (range 2 – 21) vs. 6 (1 - 28) days, respectively; p=0.020). Patients with a subsequent event had significantly higher WBC at the time of the last repeated BM aspiration, compared to patients without event (p=0.019), while there was no difference relative to the original aspiration (p=0.9). Analysis of the BM morphology at the original aspiration showed no significant difference between patients with an event vs. those in CR. However, the repeated aspiration of patients with a subsequent event had significantly better morphology (median 18.5/26 vs. 15/26 points, p=0.0012) mainly due to higher cellularity (p=0.003) and number of megakaryocytes (p=0.048). MRD levels were identical or decreased in 88% and increased in 12% of cases comparing the original aspiration to the repeated aspiration. In 63 patients (20%) the different MRD levels would lead to different treatment stratification. Higher MRD was associated with treatment failure; the best predictive values for subsequent event were obtained using the MRD results of the original aspiration (p=3.1e-07) or the highest of the detected MRD levels (p=6.0e-07). The last aspiration before proceeding with treatment had the lowest, though still a highly significant predictive value (p=8.6e-06). Corresponding results are obtained when MRD levels are substituted by final MRD risk stratification into standard, medium or high risk (p

Description: Patients with core binding factor acute myeloid leukemia (CBF-AML) benefit from more intensive chemotherapy, but whether both the t(8;21) and inv(16)/t (16;16) subtypes requires intensification remained to be determined. In the 2 successive studies (AML-BFM-1998 and AML-BFM-2004), 220 CBF-AML patients were treated using the same chemotherapy backbone, whereby reinduction with high-dose cytarabine and mitoxantrone (HAM) was scheduled for these cohorts only in study AML-BFM-1998 but not in AML-BFM-2004 against the background to minimize overtreatment. Five-year overall survival (OS) and event-free survival (EFS) were significantly higher and the cumulative incidence of relapse (CIR) lower in t(8;21) patients treated with HAM (n = 78) compared with without HAM (n = 53): OS 92% ± 3% versus 80% ± 6%, plogrank0.047, EFS 84% ± 4% versus 59% ± 7%, plogrank0.001, and CIR 14% ± 4% versus 34% ± 7%, p(gray)0.006. These differences were not seen for inv(16) (n = 43 and 46, respectively): OS 93% ± 4% versus 94% ± 4%, EFS 75% ± 7% versus 71% ± 9% and CIR 15% ± 6% versus 23% ± 8% (not significant). The subtype t(8;21), but not inv(16), was an independent predictor of worse outcome without HAM reinduction. Based on our data, a 5-year OS of 〉 90% can be expected for CBF-AML, when stratifying t(8;21), but not inv(16), patients to high-risk chemotherapy, including HAM reinduction.

Description: The definition “very-high risk” (VHR) in the setting of childhood acute lymphoblastic leukaemia (ALL) is usually adopted to identify children eligible for transplantation in first complete remission (CR1). The endpoint of this study is to assess whether, how many, and how VHR ALL children achieving CR1 and not transplanted in CR1 can be rescued in case of relapse. Eligiblity criteria to transplantation in AIEOP-BFM ALL 2000 study slightly changed overtime and differed between the BFM (Berlin Frankfurt Muenster) and AIEOP (Associazione Italiana di Ematologia ed Oncologia Pediatrica) Groups.. For this analysis VHR ALL was defined by one or more of the following criteria: induction failure (IF), high levels (≥ 1x10−3) of minimal residual disease at day 78 (VHR-MRD), clonal abnormality t(4;11), prednisone poor response associated (PPR+) with one or more of the followings: hyperleukocytosis, T-immunophenotype, ≥ 25% marrow blasts at day 15, pro-B immunophenotype. Patients carrying clonal abnormality t(9;22) were not included here since allocated in a specific study protocol since 2004. Out of 571 VHR patients, distributed among the four criteria (17%, 45%, 3%, 35%, as hierarchically listed), 249 (43%) were transplanted in CR1 (80%, 48%, 47%, 25% in each category) and 322 (57%) were not. Of the 322 patients treated with chemotherapy [5-year-EFS 53.5% (SE 3.1), 5-year-survival 66.6% (SE 2.9)], 19 died in CR1, 6 presented with a second malignancy, and 113 (35%) relapsed at a median of 15 months after diagnosis, mostly in the bone marrow (81%: 66% isolated, 15% combined) or in an extramedullary site (19%, 15% in the central nervous system) at a median time of 9 ms after diagnosis. The 3-year-survival after relapse was only 26.0% (SE 5.1) for the 113 patients overall, 38.7% (SE 7.8) for the 55 who underwent transplantation in CR2 and 16.2% (SE 5.3) for the 58 who were not transplanted in CR2. Interestingly the 3-year-survival after relapse by VHR criteria was only 7.1% (SE 9.3) in the IF, 28.6% (SE 7.1) in the VHR-MRD and 33.1% (SE 6.7) in the PPR+ subgroups. In conclusion very few VHR-ALL children could be rescued after relapse, particularly among those carrying most unfavourable criteria at the onset (IF and VHR MRD), confirming that transplantation should be performed in CR1. Transplantation in CR2 was feasible in only half of the patients and presumably their outcome was influenced by MRD response after relapse; this aspect is under investigation. New strategies are needed for this dismal subset of patients.

Description: The vast majority of relapses in childhood T-cell acute lymphoblastic leukemia (T-ALL) patients occur relatively early, usually within 2 years from diagnosis. Our previous comparative molecular analyses between diagnosis and relapse in all such “classical” T-ALL showed totally or at least partly identical T-cell receptor (TCR) gene rearrangement patterns at both disease phases. These results confirm that the relapse clone in these patients originated from the original diagnosis clone, which became resistant to the applied treatment. In contrast to these “classical” T-ALL, two patients experienced very late T-ALL recurrences and displayed completely different TCR gene rearrangement sequences between diagnosis and relapse. We hypothesized that such late “relapses” of T-ALL in fact might represent second malignancies in genetically predisposed persons. We investigated 16 T-ALL patients with late relapses, i.e. at least 2.5 years from initial diagnosis. The studies at the DNA level involved detailed comparison of TCR gene rearrangements between diagnosis and relapse (PCR-heteroduplex, sequencing and/or Southern blot analyses), the detection of gene fusions involving the TAL1 gene and/or TCR genes and comparative genomic hybridization (CGH) using high-resolution Agilent arrays. We found evidence of a common clonal origin between diagnosis and relapse in ten of the 16 patients. In one case, no clonal TCR rearrangements were detected neither at diagnosis nor at relapse. In the other five patients TCR gene rearrangement sequences had completely changed between diagnosis and relapse, suggesting a second T-ALL rather than a relapse. Moreover, three of these five patients remained in complete remission after second-line treatment, which is unusual for relapsed T-ALL. In three of the five patients with presumed second T-ALL, CGH arrays showed completely different patterns of genomic aberrations between diagnosis and relapse, while in the remaining two patients the patterns of genomic changes were vastly different, but some aberrations were similar at both disease phases. In eight patients with evidence of a common clonal origin between diagnosis and relapse at least 50% of genomic events revealed by CGH arrays were identical between diagnosis and relapse of T-ALL. We conclude that approximately one-third of late T-ALL “relapses” in fact represent second malignancies. We are currently performing further genomic analyses to identify common genetic events or common genomic features which might be related to predisposition for development of T-ALL.

Description: High-level expression of the cytokine receptor-like factor 2 gene, CRLF2, in precursor B-cell acute lymphoblastic leukemia (pB-ALL) was shown to be caused by a translocation involving the IGH@ locus or a deletion juxtaposing CRLF2 with the P2RY8 promoter. To assess its possible prognostic value, CRLF2 expression was analyzed in 555 childhood pB-ALL patients treated according to the Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster 2000 (ALL-BFM 2000) protocol. Besides CRLF2 rearrangements, high-level CRLF2 expression was seen in cases with supernumerary copies of the CRLF2 locus. On the basis of the detection of CRLF2 rearrangements, a CRLF2 high-expression group (n = 49) was defined. This group had a 6-year relapse incidence of 31% plus or minus 8% compared with 11% plus or minus 1% in the CRLF2 low-expression group (P = .006). This difference was mainly attributable to an extremely high incidence of relapse (71% ± 19%) in non–high-risk patients with P2RY8-CRLF2 rearrangement. The assessment of CRLF2 aberrations may therefore serve as new stratification tool in Berlin-Frankfurt-Münster–based protocols by identifying additional high-risk patients who may benefit from an intensified and/or targeted treatment.

Description: In trial ALL-BFM 2000, high-risk (HR) acute lymphoblastic leukemia (ALL) is defined by inadequate initial response to induction treatment [poor prednisone response on treatment day eight, non remission on treatment day 33, and/or a high load of minimal residual disease (MRD, ≥10E-3) after 12 weeks of treatment (TP2)] and/or by cytogenetics [t(4;11 or t(9;22)]. Between August 1999 and November 2006, 494 (15%) out of 3255 study patients were stratified into the HR branch of trial ALL-BFM 2000. 431 (87%) of these HR patients underwent successful MRD monitoring at TP2 with 274 (56%) patients having received additional extensive prospective MRD monitoring subsequent to TP2. Patients with an indication for stem cell transplantation (SCT) and a suitable donor were scheduled for SCT within six weeks after the third HR block of the intensive consolidation phase. The estimated 4-years event-free-survival (4y-pEFS) for the entire HR group was 68%+/−3%, estimated survival was 74%+/−3%. Patients with MRD load of ≤10E-4 at TP2 (n=231) had a 4y-pEFS of 82%+/−3%, patients with MRD levels of 10E-3 at TP2 (n=84) had a 4y-pEFS of 74%+/−6%, and patients with MRD of 〉10E-3 at TP2 (n=116) had a 4y-pEFS of 35%+/−5%. MRD-kinetics subsequent to TP2 revealed, that 85% of all patients with an MRD level of 10E-3 at TP2 continued to decrease their load below 10E-3 during the pulsatile intensive consolidation phase, whereas this was observed in only 35% of patients with an MRD level of 〉10E-3 at TP2. The 4y-pEFS of patients with an MRD load persisting at 10E-2 after application of three intensive HR blocks after TP2 was 0% if no SCT was performed, and 33%+/−11% after SCT in CR1. Our data reflect that extensive MRD measurements in HR-ALL patients allow a dynamic insight into the development of resistance, and serve as valuable tool for further clinical treatment adjustment. “MRD non-response” after three BFM HR blocks identifies a patient group in urgent need of alternative treatment elements, closely monitored by MRD, before going into SCT in CR1.