ALBERT

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: 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: 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.

Description: At acute lymphoblastic leukemia (ALL) relapse, about 40% of children can be salvaged with intensified multi-agent, high dose chemotherapy and in very high risk patients, with additional stem cell transplantation (SCT). To improve on this, the International BFM Study Group has led a consortium of 19 countries to develop the world's largest trial for relapsed ALL (IntReALL). Standard risk patients will be randomized to receive the ALL-REZ BFM 2002 or UK ALL R3 therapy and post induction will be randomised to receive the additional targeted anti-CD22 drug, Epratuzumab, during consolidation, to clear residual disease. Children with end of re-induction MRD positive bone marrow will undergo SCT following consolidation. Both ALL-REZ BFM 2002 and UK ALL R3 used MRD PCR-based quantification of clonal Ig/TCR rearrangements, with different cut offs (10-3 for ALL-REZ BFM 2002 and 10-4 for UK ALL R3) and this is the reference assay for IntReALL. However, flow MRD may also play a role for patients without PCR targets. Flow MRD relies on the discrimination of leukaemic blasts from hematogones and this can be hampered depending on the degree of haematopoietic regeneration which varies depending on the treatment protocol and is especially important after intensive induction treatment in relapsed protocols. Thus, prospective MRD quantification of patients entered onto the UKALLR3 and ALL-REZ BFM 2002 clinical trials was performed by a standardised, quality assured, 4-8colour Flow MRD assay in end of re-induction bone marrow aspirates, by laboratories in the IBFM FLOW consortium (n=221). Flow MRD in both treatment protocols was classed as a prospective biological add on study and not used for clinical decision making. Median MRD levels were 0.026 +/-9.9% SD for BFM versus 0.027+/-18% SD for UK protocols, with comparative MRD positivity rates of 45% versus 54%, respectively. Comparison with MRD levels as assessed by molecular analysis of antigen receptor gene rearrangements was performed in 170 samples (BFM,128; UK R3, 42). The Spearman rank correlation of all samples was 0.90 (p

Description: Slow early response indicates poor prognosis in childhood ALL. We aimed to evaluate if post-induction MRD levels had different prognostic impact in precursor B-cell (pB) or T-cell ALL. From 07/2000 to 06/2006, 4730 pts with ALL were enrolled in trial AIEOP-BFM ALL 2000. MRD levels were centrally measured by real-time quantitative polymerase chain reaction using the identification of clone-specific T-cell receptor and immunoglobulin gene rearrangements. MRD study time-points (TP) were treatment day 33 (TP1, end of induction) and day 78 (TP2, after consolidation). To define MRD negativity, two markers with a sensitivity of at least 10−4 were required. Patients were treated with BFM induction (protocol I-A), consolidation (I-B), extra-compartment/intensified consolidation (HD-MTX in non-high-risk patients, pulses in high-risk patients), reinduction, and maintenance. MRD analysis at one or two time points suceeded in 3707 pts; the immunophenotype was available from 3636 pts. MRD levels and corresponding estimated 5-year event-free survival (5y-pEFS) comparing pB- and T-ALL are shown in Table 1 (3yrs median follow-up). MRD response in T-ALL was slower than in pB-ALL resulting in a higher percentage of pts with high MRD load in T-ALL. In pB-ALL as well as T-ALL, high MRD levels at TP2 were well predictive to identify pts with poor prognosis. For prediction of good prognostic subgroups, TP1 was more appropriate identifying a subgroup with excellent 5y-pEFS of 〉90% in case of MRD negativity. Specificity of TP1 was poor in T-ALL if the pB-ALL criteria of MRD negativity were applied. If MRD low positive and MRD negative T-ALL pts were combined, the discrimination was as good as in pB-ALL. The optimal choice of MRD evaluation time points depends on biological factors and treatment, and is most relevant for MRD-based risk stratification. Table 1 pB-ALL T-ALL n % 5y-pEFS % (SE) n % 5y-pEFS % (SE) all 3177 100% 82.3 (1.0) 459 100% 77.2 (2.2) MRD TP1     neg 1399 44.1 92.5 (1.0) 75 16.4 94.3 (2.8)     10E-4/−5 1122 35.4 81.9 (1.7) 116 25.4 91.2 (2.8)     10E-3 393 12.4 66.4 (3.5) 110 24.1 75.3 (4.6)     ≥10E-2 256 8.1 53.2 (4.3) 156 34.1 59.8 (4.5) MRD TP2     neg 2464 77.6 87.7 (1.0) 220 47.9 91.9 (2.0)     10E-4/−5 523 16.5 68.9 (2.9) 143 31.2 76.6 (3.9)     10E-3 107 3.4 56.3 (6.5) 58 12.6 50.2 (8.1)     ≥10E-2 82 2.6 38.0 (7.3) 38 8.3 33.2 (8.3)

Description: From 07/2000 to 06/2006, 4741 eligible pts with ALL (age range 1–17 years) were enrolled in the trial AIEOP-BFM ALL 2000 (registered at http://clinicaltrials.gov with identifiers NCT 00430118 (BFM) and NCT 00613457 (AIEOP)). 3655 patients from Germany, Italy, Austria, and Switzerland entered the randomized comparison of dexamethasone (DEX) at 10 mg/m2/d vs prednisone (PDN) at 60 mg/m2/d (both given daily from d8 to d29 of induction plus 9 days tapering after a 7d prednisone prophase). Induction therapy also comprised vincristine (1.5 mg/m2) and daunorubicine (30 mg/m2) both given on d8, 15, 22, 29, as well as L-asparaginase given at 5,000 IU/m2 x8 q3d from d12. I.T. methotrexate was given thrice (in CNS-2 and CNS-3 patients for 5 times) in induction. Post-induction therapy was derived from trial ALL-BFM 95 (A. Möricke et al, BLOOD 2008) but stratification and subsequent treatment intensity was based on early response as measured by prednisone response (on d8) and MRD quantification on d33 and d78. With a median follow-up of 4.4 years, 6-year event-free survival (6y-EFS) was 84.1% (SE 1%) and 79.1% (1%) for patients randomized to receive DEX or PDN, respectively (log-rank p=0.0083). The 6-year cumulative incidence (CI) of relapse was 11% (1%) and 18% (1%) for patients randomized to receive DEX or PDN, respectively (Gray p

Description: In the AIEOP-BFM ALL2000 trial, childhood acute lymphoblastic leukemia (ALL) patients were stratified mainly according to minimal residual disease (MRD) levels at day 33 (Time Point 1, TP1) and 78 (TP2) of treatment, as detected by PCR amplification of clonotypic immunoglobulin and T-cell receptor gene rearrangements. Overall, the results confirmed that PCR-based MRD detection is an independent prognostic indicator which overrides classical risk factors. In this context, MRD measurement by flow cytometry (FCM) on day 15 has been evaluated as an earlier predictor of relapse-free outcome in children with ALL. To understand how these two methods could be applied in a MRD-based clinical protocol, we analyzed the correlation of methodologies in data sets in which PCR and FCM were simultaneously applied. We studied 3,618 BM samples from 1,570 patients (32.5% of all patients on trial) derived from day15 (n=478, 30.4%), 33 (n=1,570) and 78 (n=1,570) of remission induction therapy of the AIEOP-BFM ALL 2000 protocol. Patients were enrolled from September 2000 to June 2006 in Italy, Austria and Germany, and selection was based only on available samples for both PCR and FCM. As in most MRD-based protocols, RQ-PCR was performed on BM mononuclear cells after Ficoll gradient separation, while 4-colors FCM was done on 300,000 nucleated cells (NC) from the whole BM sample after red blood cells lyse-wash procedure. MRD levels ≥10−4 were considered as ‘positive’, while levels below this threshold, not-quantifiable or undetectable were classified as ‘negative’. Overall, qualitative concordance was observed in 2,704/3,618 samples (74.7%) measured at d15, d33 and d78. Concordances at each TP are indicated in the Table. Figure Figure Around 70% of discordant samples in all TPs were cases with low-positivity by PCR (10−4 log range) and negative by FCM, or viceversa. Concordance was also evaluated according to PCR-based MRD risk subgroups. Of note, 519/571 (90.9%) of Standard Risk by PCR-MRD (negative at d33 by 2 markers with sensitivity ≥10−4) were also FCM negative at d33; whereas 59/121 (48.8%) of cases PCR ≥10−3 at d78 (High Risk by PCR-MRD) were FCM negative. In order to evaluate the correlation of methodologies more exactly, we investigated similar cell preparations (same MNC divided for PCR and FCM), and we enhanced the resolution of FCM by using 7-colors and analyzing 500,000 MNC. Samples were collected at days 15, 33, 52, and 78 within a single center. Among a total of 266 samples, the concordance increased up to 87%. More specifically, 100 samples (37.6%) resulted undetectable by both methods; 56 samples were FCM-undetectable and PCR-positive

Description: The t(12;21) chromosomal translocation generating the TEL-AML1 fusion product is the most frequent genetic aberration of childhood acute lymphoblastic leukemia. This translocation has been shown to occur in utero and thus represents an early or even initiating event in leukemia development. However, additional molecular changes are necessary for the clinical manifestation of the disease. Despite increasing knowledge on TEL and AML1, the role of the TEL-AML1 fusion gene and its contribution to the malignant transformation is largely unknown. In particular, the mechanisms by which TEL-AML1 influences the biology of the affected cell, and thus survival and expansion, are yet to be defined. The aim of this study was to investigate the functional contribution of TEL-AML1 to the leukemic cell phenotype. We particularly sought to search for TEL-AML1 dependent genes as well as for pathways that function in a fusion gene dependent manner. The t(12;21) positive B cell precursor leukemia cell line REH was used as model system and RNA interference technology, a new and highly specific approach, was employed to block the expression of the TEL-AML1 fusion gene. The successful silencing of TEL-AML1 was demonstrated by Western blot analysis. In order to gain a broad overview of the impact of TEL-AML1 silencing upon the leukemic cell, RNA was isolated and subjected to Affymetrix gene chip analysis. In addition, we studied the proteome by high-resolution 2D electrophoresis. Expression profiling resulted in a specific pattern of differentially regulated genes after TEL-AML1 suppression with a particular emphasis on genes involved in the control of apoptosis. Genes with anti-apoptotic properties were down regulated after TEL-AML1 silencing while those that contribute to apoptosis were largely unaffected. Among the affected anti-apoptotic genes were the heat shock proteins, the most prominent representatives of which were HSP90 and HSP70. Their changes were observed at the mRNA but also at the protein level. This is of special interest, since both heat shock proteins interfere with the intrinsic as well as the extrinsic pathway of apoptosis and hence provide a powerful anti-apoptotic signal. These first data support a model in which TEL-AML1, as an early event, is functionally linked to an anti-apoptotic network and especially to the members of the heat shock protein family. Engagement of these mechanisms might rescue a cell from apoptosis and in consequence give rise to the development of a pre-leukemic clone, a reservoir for the acquisition of further mutations that might eventually give rise to leukemia. Thus, decreasing the apoptosis defence by silencing TEL-AML1 or by inhibiting the heat shock proteins might sensitize TEL-AML1 positive cells to apoptosis. This would not only be an option for future therapy of leukemias but also for the eradication of the pre-leukemic clone as a proposed source of relapse.

Description: B cell precursor (BCP) ALL is usually a monoclonal disease in which the number of IGH rearrangements per cell does not exceed the number of the IGH alleles on chromosome 14. Consequently, a clone with disomy 14 can have a maximum of two unique rearrangements. In contrast, monoclonal high hyperdiploid (HeH) cases with a trisomy 14 can harbor either a maximum of three unique or two unique rearrangements together with a third that may share particular sequences with the one or the other The pattern of IGH rearrangements in cases with trisomy 14 may therefore be misinterpreted to be oligoclonal if the chromosome 14 copy number is not known. Since oligoclonal IGH rearrangements may be instable at relapse, they generally are not used for minimal residual disease (MRD) analysis. Thus, in HeH patients seemingly oligoclonal IGH rearrangements may undeserved be skipped as MRD target. We investigated the association between IGH allele copy numbers and the IGH rearrangement patterns in 90 consecutively recruited HeH BCP ALL. This cohort was used for assessing overall frequencies. To enrich the number of small subgroups, 40 selected HeH cases were added. Cytogenetic and FISH analyses were performed according to standard procedures. IGH rearrangements were determined according to standardized ESG-MRD protocols. Even though the majority of HeH cases (78/90, 87%) had an extra chromosome 14, there was a small but distinct subgroup comprising 13% (12/90) of HeH cases with a disomy 14. Overall, IGH rearrangements were present in about 95% of leukemias representing incomplete DJH rearrangements in about 40% of cases. More than two IGH rearrangements and/or related rearrangements were found in 44% of the same HeH cohort with an overall frequency of 16% “true” oligoclonality after correction for the actual number of chromosomes 14. Of note, leukemias with only two copies of chromosome 14 revealed a significantly higher frequency of apparent oligoclonality compared to those with three copies of chromosome 14 (36% versus 13%). Monoclonal HeH leukemias with trisomy 14 could neither be distinguished from their oligoclonal counterparts nor from oligoclonal TEL-AML1 positive and “not further genetically discriminated” BCP ALLs based on the number of IGH rearrangements per cases and the type of secondary rearrangements (VH or DH to DJH or VH replacement). However, the patterns of secondary rearrangements had shifted from a predominantly VH to DJH recombination in the former towards VH replacement in the latter two groups. Our data have implications for MRD analysis, since oligoclonal patterns of IGH rearrangements account for about 25–30% of childhood BCP ALL. Hence, the interpretation of whether a particular IGH rearrangement pattern is really clonal or not may be crucial in some of these cases and may be better defined by taking into account at least the genetic subtype of the respective leukemia (i.e. hyperdiploid versus those with various fusion genes). If necessary the quality of this information can be further refined by enumerating chromosomes 14 with karyotyping or IGH alleles with interphase FISH. The data provide also insights into the biology of HeH leukemia suggesting that nondisjunction of chromosomes - leading to a HeH karyotype - affects a cell at the beginning of IGH recombination, which is a more undifferentiated B progenitor cell than the cell of origin of the TEL-AML1 positive leukemias and the group of other BCP ALLs.

Description: Background: Acute lymphoblastic leukemia (ALL) with a T cell immunophenotype accounts for about 15% of childhood ALL. While contemporary treatment protocols have improved the relapse free survival for children with T-ALL, it has for a long time been considered a malignancy with a dismal prognosis. In fact, this type of leukemia is still at a higher risk for treatment failure and early relapses than B cell precursor ALLs. Further intensification of conventional treatment or stem cell transplantation may increase the cure rate for only some subtypes of ALL but is also associated with considerable treatment-related mortality and morbidity. Thus, new treatment strategies that are less toxic and, at the same time, more effective for patients with a resistant disease, are needed. The aim of the study was to evaluate whether a specific immune reaction is triggered in children with T-ALL and to identify the respective targets. We chose SEREX (serological identification of leukemia derived antigens by recombinant expression cloning) to screen a T-ALL expression library with plasma from 18 children with T-ALL. Results: Thirteen antigens with homology to known genes that are involved in critical cellular processes were detected. Some of them have already been implicated in the induction of an immune response in a variety of cancer types. From four of these genes novel isoforms were identified and further analyzed. mRNA expression of three isoforms (HECTD1Δ, CX-ORF-15Δ and hCAP-EΔ) was restricted to more than 70% of T-ALLs (n=22) and specific antibodies against these isoforms were detected in up to 30% of patients (n=16) with the highest frequency for HECTD1Δ. The alternative splicing leads to the deletion of one glutamic acid located in a putative PEST domain thereby decreasing the PEST score suggesting a higher stability of the protein. Interestingly, the HECTD1 protein was present at high abundance in T-ALLs while is was not detectable in normal hematopoietic tissues. Since the leukemia-associated antigens detected in this study have an intracellular localization, a feature that is shared by the majority of SEREX defined antigens, the generation of immune effector responses most likely requires antigen presentation. To test this assumption, dendritic cells were loaded with HECTD1Δ protein and used for T cell stimulation. A specific T cell response was induced in vitro in all three donors, including a former T-ALL patient. Conclusion: Leukemia-associated antigens, identified by SEREX, appear to be capable of inducing both a humoral and cellular immune response in children with T-ALL. Thus, these data support further studies to establish new approaches for immunotherapy.