ALBERT

Description: Minimal residual disease (MRD) testing based on a unique Ig/TCR gene rearrangement pattern of each patient’s leukaemia turned out to be an independent tool to determine treatment response and the risk of relapse in paediatric acute lymphoblastic leukaemia (ALL). Since 07/2000, MRD information at week 5 and 12 of therapy has been used for stratification in ALL-BFM 2000 trial. In parallel, ALL IC-BFM 2002 has been designed by the International-BFM Group to test the morphological assessment of the early treatment response. Patients are stratified according to the blast proportion in peripheral blood (PB) at day 8 and in bone marrow (BM) at day 15 and 33 of therapy, age, initial WBC and the presence of BCR/ABL and MLL/AF4 fusion. One of the goals of the study is the comparison of this risk group assessment to the MRD-based criteria used in ALL-BFM 2000. In the Czech Republic, 73 patients were treated according to ALL IC-BFM 2002 protocol from 11/2002 to 12/2003, 29 in the standard-risk (SR), 35 in the intermediate-risk (IR) and 9 in the high-risk (HR) group. The SR, HR and all T-cell ALL patients were examined for clonal Ig/TCR rearrangements. RQ-PCR patient-specific systems were designed for each of these patients according to the ESG-MRD-ALL criteria. For 39 of the 40 patients tested (97.5%) at least one target with minimal sensitivity of 10(−4) was identified. MRD was evaluated in BM samples from 34 patients at several time points inclusive of the mandatory 5 and 12 week ones. Simultaneously the PB specimens of the T-ALL patients were tested. In total, 205 BM and 64 PB specimens were included. In 7 patients of 24 in the SR group, MRD positivity at week 5 and/or at week 12 was observed (ranging between 9.7x10(−4) and 1.5x10(−2)), thus identifying patients who would not qualify to the MRD-based SR group in ALL-BFM 2000 despite the identical induction regimen. In T-ALL patients, PB-MRD levels paralleled those in BM. MRD results showed no separation of MRD levels between IR- and HR-stratified T-ALL patients. These preliminary findings reveal a significant difference between the stratification results of ALL IC-BFM 2002 and ALL-BFM 2000. A fast response as measured by the morphology criterion (M1 or M2 bone marrow at day 15) together with other low-risk features does not necessarily correspond with rapid MRD clearance. The complete analysis of MRD is planned for the international consortium participating in the ALL IC-BFM 2002 protocol.

Description: More than 800 children with acute lymphoblastic leukaemia (ALL) are treated every year according to ALL IC-BFM 2002 protocol, which was designed by the International-BFM Group as a parallel to MRD-based ALL/AIEOP BFM 2000 study. The ALL-IC BFM 2002 risk group stratification comprises blast proportion in peripheral blood (PB) after 7 days of prednisone and one IT-MTX (prednisone response) and bone marrow (BM) morphology evaluation at days 15 and 33 of therapy together with age, initial WBC and presence of BCR/ABL and MLL/AF4 fusion. One of the aims of the ALL IC-BFM 2002 study is the comparison of this risk group assessment to the MRD-based criteria used in ALL-BFM 2000. We analyzed a total of 203 patients treated according to the ALL IC-BFM 2002 in the Czech Republic, Israel, Hong Kong and Uruguay for the presence of clonal antigen receptor rearrangements. MRD was evaluated in 175 patients at several time-points of therapy including mandatory points at week 5 and 12, which are used in the ALL/AIEOP BFM 2000 stratification. In total, 654 follow-up BM specimens and 80 PB samples were tested. In the univariate analysis, a good molecular response defined as MRD negativity at both week 5 and 12 was associated with the age of 1–6 years (p=0.0001), WBC

Description: Leukemias with the t(9;22) translocation resulting in BCR/ABL fusion protein expression comprise 3–5% of childhood ALL. Despite modern therapeutic regimens, their prognosis is inferior. Minimal residual disease (MRD) based on leukemia-specific immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements has become a tool influencing clinical decisions in many therapeutic trials for childhood ALL. The presence of BCR/ABL fusion gene offers a possibility of the fusion transcript detection - a faster and cheaper alternative to Ig/TCR-based MRD monitoring. Up to now, no direct comparison based on a sufficient number of samples has been done. We analyzed 350 follow-up samples from 16 children (aged 4–17 years) with BCR/ABL-positive ALL by Ig/TCR-based real-time quantitative PCR (RQ-PCR) and by reverse-transcriptase (RT) RQ-PCR for BCR/ABL transcripts. Beta-2 microglobulin housekeeping gene was used for cDNA quality normalization. WBC, age, immunophenotype and blast proportion in the bone marrow (BM) and peripheral blood (PB) showed no relation to the initial BCR/ABL level. All children expressed m-BCR/ABL transcript at the time of diagnosis; 3 of 16 children expressed both m-BCR/ABL and M-BCR/ABL transcripts representing the p190 and p210 variant of BCR/ABL protein, respectively. The expression levels of m-BCR/ABL in diagnostic samples differed up to 3 logs, being the lowest in patients expressing both variants of the fusion gene. In 38 samples from those patients, M-BCR/ABL expression was generally higher than m-BCR/ABL expression, being negative by m-BCR/ABL and positive by M-BCR/ABL in 13 samples. For further analysis we used the higher value of m- and M-BCR/ABL as the BCR/ABL MRD level. For the comparison with Ig/TCR-based method, MRD levels in follow-up samples were related to the expression levels in diagnostic samples, which were set to 1. In total, 133 (38%) and 127 (36%) samples were negative and positive by both methods, respectively. The quantitative levels differed by more than 1 log in 46 (36%) double-positive samples, being underestimated by Ig/TCR method in 25 cases and by m-BCR/ABL quantification in 21 cases. With the same sensitivity of both methods we found significantly more false-negative samples by Ig/TCR approach (70 samples) compared to BCR/ABL quantification (20 samples). Altogether, we tested 219 bone marrow (BM), 130 peripheral blood (PB) and 1 cerebrospinal fluid samples. The PB samples showed significantly worse correlation between the two methods compared to BM (p=0.02). Interestingly, some patients had higher MRD levels in PB compared to BM as shown by corresponding BM and PB samples. Our data suggest that BCR/ABL-positive childhood ALL is a biologically heterogeneous group. We show that all diagnostic samples should be screened for the simultaneous m- and M- BCR/ABL expression to avoid false-negativity when using m-BCR/ABL quantification only. In our hands, the quantification of BCR/ABL transcripts appears to be a more reliable method than the generally accepted Ig/TCR-based MRD monitoring as the number of false-negative samples by BCR/ABL quantification is significantly lower. This contention is further supported by our pilot data on transplanted patients where BCR/ABL positivity preceding transplantation seems to be a better predictor of subsequent relapse than Ig/TCR approach. Support: MSM0021620813, MZ00064203 and 62/2004 GAUK CR. KK and KM contributed equally to this work.

Description: Three types of diagnostic findings fulfill the definition of acute hybrid/biphenotypic leukemias (AHL): coexpression of multiple myeloid/lymphoid markers on lymphoid/myeloid population, coexistence of two or more distinct populations from different lineages and lineage switch prior achievement of complete remission. Aberrant expression of myeloid/lymphoid antigens is frequent among ALL and AML, respectively. A scoring system (EGIL) was developed for arbitrary distinction of “scored AHL”. Published data contain cohorts of adults or combined cohorts of children and adults. There is lack of data specifically on childhood AHL as well as on consecutive cohorts analyzed from an immunophenotypic, genetic and clinical perspective. From 09/1996 to 08/2006, immunophenotype, DNA index and BCR/ABL and MLL/AF4 fusion gene status of 898 leukemic samples of de novo (730 children), relapsed (154 samples from 122 children), and secondary AL (14 children) were centrally evaluated. In all cases classified either as “scored AHL” or with lineage switch we analyzed clonality in Ig/TCR genes and other fusions of MLL gene. De novo AL samples consisted of: 572 ALL, 118 AML, 32 “scored AHL” (28 primary ALL/My+, 4 primary AML/Ly+), 2 cases of ALL switched to AML and 6 samples not appraisable by FC (morphology ALL). Relapsed AL samples consisted of 113 ALL, 32 AML and 9 “scored AHL” (6 primary ALL/My+, 3 primary AML/Ly+). Secondary AL consisted of 3 ALL, 10 AML and 1 “scored AHL” (primary ALL/My+). AHL primarily classified as ALL cases fell into the following immuno-genetic subsets: T ALL(6), 1 hyperdiploid(1), TEL/AML1(9), BCR/ABL(3), and MLL/AF4(3); one T ALL was BCR/ABL+. The most common genetic fusion was TEL/AML1 (n.s.). The MLL/AF4 and BCR/ABL were more frequent in AHL cases (p=0.0098 and p=0.044, respectively). The incidence of hyperdiploidy was lower among AHL cases (p=0.026). Prognosis of AHL in non mature B ALL is significantly worse in AHL BCP ALL only (in 3.5 yrs follow up 84%+−1.9% in non AHL versus 61%+−14% in AHL). Separate analysis in genetic subsets revealed significant differences in relapse free survival of TEL/AML1 (AHL 41%+−22% versus non AHL 91%+−3% in 3.5 yrs of fUp, p=0.01) and MLL/AF4 subset (AHL 0%, non-hybrid ALL 85+−13% in 1st year, p=0.0082). The incidence and maturity of clonal Ig/TCR rearrangements generally reflected the immunophenotype of primary lineage, with the exception of 4/6 T ALL/My+ lacking clonal rearrangements. All 4 cases with AML/Ly+ co-expressed T lymphoid markers and no clonal Ig/TCR rearrangement was found in any of them. Among 4 patients with AML/Ly+, 3 relapses occurred in the 1st year of follow up. Conclusion: Principal lineage was determined by FC in all AHL cases. We found significantly worse prognosis of patients classified as AHL (primary BCP ALL). The prognosis of AHL (AML/Ly) should be confirmed on larger number of patients.

Description: Minimal residual disease (MRD) testing performed on bone marrow (BM) samples has become a part of the risk group stratification procedure in several of the most progressive acute lymphoblastic leukemia (ALL) treatment protocols. MRD testing of peripheral blood (PB) instead of BM is not routinely performed, although PB sampling could cause less discomfort especially in children. It is well established that the level of MRD in PB and BM correlates well in T-ALL; the data concerning B-cell precursor (BCP)-ALL remain controversial, with most studies lacking sufficient number of samples taken during the early phase of treatment with quantifiable MRD in both compartments. We simultaneously evaluated MRD in BM and PB using immunoglobulin and T-cell receptor gene-based RQ-PCR. 221 paired samples from 47 children with BCP-ALL treated according to the Berlin-Frankfurt-Muenster (BFM) ALL IC-BFM 2002 protocol were taken at diagnosis (dg, n=47), day 8 (d8, n=39), day 15 (d15, n=44), day 33 (d33, n=34), week 12 (w12, n=31) and at the end of maintenance therapy (post-MT, n=26). As in the BM at d15, patients with lower MRD in PB at d15 were more likely to achieve MRD negativity in BM at d33 in the univariate analysis (p=0.01, Mann Whitney). Patients younger than 10 yrs had lower MRD in PB at d8 and at d15 than other patients (p=0.03 and p=0.01, respectively). Unlike in BM, patients with hyperdiploidy had lower MRD in PB at d15 than other patients excluding TEL/AML1 cases (p=0.05). There were no significant associations with diagnostic white blood cell count (WBC), sex, immunophenotype (cALL/prae-B ALL) or presence of TEL/AML1 fusion at any time point. Patients with MRD

Description: The level of minimal residual disease (MRD) prior to allogeneic haematopoietic stem cell transplantation (HSCT) was shown to be an independent prognostic factor for the outcome of paediatric patients with high-risk acute lymphoblastic leukemia (ALL). Retrospective studies which used (semi-)quantitation of clone-specific immunoglobulin/T-cell receptor (Ig/TCR) rearrangements documented feasibility and practicality of such an approach. Recently, this approach was disputed by Imashuku et al (BMT 2003) due to great occurrence of the clonal evolution and generally high MRD levels prior HSCT in their cohort. In our prospective study, MRD before and after HSCT was monitored in a cohort of 36 children with ALL consecutively transplanted in our centre between VIII/2000 and VII/2004. We used a quantitative real-time PCR approach introduced and standardised by European Study Group on MRD in ALL. In 25 of 36 patients MRD level prior HSCT was assessed (9 patients lacked adequately sensitive Ig/TCR target; two lacked analysable DNA prior HSCT). Seventeen patients were MRD-negative prior HSCT (including two with MRD level below the quantitative range 10(−4)) and 8 were MRD-positive up to 9x10(−2). In the MRD-positive subgroup, 7 events (6 relapses) occurred post-transplant in striking contrast to only one relapse in MRD-negative subgroup (EFS log-rank p

Description: CCAAT/enhancer binding protein alpha (CEBPα) is one of the crucial transcription factors involved in hematopoietic differentiation and leukemogenesis. CEBPα promotes myeloid differentiation by up-regulation of lineage specific genes and by cell proliferation arrest. Epigenetic regulation of CEBPα expression through DNA methylation has been demonstrated in acute myeloid leukemia (AML) (Figueroa et al, Cancer Cell, 2010). However, only limited data are available regarding CEBPA promoter methylation and its expression in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Methylation status of CEBPA promoter (-295 to -593bp upstream of the transcription start site (TSS), 24 CpG dinucleotides) was analyzed by bisulfite sequencing. Five subgroups of BCP-ALLs were analyzed: MLL gene rearranged (n=5), hyperdiploid (n=6), mBCR-ABLpos(n=5), ETV6-RUNX1pos(n=6) and other BCP-ALLs (no hyperdiploidy, MLL gene rearrangement, BCR-ABL or ETV6-RUNX1 fusion gene (“BCP-others”, n=29)). CEBPA promoter was hypermethylated in MLL-rearranged, hyperdiploid and ETV6-RUNX1pos BCP-ALL (5/5, 6/6 and 4/6 respectively). Surprisingly CEBPA promoter was hypomethylated in all mBCR-ABLpos cases (5/5). In subgroup of other BCP-ALLs both hypermethylation (10/29) and hypomethylation of CEBPA promoter (19/29) were detected (Figure 1A). In previous study we found association of CD2 (LFA-2) aberrant expression and switch to the monocytic lineage during the early phase of treatment in BCP-ALLs (Slamova et al, ASH 2012). We were interested if a possible link between hypomethylation of CEBPA promoter correlates with aberrant expression of CD2. There was a significant association between aberrant expression of CD2 antigen and hypomethylation in CEBPA promoter in BCP-others (Fisher exact test, p

Description: Abstract 876 Immunophenotypic instability during early phase of ALL treatment is a frequent observation during flow cytometric minimal residual disease (FC MRD) monitoring. Antigens typically involved include CD10, CD20, CD34 and CD45 and these changes do not usually revoke initial disease classification and do not hamper FC MRD detection. We previously described a subtype of B-cell precursor (BCP) ALL with striking immunophenotypic instability towards monocytoid lineage within the first month of therapy (switching ALL-swALL). Blasts expressing both B and monocytoid markers emerged at early time points on days 8 and 15 of treatment while later only those blasts with pure monocytoid phenotype were present. Incidence of swALL in childhood was unexpectedly high (3-4% of all pediatric BCP ALL) as confirmed in two national reference labs. This phenomenon was associated with aberrant expression of CD2 (LFA-2) on diagnostic blasts (Mejstrikova et al, ASH 2010). The leukemic origin of monocytoid blasts was proven by the detection of clone-specific immunoreceptor gene rearrangements (Ig-TCR). No common genetics aberration was found in a cohort of swALL (n=17), MLL gene was always in germline configuration. We found an increased rate of alteration in IKZF1 gene compared to control BCP ALL cases (p=0.012). An expression analysis of the key hematopoietic regulators showed a difference in CEBPα expression, which is an important transcription factor in transdifferentiation of B cells into macrophages (Xie et al., Cell 2004). Expression of CEBPα is increased in swALL compared to other BCP ALL cases (p=0.01) already at diagnosis prior switching, however, the expression is lower compared to AML (p=0.002). We analyzed CEBPα the methylation status of the promoter region of this gene. Demethylation of CEBPα promoter region analyzed by bisulfite sequencing (295bp-594bp of promoter region) was found in 10/12 swALL cases, while it was seen in only 6/28 control BCP ALLs (Fisher test, p=0.0004). The only cases having demetylation in CEBPα were 5/5 BCR-ABLpos and 1/4 ETV6-RUNX1pos. Whole genome ERRBS method (Enhanced Reduced Representation Bisulfite Sequencing) confirmed this methylation pattern of CEBPα in 7 patients (4 swALL,3 BCP ALL). In order to establish an in vivo model to study the underlying molecular mechanisms, we transplanted ALL cells from 7 swALL patients intrafemorally into NOD-SCIDIL2Rgammanull (NSG) mice. Successfulstable engraftment was achieved only in 2 out of 7 swALL cases (28%) (Fisher test, p=0.049).Interestingly in these two cases, the 200 bp promoter region of CEBPα was methylated to some extent at diagnosis and completely methylated after engraftment into mice, suggesting the possibility of a selective advantage in this context. We treated engrafted animals with prednisolone and in both cases we observed demethylation of CEBPα promoter. Because the rate of engraftment of ALL in NSG is usually very high, these observations may indicate that the biology of this particular subset of patient is distinct. Conclusion: We described a novel subtype of BCP-ALL with the demethylation of CEBPα promoter region, increased CEBPα expression and immunophenotypic shift towards monocytic lineage during first weeks of the therapy. We identified CEBPα as the potential regulator of this lineage plasticity. Disclosures: No relevant conflicts of interest to declare.

Description: Minimal residual disease (MRD) monitoring is an essential tool for current leukaemia therapy. The only standard method for MRD monitoring in childhood ALL is the quantitative detection of clonal immunoglobulin (Ig) and T-cell receptor (TCR) genes rearrangements. The quantitative detection of fusion genes or transcripts provides an alternative option for MRD monitoring. We aimed to compare the significance of these MRD methods by parallel monitoring of fusion transcripts/genes and Ig/TCR targets during the follow-up of children from the three most common ALL genotype groups. We analysed 117, 109 and 191 bone marrow samples from 28 TEL/AML1-positive, 7 MLL fusion-positive and 16 BCR/ABL-positive patients, respectively. To keep the comparability of different MRD approaches, we used qPCR detection systems with similar sensitivity (at least 10−4), we adopted ESG-MRD-ALL principles for MRD quantification and we related the MRD level in follow-up samples to the diagnostic level for all MRD methods. We found a very good correlation of fusion transcript- and Ig/TCR-based approaches (R2=0.903) with only 7% of samples differing by more than 1 log in a cohort of TEL/AML1-positive patients. A good correlation was also found between fusion transcript- and Ig/TCR-based MRD in MLL fusion-positive patients (R2=0.8419). Only 10% of samples differed by more than 1 log, being underestimated by Ig/TCR in 4.5% and by MLL-fusion transcript in 5.5%. For the follow-up of MLL-fusion-positive patients we further employed the monitoring of MLL-fusions on genomic level. The MRD based on genomic MLLfusion genes showed a very good correlation with Ig/TCR -based method (R2=0.9124) with only 5% of samples differing by more than 1 log, and it also closely correlated with MLL-fusion transcript levels (R2=0.9195). Strikingly, in BCR/ABL-positive patients we found a limited correlation of fusion transcript-based and Ig/TCR-based MRD (R2=0.6880) with 1/3 (34%) of samples differing by more than 1 log. In contrast to the MLL cases, the underestimation of MRD by individual methods was “asymmetrical”: 8% of the discordant samples had higher MRD measured by Ig/TCR and 26% by BCR/ABL transcript. Despite identical sensitivity of both methods, in 19% of samples the MRD positivity was revealed only by BCR/ABL approach while Ig/TCR approach gave negative results. Detailed analysis showed clinical significance of the discordant BCR/ABL vs. Ig/TCR MRD information. Altogether, 13 relapses occurred during the follow-up of our cohort. We compared number of BCR/ABL and Ig/TCR -positive samples among all BM specimens taken 6 and 12 months before relapse. While the majority of samples preceding relapse were BCR/ABL-positive (14/18 and 22/36 six and twelve months before relapse) only a minority of samples showed Ig/TCR positivity (7/18 and 12/36, respectively). The non-equal distribution of the BCR/ABL and Ig/TCR-positive samples was statistically significant (p=0.04 and p=0.03 for the two time-points, respectively). Our study shows, that in TEL/AML1 and MLL fusion-positive patients, fusion gene/transcript-based MRD monitoring provides information highly concordant to the standard Ig/TCR approach and thus it is useful as a complementary method in patients with absent or inadequate Ig/TCR targets (particularly in MLL cases where clonal Ig/TCR rearrangements are rare). The situation is different in BCR/ABL patients, where the MRD information from both approaches is discordant in a high subset of samples. This result probably reflects the dissimilar biology of this ALL subtype and the fact, that BCR/ABL-positive (prae-)leukaemic stem cell is different and multilineage involvement more common. Thus, in some cases, the fusion transcript monitoring reveals the existing pool of cells that increase the risk of relapse despite the Ig/TCR negativity. We conclude that MRD in all BCR/ABL–positive patients should be monitored not only by the standard Ig/TCR approach but in parallel also by the quantitative fusion transcript-based detection. Support: MSM0021620813, MZO00064203.