AIEOP-BFM Acute Lymphoblastic Leukemia Research Articles

The Strong Prognostic Effect of Concurrent Deletions of IKZF1 and PAX5, CDKN2A, CDKN2B or PAR1 in the Absence of ERG Deletions (IKZF1plus) in Pediatric Acute Lymphoblastic Leukemia Strongly Depends on Minimal Residual Disease Burden after Induction Treatment

Technical advances in the field of genomic analyses have stimulated a large number of discovery studies on etiological and clinical endpoints in acute lymphoblastic leukemia (ALL) to provide new avenues for preventive strategies, diagnostics and treatment. Recently, deletion of IKZF1 (IKZF1del) was described as a poor prognostic factor in pediatric ALL (Mullighan CG et al., 2012). In our trial AIEOP-BFM ALL 2000 patients with IKZF1del had a lower 5-year event-free survival (EFS; 0.69±0.05 vs. 0.85±0.01; P<0.0001) compared to those without, mainly due to a higher cumulative incidence of relapses (CIR; 0.21±0.04 vs. 0.10±0.01; P=0.001) (Dörge P et al., 2013). IKZF1delwas an independent prognostic factor in this modern protocol. However, on its own, and even in association with minimal residual disease (MRD) levels, its relatively mild prognostic strength limited incorporation in clinical stratification strategies so far.The present study was undertaken to evaluate the prognostic effect of concurrent recurrent genetic aberrations in association with IKZF1del to further refine a high-risk genetic signature for pediatric ALL treated on AIEOP-BFM protocols. For this purpose, we studied a cohort of 1100 German patients with B-cell precursor ALL treated on AIEOP-BFM ALL 2000 with available characterization of genetic aberrations at initial diagnosis by MLPA (MLPA SALSA kit P335; MRC-Holland) and a PCR assay for detection of ERG deletions (ERGdel; Zaliova M et al., 2014). Validation of results was conducted by use of an independent cohort of 417 Italian patients from the same trial. BCR/ABL1-positive patients were excluded from outcome analysis.When single marker analyses were conducted, IKZF1del was the strongest determinator of outcome in the discovery as well as the validation cohort. When IKZF1del was analyzed in combination with PAX5, CDKN2A, CDKN2B, and PAR1 deletions, patients with an additional deletion to that of IKZF1 had the worst EFS and highest CIR in absence of ERGdel and, consequently, were grouped as IKZF1plus (definition: presence of IKZF1del and at least an additional deletion in PAX5, CDKN2A, CDKN2B or PAR1 in the absence of ERGdel). This group comprised 6% of B-lineage ALL patients and had a very poor clinical outcome: 5y-EFS 50%±0.06 compared to 86%±0.01 in IKZF1plus-negatives (p<0.0001); 5y-CIR 45%±0.06 compared to 11%±0.01 (p<0.0001) and was an independent prognostic factor in multivariate analyses including MRD, slow early response, prednisone response, ETV6/RUNX1 status, and WBC (≥100.000/µl) (hazard ratio for an event: 3.39; 95% CI 2.09 – 5.48; p<0.0001). Surprisingly, stratified analysis by MRD demonstrated that the effect of IKZF1plus was restricted to those patients still carrying MRD loads of at least 10E-4 after induction treatment: standard-risk group 5y-EFS 94%±0.06 compared to 38%±0.09 in intermediate-risk and 27%±0.13 in high-risk patients (p<0.0001); standard-risk group 5y-CIR 6%±0.10 compared to 62%±0.10 in intermediate risk and 55%±0.17 in high-risk patients (p<0.0001). Hierarchical clustering of gene expression profiles of IKZF1plus patients did not demonstrate association specific to the different MRD risk groups. Similarly, analysis of whole exome and transcriptome data from material at initial diagnosis in a very restricted number of patients could not demonstrate insights into the observed differences.Newly identified very poor prognostic ALL subgroup – termed IKZF1plus – represented worse outcome compared to that of IKZF1del or others as a sole marker. The differential prognostic effect of IKZF1plus at different MRD levels without currently discernable molecular explanations suggest a quantitative mechanism with higher levels of leukemic cell burden during the early treatment phases predisposing to evolution of a treatment resistant leukemic clone under exposure towards genotoxic chemotherapeutic agents. Potential explanations for these differences in treatment response may be undetected sub-clones already present at diagnosis or underlying germline genetic variation associated with treatment response. Further analyses will be required to better understand this phenomenon. However, due to its strength, the definition of IKZF1plus is likely to aid in the practical implementation of newly detected markers for risk stratification in childhood ALL in a clinical setting.Support: EU FP7 (ENCCA, TRANSCALL), IGA MZ NT/13170-4 DisclosuresNo relevant conflicts of interest to declare.

Antithrombin Plasma Levels and Fibtem Determination in Children with Acute Lymphoblastic Leukemia Undergoing Asparaginase Treatment

Introduction We have evaluated Antithrombin (AT) values and Whole Blood Rotation Thromboelastometry (ROTEM) profiles in children affected by acute lymphoblastic leukemia (ALL), followed at the Clinic of Pediatric Hematology Oncology of Padua. L-Asparaginase, used in protocols for pediatric ALL, is an inhibitor of protein synthesis. Typical collateral effects are coagulation disturbances. Guidelines for identification of patients at risk of thrombosis and hemorrhages are not yet established. AT levels are the only reliable parameter for thrombotic risk. Very low levels of fibrinogen are not correctly detected by classical Clauss method; ROTEM test using FIBTEM is considered a valid tool to identify hypofibrinogenemia and hemorrhagic risk in surgical patients. The aim of the study was to analyze coagulation patterns during treatment with Pegilated L-Asparaginase (Peg-Asp).Methods Forty-two children (25 males, 17 females; 31 pB-ALL; 11 T-ALL; 23 at standard/medium risk; 19 at high risk) were consecutively diagnosed and treated with AIEOP BFM ALL 2009 protocol from June 2012 to March 2014. They received 3 (standard/medium risk) or 8 (high risk) doses of Peg-Asp (2500UI/mq/dose). ROTEM and AT determinations were performed at 5 fixed time-points before and after each Peg-Asp administration. Prothrombin time (PT), fibrinogen plasma levels and platelets counts were recorded. Maximum Clot Firmness (MCF; normal value 9-25 mm), measuring the maximum amplitude reached in FIBTEM thromboelastogram, assessed the specific role of fibrinogen in the whole blood clot formation following platelets inhibition by Cytocalasin D.Results: 798 AT and 706 FIBTEM tests were performed; details are reported in Table. We divided abnormal MCF in 3 grade levels. Only values of MCF >9 mm had a linear correlation with fibrinogen levels. Plasma fibrinogen values <1 g/l were seen only in 45/286 (15%) cases with low MCF, without the possibility to discriminate the 3 MCF groups. Hemorrhagic risk was not suggested by PT, which was highly prolonged only in 5 cases (MCF 4-8 mm). Supplementation with AT or fibrinogen concentrates was suggested for patients with AT <50% or MCF <2 and decided on clinical basis.Twenty six children had a complete set of tests available during Induction phase (2 doses of Peg-Asp). They showed two different patterns of AT and MCF behavior. In 13 patients, mean AT levels decreased until 12-14 days after Peg-Asp, never being <50% and increased thereafter. The other thirteen patients had low AT levels from day 12 after Peg-Asp, required supplementation even before the second dose of Peg-Asp and did not show recovery until the forth week after treatment (figure 1). In 11 patients , MCF were never <9mm , without any trend to reduction. In 15 patients, who had at least 2 tests of MCF <9mm, mean MCF decreased regularly from first Peg-Asp dose until day 21, showing a trend to recovery at day 28 (figure 2).One child had a cerebral sinus thrombosis at the end of induction; one child had extensive thrombosis, strongly related to the central line used for dialysis. Thromboses were not related to fibrinogen concentrates administration or low AT levels. No bleedings were observed.Conclusions: Extensive analysis of coagulation parameters in children undergoing chemotherapy for ALL containing PEG Asp showed abnormalities of AT in 88% of patients and of MCF in 30%. These tests identified patients with significant abnormalities more closely than classical methods. Careful use of AT dosage and FIBTEM can help in managing coagulation disturbances, by identifying patients at risk and suggesting prevention measures.Table:AT and FIBTEM analysis in ALL patientsTotal N. pts / total N. testsAT42/798MCF42/706Pts with pathological testsAT<50%37 (88%)MCF<9mm13 (30%)N. of Pathological testsAT<50%158 (19%)MCF<9 mm286 (40%)MCF 5-9 mm212 (30.5%)MCF 3-4 mm56 (7%)MCF <2 mm18 (2.5%)Supplementation with AT or FCAT<50%58 units in 24 ptsMCF<2 mm18 units in 11 pts [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Next Generation Amplicon Sequencing of Immunoglobulin Heavy Chain Gene Rearrangaments for Minimal Residual Disease (MRD) Stratification in Childhood Acute Lymphoblastic Leukemia (ALL): A Comparison with Classical qPCR-Based Technique

▪IntroductionMRD is an important predictor of outcome in childhood ALL. Since 2000, MRD detected by quantitative PCR (qPCR) for immunoglobulin and T-cell receptor gene rearrangements with a minimal sensitivity of 1E-04 has been used for risk group stratification in pediatric BFM trials. Next generation sequencing (NGS) permits rapid parallel sequencing of large numbers of DNA segments. It can overcome most of the limitations of qPCR: it allows highly specific molecular detection of MRD without laborious optimization of patient-specific assays. Moreover it enables not only monitoring of malignant clone but also shows the picture of entire immune background.AimsTo develop an assay for immunoglobulin heavy chain (IgH) rearrangements detection on Ion Torrent PGM/Ion Proton platforms and compare the MRD levels with qPCR at BFM stratification timepoints.MethodsTwo round PCR was used for library preparation. Libraries were created from 450ng (equivalent of 70,000 DNA copies) of bone marrow DNA and 50ng of Human Genomic DNA (Roche). In the first round of PCR rearranged IgH genes were amplified using IGH FR3 BIOMED-2 primers (van Dongen, Leukemia 2003). In the second round the sequencing adapters and multiplex identifiers were added. Sequencing was performed on Ion Torrent PGM/Ion Proton sequencers using a 200bp chemistry. We developed a bioinformatics algorithm for detection of reads with known clonal V-D-J rearrangements within the resulting fastq files.For validation of the assay we sequenced 1E-1 to 1E-5 dilutions of diagnostic samples from 2 patients in multiplicates. The results show that the assay gives reproducible quantitative results up to 1E-4 dilution.ResultsWe sequenced 183 samples from 67 patients (52×day 15, 65×day 33, 66×day 78) with childhood ALL treated according to AIEOP-BFM ALL 2000 protocol with the median coverage 587,406 reads per sample. Eighty-three (45.4%) samples were negative by both methods. Fifteen (8.2%) samples were positive by NGS and negative by qPCR and 14 (7.7%) samples were positive by qPCR and negative by NGS. All the discordant samples had MRD levels below the sensitivity of both methods. The overall correlation of all double positive and double negative samples was very good (R2=0.93). If risk group stratification based on NGS results would be performed, 8 patients would be classified as intermediate risk (IR) instead of standard risk (SR) (one of whom relapsed) and 8 patients as SR instead of IR. One patient would be relocated from IR to slow early responders (SER) group, and two patients from SER to IR (one of them relapsed). One patient who relapsed would be classified as high risk (HR) instead of SER. All 5 patients who were MRD negative at d15 by NGS remained MRD negative in later timepoints and none of them relapsed.DiscussionWe present a cost-effective and widely adoptable NGS-based method that provides clinically relevant results in childhood ALL. NGS has a great potential to reduce the laboriousness associated with patient-specific qPCR analysis and to speed up the process of MRD detection. The sensitivity of both methods is comparable when ~500ng of DNA is used. The majority of the differences were in the samples with MRD levels below 1E-4 and most of treatment stratification changes occurred between SR/IR. However, the different stratification mostly concerned patients who did not relapse. The sensitivity of NGS could be improved if more DNA was analyzed. However, the benefit of increased MRD sensitivity is questionable due to possible overtreatment of patients with very low MRD loads after induction treatment. “Online” identification of d15 MRD negative patients reported as having an excellent prognosis previously is possible only by NGS, because optimization of patient-specific qPCR takes several weeks.The addition of 10% polyclonal DNA seems to solve the problem of MRD overestimation by NGS in samples with B-cell aplasia. At present, the main drawback of the Ig/TCR-exploring NGS methods is lack of standardization both in the experimental setting and in data analysis. Therefore, recently a European network, the EuroClonality NGS Consortium, has been formed to standardize the whole workflow of analytics, pre-analytics and bioinformatics not only for MRD quantification but also for clonality assessment in lymphoid neoplasms and for repertoire analysis.Supported by IGA NT14343, IGA NT12397, IGA NT13462-4 and GAUK 394214. DisclosuresNo relevant conflicts of interest to declare.

Minimal residual disease-based treatment is adequate for relapse-prone childhood acute lymphoblastic leukemia with an intrachromosomal amplification of chromosome 21: the experience of the ALL-BFM 2000 trial.

Recently, the UK CCLG and COG reported that an intrachromosomal amplification of chromosome 21 (iAMP21) in acute lymphoblastic leukemia (ALL) loses its adverse prognostic impact with intensified therapy. We evaluated the prognosis of iAMP21 among patients from the ALL-BFM (Berlin-Frankfurt-Münster) 2000 trial with 46 of 2 637 (2%) patients iAMP21+. 8-year event-free-survival (EFS, 64 ± 8% vs. 81 ± 1%, p=0.0026) and cumulative incidence of relapse (CIR, 29 ± 8% vs. 14 ± 1%, p=0.008) of the iAMP21 cases were significantly worse compared with non-iAMP21 patients. Within the MRD low-risk group, iAMP21 cases (n=14) had an inferior 8-year EFS (76 ± 12% vs. 92 ± 1%, p=0.0081), but no increased CIR (10 ± 10% vs. 6 ± 1%, p=0.624). Within the MRD intermediate-risk group, iAMP21 cases (n=27) had a worse 8-year EFS (56 ± 11% vs. 78 ± 2%, p=0.0077) and CIR (44 ± 11% vs. 20 ± 2%, p=0.003) with 6/10 relapses occurring after 2 years. Conclusively, we believe that there is no necessity for enrolling all iAMP21 patients into the high-risk arm of ongoing ALL-BFM trials because MRD low-risk patients have a moderate relapse risk under current therapy. Whether the increased relapse risk in MRD intermediate-risk patients can be avoided by late treatment intensification remains to be answered by the AIEOP-BFM ALL 2009 trial randomly using protracted pegylated L-asparaginase during delayed intensification and early maintenance.

Postinduction minimal residual disease monitoring by polymerase chain reaction in children with acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. Monitoring minimal residual disease (MRD) by using real-time quantitative polymerase chain reaction (RQ-PCR) provides information for patient stratification and individual risk-directed treatment. Cooperative studies have documented that measurement of blast clearance from the bone marrow during and after induction therapy identifies patient populations with different risk of relapse. We explored the possible contribution of measurements of MRD during the course of treatment. We used RQ-PCR to detect MRD in 110 unselected patients treated in Italy in the International Collaborative Treatment Protocol for Children and Adolescents With Acute Lymphoblastic Leukemia (AIEOP-BFM ALL 2000). The trial took place in AIEOP centers during postinduction chemotherapy. Results were categorized as negative, low positive (below the quantitative range [< 5 × 10(-4)]), or high positive (≥ 5 × 10(-4)). Patients with at least one low-positive or high-positive result were assigned to the corresponding subgroup. Patients who tested high positive, low positive, or negative had significantly different cumulative incidences of leukemia relapse: 83.3%, 34.8%, and 8.6%, respectively (P < .001). Two thirds of positive cases were identified within 4 months after induction-consolidation therapy, suggesting that this time frame may be most suitable for cost-effective MRD monitoring, particularly in patients who did not clear their disease at the end of consolidation. These findings provide further insights into the dynamic of MRD and the ongoing effort to define molecular relapse in childhood ALL.

Frequent and sex-biased deletion of SLX4IP by illegitimate V(D)J-mediated recombination in childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) accounts for ∼25% of pediatric malignancies. Of interest, the incidence of ALL is observed ∼20% higher in males relative to females. The mechanism behind the phenomenon of sex-specific differences is presently not understood. Employing genome-wide genetic aberration screening in 19 ALL samples, one of the most recurrent lesions identified was monoallelic deletion of the 5' region of SLX4IP. We characterized this deletion by conventional molecular genetic techniques and analyzed its interrelationships with biological and clinical characteristics using specimens and data from 993 pediatric patients enrolled into trial AIEOP-BFM ALL 2000. Deletion of SLX4IP was detected in ∼30% of patients. Breakpoints within SLX4IP were defined to recurrent positions and revealed junctions with typical characteristics of illegitimate V(D)J-mediated recombination. In initial and validation analyses, SLX4IP deletions were significantly associated with male gender and ETV6/RUNX1-rearranged ALL (both overall P < 0.0001). For mechanistic validation, a second recurrent deletion affecting TAL1 and caused by the same molecular mechanism was analyzed in 1149 T-cell ALL patients. Validating a differential role by sex of illegitimate V(D)J-mediated recombination at the TAL1 locus, 128 out of 1149 T-cell ALL samples bore a deletion and males were significantly more often affected (P = 0.002). The repeatedly detected association of SLX4IP deletion with male sex and the extension of the sex bias to deletion of the TAL1 locus suggest that differential illegitimate V(D)J-mediated recombination events at specific loci may contribute to the consistent observation of higher incidence rates of childhood ALL in boys compared with girls.

Pharmacogenomic Approaches for Tailored Anti-Leukemic Therapy in Children

Several lympholytic and cytotoxic agents are used in acute lymphoblastic leukemia (ALL) polychemotherapy. Genetic variants for cellular components involved in the pharmacokinetics and pharmacodynamics of these drugs can influence the pharmacological response, and molecular characterization of these genetic variants could be helpful for the comprehension of the mechanisms of resistance or increased sensitivity. The purpose of this review is to carry out an update of recent publications on genes that might influence ALL treatment in terms of outcome and/or toxicity and to underlie the role of genetic variants, particularly single nucleotide polymorphisms (SNP), in predicting clinical response, with particular reference to the current protocol for ALL therapy used in Italy, AIEOP-BFM ALL 2009.

Optimizing asparaginase therapy for acute lymphoblastic leukemia

Asparaginases are important agents used in the treatment of children with acute lymphoblastic leukemia (ALL). Three types of asparaginase are currently available: two are derived from Escherichia coli [native asparaginase and pegylated asparaginase (PEG-asparaginase)] and one from Erwinia chrysanthemi (crisantaspase). All three products share the same mechanism of action but have different pharmacokinetic properties, which do not make them easily interchangeable. Among the known toxicities and side-effects, allergic reactions and silent inactivation represent the most important limitations to the prolonged use of any asparaginase product, with associated reduced therapeutic effects and poorer outcomes. Routine real time monitoring can help to identify patients with silent inactivation and facilitate a switch to a different product to ensure continued depletion of asparagine, completion of the treatment schedule and maintenance of outcomes. However, the most appropriate second-line treatment is still a matter of debate. PEG-asparaginase has lower immunogenicity and a longer half-life than native Escherichia coli (E. coli) asparaginase, which makes it useful for both first-line and second-line use with a reduced number of doses. However, PEG-asparaginase displays cross-reactivity with native E. coli asparaginase that may harm its therapeutic effects. Crisantaspase does not display cross-reactivity to either of the E. coli-derived products, which has made crisantaspase the second-line treatment option in a number of recent protocols. As crisantaspase has a much shorter biological half-life than the E. coli-derived products, the appropriate dosage and administration schedule are of paramount importance in delivering treatment with this product. In the ongoing trial AIEOP-BFM ALL 2009 (Associazione Italiana Ematologia Oncologia Pediatrica - Berlin-Franklin-Munster), in which PEG-asparaginase is used first-line, one dose of PEG-asparaginase is substituted by seven doses of crisantaspase given intravenously at 20,000 IU/m2 on alternate days when clinical allergy or silent inactivation is present. Based on the indications of different protocols, lack of cross-reactivity to the E. coli-derived products and taking into consideration regulatory factors and availability, crisantaspase may be considered a viable second-line therapy.

Results of the AIEOP-BFM ALL 2000 Study for Childhood Acute Lymphoblastic Leukemia IN AIEOP High Risk Patients.

Aim:The Italian Association of Pediatric Haematology and Oncology (AIEOP) patients, diagnosed in the period September 2000-July 2006, were treated in the context of the AIEOP-BFM ALL 2000 Study. Some differences in high risk (HR) treatment and hematopoietic stem cell transplantation (HSCT) indications justify separate reporting of results obtained by AIEOP and BFM respectively. We report here the AIEOP experience. Patients and methods:Overall, 1999 AIEOP Ph negative Acute Lymphoblastic Leukemia (ALL) patients were eligible to the AIEOP-BFM ALL 2000 Study. High Risk (HR) criteria were: t(4;11) translocation, Prednisone Poor Response (PPR), no complete remission (CR) at day 33, high minimal residual disease (MRD) levels (≥10-3) at day 78 (HR-MRD). Treatment consisted of protocol I (patients were randomised to receive either dexametasone or prednisone in induction), 3 HR polychemotherapy blocks, a randomized comparison between delayed intensification based on protocol II repeated twice or protocol III repeated thrice, cranial radiotherapy (CRT), maintenance therapy for a total of 2 years of treatment. Results:311 patients were classified as being at HR (15.6% of the total ALL population) and had an overall event-free survival (EFS) and Survival of 58.7%(standard error 2.9) and 70.1%(2.7), respectively. For the 204 patients randomized to different steroids in protocol I, we observed a 5-year EFS of 62.7%(5.0) and 62.3%(4.8) and a 5-year Survival of 72.7%(4.7) and 72.8%(4.3) for dexamethasone and prednisone arm, respectively. The 5-year EFS was 44.4%(4.5) in 132 patients at HR for MRD, 36.4%(14.5) for the 11 patients at HR for t(4;11), 41.2%(11.9) for the 17 patients at HR for no CR at day 33, 74.6%(3.7) for the 151 patients at HR only for PPR. Patients at HR with <10-3 (44/258) or negative (82/258) MRD levels at day 78 had a 5-year EFS of 63.4%(7.3) and 79.4%(4.9), respectively. Among 80 patients who underwent allogeneic HSCT at a median time of 6 months from diagnosis, 68 had HR-MRD or t(4;11) or no CR at day 33. After adjusting for waiting time to transplantation, their 5-year EFS was 51.7%(6.6) compared with a 5-year EFS of 44.6%(5.8) in patients with the same features and treated given chemotherapy only (p=0.72). Conclusions:These data show that AIEOP-BFM ALL 2000 HR therapy is very effective for patients defined at HR for PPR. These patients could thus be considered for some treatment reduction, i.e. sparing most toxic therapeutic elements such as CRT. MRD identifies patients at HR (8.7% of the analysed population, 42% of HR patients), who have a poor outcome despite receiving intensive BFM treatment, including HSCT, and who may thus be eligible for innovative therapies. Disclosures:No relevant conflicts of interest to declare.

Impact of Minimal Residual Disease (MRD) on Prognosis in Children with Philadelphia Positive Acute Lymphoblastic Leukemia (ALL) Treated in the AIEOP-BFM ALL 2000 Study.

Introduction: The International “Berlin-Frankfurt-Münster” Study Group (I-BFM-SG) pioneered the evaluation of minimal residual disease (MRD) based on Immunoglobulin and T-cell Receptor gene rearrangements as PCR targets. The prospective AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica)-BFM ALL 2000 study is the largest in which standardized quantitative assessment of PCR-MRD at two time points (TP) was used for stratification in 127 centers.Objective: To assess whether PCR-MRD levels discriminate outcome in patients with childhood Philadelphia positive (Ph+) ALL treated with intensive chemotherapy.Material and Methods: Between 07–2000 and 07–2006, 79 Ph+ patients were enrolled in the AIEOP-BFM ALL 2000 study. They were eligible for the high risk (HR) treatment arm and treated with Induction (protocol IA + IB), poly-chemotherapy blocks, reinduction (by one or more Protocols II or III), followed by maintenance therapy. BM samples obtained at day 33 (Time Point 1, TP1) and 78 (TP2) of induction therapy were used for MRD analysis by patient specific PCR targets. At least one or two sensitive markers (≥ 1 × 10−4) could be determined in 62 (78.5%) and 54 (68.4%) patients, respectively. MRD-Standard Risk (SR) was defined by MRD-negative at both TP1 and TP2; MRD-HR by MRD ≥1×10−3 at TP2; MRD-Intermediate Risk (IR): all others. Median follow-up was 3 years; 5-year survival and event-free survival (EFS) (SE) estimates are given.Results: Out of 79 registered patients, 3 (3.7%) died during Induction phase, 15 (19.2%) were Prednisone-poor responders (PPR), 12 (19.2%) were resistant to phase IA and 75 (94.9%) achieved CR. Forty-six patients (58.2%) underwent hematopoietic stem cell transplantation (HSCT). Overall, EFS and Survival (SE) were 44.3%(6.5) and 61.5%(6.2), respectively. Sixty-two patients were stratified by MRD (i.e. they were alive and valuable at day 78 and had at least one sensitive PCR marker). Eleven patients (17.7%) were at MRD-SR: 8 remained in CCR (4 after BMT), 1 died in CCR and 2 relapsed at 2.7 and 5.7 years from diagnosis; 28 (45.2%) were at MRD-IR: 18 remained in CCR (14 after BMT), 1 died in CCR and two for TRM after HSCT; 7 relapsed after 0.6 to 5.1 years; 23 (37.1%) were at MRD-HR and only 4 remained in CCR (all after BMT). The relapse rate was 18% in MRD-SR, 25% in MRD-IR and 61% in MRD-HR. Of note, within the Prednisone good-response subgroup (n=61), the evaluation of MRD identified those patients (MRD-HR, n=12 out of 51 PGR, MRD stratifiable) at very high risk of relapse (6/12: 50%).Conclusions: PCR-MRD is a strong predictor for outcome in Ph+ ALL patients treated with BFM therapy. MRD response detected by PCR can tailor the selection of the best treatment (Imatinib or other tyrosine kinase inhibitors and chemotherapy with or without transplant). The prognosis in the subgroup of Ph+ALL as defined HR according to PCR-MRD detection is still very poor even after HSCT and accordingly new therapeutic strategies are needed.

Dexamethasone in Induction Can Eliminate One Third of All Relapses in Childhood Acute Lymphoblastic Leukemia (ALL): Results of An International Randomized Trial in 3655 Patients (Trial AIEOP-BFM ALL 2000)

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<0.001). The difference between the two groups was observed for isolated BM relapses (8% vs 12%), CNS-relapses (2% vs 4%) and other relapses (2% vs 3%). Higher toxicity was observed in patients treated with DEX: CI for death in induction was 2.0% for DEX and 0.9% for PDN (p=0.003). The latter value was similar to that of study ALL-BFM 95 (0.7%). CI for death in remission was similar being 2.0% and 1.6% for DEX and PDN groups. In each arm, only 0.2% did not achieve CR after intensive consolidation. Severe toxicities, mainly infections, were recorded for DEX treated patients more frequently. Three years into the trial, the DSMC and the TSC decided to halt the randomization for patients aged 10 years or older. CI for relapse/death in induction in this age group is now at 15%/4.5% for DEX, and 20%/2.4% for PDN (p=0.09/0.13). In patients less than 10 years of age, the CI for relapse/death in induction was 11%/1.4% if treated with DEX, and 18%/0.5% if treated with PDN (p<0.001/0.01). If specific biological subgroups were analyzed, a significantly lower CI of relapse for patients randomized to receive DEX was observed in T-ALL as well as TEL/AML1 positive and negative pcB-ALL patients. The reduction was most pronounced in T-ALL patients with good prednisone response after the prophase: CI for relapse in DEX treated patients (n=135) was only 6% (SE 2%) as compared to 20% (SE 4%) in PDN treated patients (n=138; p=0.003). Four patients died in the DEX arm, 3 in the PDN arm for toxicity. In TEL/AML1-positive patients with good prednisone response, the CI for relapse was 4% (SE 1%) in the DEX group and 13% (SE 2%) for PDN treated patients (p<0.001). This large difference became most evident only more than 2 years from diagnosis. The percentage of patients found negative for MRD at d33 was very similar between the randomized groups: It was 47.9% for patients treated with DEX, and 45% for patients treated with PDN. Interestingly, patients with pcB-ALL found MRD negative at d33 had significantly less subsequent relapses if treated with DEX in induction (p(Gray)=0.012). In conclusion, the use of DEX at the same dosage as applied in delayed intensification (10 mg/m2/d for 3 weeks) although associated with a greater risk of severe toxicity leads to a marked reduction of the risk of relapse, translating this into a significant benefit in terms of EFS. This was most evident in patients with in vivo sensitivity to the prednisone prephase, while the efficacy of DEX in poor responding patients was not convincing. In the future, more intensive clinical monitoring and early anti-infective interventions could render the advantage of using DEX even more evident. Moreover, other potentially toxic agents such as anthracyclines may be limited in induction for carefully selected subgroups of patients with the aim of limiting early or late toxicities.

Relapse in Childhood “Very High Risk” Acute Lymphoblastic Leukemia Treated with BFM Chemotherapy Has a Dismal Outcome Even When Treated with Transplantation.

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.

Time Point-Dependent Concordance of Flow Cytometry and RQ-PCR in the MRD Detection in Childhood ALL: The Experience of the AIEOP-BFM- ALL MRD Study Group

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. [Display omitted] 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 <10−4, while only 13 samples were PCR-positive ≥10−4 but undetectable by FCM. In summary, several methodological issues limit the concordance of FCM and PCR as used by the AIEOP-BFM MRD study group. These include differences in the material analyzed by PCR and FCM, as well as in the amount of sample input (number of cells analyzed). The recent implementation of further technical developments in the FCM procedure, are designed to increase the concordance rate. In conclusion, within BFM-based protocols, FCM cannot simply substitute the current PCR-based MRD risk stratification at the same TPs. Instead, a tailored FCM-based risk definition may be independently reliable. Moreover, the two methodologies applied at different TPs might be complementary in more advanced MRD-based patient stratification protocols. Overall, the choice of the MRD methodology largely depends on the aims of the study protocol, resources available, and treatment strategy.

Risk-adapted stratification and treatment of childhood acute lymphoblastic leukaemia

Systematic enrollment of children and adolescents with acute lymphoblastic leukaemia (ALL) into clinical trials has allowed the establishment of prognostic parameters derived from initial diagnostic findings. More important, these trials have significantly contributed to the reduction of disease recurrence as much as to the reduction of acute and late side effects. Some problems that are related to the specificity of the parameters used for risk assessment were not overcome: high tumour load by white blood cell count (WBC), age and (rare) cytogenetic subtypes (e.g. t9;22) may characterise a significant proportion of children and adolescents with high-risk ALL. Most patients who will eventually relapse do not present with characteristic features at initial diagnosis. It appears feasible through careful response assessment to identify these patients at risk of relapse, who present initially without specific features. Earlier trials of the ALL-BFM (Berlin/Frankfurt/Münster) study group and others have demonstrated that inadequate leukaemic blast reduction in the peripheral blood or bone marrow after the first few days of therapy is highly predictive of treatment failure. Using clone-specific polymerase chain reaction-based detection of minimal residual disease (MRD) as done in trial AIEOP-BFM ALL 2000 allowed a close surveillance of specific treatment elements when applied in MRD positive patients. This may facilitate innovative chemotherapy approaches and a more rational use of allogeneic haematopoetic stem cell transplantation. In addition, genetic signatures of treatment response or failure have been identified.

Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia

Detection of minimal residual disease (MRD) is the most sensitive method to evaluate treatment response and one of the strongest predictors of outcome in childhood acute lymphoblastic leukemia (ALL). The 10-year update on the I-BFM-SG MRD study 91 demonstrates stable results (event-free survival), that is, standard risk group (MRD-SR) 93%, intermediate risk group (MRD-IR) 74%, and high risk group (MRD-HR) 16%. In multicenter trial AIEOP-BFM ALL 2000, patients were stratified by MRD detection using quantitative PCR after induction (TP1) and consolidation treatment (TP2). From 1 July 2000 to 31 October 2004, PCR target identification was performed in 3341 patients: 2365 (71%) patients had two or more sensitive targets (< or =10(-4)), 671 (20%) patients revealed only one sensitive target, 217 (6%) patients had targets with lower sensitivity, and 88 (3%) patients had no targets. MRD-based risk group assignment was feasible in 2594 (78%) patients: 40% were classified as MRD-SR (two sensitive targets, MRD negativity at both time points), 8% as MRD-HR (MRD > or =10(-3) at TP2), and 52% as MRD-IR. The remaining 823 patients were stratified according to clinical risk features: HR (n=108) and IR (n=715). In conclusion, MRD-PCR-based stratification using stringent criteria is feasible in almost 80% of patients in an international multicenter trial.

Impact of Minimal Residual Disease (MRD) on Prognosis in Children with Acute Lymphoblastic Leukemia (ALL) According to WBC Count, Age and TEL/AML1 Status at Diagnosis. Results of the AIEOP-BFM ALL 2000 Study.

Minimal residual disease (MRD), the most sensitive method to evaluate treatment response, has been adopted to stratify patients in study AIEOP-BFM ALL 2000. To assess whether PCR-MRD levels discriminate outcome in patients classified by WBC count, age at diagnosis, NCI criteria (Standard Risk, SR: WBC < 50,000/cmm and age 1–9 years; High Risk, HR: all others) and TEL/AML1 status. Between 07–2000 and 07–2006, 4,730 Ph-negative patients were enrolled in AIEOP-BFM ALL 2000 study. They were treated with BFM Induction (protocol IA) consolidation (protocol IB), extra-compartment/intensified consolidation (HD-MTX in non-HR patients, blocks in HR patients), reinduction therapy (one or more Protocols II or III), followed by maintainance. BM samples obtained at weeks 5 (Time Point 1, TP1) and 12 (TP2) of induction/consolidation therapy were used for PCR-based MRD analysis of patient specific gene targets. At least 2 sensitive markers (≥ 1 x 10−4) could be determined in 3,707 (78.4%) patients. SR was defined by MRD− at both TP1 and TP2; HR by MRD ≥1x10−3 at TP2; Intermediate Risk (IR): all others. Median follow-up was 3 years; 5-year percent EFS (SE) estimates are given.Patients at MRD-SR, IR or HR had, respectively, an EFS of 93.0 (1.0), 80.5 (1.5) and 43.4 (6.0) in patients with WBC <50,000/cmm vs 90.4 (2.6), 72.4 (3.0) and 47.0 (5.1) in patients with WBC ≥50,000/cmm. Patients at MRD SR, IR or HR had, respectively, EFS of 93.6 (1.0), 80.3 (1.5) and 44.1 (5.4) if aged 1–9 years vs 87.2 (3.4), 73.9 (3.0) and 49.3 (5.2) if aged ≥10 years. Patients at SR by NCI criteria [N= 2,355, EFS of 85.3 (1.0)] were stratified by PCR-MRD as SR (N=1046; 44.4%), IR (N=1198; 50.9%), or HR (N=111; 4.7%). EFS in these subgroups was 93.9 (1.0), 81.3 (1.6) and 43.9 (7.2), respectively (p<0.001). In patients at HR by NCI criteria [N=1,352, EFS of 75.6 (1.6)], 403 (29.8%), 774 (57.3%) and 175 (12.9%) respectively were at SR IR and HR by MRD. EFS was 89.4 (2.2) in MRD SR, 74.7 (2.3) in MRD IR and 47.9 (4.2) in MRD HR patients (p<0.001). Of 3,707 study patients, 3,410 were investigated for TEL/AML1 status: 771 (22.6%) were positive and 2,639 were negative. TEL/AML1+ patients were at SR (N=444; 57.6%) or IR (N=317; 41.1%) or HR (N=10; 1.3%) by PCR-MRD; EFS in this subgroup was 94.4% (1.5), 80% (3.7) and 60% (18.4), respectively (p<0.001). TEL/AML1− patients at SR (N=887; 33.6%) or IR (N=1497; 56.7%) or HR (N=255; 9.7%) had an EFS of 91.6% (1.3), 78.5% (1.4) and 45.7% (4.5), respectively (p<0.001). PCR-MRD in patients treated with BFM-oriented therapy overcomes the prognostic value of “historical” factors such as WBC count, age, NCI criteria or TEL/AML1 status, as it markedly discriminates prognosis within each subgroup defined by these variables. Study design for contemporary risk-directed therapy of childhood ALL should incorporate a technique for MRD determination.

Early Evaluation of Bone Marrow Minimal Residual Disease by Flowcytometry on Day 15 Is Feasible on a Multicenter Basis and Bears Strong Prognostic Value in Childhood Acute Lymphoblastic Leukemia. The AIEOP-BFM Experience.

Early measurement of blast clearance is a relevant prognostic indicator in childhood acute lymphoblastic leukemia (ALL). To this purpose we measured, by four-colour flowcytometry (FC), the percentage of blast cells in bone marrow samples from Italian patients enrolled in the multicentre AIEOP-BFM ALL 2000 trial. Samples were collected on day 15 (after 14 days of steroids, and one dose of IT-MTX, vincristine, daunorubicine, asparaginase) and shipped overnight to the reference laboratory. The data were compared to PCR-MRD performed, by study design, on day +33 and +78 BM samples. We report the results of patients enrolled between December 2000 and October 2004. The 561 patients studied were not different from the remaining ones (with no available material) including their cumulative incidence of relapse (SE): 17.3% (1.9) vs. 18.1% (1.5) in 850 patients not studied. According to the results of FC-MRD, 5 groups were defined: negative (blast count <0.01%, n=143), <0.1% (n=94), <1% (n=149), 1–10% (n=119), >10% (n=56). Their cumulative 5-year risk of relapse was: 4.1% (1.9), 9.3% (4.0), 14.3% (3.2), 26.5% (5.5), 53.7% (7.4), respectively. By PCR-MRD, the same patients were stratified as follows: 177 were standard risk and had 5-year risk of relapse of 4.1% (1.7), 233 at intermediate risk had a relapse risk of 24.2% (3.4), 37 at high risk had a relapse risk of 58.1% (9); the remaining 124 patients (21.6%) were not stratified by PCR-MRD due to lack of 2 sensitive (≥10−4) markers. Of 177 patients classified as standard risk by PCR (double negative), 110 fell within the 2 subgroups with lower FC-MRD (<0.1%), 46 had <1%, 19 had <10%, only 2 >10% of blasts. Of the 233 patients stratified as PCR-MRD intermediate risk (d78 <10−3), FC-MRD related groups had the following probabilities of EFS: 93.5% (3.6; n=47), 83.3%(8.0; n=30), 80.5%(5.1; n=70), 66.5%(10.8; n=57), 39.2%(11.8; n=29). We conclude that very early measurement of FC-MRD on day 15 bone marrow is feasible in our multicentre cooperative setting. On the basis of our data we suggest the following risk groups: standard, when <0.1% blasts on day 15 BM; intermediate for 0.1 to <10%; high, for >10% blasts. These groups had a risk of relapse of 6.2% (1.9), 19.5% (3), and 53.7% (7.4), respectively. Since it is fast, reproducible, relatively cheap and applicable to virtually all patients, our group decided to apply it prospectively on all ALL patients to integrate PCR-based stratification. Our findings showed that:i.early (d15) MRD detection by FCM identifies different patients than PCR on d33 and d78;ii.FCM may be very useful to identify earlier the highly sensitive ALL with low relapse risk (even though long-term follow-up is still missing), whereas later timepoints may be accessible for PCR and the identification of HR patients.

Drug-induced immunophenotypic modulation in childhood ALL: implications for minimal residual disease detection

Assessment of minimal residual disease (MRD) by flow cytometry is considered to be based on the reproducibility of the leukemic immunophenotype detected at diagnosis. However, we previously noticed modulation of surface antigen expression in acute lymphoblastic leukemia (ALL) during the early treatment. Hence, we investigated this in 30 children with B-cell precursor ALL consecutively enrolled in the AIEOP-BFM ALL 2000 protocol. Quantitative expression of seven antigens useful in MRD monitoring was studied at diagnosis and compared to that measured at different time points of remission induction therapy. Downmodulation in the expression of CD10 and CD34 occurred at follow-up. By contrast, upmodulation of CD19, CD20, CD45RA, and CD11a was observed, while the expression of CD58 remained stable. Despite this, we could unambiguously discriminate leukemic cells from normal residual B cells. This holds true when bone marrow (BM) samples from similarly treated T-ALL patients, but not from healthy donors, were used as reference. Our results indicate that immunophenotypic modulation occurs in ALL during the early phases of BFM-type protocols. However, the accuracy of MRD detection by flow cytometry seems not negatively affected if adequate analysis protocols are employed. Investigators should take this phenomenon into account in order to avoid pitfalls in flow cytometric MRD studies.