Eligibility for allogeneic transplantation in very high risk childhood acute lymphoblastic leukemia: the impact of the waiting time.

The advantage of allogeneic transplant from compatible related donors versus chemotherapy in children with very-high-risk acute lymphoblastic leukemia in first complete remission was previously demonstrated in an international prospective trial. This study quantified the impact of time elapsed in first remission in the same cohort. Of 357 pediatric patients with very-high-risk acute lymphoblastic leukemia, 259 received chemotherapy, 55 transplantation from compatible related and 43 from unrelated donors. The 5-year disease-free survival was 44.2% overall and 42.5% for chemotherapy only patients. The chemotherapy conditional 5-year disease-free survival increased to 44.4%, 47.6%, 51.7%, and 60.4% in patients who maintained their first remission for at least 3, 6, 9, and 12 months respectively. The overall outcome was superior to that obtained with chemotherapy-only at any time-point. The relative advantage of transplant from compatible related donors in very-high-risk childhood acute lymphoblastic leukemia was consistent for any time elapsed in first remission

Invasive fungal diseases impact on outcome of childhood ALL - an analysis of the international trial AIEOP-BFM ALL 2009

In children with acute lymphoblastic leukemia (ALL), risk groups for invasive fungal disease (IFD) with need for antifungal prophylaxis are not well characterized, and with the advent of new antifungal compounds, current data on outcome are scarce. Prospectively captured serious adverse event reports of children enrolled in the international, multi-center clinical trial AIEOP-BFM ALL2009 were screened for proven/probable IFD, defined according to the updated EORTC/MSG consensus definitions. In a total of 6136 children (median age 5.2 years), 224 proven/probable IFDs (65 yeast and 159 mold) were reported. By logistic regression, the risk for proven/probable IFDs was significantly increased in children ≥12 years and those with a blast count ≥10% in the bone marrow on day 15 (P < 0.0001 each). Proven/probable IFDs had a 6-week and 12-week mortality of 10.7% and 11.2%, respectively. In the multivariate analysis, the hazard ratio for event-free and overall survival was significantly increased for proven/probable IFD, age ≥12 years, and insufficient response to therapy (P < 0.001, each). Our data define older children with ALL and those with insufficient treatment-response at high risk for IFD. As we show that IFD is an independent risk factor for event-free and overall survival, these patients may benefit from targeted antifungal prophylaxis

Bone Marrow Stromal Cell Regeneration Profile in Treated B-Cell Precursor Acute Lymphoblastic Leukemia Patients:Association with MRD Status and Patient Outcome

SIMPLE SUMMARY: For the last 20 years, measurable residual disease (MRD) has proven to be a strong prognostic factor in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effects of therapy on the bone marrow (BM) microenvironment and their potential relationship with MRD and patient outcome still remain to be evaluated. Here, we show that mesenchymal stem cells (MSC) and endothelial cells (EC) are constantly present at relatively low frequencies in normal BM and in most follow-up BM samples from treated BCP-ALL patients. Of note, their levels are independent of the MRD status. From the prognostic point of view, an increased percentage of EC among stromal cells (EC plus MSC) at day +78 of therapy was associated with shorter disease free survival (DFS), independently of the MRD status both in childhood and in adult BCP-ALL. Thus, an abnormally high EC/MSC distribution at day +78 of therapy emerges as an adverse prognostic factor, independent of MRD in BCP-ALL. ABSTRACT: For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of therapy emerged as an independent unfavorable prognostic factor for DFS in childhood BCP-ALL in the discovery cohort—hazard ratio (95% confidence interval) of 2.50 (1–9.66); p = 0.05—together with the BM MRD status (p = 0.031). Further investigation of the predictive value of the combination of these two variables (%EC within stromal cells and MRD status at day +78) allowed classification of BCP-ALL into three risk groups with median DFS of: 3.9, 3.1 and 1.1 years, respectively (p = 0.001). These results were confirmed in two validation cohorts of childhood BCP-ALL (n = 74) (p = 0.001) and adult BCP-ALL (n = 40) (p = 0.004) treated at different centers. In summary, our findings suggest that an imbalanced EC/MSC ratio in BM at day +78 of therapy is associated with a shorter DFS of BCP-ALL patients, independently of their MRD status. Further prospective studies are needed to better understand the pathogenic mechanisms involved

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts

2009

Design and Methods Patients The study cohort was constituted by 410 non-Down syndrome, non-T, Philadelphia chromosome-negative, B-cell precursor ALL patients consecutively enrolled in the AIEOP-BFM ALL2000 study in AIEOP Centers from February 2003 to July 2005, who were included in the previous study on CRLF2 alterations and for whom DNA was still available. 19 P2RY8-CRLF2 rearrangement was tested by reverse transcriptase polymerase chain reaction analysis in 372 (90.7%) patients. The project was approved by the AIEOP ALL Scientific Committee. Risk group definitions and treatment outlines have already been reported DNA copy number variations IKZF1 deletions, together with deletions in other genes (CDKN2A/B, PAX5, ETV6, BTG1, RB1 and EBF1) were investigated by multiplex ligation-dependent probe amplification (MLPA) using the Salsa MLPA P335-A3 ALL-IKZF1 kit (MRC-Holland, Amsterdam, the Netherlands) according to the manufacturer&apos;s instructions. Samples from pediatric ALL patients in complete remission were used as wild-type controls. Statistical analysis Event-free survival time was calculated from the date of diagnosis to the date of an event, which was resistance, relapse, death or second neoplasm, whichever occurred first. Patients were censored at last follow-up if no events occurred. Event-free survival was estimated according to Kaplan-Meier, and compared using the log-rank test. The cumulative incidence of relapse at 5 years was estimated by adjusting for competing risks of other events and compared using Gray&apos;s test. Results IKZF1 deletions at diagnosis IKZF1 deletions were detected in 54/410 cases (13.2%), in keeping with incidence data reported in the literature. The clinical characteristics of the patients are presented in Using the MLPA technique we further analyzed the presence of copy number variations of other genes frequently deleted in B-cell precursor ALL and known to be involved in lymphoid development (PAX5, ETV6, EBF1) or in cell cycle regulation (CDKN2A/B, BTG1, RB1). 10,20-22 We did not find statistically significant differences in the incidence of these genetic alterations in children positive or negative for IKZF1 deletions (Online Prognostic impact of IKZF1 deletions Compared to patients without a deletion of IKZF1, those with a deletion of part or all of this gene had an inferior event-free survival [70.2% (6.2) versus 85.2% (1.9) at 5 years, P=0.007] and a significantly higher cumulative incidence of relapse [24.2% (5.9) versus 13.1% (1.8) at 5 years, P= 0.049] ( These data are in accordance with those from other studies reported in the literature, in particular with those recently published by Dorge et al. who analyzed ALL patients enrolled in the AIEOP-BFM ALL2000 study in Germany [event-free survival 69% (5) versus 85% (1), P=&lt;0.001 and cumulative incidence of relapse 21% (4) versus 10% © F e r r a t a S t o r t i F o u n d a t i o n N o c o m m e r c i a l u s e deleted patients. These events contributed to the statistical significance of the difference in the event-free survival. In both Cox model analyses, the P2RY8-CRLF2 aberration and risk group were significantly associated with outcome. Of note, when individually analyzed, the IKFZ1 deletion had a statistically significant effect on event-free survival and relapse, in keeping with results in We further analyzed the prognostic value of IKZF1 deletions within the subgroups according to protocol stratification. IKZF1 deletions were less frequent within the standard-risk group, being found in 8 out 117 standard-risk patients (6.8%), 42 out of 264 intermediate-risk patients (15.9%) and 4 out of 29 high-risk patients (13.8%) ( Discussion Previous studies Indeed, overall event-free survival for patients with IKZF1 deletions, after excluding the confounding effects of Down syndrome, T-immunophenotype and Philadelphia chromosome-positive patients, was around 70% at 5 years also in our experience. The three patients with IKZF1 deletions who were at high risk and relapsed had poor response to treatment (high minimal residual disease levels) and accordingly were all eligible for transplantation, thus identification of IKZF1 deletions would not have contributed to a better stratification. In the intermediate risk group, with a 5-year event-free survival of 70%, treatment intensification could be justified to improve results. In our context, the recent AIEOP-BFM ALL 2009 study, with a more intensive use of L-asparaginase, might already provide a benefit that reduces the impact of an IKZF1 deletion. This is especially true if we consider that in our study cohort the difference in the cumulative incidence of relapse was not so marked, being approximately 7% in the intermediate risk subgroup and 11% overall. This, as well as the lower number of events in the multivariate analysis, may explain why the presence of IKZF1 deletions is an independent prognostic factor for event-free survival but not for the hazard of relapse alone. In conclusion, based on our data, the suitability of IKZF1 deletions as an additional stratification marker for Philadelphia chromosome-negative, B-cell precursor ALL patients remains questionable, at least until new target therapy becomes available. (NoE-2011-261474). Acknowledgments The authors would like to thank Simona Songia, Lilia Corral, Eugenia Mella, Tiziana Villa (Monza), Elena Seganfreddo and Katia Polato (Padova) for AIEOP minimal residual disease monitoring and all medical doctors of the AIEOP centers. This work was supported by grants from: Fondazione Tettamanti (Monza), Fondazione Città della Speranza (Padova), Associazione Italiana Ricerca sul Cancro (AIRC) (to GB, AB, MGV, GteK and GC), MIUR (to GB and AB), Fondazione Cariplo (to AB, GC and GteK), and CARIPARO project of excellence (to GteK). This work was (partly) funded by the European Commission (FP7) under the contract ENCCA Authorship and Disclosure