ETV6::RUNX1-like ALL is defined by a gene expression signature similar to that of ETV6::RUNX1-positive ALL and absence of all genetic subtype-defining aberrations, including the ETV6::RUNX1 fusion. Within the International BFM Study Group, we assembled and analyzed a cohort of 100 patients (including 97 children) with ETV6::RUNX1-like ALL. We describe their diverse genetic landscape, centered around ETV6 aberrations with frequent IKZF1 disruptions, as previously shown, but including various rare non-ETV6/non-IKZF1 gene fusions, and rearrangements of CRLF2 (CRLF2r). We show that ETV6 and IKZF1 aberrations do not occur exclusively in this subtype, which hampers its classification based solely on genomic data. We confirm our previous observation of a strong association of the CD27-positive/CD44low-negative immunophenotype with ETV6::RUNX1(-like) subtype. Compared to ETV6::RUNX1-positive ALL, patients with ETV6::RUNX1-like ALL are younger, have higher white blood cell counts at diagnosis, and have an inferior early treatment response. While overall survival is comparable, event-free survival is significantly lower in patients with ETV6::RUNX1-like ALL, with NCI risk, early treatment response, IKZF1 deletions, CRLF2r, and JAK2 mutations having prognostic relevance. Notably, Down syndrome is highly prevalent and associated with a worse outcome in ETV6::RUNX1-like ALL. In conclusion, we provide biological, demographic, and clinical characteristics of the largest ETV6::RUNX1-like cohort presented to date.

INTRODUCTION:We aimed to describe T lymphocyte reconstitution during the first 100 days after allogeneic hematopoietic stem cell transplantation (HSCT) to identify differences associated with graft-versus-host disease (GvHD) manifestation. PROCEDURE:Eighty-three children who received transplants from unrelated donors were included in this single-institution study. Peripheral blood stem cells (PBSC) grafts and anti-T lymphocyte immunoglobulin (ATLG) were used. RESULTS:We observed greater proportions of CD8+ stem cell memory (SCMs) lymphocytes on day 28 in acute GvHD (aGvHD) patients and of CD4+ SCMs (CD3+TCRαβ+CD4+CD45RO-or CD45RA+CCR7+or CD62L+CD95+) on days 60 and 100 in chronic GvHD (cGvHD) patients. A greater proportion (≥5% CD4+ cells) of CD4+ SCMs on day 100 in patients with aGvHD was associated with a significantly greater cGvHD cumulative incidence (CI) (two-year value = 80.0% ± 0.9% vs. 27.1% ± 0.6%). Furthermore, a greater risk of non-relapse mortality (NRM) (two-year value = 16.1% ± 0.6% vs. 0%) and a lower risk of relapse (CI of relapse) (two-year value = 0% vs. 18.4% ± 0.3%) were observed in the group with a higher CD4+ SCM proportion on day 100, together with a lower probability of GvHD-free, immunosuppressive treatment-free, relapse-free two-year survival (GIRFS) (two-year value = 55.0% ± 11.1% vs.78.4% ± 7.3%). CONCLUSION:CD4+ SCM frequency is a potential biomarker of allogeneic immune response intensity and cGvHD risk in the early posttransplantation period.

BACKGROUND:Auer rods (AuRs) are prominent intracellular structures found almost exclusively in myeloid cell malignancies, such as acute myeloid leukemia (AML), chronic and juvenile myelomonocytic leukemia and myelodysplastic syndrome. Extremely rare AuRs have been reported in patients with acute lymphoblastic leukemia (ALL) or among ambiguous lineage leukemia patients with a dominantly lymphoblastic immunophenotype. PROCEDURE:We report diagnostic and follow-up data of an international cohort of 11 children suffering from leukemias with AuRs and with significant presence of T and myeloid markers, majority of whom categorized as early T-cell precursor (ETP, n = 7); or T-ALL (ETP status unknown, n = 2), ALAL (acute leukemia of ambiguous lineage, n = 1), and AML reclassified from ALAL (n = 1). We described other diagnostic details and treatment types and responses. Moreover, we summarize previously published data. RESULTS:Among the four patients who started and remained on ALL-type therapy, all were in the first complete remission, whereas both patients who started and remained on AML-type therapy relapsed and died. Of the patients who followed either a combined ALL/AML protocol (Interfant 06) or who switched from one of the two types of therapy to the other, one patient died, and the remaining four were in first complete remission at the most recent follow-up. We also searched for similar cases in the literature and found only three additional children with nonmyeloid leukemia and AuRs and 10 adults with this type of leukemia. CONCLUSIONS:Briefly, ALL- or combined ALL/AML-type therapy may be effective for treating AuR-positive leukemia patients with a lymphoid immunophenotype.

Measurable residual disease (MRD) monitoring in childhood acute myeloid leukemia (AML) is used to assess response to treatment and for early detection of imminent relapse. In childhood AML, MRD is typically evaluated using flow cytometry, or by quantitative detection of leukemia-specific aberrations at the mRNA level. Both methods, however, have significant limitations. Recently, we demonstrated the feasibility of MRD monitoring in selected subgroups of AML at the genomic DNA (gDNA) level. To evaluate the potential of gDNA-based MRD monitoring across all AML subtypes, we conducted a comprehensive analysis involving 133 consecutively diagnosed children. Integrating next-generation sequencing into the diagnostic process, we identified (presumed) primary genetic aberrations suitable as MRD targets in 97% of patients. We developed patient-specific quantification assays and monitored MRD in 122 children. The gDNA-based MRD monitoring via quantification of primary aberrations with a sensitivity of at least 10 −4 was possible in 86% of patients; via quantification with sensitivity of 5 × 10 −4 , of secondary aberrations, or at the mRNA level in an additional 8%. Importantly, gDNA-based MRD exhibited independent prognostic value at early time-points in patients stratified to intermediate-/high-risk treatment arms. Our study demonstrates the broad applicability, feasibility, and clinical significance of gDNA-based MRD monitoring in childhood AML.

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) represents a rare and clinically and genetically heterogeneous disease that constitutes 10–15% of newly diagnosed pediatric ALL cases. Despite improved outcomes of these children, the survival rate after relapse is extremely poor. Moreover, the survivors must also endure the acute and long-term effects of intensive therapy. Although recent studies have identified a number of recurrent genomic aberrations in pediatric T-ALL, none of the changes is known to have prognostic significance. The aim of our study was to analyze the cytogenomic changes and their various combinations in bone marrow cells of children with T-ALL and to correlate our findings with the clinical features of the subjects and their treatment responses. Results We performed a retrospective and prospective comprehensive cytogenomic analysis of consecutive cohort of 66 children (46 boys and 20 girls) with T-ALL treated according to BFM-based protocols and centrally investigated cytogenetics and immunophenotypes. Using combinations of cytogenomic methods (conventional cytogenetics, FISH, mFISH/mBAND, arrayCGH/SNP and MLPA), we identified chromosomal aberrations in vast majority of patients (91%). The most frequent findings involved the deletion of CDKN2A/CDKN2B genes (71%), T-cell receptor (TCR) loci translocations (27%), and TLX3 gene rearrangements (23%). All chromosomal changes occurred in various combinations and were rarely found as a single abnormality. Children with aberrations of TCR loci had a significantly better event free (p = 0.0034) and overall survival (p = 0.0074), all these patients are living in the first complete remission. None of the abnormalities was an independent predictor of an increased risk of relapse. Conclusions We identified a subgroup of patients with TCR aberrations (both TRA/TRD and TRB), who had an excellent prognosis in our cohort with 5-year EFS and OS of 100%, regardless of the presence of other abnormality or the translocation partner. Our data suggest that escalation of treatment intensity, which may be considered in subsets of T-ALL is not needed for nonHR (non-high risk) patients with TCR aberrations.

Monitoring of minimal residual disease (MRD) in childhood acute myeloid leukemia (AML) can assess response to treatment and predict relapse. Current MRD monitoring methods in childhood AML - flow cytometry and quantitation of fusion transcripts - have significant shortcomings and do not cover the entire spectrum of patients. We demonstrated (Lukes et al, Hemasphere 2020) the feasibility of MRD monitoring using DNA-based primary targets in selected subgroups of AML. Now we extended this approach to the unselected consecutive population of pediatric AML. We included 133 children (0-18) out of 135 consecutively diagnosed with AML in Czechia (2012-2022), all treated on non-MRD-based protocols. For genetic characterization, we used algorithm consisting of screening for recurrent gene fusions and mutations by PCR and targeted-NGS followed by transcriptome sequencing (WTS). Targeted-NGS (or PCR) was performed to identify genomic fusion sequences. 130 patients were eligible for MRD monitoring (3 patients died early). MRD was preferentially monitored by DNA-based qPCR (alternatively by amplicon NGS or qRT-PCR). MRD levels were expressed relative to diagnosis. Primary genetic aberration was found by targeted screening in 102 children; remaining 31 were further investigated using WTS. In 27 children, WTS identified rare, novel or atypical primary genetic aberrations not covered by the targeted screening. Primary genetic aberration was found in 97% of cases (129/133). Majority of AMLs (81%) were classified into common subtypes: AML with KMT2Ar, PML::RARA, RUNX1::RUNX1T1, CBFB::MYH11, mutation (m) of GATA1 , CEBPA or NPM1. Recurrent but rare aberrations in pediatric AML were found in 16 patients: UBTFm, RUNX1m, HOXA10 translocation, KAT6A::CREBBP, KAT6A::LEUTX, DEK::NUP214, BCR::ABL1, NUP98::NSD1 and CBFA2T3::GLIS2. In 5 patients, fusion genes were found, previously described sporadically (SPFQ::ZFP36L216, XPO1::TNRC1817) or not at all in AML (ETV6::CTNNB1, FUS::FEV, ZEB2::RUNX1). Only secondary aberrations were identified in remaining 4 patients. In 82 patients (of 84 examined), genomic fusion sequence was found and qPCR designed and used. Of 41 children carrying genetic aberrations other than fusions, in 29 quantification system was successfully implemented. Altogether, quantification system for MRD monitoring was established in 122 patients, with primary aberrations as DNA targets in 116 patients, reaching sensitivity of 10 -4 in 112 and 5x10 -4 in 4 patients. Four and two children were monitored with sensitivity of 10 -4 using secondary aberrations as DNA targets or by qRT-PCR-based quantification of fusion transcripts, respectively. MRD clearance significantly differed among genetic subtypes: while GATA1m AML had the fastest response, in contrast to patients with prognostically favorable genetic subtypes - CBFB::MYH11, RUNX1::RUNX1T1, CEBPAm and NPM1m - who were treated predominantly on the standard risk (SR) arm of the AML-BFM 2012 Registry Protocol and none achieved molecular remission (mREM) at D28 (70% vs 0%, p < 0.0001). Significant proportion of patients (25-89% within individual subtypes) did not achieve mREM even after the last block of chemotherapy. The response of patients with KMT2Ar AML and AML classified into remaining subtypes (predominantly intermediate- and high-risk) was overall faster compared to prognostically favorable subtypes on SR arm (KMT2Ar D28 mREM 33% vs 0% at D28, p < 0.0001). KMT2A::MLLT10/3 had faster clearance then other KMT2Ar (mREM D28 44% vs. 0% in other KMT2Ar, p = 0.02). In a multivariate analysis including (cyto)genetic risk and treatment, D28 MRD was the only significant predictor of outcome using both the 10 -3 (p = 0.006 for EFS and 0.012 for OS) and 10 -2 levels (p = 0.004 for EFS and 0.01 for OS) for stratification. Similarly, in an alternative multivariate analysis model, D56 MRD (positive at any level versus negative) was also the only significant predictor of outcome independent of risk and treatment (p = 0.005 for EFS and 0.01 for OS) In summary, we present a strategy for MRD monitoring in pediatric AML that is technologically feasible, real-life applicable to vast majority of all patients, and has clear prognostic significance. Supported by National Institute of Cancer Research No. LX22NPO5102 funded by the European Union-Next Generation EU, and AZV grant NU20-07-00322.

Background: Central nervous system (CNS) involvement at diagnosis of acute lymphoblastic leukemia (ALL) represents a risk factor for CNS relapse. Currently, examination of the cerebrospinal fluid (CSF) is performed and evaluated quantitatively using a counting chamber and qualitatively using cytomorphological analysis of cytospin. Recently, flow cytometry (FC) evaluation of CSF was shown to provide more reliable results and to predict relapse risk (de Haas et al., 2021, Thastrup et al., 2020). Independently of these studies, we applied a lyse-no wash technique aiming at minimizing cell loss during preparation and at improved cell concentration estimate. Aims: Our aim was to compare our findings with morphological criteria and published data and correlate them with clinical outcome in pediatric patients with ALL. Methods: We included pediatric patients with newly diagnosed ALL (04/2014 – 01/2022), whose CSF examination was performed in CLIP laboratory (n=208). Patients were classified as B cell precursor ALL (BCP ALL; 85%) and T ALL (15%) and treated according to following protocols: AIEOP-BFM ALL 2009 (n=118), AIEOP-BFM ALL 2017 (n=71), EsPhALL(n=8), Interfant06 (n=5), off-protocol (n=6). During follow up, 14 patients relapsed (5 CNS incl. combined, 9 non-CNS). CSF (drawn into tube with no fixative agent and processed within 2 hours) was concentrated by centrifugation and incubated with combination of antibodies (CD20/CD10/CD45/CD34/CD19/CD3/Syto41 in BCP ALL and CD4/CD99/CD5/CD3/CD7/CD16 + 56/CD8/CD45/Syto41 in T ALL). Residual erythrocytes were lysed with NH4Cl solution, followed by immediate acquisition on BD FACS Lyric or BD LSRII cytometers. Blasts were quantified to initial CSF volume. Cluster of 20 events was required to assign samples as FC positive (FC+). In parallel, CNS status was concluded according to morphological criteria, being classified as CNS1 (no blasts), CNS2a/b (≤5 WBC/μL with blasts), CNS2c/CNS3 (≥5 WBC/μL with blasts) according to treatment protocol guidelines. Continuous variables were compared with the Mann–Whitney test, survival data were analyzed using Mantel-Cox test. P values<0.05 were considered as significant. Results: Using FC, we identified atypical blasts in 50 out of the 208 analyzed samples (24%). Median of analyzed CSF volume was 367µL (range 143-1033µL), which enabled median sensitivity 0.054 events/µL (range 0.02-0.14ev/µL). Mean blast concentration was 0.47ev/µL (range 0-25ev/µL) in all samples and 2 ev/µL (range 0.01-25ev/µL, median 0.53ev/µL) in FC+ samples. We observed significantly higher blast concentration in patients with T ALL, in patients with CNS2 and CNS3 and those who subsequently relapsed. Patients with FC+ had significantly lower relapse-free survival than FC- patients (55% vs 94%), which provided better separation than CNS status (68% vs.85% for CNS2/3 vs. CNS1, respectively). Image:Summary/Conclusion: Using fresh CSF sample with lyse-no wash protocol enabled us to detect approximately 10x higher blast count (0.24ev/µL in FC+CNS1 and 1.25ev/µL in FC+CNS2/3) than was recently published (0.015ev/µL in FC+CNS1 and 0.073ev/µL in FC+CNS2/3 in Thastrup et al., 2020), and thus it better correlates to cytomorphological data. Interestingly, the proportion of FC+ patients in our cohort is comparable to (24% vs 25%), showing that although in our method we lose fewer cells, we identify the same proportion of FC+ patients. The presented study shows a higher impact of FC positivity on relapse risk than described previously. The disadvantage of our method is a need for a fresh sample, which is investigated locally. Supported by UNCE/MED/015, NU20J-07-00028.

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a clinically and genetically heterogeneous disease that constitutes 10%–15% of newly diagnosed pediatric ALL cases and is caused by the accumulation of genetic abnormalities that alter the mechanisms controlling normal T-cell development. The most common structural aberrations involve rearrangements of the T-cell receptor (TCR) loci – TRA/TRD (14q11) and TRB (7q34), which are found in ~25% of patients. These aberrations are the main factors initiating the events in T-ALL carcinogenesis and juxtapose TCR genes and protooncogenes that encode pivotal transcription factors, leading to their aberrant expression. The outcomes of children with T-ALL significantly improved on intensified protocols, however, ~20% of patients relapse and die owing to acquired therapy resistance. Moreover, survivors also endure the acute and long-term effects of intensive toxic chemotherapy. Aims: To analyze TCR loci rearrangements and associated genetic abnormalities in children with T-ALL treated according to BFM-based protocols, and to correlate our findings with the clinical features and the treatment responses. Methods: The bone marrow samples of 66 children diagnosed between 1996-2017 (46 boys, 20 girls, age median 7.9 years, follow-up median 86.4 months) were analyzed with cytogenomic methods. For the detection of TCR loci rearrangements and other recurrent aberrations of the genes TLX3, CDKN2A/CDKN2B and ABL1 interphase FISH (DAKO, Abbott Molecular) was used. MLPA analysis with ALL-IKZF1 probemix (MRC-Holland) was performed to detect deletions/amplifications of additional genes. Complex karyotypes were analyzed with multicolor FISH (MetaSystems) or array CGH/SNP (Agilent). Differences in OS and EFS were assessed using Kaplan-Meier method and the Mantel-Cox test. Results: Rearrangements of TCR loci were detected in 18/66 (27%) patients. Translocations involving the TRA/TRD locus were demonstrated in nine children, the TRB locus in five and the simultaneous occurrence of these aberrations in two cases, respectively. In 10/18 patients, known recurrent chromosomal translocations affecting the oncogenes TAL1 (3x), LMO2 (3x), TLX1 (2x), TAL2 (1x) and MYC (1x) were identified. In the remaining cases, TCR rearrangements were cryptic or were a part of complex karyotype. Additional chromosomal aberrations were detected in all but one patient with the deletion of CDKN2A gene being the most frequent one. Immunophenotypic data classified the patients into cortical-T (12x) and mature-T (5x) groups (1x no data). Children with aberrations of TCR loci had a significantly better prognosis - EFS (p=0.011) and OS (p=0.0074), all patients are living in the first complete remission. Summary/Conclusion: Aberrations of T-cell receptors form a genetically heterogeneous group of rearrangements, leading to aberrant expression of oncogenes involved in T-cell maturation and proliferation. They mostly occur with abnormalities of genes involved in the regulation of the cell cycle and/or signaling pathways, confirming the multistep process of T-ALL pathogenesis. In our cohort, patients with TCR rearrangements had an excellent prognosis regardless of the presence of other aberrations. Although data on larger series are clearly required, we suppose, that these children could benefit from less-intensive therapy because they may experience fewer therapeutic consequences of toxic treatment. Supported by MH CZ-DRO-VFN64165.

Successful management of relapse is critical to improve outcomes of children with acute myeloid leukemia (AML). We evaluated response, survival and prognostic factors after a second relapse of AML. Among 1222 pediatric patients of the population-based AML-Berlin–Frankfurt–Munster (BFM) study group (2004 until 2017), 73 patients met the quality parameters for inclusion in this study. Central review of source documentation warranted the accuracy of reported data. Treatment approaches included palliation in 17 patients (23%), intensive therapy with curative intent (n = 46, 63%) and other regimens (n = 10). Twenty-five patients (35%) received hematopoietic stem cell transplantation (HSCT), 21 of whom (88%) had a prior HSCT. Survival was poor, with a five-year probability of overall survival (pOS) of 15 ± 4% and 31 ± 9% following HSCT (n = 25). Early second relapse (within one year after first relapse) was associated with dismal outcome (pOS 2 ± 2%, n = 44 vs. 33 ± 9%, n = 29; p < 0.0001). A third complete remission (CR) is required for survival: 31% (n = 14) of patients with intensive treatment achieved a third CR with a pOS of 36 ± 13%, while 28 patients (62%) were non-responders (pOS 7 ± 5%). In conclusion, survival is poor but possible, particularly after a late second relapse and an intensive chemotherapy followed by HSCT. This analysis provides a baseline for future treatment planning.

Post-relapse therapy remains critical for survival in children with acute myeloid leukemia (AML). We evaluated survival, response and prognostic variables following relapse in independent cooperative group studies conducted by COG and the population-based AML-BFM study group. BFM included 197 patients who relapsed after closure of the last I-BFM relapse trial until 2017, while COG included 852 patients who relapsed on the last Phase 3 trials (AAML0531, AAML1031). Overall survival at 5 years (OS) was 42 ± 4% (BFM) and 35 ± 2% (COG). Initial high-risk features (BFM 32 ± 6%, COG 26 ± 4%) and short time to relapse (BFM 29 ± 4%, COG 25 ± 2%) predicted diminished survival. In the BFM dataset, there was no difference in OS for patients who had a complete remission with full hematopoietic recovery (CR) following post-relapse re-induction compared to those with partial neutrophil and platelet recovery (CRp and CRi) only (52 ± 7% vs. 63 ± 10%, p = 0.39). Among 90 patients alive at last follow-up, 87 had received a post-relapse hematopoietic stem cell transplant (HSCT). OS for patients with post-relapse HSCT was 54 ± 4%. In conclusion, initial high-risk features and early relapse remain prognostic. Response assessment with full hematopoietic recovery following initial relapse therapy does not predict survival. These data indicate the need for post-relapse risk stratification in future studies of relapse therapies.

BACKGROUND: Children with high risk acute myeloid leukemia (AML) still experience consistently high rates of relapse. Survival after first relapse increased from 21% between 1987 and 1997 up to 39% in recent studies. However, since 2009, there have been no publications on subsequent large pediatric AML relapse trials. As the indications for HSCT during first-line treatment have been extended since then, the current survival of these patients at relapse remains unclear. Herein, we report outcome results from the BFM and COG study group, which represents the largest available dataset analyzed for post-relapse survival. PATIENTS AND METHODS: Pediatric patients with first relapse of AML (no Down syndrome, secondary leukemia or FAB M3) have been analyzed from two large study groups with patients from the United States, Canada, Australia, New Zealand, Germany, Austria, Czech Republic and Switzerland. Out of 1222 patients in the BFM cohort (AML-BFM study 2004, registry 2012 and study 2012), 350 experienced at least one relapse and 197 of those had a first relapse after closure of the last I-BFM relapse trial (04/2009 through 2017). Within the Children's Oncology Group (COG) Phase 3 trials (AAML0531 and AAML1031, n=2119) 852 pediatric patients suffered a relapse. Five-year probability of overall survival (pOS) and event-free survival (pEFS) were calculated according to Kaplan-Meier. EFS was calculated for the BFM cohort as time from relapse to the next event (second relapse, death, failure to achieve a second remission or secondary malignancy) or until last follow-up, while OS reflects the time from relapse until death or last follow-up. The Cox proportional hazards model was used for multivariate analysis of outcomes. Living patients were censored at last follow-up with a median follow-up after relapse of 4·2 years (BFM) and 4·8 years (COG). Data have been frozen at 03/27/2020 (BFM) and 03/31/2020 (COG). RESULTS: In the 197 patients with relapse after closure of the last BFM relapse trial (04/2009 through 2017) the pOS at 5 years was 42±4% (BFM). The 5-year pOS in patients relapsing after COG trials 2006-2018 was 35±2% (n=852). Patients experiencing a relapse between 2014 to 2017 had a pOS of 49±6% (BFM, n=78) and 40±3% (COG, n=333). Risk classification at initial diagnosis and a short time from diagnosis to relapse predicted a diminished survival probability in both cohorts (see Table 1). However, the absence of full hematopoietic regeneration of the bone marrow after re-induction did not predict survival: Within the BFM dataset, a subgroup analysis in all patients receiving DNX-FLA (n=156) have been performed. Initial characteristics are comparable to the total cohort. Among these patients 147 were evaluable for response (7 excluded due to early death before evaluation, 2 for insufficient data). Eighty-nine (57%) achieved a CR (n=69) or CRp (n=20) and 52 (33%) no response. Overall survival was superior for patients with a CR/CRp (54±6% (CR/CRp) vs. 32±7% (No CR/CRp); p=0·0064), but long-term survival was still possible even with a poor re-induction response. Patients with a CRp had a comparable survival to those with a CR after a second re-induction (pOS 60±11% (CRp) vs. pOS 52±7% (CR); p=0·57). Patients with >5% leukemic blasts (n=32) had the lowest survival (pOS 27±9%). The 5-year pEFS for this cohort was 29±4% (pEFS 50±6% (CR) vs. pEFS 50±11% (CRp)). The analysis of post-relapse treatment showed that the vast majority of patients who survive had a HSCT following relapse. By landmark analysis, survival was significantly higher in patients with subsequent HSCT compared to that of non-transplanted patients (BFM: pOS 53±4%, n=154 vs. pOS 5±5%, n=21; p(Mantel-Byar)=0·0002). CONCLUSION: This is the largest report to date on post relapse survival in children with AML. Our analysis confirmed previously described risk factors for poor survival while also highlighting new findings contrary to established standards. Strikingly, the absence of full hematopoietic regeneration of the bone marrow after re-induction did not predict survival at first relapse, thereby questioning the current value of the established International Working Group Criteria published by Cheson et al for response-evaluation in pediatric AML. As the international pediatric AML community embarks on collaborative efforts to evaluate new therapies in children with relapsed AML, a comprehensive review of post relapse survival is critical. Disclosures Bourquin: Servier: Other: Travel Support. Reinhardt:Novartis: Membership on an entity's Board of Directors or advisory committees; CLS Behring: Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Early treatment response is a widely accepted herald of curability of acute lymphoblastic leukemia (ALL) in children [1]. While complete remission is expected to be achieved by the end of induction...

In order to identify novel prognostic markers and actionable targets, we performed whole-exome/-transcriptome sequencing of relapsed childhood B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) diagnosed in the Czech Republic. In patients with “B-other ALL” (BCP-ALL negative for ETV6-RUNX1, BCR-ABL1, TCF3-PBX1, and KMT2A rearrangement, hyperdiploidy [51-67 chromosomes] and hypodiploidy [<44 chromosomes]), we found recurrent mutations of codons H1038 and Q1072 of the zinc finger E-box binding homeobox 2 (ZEB2) gene. The ZEB2 gene is located on 2q22.3 and encodes a transcriptional repressor implicated in the pathogenesis of early T-cell ALL, possibly via deregulation of IL7R-JAK/STAT signaling. It is known that codons H1038 and Q1072 are located in the C-terminal zinc finger coding-region (DNA binding region), but the impact of the mutations (ZEB2mut) on ZEB function is unknown. Although recurrence of ZEB2mut in BCP-ALL is being traced in a growing number of genomic studies, 6 the biological and clinical roles of these mutations in BCP-ALL have not been described so far. In this study, we aimed to determine the frequency and clinical relevance of ZEB2mut in childhood B-other ALL. Using whole-exome sequencing, RNA-sequencing and amplicon sequencing of DNA or complementary DNA (cDNA), we analyzed 231 and 36 Czech children with newly manifesting and relapsed B-other ALL, respectively (the so-called “discovery cohorts”), consecutively diagnosed between November 2002 and December 2017 and treated according to several successive Berlin-FrankfurtMünster (BFM)-based protocols (see Online Supplementary Data). We detected ZEB2mut in 3.8% (9/231) of newly diagnosed B-other ALL. ZEB2mut was associated with significantly worse event-free survival and a higher relapse rate (Figure 1A and B). In accordance with the higher relapse rate, we found significant enrichment of ZEB2mut in the discovery relapse cohort (8/36, 29%; P=0.0005). Interestingly, two out of the total nine relapses were isolated extramedullary relapses and one was a combined extramedullary and bone marrow relapse. To validate these findings, we analyzed 626 and 102 children with newly diagnosed and relapsed B-other ALL diagnosed and treated in Germany (the so-called “validation cohorts”). The frequency of ZEB2mut in the validation cohort of newly manifesting leukemias (enrolled into the Associazione Italiana di Ematologia e Oncologia Pediatrica [AIEOP]-BFM ALL 2000 and 2009 protocols) was 2.7% (17/626). While ZEB2mut was associated with a significantly higher relapse frequency in the patients enrolled into the AIEOP-BFM ALL 2000 study (Figure 1C and D), only 1/12 ZEB2mut patients enrolled in the AIEOP-BFM ALL 2009 study relapsed and two patients died without relapse. In the validation relapse cohort, ZEB2mut frequency was 4.9% (5/102), which was signif-

Background:T‐cell acute lymphoblastic leukemia (T‐ALL) is a clinically and genetically heterogeneous disease that comprises 10‐15% of newly diagnosed pediatric ALL. Despite improved survival rates of children with T‐ALL, relapse occurs in almost 20% of patients and is associated with a dismal subsequent outcome. Therefore, it is of vital importance to identify children already early on during treatment who are at increased risk of such an event. Although several clinical and genetic factors were identified, prognostic implication of majority of them remains unclear. Therefore, the patients risk stratification is currently based only on minimal residual disease (MRD) monitoring.Aims:We performed retrospective and/or prospective comprehensive molecular cytogenomic analysis of bone marrow cells of children with T‐ALL with the aim to determine recurrent chromosomal aberrations and to identify those with significant impact on event free (EFS) and overall survival (OS).Methods:Samples of all patients were analyzed at the time of diagnosis with combination of conventional and molecular cytogenomic methods. For detection of the most frequent known chromosomal changes, i.e. rearrangements of TCR loci and TLX3 gene, deletion of CDKN2A and amplification of ABL1, interphase FISH with locus‐specific probes (Dako, Abbott Molecular) was used. MLPA analysis using SALSA MLPA ALL‐IKZF1 probemix (MRC‐Holland) was performed to detect deletions/amplifications of additional genes. Complex chromosomal rearrangements were proved with multicolor FISH and multicolor banding (24XCyte/XCyte Probe Kit; MetaSystems) or array CGH/SNP (SurePrint G3 Cancer CGH + SNP 4x180K, Agilent). For OS and EFS Kaplan‐Meier analysis with Mantel Cox test was done.Results:During the years 1996‐2017 we examined archived material of 68 children with T‐ALL (21 girls and 47 boys, median age 8.25 years), treated according to ALL Interfant and/or BFM based protocols. Median follow up was 9.5 years. In total, chromosomal aberrations were detected in 91% of patients. The most frequent were deletion of CDKN2A gene in 47/68 patients (27x homozygous, 20x heterozygous), rearrangements of TCR loci in 18/68 children (11x TRA/TRD, 7x TRB) and TLX3 gene rearrangement in 15/68 cases. Complex chromosomal aberrations were proved in 14/68 children. All chromosomal changes occurred in various combinations, however TLX3 aberrations never appeared along with TCR loci rearrangements. 48 patients are living in the first (45x)/second (3x) complete remission, 16 patients relapsed, 19 children died. Best outcome (EFS and OS) was associated with TRA/TRD locus translocations (p < 0.05). Conversely, patients with TLX3 rearrangement had significantly worse OS (p < 0.05).Summary/Conclusion:Considering the poor survival after relapse, it is of utmost importance to identify children at an increased risk of this event. MRD monitoring at different time points is a strong prognostic indicator and is used for risk stratification. In addition, rearrangements of TLX3 gene correlated with poor outcome in contrast to TRA/TRD translocations, which correlated with a more favorable course of the disease.Supported by grant RVO‐VFN64165, GACR P302/12/G157.

Východiska: Cilem studie bylo zhodnotit trendy incidence a mortality zhoubných nadorů u děti a adolescentů v obdobi 1994–2016 v Ceske republice. Material a metody: Udaje o novotvarech děti evidovane v Narodnim onkologickem registru byly validovany pomoci klinicke databaze dětske onkologie a dale kombinovany s udaji Narodniho registru hospitalizovaných a Listu o prohlidce zemřeleho. Z těchto validovaných dat byly zjistěny hodnoty incidence novotvarů. Ke sledovani dlouhodobeho trendu mortality byla použita data z Listu o prohlidce zemřeleho. Trendy incidence a mortality byly hodnoceny průměrnou rocni procentualni změnou. Výsledky: Trend věkově standardizovane incidence zhoubných nadorů u děti ve věku 0–19 let vykazuje statisticky významný dlouhodobý mirný narůst nových připadů průměrně o +0,5 % rocně (p < 0,01), u divek o +0,6 %, zatimco u chlapců statisticky nevýznamný pokles o −0,1 %. Nejvýraznějsi statisticky významný průměrný rocni narůst incidence u děti ve věku 0–14 let byl pozorovan u jiných malignich epitelialnich novotvarů a malignich melanomů (+4,9 %; p < 0,01), ale take u onemocněni CNS (+1,3 %; p < 0,05). Naopak statisticky významný průměrný rocni pokles incidence u děti ve věku 0–14 let vykazuji lymfomy (−2,1 %; p < 0,01). U adolescentů ve věku 15–19 let byl pozorovan statisticky významný průměrný rocni narůst incidence u jiných malignich epitelialnich novotvarů a malignich melanomů (+5,2 %; p < 0,01), ale take u onemocněni CNS (+1,5 %; p < 0,05). Trend mortality zhoubných nadorů vykazuje statisticky významný dlouhodobý pokles průměrně o −5,1 % rocně u děti ve věku 0–14 let (p < 0,01), a o −3,7 % rocně u adolescentů (p < 0,01). Zavěr: Dostupna data umožňuji analyzovat dlouhodobe trendy incidence a mortality zhoubných nadorů u děti a adolescentů.

BACKGROUND Our study aimed to evaluate incidence and mortality trends for childhood and adolescent cancers in the period 1994-2016 in the Czech Republic. MATERIAL AND METHODS Data on childhood cancers, which are recorded in the Czech National Cancer Registry, were validated using a clinical database of childhood cancer patients and combined with data from the National Register of Hospitalised Patients and with data from death certificates. These validated data were used to establish cancer incidence. Data from death certificates were used to evaluate long-term trends in mortality. Incidence and mortality trends were assessed by the average annual percentage change. RESULTS The age-standardised incidence trend for childhood cancers (i.e. those diagnosed in patients aged 0-19 years) showed a statistically significant slight long-term increase in the number of new cases, +0.5% annually on average (p < 0.01), more specifically an increase of +0.6% in girls and a statistically insignificant decrease of 0.1% in boys. In children aged 0-14 years, other malignant epithelial neoplasms and malignant melanomas showed the largest statistically significant average annual increase in incidence (+4.9%; p < 0.01), followed by central nervous system neoplasms (+1.3%; p < 0.05). Lymphomas, by contrast, showed a statistically significant average annual decrease in incidence in children aged 0-14 years (2.1%; p < 0.01). In adolescents aged 15-19 years, other malignant epithelial neoplasms and malignant melanomas also showed a statistically significant average annual increase in incidence (+5.2%; p < 0.01), followed by central nervous system neoplasms (+1.5%; p < 0.05). Mortality trends showed a statistically significant long-term decrease: on average, 5.1% annually in children aged 0-14 years (p < 0.01), and 3.7% annually in adolescents aged 15-19 years (p < 0.01). CONCLUSION Available data make it possible to analyse long-term trends in childhood cancer incidence and mortality.