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Detection of minimal residual disease (MRD) pre- and post-hematopoietic cell transplantation (HCT) for pediatric acute lymphoblastic leukemia (ALL) has been associated with relapse and poor survival. Published studies have had insufficient numbers to: (1) compare the prognostic value of pre-HCT and post-HCT MRD; (2) determine clinical factors post-HCT associated with better outcomes in MRD+ patients; and (3) use MRD and other clinical factors to develop and validate a prognostic model for relapse in pediatric patients with ALL who undergo allogeneic HCT. To address these issues, we assembled an Cl international database including sibling (n = 191), unrelated (n = 259), mismatched (n = 56), and cord blood (n = 110) grafts given after myeloablative conditioning. Although high and m very high MRD pre-HCT were significant predictors in univariate analysis, with bivariate analysis using MRD pre-HCT and post-HCT, MRD pre-HCT at any level was less predictive than even low-level MRD post-HCT. Patients with MRD pre-HCT must become MRD low/negative at 1 to 2 months and negative within 3 to 6 months after HCT for successful therapy. Factors associated with improved outcome of patients with detectable MRD post-Ha included acute graft-versus-host disease. We derived a risk score with an MRD cohort from Europe, North America, and Australia using negative predictive characteristics (late disease status, non-total body irradiation regimen, and MRD [high, very high]) defining good, intermediate, and poor risk groups with 2-year cumulative incidences of relapse of 21%, 38%, and 47%, respectively. We validated the score in a second, more contemporaneous cohort and noted 2-year cumulative incidences of relapse of 13%, 26%, and 47% (P < .001) for the defined risk groups.

BACKGROUND AND OBJECTIVES: Image segmentation represents one of the most challenging issues in medical image analysis to distinguish among different adjacent tissues in a body part. In this context, appropriate image pre-processing tools can improve the result accuracy achieved by computer-assisted segmentation methods. Taking into consideration images with a bimodal intensity distribution, image binarization can be used to classify the input pictorial data into two classes, given a threshold intensity value. Unfortunately, adaptive thresholding techniques for two-class segmentation work properly only for images characterized by bimodal histograms. We aim at overcoming these limitations and automatically determining a suitable optimal threshold for bimodal Magnetic Resonance (MR) images, by designing an intelligent image analysis framework tailored to effectively assist the physicians during their decision-making tasks. METHODS: In this work, we present a novel evolutionary framework for image enhancement, automatic global thresholding, and segmentation, which is here applied to different clinical scenarios involving bimodal MR image analysis: (i) uterine fibroid segmentation in MR guided Focused Ultrasound Surgery, and (ii) brain metastatic cancer segmentation in neuro-radiosurgery therapy. Our framework exploits MedGA as a pre-processing stage. MedGA is an image enhancement method based on Genetic Algorithms that improves the threshold selection, obtained by the efficient Iterative Optimal Threshold Selection algorithm, between the underlying sub-distributions in a nearly bimodal histogram. RESULTS: The results achieved by the proposed evolutionary framework were quantitatively evaluated, showing that the use of MedGA as a pre-processing stage outperforms the conventional image enhancement methods (i.e., histogram equalization, bi-histogram equalization, Gamma transformation, and sigmoid transformation), in terms of both MR image enhancement and segmentation evaluation metrics. CONCLUSIONS: Thanks to this framework, MR image segmentation accuracy is considerably increased, allowing for measurement repeatability in clinical workflows. The proposed computational solution could be well-suited for other clinical contexts requiring MR image analysis and segmentation, aiming at providing useful insights for differential diagnosis and prognosis.

The prognostic relevance of CRLF2 -rearrangements in childhood acute B-cell precursor lymphoblastic leukaemia (ALL), was assessed by a comparative analysis of 114 non-Down-syndrome patients (99 P2RY8-CRLF2+ , 15 IGH@-CRLF2+ ), 76 from the AIEOP-BFM ALL 2000 and 38 from the MRC ALL97 trials. The 6-year cumulative relapse incidence of P2RY8-CRLF2+ patients treated on the two trials was not statistically different: 0·37 ± 0·06 vs. 0·25 ± 0·08 (P = 0·194). In contrast, 0/9 IGH@-CRLF2+ AIEOP-BFM, but 5/6 ALL97 patients relapsed. Conclusively, P2RY8-CRLF2+ patients had an intermediate protocol-independent outcome while the different prognosis of IGH@-CRLF2+ patients could be related to the different structures of the applied treatment protocols.

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR

Aim: In the AIEOP-BFM 2000 trial, 15% of pediatric patients treated according to risk-adapted polychemotherapeutic regimens relapsed. The present study aimed to investigate the influence of GST-M1 and GST-T1 deletions on clinical outcome of children with acute lymphoblastic leukemia treated according to the AIEOP-BFM ALL 2000 study protocol. Materials & methods: A novel-design, two-phase study was applied to select a subsample of 614 children to be genotyped for the deletions of GST genes. Cumulative incidence of relapse was then estimated by weighted Kaplan–Meier analysis, and the Cox model was applied to evaluate the effect of GST-M1 and GST-T1 isoenzyme deletions on relapse. Results: No overall effect was found, but the GST-M1 deletion was associated with better clinical outcome within prednisone poor-responder patients (hazard ratio [HR]: 0.45; 95% CI: 0.23–0.91; p = 0.026), whereas the GST-T1 deletion was associated with worse outcome in the standard-risk group (HR: 4.62; 95% CI: 1.04–20.6; p = 0.045) and within prednisone good responders (HR: 1.62; 95% CI: 1.02–2.58; p = 0.041). Conclusion: Our results show that GST-M1 and GST-T1 homozygous deletions have opposite correlation with relapse, the former being protective and the latter unfavourable in specific subsets of acute lymphoblastic leukemia patients. Original submitted 1 August 2012; Revision submitted 27 September 2012

Somatic mutations in PTPN11, the gene encoding the transducer SHP-2, have emerged as a novel class of lesions that upregulate RAS signalling and contribute to leukaemogenesis. In a recent study of 69 children and adolescents with de novo acute myeloid leukaemia (AML), we documented a non-random distribution of PTPN11 mutations among French - American British (FAB) subtypes. Lesions were restricted to FAB-M5 cases, where they were relatively common ( four of 12 cases). Here, we report on the results of a molecular screening performed on 181 additional unselected patients, enrolled in participating institutions of the Associazione Italiana Ematologia Oncologia Pediatrica - AML Study Group, to provide a more accurate picture of the prevalence, spectrum and distribution of PTPN11 mutations in childhood AML and to investigate their clinical relevance. We concluded that PTPN11 defects do not represent a frequent event in this heterogeneous group of malignancies ( 4 center dot 4%), although they recur in a considerable percentage of patients with FAB-M5 ( 18%). PTPN11 lesions rarely occur in other subtypes. Within the FAB- M5 group no clear association of PTPN11 mutations with any clinical variable was evident. Nearly two third of the patients with this subtype were found to harbour an activating mutation in PTPN11, NRAS, KRAS2 or FLT3.

Acute lymphoblastic and myeloid leukemia (ALL, AML) are neoplasms characterized by a clonal, abnormal and self-maintaining proliferation of hematopoietic cells. These acute leukemias can be categorized into several subtypes, based principally on phenotype and according to specific genetic

Abstract 1678 Introduction: The IDH1 gene encodes for NADP+-dependent isocitrate dehydrogenase 1 enzyme, which catalyzes the oxydative decarboxylation of isocitrate to α-ketoglutarate. Acquired somatic mutations of the R132 residue of IDH1 have been detected in adults with de novo AML, while so far no IDH1 mutations were detected in a series of 257 children in USA. The role of IDH1 mutations in the pathogenesis of AML is still unclear. Material and Methods: From 01/12/2002 to 31/12/2007, 205 childhood AML patients were enrolled into the multicentric AIEOP-LAM 2001/02 protocol. Among them, we analyzed the prevalence of IDH1 mutations in 165 patients for whom material was available. IDH1 gene mutations have been analyzed by PCR amplification and sequencing of the exon 4. The clinical and biological features of the analyzed and not analyzed population were not statistically different. Results: In this series of childhood AML cases, 4 out of 165 cases (2.4%) were positive for IDH1 mutations. All patients were male, the age at diagnosis ranged from 3 to 14 years, while the WBC count at diagnosis ranged from 8750 to 233970 WBC/ml. Three of them had FAB M1 and one M2; none of them had localization in the central nervous system, while one had lymph nodes involvement. Two of the 4 children with the IDH1 mutation had a normal karyotype while two carried different clonal translocation. One patient carried the FLT3-ITD mutation at diagnosis, whereas no other known associated mutations were found. Based on cytogenetics, all of them were classified within the high risk group. Complete remission was achieved in all cases, and all but one received BMT. Two patients had a medullary relapse and all are alive, after 20, 26, 33 and 33 months from BMT. All patients carried the R132H IDH1 mutation. The mutation was specific of the leukemia cells, being absent in the remission phase of all patients. Interestingly, the R132H mutation was detected at the relapse stage of one patient, but not at the relapse of the second patient, suggesting that IDH1 mutations could represent a secondary lesion in the pathogenesis of leukemia. Because 2/4 IDH1 mutated cases in the sequential screening had a normal karyotype, we extended the mutational screening to all Italian childhood cases diagnosed as AML with normal karyotype from 13/10/2000 to 15/04/2010. Out of the additional 97 cases with normal karyotype, only 1 carried a IDH1 mutation (R132H). Conclusions: In summary, we showed that IDH1 gene mutation can be detected also in pediatric AML, with an estimate prevalence of 2.4% (4/165) in the Italian series. The low prevalence does not allow any prediction on the outcome, although all patients are alive at different time after BMT, even in the presence of FLT3-ITD mutation (one patient). The clinical and biological characteristics of the mutated patients seemed not to be different from the overall childhood AML population, and similar to the adult IDH1 mutated cases. The R132 mutation is the only pediatric mutation detected so far. The extended series to a total number of 186 childhood AML with normal karyotype identified 3/186 mutations (1.6%) in this specific subgroup. Therefore, it does not seem that IDH1 mutation is more prevalent in normal karyotype. In addition, even considering that the IDH1 mutation could not be present at the relapse (like it happens for FLT3 mutations), it is questionable the role of these abnormalities, and whether those mutations in ‘normal cytogenetic’ subgroup could be sufficient for the clinical disease emergence, or further events must be discovered in the complex and multistep pathogenesis of leukemia. Disclosures: No relevant conflicts of interest to declare.

PURPOSE Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of KMT2A gene rearrangements and poor outcome. We evaluated the value of minimal residual disease (MRD) in infants with KMT2A-rearranged ALL treated within the Interfant-06 protocol, which compared lymphoid-style consolidation (protocol IB) versus myeloid-style consolidation (araC, daunorubicin, etoposide/mitoxantrone, araC, etoposide). MATERIALS AND METHODS MRD was measured in 249 infants by DNA-based polymerase chain reaction of rearranged KMT2A, immunoglobulin, and/or T-cell receptor genes, at the end of induction (EOI) and end of consolidation (EOC). MRD results were classified as negative, intermediate (< 5 × 10−4), and high (≥ 5 × 10−4). RESULTS EOI MRD levels predicted outcome with 6-year disease-free survival (DFS) of 60.2% (95% CI, 43.2 to 73.6), 45.0% (95% CI, 28.3 to 53.1), and 33.8% (95% CI, 23.8 to 44.1) for infants with negative, intermediate, and high EOI MRD levels, respectively ( P = .0039). EOC MRD levels were also predictive of outcome, with 6-year DFS of 68.2% (95% CI, 55.2 to 78.1), 40.1% (95% CI, 28.1 to 51.9), and 11.9% (95% CI, 2.6 to 29.1) for infants with negative, intermediate, and high EOC MRD levels, respectively ( P < .0001). Analysis of EOI MRD according to the type of consolidation treatment showed that infants treated with lymphoid-style consolidation had 6-year DFS of 78.2% (95% CI, 51.4 to 91.3), 47.2% (95% CI, 33.0 to 60.1), and 23.2% (95% CI, 12.1 to 36.4) for negative, intermediate, and high MRD levels, respectively ( P < .0001), while for myeloid-style–treated patients the corresponding figures were 45.0% (95% CI, 23.9 to 64.1), 41.3% (95% CI, 23.2 to 58.5), and 45.9% (95% CI, 29.4 to 60.9). CONCLUSION This study provides support for the idea that induction therapy selects patients for subsequent therapy; infants with high EOI MRD may benefit from AML-like consolidation (DFS 45.9% v 23.2%), whereas patients with low EOI MRD may benefit from ALL-like consolidation (DFS 78.2% v 45.0%). Patients with positive EOC MRD had dismal outcomes. These findings will be used for treatment interventions in the next Interfant protocol.

BACKGROUND: Chimeric antigen receptor (CAR) T cell immunotherapy has resulted in complete remission (CR) and durable response in highly refractory patients. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. METHODS: We report the early results of a phase I/II trial in B cell acute lymphoblastic leukemia (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) using donor-derived CD19 CAR T cells generated with the Sleeping Beauty (SB) transposon and differentiated into cytokine-induced killer (CIK) cells. RESULTS: The cellular product was produced successfully for all patients from the donor peripheral blood (PB) and consisted mostly of CD3(+) lymphocytes with 43% CAR expression. Four pediatric and 9 adult patients were infused with a single dose of CAR T cells. Toxicities reported were 2 grade I and 1 grade II cytokine-release syndrome (CRS) cases at the highest dose in the absence of graft-versus-host disease (GVHD), neurotoxicity, or dose-limiting toxicities. Six out of 7 patients receiving the highest doses achieved CR and CR with incomplete blood count recovery (CRi) at day 28. Five out of 6 patients in CR were also minimal residual disease negative (MRD(–)). Robust expansion was achieved in the majority of the patients. CAR T cells were measurable by transgene copy PCR up to 10 months. Integration site analysis showed a positive safety profile and highly polyclonal repertoire in vitro and at early time points after infusion. CONCLUSION: SB-engineered CAR T cells expand and persist in pediatric and adult B-ALL patients relapsed after HSCT. Antileukemic activity was achieved without severe toxicities. TRIAL REGISTRATION: ClinicalTrials.gov NCT03389035. FUNDING: This study was supported by grants from the Fondazione AIRC per la Ricerca sul Cancro (AIRC); Cancer Research UK (CRUK); the Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC); Ministero Della Salute; Fondazione Regionale per la Ricerca Biomedica (FRRB).

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory multisystemic disease. Familial hemophagocytic lymphohistiocytosis (FHL) represents a group of rare autosomal recess...

Whole-slide imaging and virtual microscopy are useful tools implemented in the routine pathology workflow in the last 10 years, allowing primary diagnosis or second-opinions (telepathology) and demonstrating a substantial role in multidisciplinary meetings and education. The regulatory approval of this technology led to the progressive digitalization of routine pathological practice. Previous experiences on renal biopsies stressed the need to create integrate networks to share cases for diagnostic and research purposes. In the current paper, we described a virtual lab studying the routine renal biopsies that have been collected from 14 different Italian Nephrology centers between January 2014 and December 2019. For each case, light microscopy (LM) and immunofluorescence (IF) have been processed, analysed and scanned. Additional pictures (eg. electron micrographs) along with the final encrypted report were uploaded on the web-based platform. The number and type of specimens processed for every technique, the provisional and final diagnosis, and the turnaround-time (TAT) have been recorded. Among 826 cases, 4.5% were second opinion biopsies and only 4% were suboptimal/inadequate for the diagnosis. Transmission electron microscopy (TEM) has been performed on 41% of cases, in 22% changing the final diagnosis, in the remaining 78% contributed to the better definition of the disease. For light microscopy and IF the median TAT was of 2 working days, with only 8.6% with a TAT longer than 5 days. For TEM, the average TAT was 26 days (IQR 6–64). In summary, we systematically reviewed the 6-years long nephropathological experience of an Italian renal pathology service, where digital pathology is a definitive standard of care for the routine diagnosis of glomerulonephritides.

Background The outcome of infants with KMT2A-germline ALL is much better than of infants with KMT2A-rearranged ALL, but still worse than of non-infant ALL patients. Here, we describe the outcome and prognostic factors for infants with KMT2A-germline ALL treated on Interfant-06 protocol. Methods 167 infants with KMT2A-germline ALL were enrolled in Interfant-06. Univariate analysis on prognostic factors (age, WBC at diagnosis, prednisolone response and CD10 expression) was performed on KMT2A-germline infants in complete remission at the end of induction (EOI) (n=163). Bone marrow minimal residual disease (MRD) was measured in 73 patients, by real-time quantitative PCR of immunoglobulin genes and/or T-cell receptor genes, at various time points (EOI, n= 68, end of consolidation (EOC) n= 56, and before OCTADAD (n=57)). MRD results were classified as negative, intermediate (< 5*10 -4), and high (≥5*10 -4). Genetic data on NUTM1 rearrangements and MRD were available in 53 patients. Results The 6-year event free survival [SE] and overall survival [ SE] was 73.9% (3,6) and 87.2% (2.7). 28 of 31 (90%) relapses occurred early, within 2 years of diagnosis. Treatment related mortality was 3.6%. Age 6 months of age (p=0.04). Of the MRD timepoints, MRD at end of induction (EOI) was most prognostic for outcome. At EOI, 76.5% (n=52/68) of patients were either MRD negative (41.2%, n=28) or intermediate MRD (35.3%, n=24). 23.5% (n=16) had high EOI MRD, which was associated with significantly lower 6-year DFS (SE), compared to patients with intermediate or negative EOI MRD (61.4% (12.4), 76.4% (11.3) and 87.9% (6.6), respectively; p=0.02, Figure 1a). At EOC, 55.4% (n=31/56) of patients were MRD negative. Outcome by MRD levels at EOC was not significantly different (p=0.24); the 6-year DFS (SE) of negative and intermediate EOC MRD patients was 89.0% (6.0) and 72.7 % (10.6), respectively, while only one of the 5 patients with high EOC MRD relapsed in BM and CNS (Figure 1b). MRD data for both EOI and EOC were available for 55 patients. Of these patients, 18 were MRD negative at EOI and EOC, with a 6-year DFS 93.3% (SE, 6.4). Five patients had negative EOI MRD, but showed intermediate EOC MRD levels; none of these patients relapsed. There were 12 of 55 patients who were MRD positive at EOI and became MRD negative at EOC. These patients had a 6-year DFS of 82.5 % (SE, 11.3). Patients with detectable disease at both timepoints had a 6-year DFS of 68.3% (SE,10.8) (n=20, Figure 1c). At the end of MARMA (TP5), 77.2% (n=44/57) of patients were MRD negative. MRD at TP5 was significantly related to DFS (Figure 1d); the 6-year DFS was 89.6% (SE, 5.0) for MRD negative patients, compared to 65.6% (SE, 14.0) for patients with intermediate MRD levels (p= 0.039). In the current study, NUTM1 status was known in 53 patients with MRD data. Of them, 13 harbored a NUTM1-rearrangement. Seven out of 11 (63.6%) were aged< 6 months and 6 out of 42 (14.3%) were older. None of them relapsed, despite positive EOI MRD detected in 8 cases. Conclusion We conclude that young age at diagnosis and low EOI MRD are favorable prognostic factors in infants with KMT2A-germline ALL. However, the prognostic value of MRD is not as strong as in infants with KMT2A-rearranged ALL or older children with ALL. This can partly be explained by the differences in genetic makeup of infants with KMT2A-germline ALL, thus supporting the hypothesis that in the future a combined MRD- and genetic-based stratification of KMT2A-g infants might be considered Figure 1 Figure 1. Disclosures Biondi: Colmmune: Honoraria; Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria. Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharamceutical: Speakers Bureau; Takeda: Speakers Bureau. Silverman: Takeda, Servier, Syndax, Jazz Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Schrappe: Novartis: Honoraria, Other: research support; SigmaTau: Other: research support; Amgen: Other: research support; Servier: Honoraria; Novartis: Honoraria; JazzPharma: Honoraria; JazzPharma: Honoraria, Other: research support; SHIRE: Other: research support; Servier: Honoraria, Other: research support.

Comparison of treatment strategies in de novo pediatric acute lymphoblastic leukemia (ALL) requires standardized measures of efficacy. Key parameters that define disease-related events, including complete remission (CR), treatment failure (TF; not achieving CR), and relapse (loss of CR) require an updated consensus incorporating modern diagnostics. We collected the definitions of CR, TF, and relapse from recent and current pediatric clinical trials for the treatment of ALL, including the key components of response evaluation (timing, anatomic sites, detection methods, and thresholds) and found significant heterogeneity, most notably in the definition of TF. Representatives of the major international ALL clinical trial groups convened to establish consensus definitions. CR should be defined at a time point no earlier than at the end of induction and should include the reduction of blasts below a specific threshold in bone marrow and extramedullary sites, incorporating minimal residual disease (MRD) techniques for marrow evaluations. TF should be defined as failure to achieve CR by a prespecified time point in therapy. Relapse can only be defined in patients who have achieved CR and must include a specific threshold of leukemic cells in the bone marrow confirmed by MRD, the detection of central nervous system leukemia, or documentation of extramedullary disease. Definitions of TF and relapse should harmonize with eligibility criteria for clinical trials in relapsed/refractory ALL. These consensus definitions will enhance the ability to compare outcomes across pediatric ALL trials and facilitate development of future international collaborative trials.

Contains fulltext : 248362.pdf (Publisher’s version ) (Open Access) INTRODUCTION: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. METHODS: In this study, we performed a genomic analysis of diagnosis-relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. RESULTS: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. CONCLUSION: Comprehensive genomic characterization of diagnosis-relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer with the majority of cases being of B lineage [1]. B-cell precursor ALL (BCP-ALL) is a heterogeneous disease in which risk ass...

Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq: PQ-2017#305529/2017-0 Deutsche Forschungsgemeinschaft, DFG: MA 1876/12-1 Alexander von Humboldt-Stiftung: 88881.136091/2017-01 RVO-VFN64165, 26/203.214/2017 2018.070.1 Associazione Italiana per la Ricerca sul Cancro, AIRC: IG2015, 17593 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES Cancer Australia: PdCCRS1128727 Cancerfonden Barncancerfonden VetenskapsrÃ¥det, VR Crafoordska Stiftelsen Knut och Alice Wallenbergs Stiftelse Lund University Medical Faculty Foundation Xiamen University, XMU 2014S06 17-74-30019 C7838/A15733 Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, SNSF: 31003A_140913 CNIB Institut National Du Cancer, INCa R01 NCI CA167824 National Institutes of Health, NIH: S10OD018522 2016/2017, 02R/2016 AU 525/1-1 Deutschen Konsortium für Translationale Krebsforschung, DKTK 70112951 Smithsonian Institution, SI Israel Science Foundation, ISF Austrian Science Fund, FWF: W1212 SFB-F06107, SFB-F06105 Acknowledgements BAL received a fellowship provided by CAPES and the Alexander von Humboldt Foundation (#88881.136091/2017-01). ME is supported by CNPq (PQ-2017#305529/2017-0) and FAPERJ-JCNE (#26/203.214/2017) research scholarships, and ZZ by grant RVO-VFN64165. GC is supported by the AIRC Investigator grant IG2015 grant no. 17593 and RS by Cancer Australia grant PdCCRS1128727. This work was supported by grants to RM from the “Georg und Franziska Speyer’sche Hochsschulstiftung”, the “Wilhelm Sander foundation” (grant 2018.070.1) and DFG grant MA 1876/12-1. Acknowledgements This work was supported by The Swedish Childhood Cancer Foundation, The Swedish Cancer Society, The Swedish Research Council, The Knut and Alice Wallenberg Foundation, BioCARE, The Crafoord Foundation, The Per-Eric and Ulla Schyberg Foundation, The Nilsson-Ehle Donations, The Wiberg Foundation, and Governmental Funding of Clinical Research within the National Health Service. Work performed at the Center for Translational Genomics, Lund University has been funded by Medical Faculty Lund University, Region Skåne and Science for Life Laboratory, Sweden. Acknowledgements This work was supported by the Fujian Provincial Natural Science Foundation 2016S016 China and Putian city Natural Science Foundation 2014S06(2), Fujian Province, China. Alexey Ste-panov and Alexander Gabibov were supported by Russian Scientific Foundation project No. 17-74-30019. Jinqi Huang was supported by a doctoral fellowship from Xiamen University, China. Acknowledgments This work was supported by the Swiss National Science Foundation (grant 31003A_140913; OH) and the Cancer Research UK Experimental Cancer Medicine Centre Network, Cardiff ECMCI, grant C7838/A15733. We thank N. Carpino for the Sts-1/2 double-KO mice. Acknowledgements This work was supported by the French National Cancer Institute (INCA) and the Fondation Française pour la Recherche contre le Myélome et les Gammapathies (FFMRG), the Intergroupe Francophone du Myélome (IFM), NCI R01 NCI CA167824 and a generous donation from Matthew Bell. This work was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai. Research reported in this paper was supported by the Office of Research Infrastructure of the National Institutes of Health under award number S10OD018522. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank the Association des Malades du Myélome Multiple (AF3M) for their continued support and participation. Where authors are identified as personnel of the International Agency for Research on Cancer / World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer / World Health Organization. We are indebted to all members of our groups for useful discussions and for their critical reading of the manuscript. Special thanks go to Silke Furlan, Friederike Opitz and Bianca Killing. F.A. is supported by the Deutsche For-schungsgemeinschaft (DFG, AU 525/1-1). J.H. has been supported by the German Children’s Cancer Foundation (Translational Oncology Program 70112951), the German Carreras Foundation (DJCLS 02R/2016), Kinderkrebsstiftung (2016/2017) and ERA PerMed GEPARD. Support by Israel Science Foundation, ERA-NET and Science Ministry (SI). A. B. is supported by the German Consortium of Translational Cancer Research, DKTK. We are grateful to the Jülich Supercomputing Centre at the Forschungszemtrum Jülich for granting computing time on the supercomputer JURECA (NIC project ID HKF7) and to the “Zentrum für Informations-und Medientechnologie” (ZIM) at the Heinrich Heine University Düsseldorf for providing computational support to H. G. The study was performed in the framework of COST action CA16223 “LEGEND”. Funding The work was supported by the Austrian Science Fund FWF grant SFB-F06105 to RM and SFB-F06107 to VS and FWF grant W1212 to VS.

Minimal residual disease (MRD) is the most powerful prognostic factor in pediatric acute lymphoblastic leukemia (ALL). Real-time quantitative polymerase chain reaction (RQ-PCR) represents the gold standard for molecular MRD assessment and risk-based stratification of front-line treatment. In the protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP) and the Berlin-Frankfurth-Munschen (BFM) group AIEOP-BFM ALL2009 and ALL2017, B-lineage ALL patients with high RQ-PCR-MRD at day+33 and positive at day+78 are defined slow early responders (SERs). Based on results of the AIEOP-BFM ALL2000 study, these patients are treated as high-risk also when positive MRD signal at day +78 is below the lower limit of quantification of RQ-PCR ("positive not-quantifiable," POS-NQ). To assess whether droplet digital polymerase chain reaction (ddPCR) could improve patients' risk definition, we analyzed MRD in 209 pediatric B-lineage ALL cases classified by RQ-PCR as POS-NQ and/or negative (NEG) at days +33 and/or +78 in the AIEOP-BFM ALL2000 trial. ddPCR MRD analysis was performed on 45 samples collected at day +78 from SER patients, who had RQ-PCR MRD ≥ 5.0 * 10-4 at day+33 and POS-NQ at day+78 and were treated as medium risk (MR). The analysis identified 13 of 45 positive quantifiable cases. Most relapses occurred in this patients' subgroup, while ddPCR NEG or ddPCR-POS-NQ patients had a significantly better outcome (P < 0.001). Overall, in 112 MR cases and 52 standard-risk patients, MRD negativity and POS-NQ were confirmed by the ddPCR analysis except for a minority of cases, for whom no differences in outcome were registered. These data indicate that ddPCR is more accurate than RQ-PCR in the measurement of MRD, particularly in late follow-up time points, and may thus allow improving patients' stratification in ALL protocols.

DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokinesis 8 (DOCK8) gene. The disease has a wide clinical spectrum encompassing recurrent infections (candidiasis, viral and bacterial infections), virally driven malignancies and immune dysregulatory features, including autoimmune (cytopenia and vasculitis) as well as allergic disorders (eczema, asthma, and food allergy). Hypomorphic function and somatic reversion of DOCK8 has been reported to result in incomplete phenotype without IgE overproduction. Here we describe a case of DOCK8 deficiency in a 8-year-old Caucasian girl. The patient’s disease was initially classified as autoimmune thrombocytopenia, which then evolved toward a combined immunodeficiency phenotype with recurrent infections, persistent EBV infection and lymphoproliferation. Two novel variants (one deletion and one premature stop codon) were characterized, resulting in markedly reduced, but not absent, DOCK8 expression. Somatic reversion of the DOCK8 deletion was identified in T cells. Hypomorphic function and somatic reversion were associated with restricted T cell repertoire, decreased STAT5 phosphorylation and impaired immune synapse functioning in T cells. Although the patient presented with incomplete phenotype (absence of markedly increase IgE and eosinophil count), sclerosing cholangitis was incidentally detected, thus indicating that hypomorphic function and somatic reversion of DOCK8 may delay disease progression but do not necessarily prevent from severe complications.

Introduction Acute lymphoblastic leukemia (ALL) is the first pediatric cancer where the assessment of early response to therapy by minimal residual disease (MRD) monitoring has demonstrated its importance to improve risk-based treatment approaches. The most standardized tools to study MRD in ALL are multiparametric flow cytometry (MFC) and realtime-quantitative polymerase chain reaction (RQ-PCR) amplification-based methods. In recent years, MRD measurement has reached greater levels of sensitivity and standardization through international laboratory networks collaboration. Areas covered We herewith describe how to assess and apply the prognostic impact of MRD in treatment decisions, with specific focus on pediatric ALL. We also highlight the role of MRD monitoring in the context of genetically homogeneous subgroups of pediatric ALL. However, some queries remain to be addressed and emerging technologies hold the promise of improving MRD detection in ALL patients. Expert opinion Emerging technologies, like next generation flow cytometry, droplet digital PCR, and next generation sequencing appear to be important methods for assessing MRD in pediatric ALL. These more specific and/or sensitive MRD monitoring methods may help predict relapse with greater accuracy, and are currently being used in clinical trials to improve pediatric ALL patient outcome by optimizing patient stratification and earlier MRD-based interventional therapy.

Agammaglobulinemia is the most profound primary antibody deficiency that can occur due to an early termination of B-cell development. We here investigated 3 novel patients, including the first known adult, from unrelated families with agammaglobulinemia, recurrent infections, and hypertrophic cardiomyopathy (HCM). Two of them also presented with intermittent or severe chronic neutropenia. We identified homozygous or compound-heterozygous variants in the gene for folliculin interacting protein 1 (FNIP1), leading to loss of the FNIP1 protein. B-cell metabolism, including mitochondrial numbers and activity and phosphatidylinositol 3-kinase/AKT pathway, was impaired. These defects recapitulated the Fnip1−/− animal model. Moreover, we identified either uniparental disomy or copy-number variants (CNVs) in 2 patients, expanding the variant spectrum of this novel inborn error of immunity. The results indicate that FNIP1 deficiency can be caused by complex genetic mechanisms and support the clinical utility of exome sequencing and CNV analysis in patients with broad phenotypes, including agammaglobulinemia and HCM. FNIP1 deficiency is a novel inborn error of immunity characterized by early and severe B-cell development defect, agammaglobulinemia, variable neutropenia, and HCM. Our findings elucidate a functional and relevant role of FNIP1 in B-cell development and metabolism and potentially neutrophil activity.

B-cell precursor (BCP) acute lymphoblastic leukemia (BCP-ALL) in infants (ie, children age

Background: ABL-class fusion genes other than BCR–ABL1 have been identified in approximately 3% of children with newly diagnosed acute lymphocytic leukaemia, and studies suggest that leukaemic cells carrying ABL-class fusions can be targeted successfully by tyrosine-kinase inhibitors. We aimed to establish the baseline characteristics and outcomes of paediatric patients with ABL-class fusion B-cell acute lymphocytic leukaemia in the pre-tyrosine-kinase inhibitor era. Methods: This multicentre, retrospective, cohort study included paediatric patients (aged 1–18 years) with newly diagnosed ABL-class fusion (ABL1 fusion-positive, ABL2 fusion-positive, CSF1R fusion-positive, and PDGFRB fusion-positive) B-cell acute lymphocytic leukaemia enrolled in clinical trials of multidrug chemotherapy done between Oct 3, 2000, and Aug 28, 2018, in which tyrosine-kinase inhibitors had not been given as a first-line treatment. Patients from 14 European, North American, and Asia-Pacific study groups of the Ponte di Legno group were included. No patients were excluded, and patients were followed up by individual study groups. Through the Ponte di Legno group, we collected data on the baseline characteristics of patients, including IKZF1, PAX5, and CDKN2A/B deletion status, and whether haematopoietic stem cell transplantation (HSCT) had been done, as well as treatment outcomes, including complete remission, no response, relapse, early death, and treatment-related mortality, response to prednisone, and minimal residual disease (MRD) at end of induction therapy. 5-year event-free survival and 5-year overall survival were estimated by use of Kaplan-Meier methods, and the 5-year cumulative incidence of relapse was calculated by use of a competing risk model. Findings: We identified 122 paediatric patients with newly diagnosed ABL-class fusion B-cell acute lymphocytic leukaemia (77 from European study groups, 25 from North American study groups, and 20 from Asia-Pacific study groups). 64 (52%) of 122 patients were PDGFRB fusion-positive, 40 (33%) were ABL1 fusion-positive, ten (8%) were CSF1R fusion-positive, and eight (7%) were ABL2 fusion-positive. In all 122 patients, 5-year event-free survival was 59·1% (95% CI 50·5–69·1), 5-year overall survival was 76·1% (68·6–84·5), and the 5-year cumulative incidence of relapse was 31·0% (95% CI 22·4–40·1). MRD at the end of induction therapy was high (≥10−2 cells) in 61 (66%) of 93 patients, and most prevalent in patients with ABL2 fusions (six [86%] of 7 patients) and PDGFRB fusion-positive B-cell acute lymphocytic leukaemia (43 [88%] of 49 patients). MRD at the end of induction therapy of 10−2 cells or more was predictive of an unfavourable outcome (hazard ratio of event-free survival in patients with a MRD of ≥10−2 vs those with a MRD of

Although new techniques (i.e., droplet digital-PCR, next-generation sequencing, advanced flow cytometry) are being developed, DNA-based allele-specific real-time quantitative (RQ)-PCR is still the gold standard for sensitive and accurate immunoglobulin/T cell receptor (IG/TR)-based minimal residual disease (MRD) monitoring, allowing the detection of up to 1 leukemic cell in 100,000 normal lymphoid cells. We herewith describe the standard PCR procedure which has been developed and standardized (with minor modification in single labs) through the last 20years of activity of the EuroMRD Consortium, a volunteer activity of expert laboratories that is continuously providing education, standardization, quality control rounds, and guidelines for interpretation of RQ-PCR data.

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This paper was funded by grants to MP by the Spanish Ministry of Science, Innovation and Universities (SAF2017-87990-R and EUR2019-103835) and elaborated at the Josep Carreras Leukaemia Research Institute (IJC, Badalona, Barcelona) and IDIBELL Research Institute (L’Hospitalet de Llobregat, Barcelona). OdB is funded by a Juan de la Cierva—Formación fellowship from the Spanish Ministry of Science, Innovation and Universities (FJCI-2017-32430). AM is funded by the Spanish Ministry of Science, Innovation and Universities, which is part of the Agencia Estatal de Investigación (AEI), through grant PRE2018-083183 (cofunded by the European Social Fund). Work in PM and CB’s lab was supported by the European Research Council (CoG-2014646903), the Spanish Ministry of Economy and Competitiveness (SAF-2016-80481-R), Uno entre Cien Mil Foundation, the Leo Messi Foundation, the Asociación Española Contra el Cáncer (AECC-CI-2015), and the ISCIII/FEDER (PI17/01028). ¡

Risk-adapted therapy has significantly contributed to improved survival rates in pediatric acute lymphoblastic leukemia (ALL) and reliable detection of chromosomal aberrations is mandatory for risk group stratification. This study evaluated the applicability of panel-based RNA sequencing and array CGH within the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial. In a consecutive cohort of 117 children with B cell precursor (BCP) ALL, array analysis identified twelve cases with an IKZF1plus profile of gene deletions and one case of masked hypodiploidy. Genetic markers BCR-ABL1 (n = 1), ETV6-RUNX1 (n = 25), and rearrangements involving KMT2A (n = 3) or TCF3 (n = 3) were assessed by established conventional techniques such as karyotyping, FISH, and RT-PCR. Comparison of these results with RNA sequencing analysis revealed overall consistency in n=115/117 cases, albeit with one undetected AFF1-KMT2A fusion in RNA sequencing and one undetected ETV6-RUNX1 fusion in conventional analyses. The combined application of RNA sequencing, FISH, and CGH+SNP array reliably detected all genetic markers necessary for risk stratification and will be used as the diagnostic standard workflow for BCP-ALL patients enrolled in the AIEOP-BFM ALL 2017 study. Prospectively, consistent collection of genome-wide CGH+SNP array as well as RNA sequencing data will be a valuable source to elucidate new prognostic lesions beyond established markers of pediatric ALL. In this respect, RNA sequencing identified various gene fusions in up to half of the IKZF1plus (n = 6/12) and B-other (n = 19/36) cases but not in cases with hyperdiploid karyotypes (n = 35). Among these fusions, this study reports several previously undescribed in frame PAX5 fusions, including PAX5-MYO1G and PAX5-NCOA6.

Minimal residual disease (MRD) analysis has become a powerful indicator to refine therapy in acute lymphoblastic leukemia (ALL). Here, we present an MRD detection based on the next-generation sequencing of PTEN exon 7 mutations (NGS-PTEN) in 30 pediatric T-cell ALL patients. By comparing the NGS-PTEN results with current quantitative PCR of antigen receptor gene rearrangements (qPCR-Ig/TR), an overall concordance of 80% was found between the two methods. However, the NGS-PTEN qualified a lower number of high-risk patients than qPCR-Ig/TR. These findings suggest that NGS-PTEN is a promising tool that could potentially be used to support current MRD methodologies for T-ALL patients.

ABL-class fusions other than BCR-ABL1 characterize around 2-3% of precursor B-cell acute lymphoblastic leukemia. Case series indicated that patients suffering from these subtypes have a dismal outcome and may benefit from the introduction of tyrosine kinase inhibitors. We analyzed clinical characteristics and outcome of 46 ABL-class fusion positive cases other than BCR-ABL1 treated according to AIEOP-BFM (Associazione Italiana di Ematologia-Oncologia Pediatrica-Berlin-Frankfurt-Munster) ALL 2000 and 2009 protocols; 13 of them received a tyrosine kinase inhibitor (TKI) during different phases of treatment. ABL-class fusion positive cases had a poor early treatment response: minimal residual disease levels of ≥5×10-4 were observed in 71.4% of patients after induction treatment and in 51.2% after consolidation phase. For the entire cohort of 46 cases, the 5-year probability of event-free survival was 49.1+8.9% and that of overall survival 69.6+7.8%; the cumulative incidence of relapse was 25.6+8.2% and treatment-related mortality (TRM) 20.8+6.8%. One out of 13 cases with TKI added to chemotherapy relapsed while eight of 33 cases without TKI treatment suffered from relapse, including six in 17 patients who had not received hematopoietic stem cell transplantation. Stem cell transplantation seems to be effective in preventing relapses (only three relapses in 25 patients), but was associated with a very high TRM (6 patients). These data indicate a major need for an early identification of ABL-class fusion positive acute lymphoblastic leukemia cases and to establish a properly designed, controlled study aimed at investigating the use of TKI, the appropriate chemotherapy backbone and the role of hematopoietic stem cell transplantation. (Registered at: clinicaltrials.gov identifier: NTC00430118, NCT00613457, NCT01117441).

MLL-rearranged acute lymphoblastic leukemia (ALL) occurring in infants is a rare but very aggressive leukemia, typically associated with a dismal prognosis. Despite the development of specific therapeutic protocols, infant patients with MLL-rearranged ALL still suffer from a low cure rate. At present, novel therapeutic approaches are urgently needed. Recently, the use of small molecule inhibitors targeting the epigenetic regulators of the MLL complex emerged as a promising strategy for the development of a targeted therapy. Herein, we have investigated the effects of bromodomain and extra-terminal (BET) function abrogation in a preclinical mouse model of MLL-AF4+ infant ALL using the BET inhibitor I-BET151. We reported that I-BET151 is able to arrest the growth of MLL-AF4+ leukemic cells in vitro, by blocking cell division and rapidly inducing apoptosis. Treatment with I-BET151 in vivo impairs the leukemic engraftment of patient-derived primary samples and lower the disease burden in mice. I-BET151 affects the transcriptional profile of MLL-rearranged ALL through the deregulation of BRD4, HOXA7/HOXA9, and RUNX1 gene networks. Moreover, I-BET151 treatment sensitizes glucocorticoid-resistant MLL-rearranged cells to prednisolone in vitro and is more efficient when used in combination with HDAC inhibitors, both in vitro and in vivo. Given the aggressiveness of the disease, the failure of the current therapies and the lack of an ultimate cure, this study paves the way for the use of BET inhibitors to treat MLL-rearranged infant ALL for future clinical applications. Mol Cancer Ther; 17(8); 1705–16. ©2018 AACR.

The prognostic value of minimal residual disease (MRD) in Philadelphia-chromosome-positive (Ph+) childhood acute lymphoblastic leukemia (ALL) treated with tyrosine kinase inhibitors is not fully established. We detected MRD by real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged immunoglobulin/T-cell receptor genes (IG/TR) and/or BCR/ABL1 fusion transcript to investigate its predictive value in patients receiving Berlin-Frankfurt-Münster (BFM) high-risk (HR) therapy and post-induction intermittent imatinib (the European intergroup study of post-induction treatment of Philadelphia-chromosome-positive acute lymphoblastic leukemia (EsPhALL) study). MRD was monitored after induction (time point (TP)1), consolidation Phase IB (TP2), HR Blocks, reinductions, and at the end of therapy. MRD negativity progressively increased over time, both by IG/TR and BCR/ABL1. Of 90 patients with IG/TR MRD at TP1, nine were negative and none relapsed, while 11 with MRD

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

// Elena Vendramini 1, 2, 3 , Marco Giordan 1 , Emanuela Giarin 1 , Barbara Michielotto 1 , Grazia Fazio 4 , Gianni Cazzaniga 4 , Andrea Biondi 4 , Daniela Silvestri 4 , Maria Grazia Valsecchi 5 , Martina U. Muckenthaler 6 , Andreas E. Kulozik 6 , Valter Gattei 7 , Shai Izraeli 2, 3 , Giuseppe Basso 1 and Geertruy te Kronnie 1 1 Department of Women’s and Children’s Health, University of Padova, Padova, Italy 2 Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel 3 Tel Aviv University, Tel Aviv, Israel 4 Centro Ricerca Tettamanti, Clinica Pediatrica, University of Milano-Bicocca, Monza, Italy 5 School of Medicine and Surgery, University of Milano-Bicocca, Milano, Italy 6 Department of Pediatric Oncology Hematology, University of Heidelberg, Heidelberg, Germany 7 Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano (PN), Italy Correspondence to: Elena Vendramini, email: elena.vendramini@gmail.com Keywords: B cell precursor acute lymphoblastic leukemia, ERG aberrations, noncoding RNAs, miR-125, SNORD116 Received: June 29, 2016 Accepted: March 04, 2017 Published: March 21, 2017 ABSTRACT ERG-related leukemia is a B cell precursor acute lymphoblastic leukemia (BCP ALL) subtype characterized by aberrant expression of DUX4 and ERG transcription factors, and highly recurrent ERG intragenic deletions. ERG-related patients have remarkably favorable outcome despite a high incidence of inauspicious IKZF1 aberrations. We describe clinical and genomic features of the ERG-related cases in an unselected cohort of B-other BCP ALL pediatric patients enrolled in the AIEOP ALL 2000 therapeutic protocol. We report a small noncoding RNA signature specific of ERG-related group, with up-regulation of miR-125b-2 cluster on chromosome 21 and several snoRNAs in the Prader-Willi locus at 15q11.2, including the orphan SNORD116 cluster.

Williams syndrome (WBS) is a rare neurodevelopmental disorder with specific phenotypic characteristics and cardiac abnormalities, but is not considered as a cancer predisposing condition. However, in rare cases, malignancies have been described in patients with WBS, with hematologic cancer (mainly Burkitt Lymphoma and Acute Lymphoblastic Leukemia) as the most represented. We report here the case of a boy with WS and B-NHL. This is the unique case within the large cohort of patients (n = 117) followed in our institution for long time (mean clinical follow-up, 13 years). We herewith propose that the BCL7B gene, located in the chromosomal region commonly deleted in Williams syndrome, could potentially have a role in this particular association.

Introduction: Human herpes virus 8-related lymphoproliferative disorders are a complex and heterogeneous group of entities and some of them are eminently diagnosed by cytopathology. In a routine laboratory, these lesions account for less than 1% of the effusion fluids samples. However, they represent up to 30% of all the lymphoma diagnosis from effusion cytological samples and their consideration in the diagnostic flow chart is mandatory, especially in human immunodeficiency virus-positive patients. Methods: A retrospective series of cytological specimens from cavity effusions (n=605) were analysed. Five human herpes virus 8-related lymphoproliferative processes were recruited. A combination of morphological criteria (enhanced with May-Grünwald Giemsa staining), cell block-based immunocytochemistry and flow cytometry were undertaken for final characterisation. Results: The identification of malignant cells may be difficult. Some specimens are particularly rich, easily leading to suspect a lymphoproliferative process, whereas in other cases, the presence of abundant reactive mesothelial cells, histiocytes, neutrophils, small reactive T and B lymphocytes may obscure the neoplastic process. The biological behaviour may be very heterogeneous and a standardised therapy for these cases is still lacking, although some patients may benefit from antiretroviral therapy in a human immunodeficiency virus setting. Conclusions: The present case series highlights some characteristic findings of these entities to reaffirm useful cytopathological diagnostic criteria, stressing the crucial role of the appropriate technical processing of effusion fluids to obtain the best performances.

Cornelia de Lange syndrome (CdLS) is a rare autosomal-dominant genetic disorder characterised by prenatal and postnatal growth and mental retardation, facial dysmorphism and upper limb abnormalities. Germline mutations of cohesin complex genes SMC1A, SMC3, RAD21 or their regulators NIPBL and HDAC8 have been identified in CdLS as well as somatic mutations in myeloid disorders. We describe the first case of a paediatric patient with CdLS with B-cell precursor Acute Lymphoblastic Leukaemia (ALL). The patient did not show any unusual cytogenetic abnormality, and he was enrolled into the high risk arm of AIEOP-BFM ALL2009 protocol because of slow early response, but 3 years after discontinuation, he experienced an ALL relapse. We identified a heterozygous mutation in exon 46 of NIPBL, causing frameshift and a premature stop codon (RNA-Targeted Next generation Sequencing Analysis). The analysis of the family indicated a de novo origin of this previously not reported deleterious variant. As for somatic cohesin mutations in acute myeloid leukaemia, also this ALL case was not affected by aneuploidy, thus suggesting a major impact of the non-canonical role of NIPBL in gene regulation. A potential biological role of NIPBL in leukaemia has still to be dissected.

© 2019 Ferrata Storti Foundation., B-cell acute lymphoblastic leukemia is the commonest childhood cancer. In infants, B-cell acute lymphoblastic leukemia remains fatal, especially in patients with t(4;11), present in ~80% of cases. The pathogenesis of t(4;11)/KMT2A-AFF1+ (MLL-AF4+) infant B-cell acute lymphoblastic leukemia remains difficult to model, and the pathogenic contribution in cancer of the reciprocal fusions resulting from derivative translocated-chromosomes remains obscure. Here, “multi-layered” genome-wide analyses and validation were performed on a total of 124 de novo cases of infant B-cell acute lymphoblastic leukemia uniformly diagnosed and treated according to the Interfant 99/06 protocol. These patients showed the most silent mutational landscape reported so far for any sequenced pediatric cancer. Recurrent mutations were exclusively found in K-RAS and N-RAS, were subclonal and were frequently lost at relapse, despite a larger number of non-recurrent/non-silent mutations. Unlike non-MLL-rearranged B-cell acute lymphoblastic leukemias, B-cell receptor repertoire analysis revealed minor, non-expanded B-cell clones in t(4;11)+ infant B-cell acute lymphoblastic leukemia, and RNA-sequencing showed transcriptomic similarities between t(4;11)+ infant B-cell acute lymphoblastic leukemias and the most immature human fetal liver hematopoietic stem and progenitor cells, confirming a “pre-VDJ” fetal cellular origin for both t(4;11) and RASmut. The reciprocal fusion AF4-MLL was expressed in only 45% (19/43) of the t(4;11)+ patients, and HOXA cluster genes are exclusively expressed in AF4-MLL-expressing patients. Importantly, AF4-MLL/HOXA-expressing patients had a significantly better 4-year event-free survival (62.4% vs. 11.7%, P=0.001), and overall survival (73.7 vs. 25.2%, P=0.016). AF4-MLL expression retained its prognostic significance when analyzed in a Cox model adjusting for risk stratification according to the Interfant-06 protocol based on age at diagnosis, white blood cell count and response to prednisone. This study has clinical implications for disease outcome and diagnostic risk-stratification of t(4;11)+ infant B-cell acute lymphoblastic leukemia., This work was supported by the European Research Council (CoG-2014- 646903 to PM; and StG-2014-637904 to IV), the Spanish Ministry of Economy and Competitiveness (SAF-SAF2013- 43065 to PM and SAF2016-76758-R to IV), the Asociación Española Contra el Cáncer (AECC-CI-2015), FERO Foundation, and the ISCIII (PI14-01191) to CB. PM/IV also acknowledge financial support from The Obra Social La CaixaFundaciò Josep Carreras, The Inocente Inocente Foundation, Fundación Ramón Areces and The Generalitat de Catalunya (SGR330). IR was supported by a Programme Grant from Bloodwise (LLR 13001) and by the Oxford NIHR Biomedical Centre based at Oxford University Hospitals NHS Trust and University of Oxford. PM is an investigator of the Spanish Cell Therapy cooperative network (TERCEL). AR was supported by a Clinician Scientist Fellowship from Bloodwise (14041).

Background The aim of this study was to assess whether minimal residual disease (MRD) at the end of induction front-line treatment can serve as a surrogate endpoint for event-free survival (EFS) in childhood B-lineage acute lymphoblastic leukemia. Methods The analysis was based on individual data of 4830 patients from two large phase III trials that asked a randomized question on the effect of different corticosteroids (dexamethasone vs prednisone) during induction chemotherapy on EFS. The association between MRD classified in three ordered categories [negative = 0, low positive = (>0 and Results MRD at the end of induction was a poor surrogate for treatment effect on EFS at the trial level, with Rtrial2 = 0.09 (95% confidence interval [CI] = 0.00 to 0.29), whereas at the individual level it was strongly associated with EFS, with an odds ratio of 3.90 (95% CI = 3.35 to 4.44) of failure for patients with higher compared with lower MRD levels. Additional sensitivity and relevant subgroup analyses confirmed these findings at both trial- and patient-level association. Conclusions Although MRD is a robust biomarker highly predictive of outcome for individual patients, clinicians and regulatory bodies should be cautious in using early MRD response in the context of complex multiagent acute lymphoblastic leukemia therapy as an early surrogate endpoint to predict the effect of a randomized treatment intervention on long-term EFS.

Aim: To identify genetic markers associated with vincristine-induced peripheral neuropathy (VIPN) in childhood acute lymphoblastic leukemia. Patients & methods: Whole-exome sequencing data were combined with exome-wide association study to identify predicted-functional germline variants associated with high-grade VIPN. Genotyping was then performed for top-ranked signals (n = 237), followed by validation in independent replication group (n = 405). Results: Minor alleles of rs2781377/SYNE2 (p = 0.01) and rs10513762/MRPL47 (p = 0.01) showed increased risk, whereas that of rs3803357/BAHD1 had a protective effect (p = 0.007). Using a genetic model based on weighted genetic risk scores, an additive effect of combining these loci was observed (p = 0.003). The addition of rs1135989/ACTG1 further enhanced model performance (p = 0.0001). Conclusion: Variants in SYNE2, MRPL47 and BAHD1 genes are putative new risk factors for VIPN in childhood acute lymphoblastic leukemia.

Infusion of patient-derived CD19-specific chimeric antigen receptor (CAR) T cells engineered by viral vectors achieved complete remission and durable response in relapsed and refractory (r/r) B-lineage neoplasms. Here, we expand on those findings by providing a preclinical evaluation of allogeneic non-viral cytokine-induced killer (CIK) cells transfected with the Sleeping Beauty (SB) transposon CD19CAR (CARCIK-CD19). Specifically, thanks to a large-scale 18-day manufacturing process, it was possible to achieve stable CD19CAR expression (62.425 ± 6.399%) and efficient T-cell expansion (23.36 ± 3.00-fold). Frozen/thawed CARCIK-CD19 remained fully functional both in vitro and in an established patient-derived xenograft (PDX) of MLL-ENL rearranged acute lymphoblastic leukemia (ALL). CARCIK-CD19 showed a dose-dependent antitumor response and prolonged persistence in a PDX, bearing the feature of a Philadelphia-like ALL with PAX5/AUTS2 translocation, and in a survival model of lymphoma, achieving complete eradication of disseminated tumors. Finally, the infusion of CARCIK-CD19 proved to be safe and well tolerated in a biodistribution and toxicity model. The infused cells persisted in the hematopoietic and post-injection perfused organs until the end of the study and consisted of CD8+, CD56+, and CAR+ T cells. Overall, these findings provide important implications for non-viral technology and the proof-of-concept that donor-derived CARCIK-CD19 are indeed effective against relapsed ALL, a possibility that will be tested in Phase I/II clinical trials after allogeneic hematopoietic stem-cell transplantation

BACKGROUND: The EsPhALL2004 randomised trial showed a 10% advantage in disease-free survival for short, discontinuous use of imatinib after induction compared with no use of imatinib in patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia receiving Berlin-Frankfurt-Münster chemotherapy and haemopoietic stem-cell transplantation (HSCT). Other contemporary studies showed an advantage from continuous protracted exposure to imatinib, challenging the indications to transplant. The EsPhALL2010 study was designed to assess whether imatinib given from day 15 of induction and continuously throughout chemotherapy led to a different outcome to that obtained in EsPhALL2004, despite decreasing the number of patients having HSCT.METHODS: This prospective, intergroup, open-label, single-arm clinical trial (EsPhALL2010) was done at 11 study groups across Europe, Chile, and Hong Kong. Patients aged 1-17 years with the translocation t(9;22)(q34;q11) who were recruited into national front-line trials for acute lymphoblastic leukaemia were eligible for this trial. Patients with abnormal renal or hepatic function or an active systemic infection were ineligible. Patients received imatinib 300 mg/m2 continuously from day 15 of induction during chemotherapy. Eligibility to HSCT depended on early morphological response and minimal residual disease. Imatinib was recommended throughout the first year after transplant. The co-primary endpoints were event-free survival and overall survival. All analyses were done in the intention-to-treat population. The trial is registered with the European Clinical Trials Database (EudraCT 2004-001647-30) and with ClinicalTrials.gov (NCT00287105) and is completed.FINDINGS: 158 patients were screened for eligibility, of whom 155 were enrolled between Jan 1, 2010, and Dec 31, 2014. 151 (97%) patients achieved first complete remission after induction and four after the consolidation phase, with 102 (66%) patients categorised as good risk and 53 (34%) as poor risk according to EsPhALL risk stratification criteria. 59 (38%) patients had HSCT during their first complete remission. 40 (26%) patients relapsed and 41 (26%) patients died during the study (25 [61%] during complete continuous remission, and 16 [39%] after relapse). The 5-year event-free survival was 57·0% (95% CI 48·5-64·6) and 5-year overall survival was 71·8% (63·5-78·5). 154 serious adverse events were reported in 80 (52%) of 155 patients. The most common toxicity was infection (61 [39%] patients, mostly bacterial); gastrointestinal disorders occurred in ten (6%) patients and osteonecrosis in eight (5%). Serious adverse events occurred mainly during high-risk blocks and delayed intensifications, including 14 fatal events (one in the consolidation phase, six in high-risk blocks, six in first delayed intensification, and one in second delayed intensification).INTERPRETATION: Although HSCT was done in a smaller proportion of patients in EsPhALL2010 than in EsPhALL2004, event-free and overall survival were similar between the two studies. Our data suggest that imatinib given early and continuously with intensive chemotherapy might increase toxicity.FUNDING: Projet Hospitalier de Recherche Clinique-Cancer and Novartis France; Bloodwise and Cancer Research UK; Ministry of Health, Czech Republic.

Relapse remains the leading cause of treatment failure in children with acute lymphoblastic leukaemia (ALL) undergoing allogeneic haematopoietic stem cell transplantation (HSCT). We retrospectively investigated the prognostic role of minimal residual disease (MRD) before and after HSCT in 119 children transplanted in complete remission (CR). MRD was measured by polymerase chain reaction in bone marrow samples collected pre-HSCT and during the first and third trimesters after HSCT (post-HSCT1 and post-HSCT3). The overall event-free survival (EFS) was 50%. The cumulative incidence of relapse and non-relapse mortality was 41% and 9%. Any degree of detectable pre-HSCT MRD was associated with poor outcome: EFS was 39% and 18% in patients with MRD positivity

Aim: We have previously reported an association of dihydrofolate reductase promoter polymorphisms with reduced event-free survival in childhood acute lymphoblastic leukemia (ALL) patients treated with Dana Farber Cancer Institute protocol. Here, we assessed whether these associations are applicable to other protocol, based on different methotrexate doses. Methods: Genotypes for six tag polymorphisms and resulting haplotypes were analyzed for an association with ALL outcome. Results: The association was found with the polymorphisms A-680C, A-317G and C-35T in high-risk group patients. Carriers of haplotype *1 had a remarkably higher risk of events compared with noncarriers and a lower probability of event-free survival (21.4 vs 81.3%). Conclusion: The role of DHFR variants in predicting the outcome of childhood ALL extends beyond single-treatment protocol and can be useful biomarker in personalizing treatment.

Purpose Somatic deletions that affect the lymphoid transcription factor–coding gene IKZF1 have previously been reported as independently associated with a poor prognosis in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). We have now refined the prognostic strength of IKZF1 deletions by analyzing the effect of co-occurring deletions. Patients and Methods The analysis involved 991 patients with BCP ALL treated in the Associazione Italiana Ematologia ed Oncologia Pediatrica–Berlin-Frankfurt-Muenster (AIEOP-BFM) ALL 2000 trial with complete information for copy number alterations of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, Xp22.33/Yp11.31 (PAR1 region; CRLF2, CSF2RA, and IL3RA), and ERG; replication of findings involved 417 patients from the same trial. Results IKZF1 deletions that co-occurred with deletions in CDKN2A, CDKN2B, PAX5, or PAR1 in the absence of ERG deletion conferred the worst outcome and, consequently, were grouped as IKZF1plus. The IKZF1plus group comprised 6% of patients with BCP ALL, with a 5-year event-free survival of 53 ± 6% compared with 79 ± 5% in patients with IKZF1 deletion who did not fulfill the IKZF1plus definition and 87 ± 1% in patients who lacked an IKZF1 deletion ( P ≤ .001). Respective 5-year cumulative relapse incidence rates were 44 ± 6%, 11 ± 4%, and 10 ± 1% ( P ≤ .001). Results were confirmed in the replication cohort, and multivariable analyses demonstrated independence of IKZF1plus. The IKZF1plus prognostic effect differed dramatically in analyses stratified by minimal residual disease (MRD) levels after induction treatment: 5-year event-free survival for MRD standard-risk IKZF1plus patients was 94 ± 5% versus 40 ± 10% in MRD intermediate- and 30 ± 14% in high-risk IKZF1plus patients ( P ≤ .001). Corresponding 5-year cumulative incidence of relapse rates were 6 ± 6%, 60 ± 10%, and 60 ± 17% ( P ≤ .001). Conclusion IKZF1plus describes a new MRD-dependent very-poor prognostic profile in BCP ALL. Because current AIEOP-BFM treatment is largely ineffective for MRD-positive IKZF1plus patients, new experimental treatment approaches will be evaluated in our upcoming trial AIEOP-BFM ALL 2017.