IKZF1 gene deletions drive resistance to cytarabine in B-cell precursor acute lymphoblastic leukemia.

IKZF1-deletions occur in 10-15% of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and predict a poor outcome. However, the impact of IKZF1-loss on sensitivity to drugs used in contemporary treatment protocols has remained underexplored. Here we show in experimental models and in patients that loss of IKZF1 promotes resistance to AraC, a key component of both upfront and relapsed treatment protocols. We attribute this resistance, in part, to diminished import and incorporation of cytarabine (AraC) due to reduced expression of the solute carrier hENT1. Moreover, we find elevated mRNA expression of Evi1, a known driver of therapy resistance in myeloid malignancies. Finally, a kinase directed CRISPR/Cas9-screen identified that inhibition of either mediator kinases CDK8/19 or casein kinase 2 can restore response to AraC. We conclude that this high-risk patient group could benefit from alternative antimetabolites, or targeted therapies that resensitize the cells to AraC.

Continuous PEGasparaginase Dosing Reduces Hypersensitivity Reactions in Pediatric ALL: A Dutch Childhood Oncology Group ALL11 Randomized Trial.

PURPOSE: The primary objective of this randomized study was to determine whether a continuous dosing schedule (without the asparaginase-free interval) would result in less hypersensitivity reactions to PEGasparaginase (PEGasp) compared with the standard noncontinuous dosing schedule. METHODS: Eight hundred eighteen patients (age 1-18 years) with ALL were enrolled in the Dutch Childhood Oncology Group-ALL11 protocol and received PEGasp. Three hundred twelve patients stratified in the medium-risk arm were randomly assigned to receive 14 individualized PEGasp doses once every two weeks in either a noncontinuous or continuous schedule after the first three doses in induction (EudraCT: 2012-000067-25). Hypersensitivity reactions were defined as allergies, allergic-like reactions, and silent inactivation. Secondary end points were other asparaginase-related toxicities, asparaginase activity and antibody levels, and outcome. RESULTS: During induction, 27 of 818 patients (3.3%) experienced hypersensitivity reactions. After random assignment, 4 of 155 (2.6%) in the continuous treatment arm versus 17 of 157 (10.8%) patients in the noncontinuous treatment arm had hypersensitivity reactions (P < .01), of which two (1.3%) versus 13 (8.3%) were inactivating reactions (P < .01). The occurrence of inactivating hypersensitivity reactions was seven times lower in the continuous arm (odds ratio, 0.15 [0.032-0.653]). In addition, antibody levels were significantly lower in the continuous arm (P < .01). With exception of a lower incidence of increased amylase in the continuous arm, there were no significant differences in total number of asparaginase-associated toxicities between arms. However, the timing of the toxicities was associated with the timing of the asparaginase administrations. No difference in 5-year cumulative incidence of relapse, death, or disease-free survival was found between both treatment arms. CONCLUSION: A continuous dosing schedule of PEGasp is an effective approach to prevent antibody formation and inactivating hypersensitivity reactions. The continuous PEGasp schedule did not increase toxicity and did not affect the efficacy of the therapy.

Improved Outcome for ALL by Prolonging Therapy for IKZF1 Deletion and Decreasing Therapy for Other Risk Groups.

PURPOSE: The ALL10 protocol improved outcomes for children with ALL by stratifying and adapting therapy into three minimal residual disease-defined risk groups: standard risk, medium risk (MR), and high risk. IKZF1-deleted (IKZF1del) ALL in the largest MR group still showed poor outcome, in line with protocols worldwide, accounting for a high number of overall relapses. ALL10 showed high toxicity in Down syndrome (DS) and excellent outcome in ETV6::RUNX1 ALL. Poor prednisone responders (PPRs) were treated as high risk in ALL10. In ALL11, we prolonged therapy for IKZF1del from 2 to 3 years. We reduced therapy for DS by omitting anthracyclines completely, for ETV6::RUNX1 in intensification, and for PPR by treatment as MR. METHODS: Eight hundred nineteen patients with ALL (age, 1-18 years) were enrolled on ALL11 and stratified as in ALL10. Results were compared with those in ALL10. RESULTS: The five-year overall survival (OS), event-free survival (EFS), cumulative risk of relapse (CIR), and death in complete remission on ALL11 were 94.2% (SE, 0.9%), 89.0% (1.2), 8.2% (1.1), and 2.3% (0.6), respectively. Prolonged maintenance for IKZF1del MR improved 5-year CIR by 2.2-fold (10.8% v 23.4%; P = .035) and EFS (87.1% v 72.3%; P = .019). Landmark analysis at 2 years from diagnosis showed a 2.9-fold reduction of CIR (25.6%-8.8%; P = .008) and EFS improvement (74.4%-91.2%; P = .007). Reduced therapy did not abrogate 5-year outcome for ETV6::RUNX1 (EFS, 98.3%; OS, 99.4%), DS (EFS, 87.0%; OS, 87.0%), and PPR (EFS, 81.1%; OS, 94.9%). CONCLUSION: Children with IKZF1del ALL seem to benefit from prolonged maintenance therapy. Chemotherapy was successfully reduced for patients with ETV6::RUNX1, DS, and PPR ALL. It has to be noted that these results were obtained in a nonrandomized study using a historical control group.

The Prognostic Effect of IKZF1 Deletions in ETV6::RUNX1 and High Hyperdiploid Childhood Acute Lymphoblastic Leukemia.

IKZF1 deletions are an established prognostic factor in childhood acute lymphoblastic leukemia (ALL). However, their relevance in patients with good risk genetics, namely ETV6::RUNX1 and high hyperdiploid (HeH), ALL remains unclear. We assessed the prognostic impact of IKZF1 deletions in 939 ETV6::RUNX1 and 968 HeH ALL patients by evaluating data from 16 trials from 9 study groups. Only 3% of ETV6::RUNX1 cases (n = 26) were IKZF1-deleted; this adversely affected survival combining all trials (5-year event-free survival [EFS], 79% versus 92%; P = 0.02). No relapses occurred among the 14 patients with an IKZF1 deletion treated on a minimal residual disease (MRD)-guided protocols. Nine percent of HeH cases (n = 85) had an IKZF1 deletion; this adversely affected survival in all trials (5-year EFS, 76% versus 89%; P = 0.006) and in MRD-guided protocols (73% versus 88%; P = 0.004). HeH cases with an IKZF1 deletion had significantly higher end of induction MRD values (P = 0.03). Multivariate Cox regression showed that IKZF1 deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (hazard ratio of relapse rate [95% confidence interval]: 2.48 [1.32-4.66]). There was no evidence to suggest that IKZF1 deletions affected outcome in the small number of ETV6::RUNX1 cases in MRD-guided protocols but that they are related to higher MRD values, higher relapse, and lower survival rates in HeH ALL. Future trials are needed to study whether stratifying by MRD is adequate for HeH patients or additional risk stratification is necessary.

Immune Response following BNT162b2 mRNA COVID-19 Vaccination in Pediatric Cancer Patients.

COVID-19 vaccinations are recommended for children with cancer but data on their vaccination response is scarce. This study assesses the antibody and T-cell response following a 2- or 3-dose vaccination with BNT162b2 mRNA COVID-19 vaccine in children (5-17 years) with cancer. For the antibody response, participants with a serum concentration of anti-SARS-CoV-2 spike 1 antibodies of >300 binding antibody units per milliliter were classified as good responders. For the T-cell response, categorization was based on spike S1 specific interferon-gamma release with good responders having >200 milli-international units per milliliter. The patients were categorized as being treated with chemo/immunotherapy for less than 6 weeks (Tx < 6 weeks) or more than 6 weeks (Tx > 6 weeks) before the first immunization event. In 46 patients given a 2-dose vaccination series, the percentage of good antibody and good T-cell responders was 39.3% and 73.7% in patients with Tx < 6 weeks and 94.4% and 100% in patients with Tx > 6 weeks, respectively. An additional 3rd vaccination in 16 patients with Tx < 6 weeks, increased the percentage of good antibody responders to 70% with no change in T-cell response. A 3-dose vaccination series effectively boosted antibody levels and is of value for patients undergoing active cancer treatment.

Outcome of chimeric antigen receptor T-cell therapy following treatment with inotuzumab ozogamicin in children with relapsed or refractory acute lymphoblastic leukemia.

Chimeric antigen receptor T cells targeting CD19 (CART-19) have shown remarkable efficacy for relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We investigated whether prior use of inotuzumab ozogamicin (InO), an anti-CD22 antibody conjugated to calicheamicin, may impact CAR T-cell manufacturing or efficacy via pre-CART-19 depletion of the B-cell compartment. In this international, retrospective analysis, 39 children and young adults receiving InO before (n = 12) and/or after (n = 27) T-cell apheresis as bridging therapy to CART-19 treatment were analyzed. Median age at infusion was 13 years (range 1.4-23 years). Thirty-four out of 39 patients (87.2%) obtained complete remission. With a median follow-up of 18.2 months after CART-19 infusion, 12-month event-free survival (EFS) was 53.3% (95% confidence interval (CI): 38.7-73.4) and overall survival (OS) was 77.8% (95% CI: 64.5-93.9). Seventeen patients (44%) relapsed with a median of 159 days (range 28-655) after CART-19 infusion. No difference in day 28 minimal residual disease negative complete response rate, 12-month OS/EFS, or incidence of CD19-positive or -negative relapses was observed among patients receiving InO before or after apheresis. Compared to published data for patients treated with CART-19 therapy without prior InO exposure, response and OS/EFS for patients treated with InO prior to CART-19 are similar.

Fludarabine exposure predicts outcome after CD19 CAR T-cell therapy in children and young adults with acute leukemia.

The addition of fludarabine to cyclophosphamide as a lymphodepleting regimen prior to CD19 chimeric antigen receptor (CAR) T-cell therapy significantly improved outcomes in patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). Fludarabine exposure, previously shown to be highly variable when dosing is based on body surface area (BSA), is a predictor for survival in allogeneic hematopoietic cell transplantation (allo-HCT). Hence, we hypothesized that an optimal exposure of fludarabine might be of clinical importance in CD19 CAR T-cell treatment. We examined the effect of cumulative fludarabine exposure during lymphodepletion, defined as concentration-time curve (AUC), on clinical outcome and lymphocyte kinetics. A retrospective analysis was conducted with data from 26 patients receiving tisagenlecleucel for r/r B-ALL. Exposure of fludarabine was shown to be a predictor for leukemia-free survival (LFS), B-cell aplasia, and CD19-positive relapse following CAR T-cell infusion. Minimal event probability was observed at a cumulative fludarabine AUCT0-∞ ≥14 mg*h/L, and underexposure was defined as an AUCT0-∞ <14 mg*h/L. In the underexposed group, the median LFS was 1.8 months, and the occurrence of CD19-positive relapse within 1 year was 100%, which was higher compared with the group with an AUCT0-∞ ≥14 mg*h/L (12.9 months; P < .001; and 27.4%; P = .0001, respectively). Furthermore, the duration of B-cell aplasia within 6 months was shorter in the underexposed group (77.3% vs 37.3%; P = .009). These results suggest that optimizing fludarabine exposure may have a relevant impact on LFS following CAR T-cell therapy, which needs to be validated in a prospective clinical trial.

Clonal dynamics in pediatric B-cell precursor acute lymphoblastic leukemia with very early relapse.

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.

Risk factors and outcomes in children with high-risk B-cell precursor and T-cell relapsed acute lymphoblastic leukaemia: combined analysis of ALLR3 and ALL-REZ BFM 2002 clinical trials.

AIM: Outcomes of children with high-risk (HR) relapsed acute lymphoblastic leukaemia (ALL) (N = 393), recruited to ALLR3 and ALL-REZ BFM 2002 trials, were analysed. Minimal residual disease (MRD) was assessed after induction and at predetermined time points until haematopoietic stem cell transplantation (SCT). METHODS: Genetic analyses included karyotype, copy-number alterations and mutation analyses. Ten-year survivals were analysed using Kaplan-Meier and Cox models for multivariable analyses. RESULTS: Outcomes of patients were comparable in ALLR3 and ALL-REZ BFM 2002. The event-free survival of B-cell precursor (BCP) and T-cell ALL (T-ALL) was 22.6% and 26.2% (P = 0.94), respectively, and the overall survival (OS) was 32.6% and 28.2% (P = 0.11), respectively. Induction failures (38%) were associated with deletions of NR3C1 (P = 0.002) and BTG1 (P = 0.03) in BCP-ALL. The disease-free survival (DFS) and OS in patients with good vs poor MRD responses were 57.4% vs 22.6% (P < 0.0001) and 57.8% vs 32.0% (P = 0.0004), respectively. For BCP- and T-ALL, the post-SCT DFS and OS were 42.1% and 56.8% (P = 0.26) and 51.6% and 55.4% (P = 0.67), respectively. The cumulative incidences of post-SCT relapse for BCP- and T-ALL were 36.9% and 17.8% (P = 0.012) and of death were 10.7% and 25.5% (P = 0.013), respectively. Determinants of outcomes after SCT were acute graft versus host disease, pre-SCT MRD (≥10-3), HR cytogenetics and TP53 alterations in BCP-ALL. CONCLUSION: Improvements in outcomes for HR ALL relapses require novel compounds in induction therapy to improve remission rates and immune targeted therapy after induction to maintain remission after SCT. TRIAL REGISTRATION: ALLR3: NCT00967057; ALL REZ-BFM 2002: NCT00114348.

Unravelling the Sequential Interplay of Mutational Mechanisms during Clonal Evolution in Relapsed Pediatric Acute Lymphoblastic Leukemia.

Pediatric acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and is characterized by clonal heterogeneity. Genomic mutations can increase proliferative potential of leukemic cells and cause treatment resistance. However, mechanisms driving mutagenesis and clonal diversification in ALL are not fully understood. In this proof of principle study, we performed whole genome sequencing of two cases with multiple relapses in order to investigate whether groups of mutations separated in time show distinct mutational signatures. Based on mutation allele frequencies at diagnosis and subsequent relapses, we clustered mutations into groups and performed cluster-specific mutational profile analysis and de novo signature extraction. In patient 1, who experienced two relapses, the analysis unraveled a continuous interplay of aberrant activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity. The associated signatures SBS2 and SBS13 were present already at diagnosis, and although emerging mutations were lost in later relapses, the process remained active throughout disease evolution. Patient 2 had three relapses. We identified episodic mutational processes at diagnosis and first relapse leading to mutations resembling ultraviolet light-driven DNA damage, and thiopurine-associated damage at first relapse. In conclusion, our data shows that investigation of mutational processes in clusters separated in time may aid in understanding the mutational mechanisms and discovery of underlying causes.

Multiclonal complexity of pediatric acute lymphoblastic leukemia and the prognostic relevance of subclonal mutations

Genomic studies of pediatric acute lymphoblastic leukemia (ALL) have shown remarkable heterogeneity in initial diagnosis, with multiple (sub)clones harboring lesions in relapse-associated genes. However, the clinical relevance of these subclonal alterations remains unclear. We assessed the clinical relevance and prognostic value of subclonal alterations in the relapse-associated genes IKZF1, CREBBP, KRAS, NRAS, PTPN11, TP53, NT5C2, and WHSC1 in 503 ALL cases. Using Molecular Inversion Probe sequencing and breakpoint-spanning PCR we reliably detected alterations below 1% allele frequency. We identified 660 genomic alterations in 285 diagnosis samples of which 495 (75%) were subclonal. RAS pathway mutations were common, particularly in minor subclones, and comparisons between RAS hotspot mutations revealed differences in their capacity to drive clonal expansion in ALL. We did not find an association of subclonal alterations with unfavorable outcome. Particularly for IKZF1, an established prognostic marker in ALL, all clonal but none of the subclonal alterations were preserved at relapse. We conclude that, for the genes tested, there is no basis to consider subclonal alterations detected at diagnosis for risk group stratification of ALL treatment.

Examining the Potential Relationship Between Multidisciplinary Team Meetings and Patient Survival in Pediatric Oncology Settings: A Systematic Review.

The organization of multidisciplinary team meetings (MTMs) has become standard practice in pediatric oncology and is widely felt to improve communication, knowledge, and patient care. Although the impact of MTMs on survival in adult oncology has been extensively researched, the potential benefits of survival for pediatric cancer patients are still unclear. This systematic review aimed to examine the impact of MTMs on survival in pediatric oncology settings. Relevant studies were identified by searching MEDLINE/PubMed, EMBASE, and the Cochrane Library databases up to January 2020, resulting in 325 unique records. After the title/abstract and full-text screening, 5 studies were included. All of the included studies (one prospective and 4 retrospective cohort studies) described a difference in overall or event-free survival when comparing patients who were discussed in MTMs with non-MTM patients. This association was statistically significant in 3 studies. The quality of the studies was strongly affected by their design. Because of the small number of studies in combination with high clinical and methodological heterogeneity, this review was unable to definitively assert a causal relationship between MTMs and survival in pediatric cancer patients. Further research is needed to explore this relationship and allow cost-benefit analyses, so that time and resources are optimally spent to deliver the best possible care to childhood cancer patients.

Use of quality indicators in neuroblastoma treatment: A feasibility assessment.

BACKGROUND: Quality indicators (QIs) may be used to monitor the quality of neuroblastoma (NBL) care during treatment, in addition to survival and treatment toxicity, which can only be evaluated in the years after treatment. The present study aimed to assess the feasibility of a new set of indicators for the quality of NBL therapy. PROCEDURE: Seven QIs have been proposed based on literature and consensus of experts: (a) duration of complete diagnostic work-up, (b) prescription of thyroid prophylaxis before metaiodobenzylguanidine imaging, (c) treatment intensity, (d) use of tumor board meetings, (e) number of outpatient visits and sedation procedures during follow-up, (f) protocolled follow-up, and (g) required apheresis sessions. A retrospective data analysis from October 2014 to November 2017 including all patients with NBL in the centralized Princess Máxima Center in the Netherlands was performed to assess these parameters and determine practicality of measurement. RESULTS: A total number of 72 patients (aged between 2 weeks and 15 years) were analyzed. Adherence to all QIs could be determined for all eligible patients using their electronic medical records. Three indicators were compared over time, and an increase in adherence was observed. CONCLUSIONS: Assessment of QIs in neuroblastoma treatment is feasible. Seven new QIs were found to be feasible to measure and showed improvement over time for three indicators. Monitoring of these QIs during treatment may provide tools for quality improvement activities and comparisons of treatment quality over time or between centers. Further study is required to investigate their association with long-term outcomes.

Cost-effectiveness of Anti-CD19 chimeric antigen receptor T-Cell therapy in pediatric relapsed/refractory B-cell acute lymphoblastic leukemia. A societal view.

INTRODUCTION: In several studies, the chimeric antigen receptor T-cell therapy tisagenlecleucel demonstrated encouraging rates of remission and lasting survival benefits in pediatric patients with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL). We assessed the cost-effectiveness of tisagenlecleucel (list price: 320 000 EUR) among these patients when compared to clofarabine monotherapy (Clo-M), clofarabine combination therapy (Clo-C), and blinatumomab (Blina) from both a healthcare and a societal perspective. We also assessed future medical and future non-medical consumption costs. METHODS: A three-state partitioned survival model was used to simulate a cohort of pediatric patients (12 years of age) through different disease states until the end of life (lifetime horizon). Relevant outcomes were life years, quality-adjusted life years (QALYs), healthcare costs, societal costs, and the incremental cost-effectiveness ratio (ICER). Uncertainty was explored through deterministic and probabilistic sensitivity analyses as well as through several scenario analyzes. RESULTS: Total discounted costs for tisagenlecleucel were 552 679 EUR from a societal perspective, which was much higher than the total discounted costs from a healthcare perspective (ie, 409 563 EUR). Total discounted societal costs for the comparator regimens ranged between 160 803 EUR for Clo-M and 267 259 EUR for Blina. Highest QALYs were estimated for tisagenlecleucel (11.26), followed by Blina (2.25), Clo-C (1.70) and Clo-M (0.74). Discounted societal ICERs of tisagenlecleucel ranged between 31 682 EUR/QALY for Blina and 37 531 EUR/QALY for Clo-C and were considered cost-effective with a willingness-to-pay (WTP) threshold of 80 000 EUR/QALY. None of the scenarios exceeded this threshold, and more than 98% of the iterations in the probabilistic sensitivity analysis were cost-effective. DISCUSSION: At the current price and WTP threshold, tisagenlecleucel is cost-effective from both a healthcare and a societal perspective. Nevertheless, long-term effectiveness data are needed to validate the several assumptions that were necessary for this model.

Upfront Treatment Influences the Composition of Genetic Alterations in Relapsed Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

Genomic alterations in relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) may provide insight into the role of specific genomic events in relapse development. Along this line, comparisons between the spectrum of alterations in relapses that arise in different upfront treatment protocols may provide valuable information on the association between the tumor genome, protocol components and outcome. Here, we performed a comprehensive characterization of relapsed BCP-ALL cases that developed in the context of 3 completed Dutch upfront studies, ALL8, ALL9, and ALL10. In total, 123 pediatric BCP-ALL relapses and 77 paired samples from primary diagnosis were analyzed for alterations in 22 recurrently affected genes. We found pronounced differences in relapse alterations between the 3 studies. Specifically, CREBBP mutations were observed predominantly in relapses after treatment with ALL8 and ALL10 which, in the latter group, were all detected in medium risk-treated patients. IKZF1 alterations were enriched 2.2-fold (p = 0.01) and 2.9-fold (p < 0.001) in ALL8 and ALL9 relapses compared to diagnosis, respectively, whereas no significant enrichment was found for relapses that were observed after treatment with ALL10. Furthermore, IKZF1 deletions were more frequently preserved from a major clone at diagnosis in relapses after ALL9 compared to relapses after ALL8 and ALL10 (p = 0.03). These data are in line with previous studies showing that the prognostic value of IKZF1 deletions differs between upfront protocols and is particularly strong in the ALL9 regimen. In conclusion, our data reveal a correlation between upfront treatment and the genetic composition of relapsed BCP-ALL.

Anti-GD2 antibody and Vorinostat immunocombination therapy is highly effective in an aggressive orthotopic neuroblastoma model.

Neuroblastoma is a childhood malignancy and in the majority of patients, the primary tumor arises in one of the adrenal glands. Neuroblastoma cells highly express the disialoganglioside GD2, which is the primary target for the development of neuroblastoma immunotherapy. Anti-GD2 mAbs have shown clinical efficacy and are integrated into standard treatment for high-risk neuroblastoma patients. We previously reported synergy between the HDAC inhibitor Vorinostat and anti-GD2 mAbs in a heterotopic, subcutaneous growing neuroblastoma model. Additionally, we have previously developed an orthotopic intra-adrenal neuroblastoma model showing more aggressive tumor growth. Here, we report that anti-GD2 mAb and Vorinostat immunocombination therapy is even more effective in suppressing neuroblastoma growth in the aggressive orthotopic model, resulting in increased animal survival. Intra-adrenal tumors from mice treated with Vorinostat were highly infiltrated with myeloid cells, including macrophages, displaying increased MHCII and Fc-receptor expression. Collectively, these data provide a strong rationale for clinical testing of anti-GD2 mAbs with concomitant Vorinostat in neuroblastoma patients.

Outcomes of patients with childhood B-cell precursor acute lymphoblastic leukaemia with late bone marrow relapses: long-term follow-up of the ALLR3 open-label randomised trial.

BACKGROUND: The ALLR3 trial investigated outcomes of children with B-cell precursor acute lymphoblastic leukaemia who had late bone marrow relapses. We analysed long-term follow-up outcomes of these patients. METHODS: ALLR3 was an open-label randomised clinical trial that recruited children aged 1-18 years with B-cell precursor acute lymphoblastic leukaemia who had late bone marrow relapses. Eligible patients were recruited from centres in Australia, Ireland, the Netherlands, New Zealand, and the UK. Patients were randomly assigned from Jan 31, 2003, to Dec 31, 2007, and the trial closed to recruitment on Oct 31, 2013. Randomly assigned patients were allocated to receive either idarubicin or mitoxantrone in induction by stratified concealed randomisation; after randomisation stopped in Dec 31, 2007, all patients were allocated to receive mitoxantrone. After three blocks of therapy, patients with high minimal residual disease (≥10-4 cells) at the end of induction were allocated to undergo allogeneic stem-cell transplantation and those with low minimal residual disease (<10-4 cells) at the end of induction were allocated to receive chemotherapy. Minimal residual disease level was measured by real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements. The primary endpoint of the original ALLR3 clinical trial was progression-free survival of randomly assigned patients. The primary endpoint of this long-term follow-up analysis was progression-free survival of patients with late bone marrow relapses stratified by minimal residual disease level. Outcomes were correlated with age, site, time to recurrence, and genetic subtypes, and analysed by both intention to treat and actual treatment received. This trial is registered on the ISRCTN registry, number ISRCTN45724312, and on ClinicalTrials.gov, number NCT00967057. FINDINGS: Between Feb 2, 2003, and Oct 28, 2013, 228 patients with B-cell precursor acute lymphoblastic leukaemia and late bone marrow relapses were treated. After a median follow-up of 84 months (IQR 48-109), progression-free survival of all randomly assigned patients was 60% (95% CI 54-70). 220 patients achieved second complete remission, and minimal residual disease was evaluable in 192 (87%). 110 patients with late bone marrow relapses and high minimal residual disease at the end of induction were allocated to undergo stem-cell transplantation, and 82 patients with low minimal residual disease at the end of induction were allocated to receive chemotherapy. In the patients allocated to undergo stem-cell transplantation, four relapses and three deaths were reported before the procedure, and 11 patients were not transplanted. Of the 92 patients transplanted, 58 (63%) remained in second complete remission, 13 (14%) died of complications, and 21 (23%) relapsed after stem-cell transplantation. In patients allocated to receive chemotherapy, one early treatment-related death was reported and 11 patients were transplanted. Of the 70 patients who continued on chemotherapy, 49 (70%) remained in second complete remission, two (3%) died of complications, and 19 (27%) relapsed. Progression-free survival at 5 years was 56% (95% CI 46-65) in those with high minimal residual disease and 72% (60-81) in patients with low minimal residual disease (p=0·0078). Treatment-related serious adverse events were not analysed in the long-term follow-up. INTERPRETATION: Patients with B-cell precursor acute lymphoblastic leukaemia with late bone marrow relapses and low minimal residual disease at end of induction had favourable outcomes with chemotherapy without undergoing stem-cell transplantation. Patients with high minimal residual disease benefited from stem-cell transplantation, and targeted therapies might offer further improvements in outcomes for these patients. FUNDING: Bloodwise (Formerly Leukaemia and Lymphoma Research) UK, Cancer Research UK, Sporting Chance Cancer Foundation, National Health and Medical Research Council Australia, KindreneKankervrij Netherlands, European Union Seventh Framework Programme, India Alliance Wellcome DBT Margdarshi Fellowship.

Combined sialic acid and histone deacetylase (HDAC) inhibitor treatment up-regulates the neuroblastoma antigen GD2.

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is only sparsely expressed on healthy tissue. GD2 is a primary target for the development of immunotherapy for neuroblastoma. Immunotherapy with monoclonal anti-GD2 antibodies has proven safety and efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. Strategies to modulate GD2 expression in neuroblastoma could further improve anti-GD2-targeted immunotherapy. Here, we report that the cellular sialylation pathway, as well as epigenetic reprogramming, strongly modulates GD2 expression in human and mouse neuroblastoma cell lines. Recognition of GD2 by the 14G2a antibody is sialic acid-dependent and was blocked with the fluorinated sialic acid mimetic Ac53FaxNeu5Ac. Interestingly, sialic acid supplementation using a cell-permeable sialic acid analogue (Ac5Neu5Ac) boosted GD2 expression without or with minor alterations in overall cell surface sialylation. Furthermore, sialic acid supplementation with Ac5Neu5Ac combined with various histone deacetylase (HDAC) inhibitors, including vorinostat, enhanced GD2 expression in neuroblastoma cells beyond their individual effects. Mechanistic studies revealed that Ac5Neu5Ac supplementation increased intracellular CMP-Neu5Ac concentrations, thereby providing higher substrate levels for sialyltransferases. Furthermore, HDAC inhibitor treatment increased mRNA expression of the sialyltransferases GM3 synthase (ST3GAL5) and GD3 synthase (ST8SIA1), both of which are involved in GD2 biosynthesis. Our findings reveal that sialic acid analogues and HDAC inhibitors enhance GD2 expression and could potentially be employed to boost anti-GD2 targeted immunotherapy in neuroblastoma patients.

Tumor suppressor BTG1 limits activation of BCL6 expression downstream of ETV6-RUNX1.

Translocation t(12;21) (p13;q22), giving rise to the ETV6-RUNX1 fusion gene, is the most common genetic abnormality in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation usually arises in utero, but its expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop. Deletions affecting the transcriptional coregulator BTG1 are frequently observed in ETV6-RUNX1-positive leukemia. Here we report that Btg1 deficiency enhances the self-renewal capacity of ETV6-RUNX1-positive mouse fetal liver-derived hematopoietic progenitors (FL-HPCs). Combined expression of the fusion protein and a loss of BTG1 drive upregulation of the proto-oncogene Bcl6 and downregulation of BCL6 target genes, such as p19Arf and Tp53. Similarly, ectopic expression of BCL6 promotes the self-renewal and clonogenic replating capacity of FL-HPCs, by suppressing the expression of p19Arf and Tp53. Together these results identify BCL6 as a potential driver of ETV6-RUNX1-mediated leukemogenesis, which could involve loss of BTG1-dependent suppression of ETV6-RUNX1 function.