Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

Four Additional Doses of PEG-L-Asparaginase During the Consolidation Phase in the AIEOP-BFM ALL 2009 Protocol Do Not Improve Outcome and Increase Toxicity in High-Risk ALL: Results of a Randomized Study.

PURPOSE: The AIEOP-BFM ALL 2009 protocol included, at the end of the induction phase, a randomized study of patients with high-risk (HR) ALL to investigate if an intensive exposure to pegylated L-asparaginase (PEG-ASNASE, 2,500 IU/sqm once a week × 4) on top of BFM consolidation phase IB allowed us to decrease minimal residual disease (MRD) and improve outcome. PATIENTS AND METHODS: A total of 1,097 patients presented, from June 2010 to February 2017, with one or more of the following HR criteria: KMT2A::AFF1 rearrangement, hypodiploidy, prednisone poor response, poor bone marrow response at day 15 (Flow MRD ≥10%), or no complete remission (CR) at the end of induction. Of them, 809 (85.1%) were randomly assigned to receive (404) or not receive (405) four weekly doses of PEG-ASNASE. RESULTS: By intention to treat (ITT) analysis, there was no significant difference in the proportion of patients with polimerase chain reaction MRD ≥5 × 10-4 at the end of phase IB in the experimental versus control arm (13.9% v 17.0%, P = .25). The 5-year event-free survival (median follow-up 6.3 years) by ITT in the experimental and control arms was 70.4% (2.3) versus 75.0% (2.2; P = .18), and the 5-year overall survival was 81.5% (2.0) versus 84.0% (1.9; P = .25), respectively. The corresponding 5-year cumulative incidence of death in CR was 9.5% (1.5) versus 5.7% (1.2; P = .08), and that of relapse was 17.7% (1.9) versus 17.2% (1.9), respectively (P = .94). Adverse reactions in phase IB occurred in 22.2% and 8.9% of patients in the experimental and control arm, respectively (P < .001). CONCLUSION: Additional PEG-ASNASE in phase IB did not translate into a benefit for decreasing relapse incidence but was associated with higher toxicity. Further improvements with conventional chemotherapy might be difficult in the context of intensive treatment protocols.

High-throughput screening as a drug repurposing strategy for poor outcome subgroups of pediatric B-cell precursor Acute Lymphoblastic Leukemia.

Although a great cure rate has been achieved for pediatric BCP-ALL, approximately 15% of patients do not respond to conventional chemotherapy and experience disease relapse. A major effort to improve the cure rates by treatment intensification would result in an undesirable increase in treatment-related toxicity and mortality, raising the need to identify novel therapeutic approaches. High-throughput (HTP) drug screening enables the profiling of patients' responses in vitro and allows the repurposing of compounds currently used for other diseases, which can be immediately available for clinical application. The aim of this study was to apply HTP drug screening to identify potentially effective compounds for the treatment of pediatric BCP-ALL patients with poor prognosis, such as patients with Down Syndrome (DS) or carrying rearrangements involving PAX5 or KMT2A/MLL genes. Patient-derived Xenografts (PDX) samples from 34 BCP-ALL patients (9 DS CRLF2r, 15 PAX5r, 10 MLLr), 7 human BCP-ALL cell lines and 14 hematopoietic healthy donor samples were screened on a semi-automated HTP drug screening platform using a 174 compound library (FDA/EMA-approved or in preclinical studies). We identified 9 compounds active against BCP-ALL (ABT-199/venetoclax, AUY922/luminespib, dexamethasone, EC144, JQ1, NVP-HSP990, paclitaxel, PF-04929113 and vincristine), but sparing normal cells. Ex vivo validations confirmed that the BCL2 inhibitor venetoclax exerts an anti-leukemic effect against all three ALL subgroups at nanomolar concentrations. Overall, this study points out the benefit of HTP screening application for drug repurposing to allow the identification of effective and clinically translatable therapeutic agents for difficult-to-treat childhood BCP-ALL subgroups.

miR-126 identifies a quiescent and chemo-resistant human B-ALL cell subset that correlates with minimal residual disease.

Complete elimination of B-cell acute lymphoblastic leukemia (B-ALL) by a risk-adapted primary treatment approach remains a clinical key objective, which fails in up to a third of patients. Recent evidence has implicated subpopulations of B-ALL cells with stem-like features in disease persistence. We hypothesized that microRNA-126, a core regulator of hematopoietic and leukemic stem cells, may resolve intratumor heterogeneity in B-ALL and uncover therapy-resistant subpopulations. We exploited patient-derived xenograft (PDX) models with B-ALL cells transduced with a miR-126 reporter allowing the prospective isolation of miR-126(high) cells for their functional and transcriptional characterization. Discrete miR-126(high) populations, often characterized by MIR126 locus demethylation, were identified in 8/9 PDX models and showed increased repopulation potential, in vivo chemotherapy resistance and hallmarks of quiescence, inflammation and stress-response pathway activation. Cells with a miR-126(high) transcriptional profile were identified as distinct disease subpopulations by single-cell RNA sequencing in diagnosis samples from adult and pediatric B-ALL. Expression of miR-126 and locus methylation were tested in several pediatric and adult B-ALL cohorts, which received standardized treatment. High microRNA-126 levels and locus demethylation at diagnosis associate with suboptimal response to induction chemotherapy (MRD > 0.05% at day +33 or MRD+ at day +78).

Impact of Mercaptopurine Metabolites on Disease Outcome in the AIEOP-BFM ALL 2009 Protocol for Acute Lymphoblastic Leukemia.

In the maintenance phase of Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP)- Berlin-Frankfurt-Muenster (BFM) acute lymphoblastic leukemia (ALL) 2009 protocol, mercaptopurine (MP) is given at the planned dose of 50 mg/m2 /day; however, dose adjustments are routinely performed to target patients' white blood cells to the optimal range of 2,000-3,000 cells/µL. Pediatric patients with ALL (n = 290, age: median (1st-3rd quartile): 4.8 (3.0-8.1) years; boys: 56.9%) were enrolled mainly in 4 medium-large Italian pediatric hospitals; 14.1% of patients relapsed after a median (1st-3rd quartile) follow-up time of 4.43 (3.82-5.46) years from maintenance beginning. MP metabolites (thionucleotide (TGN) and methyl-derivatives (MMPN)) were measured in the erythrocytes of 387 blood samples of 200 patients by high performance liquid chromatography with ultraviolet detection. Single-nucleotide polymorphisms (SNPs; (rs1800462, rs1800460, and rs1142345 in TPMT gene, rs116855232 in NUDT15, rs1127354, rs7270101, rs6051702 in ITPA, and rs2413739 in PACSIN2) were characterized by Taqman SNP genotyping assays. Cox proportional hazard models did not show an impact of TGN levels and variability on relapse. In contrast, after multivariate analysis, relapse hazard ratio (HR) increased in children with ALL of the intermediate risk arm compared with those in standard risk arm (3.44, 95% confidence interval (CI), 1.31-9.05, P = 0.012), and in carriers of the PACSIN2 rs2413739 T allele compared with those with the CC genotype (heterozygotes CT: HR, 2.32, 95% CI, 0.90-5.97, P = 0.081; and homozygous TT: HR, 4.14, 95% CI, 1.54-11.11, P = 0.005). Future studies are needed to confirm the lack of impact of TGN levels and variability on relapse in the AIEOP-BFM ALL trials, and to clarify the mechanism of PACSIN2 rs2413739 on outcome.

Incidence and Characteristics of Hypersensitivity Reactions to PEG-asparaginase Observed in 6136 Children With Acute Lymphoblastic Leukemia Enrolled in the AIEOP-BFM ALL 2009 Study Protocol.

The incidence of hypersensitivity reactions (HSRs) to PEG-asparaginase (PEG-ASNase) was evaluated in 6136 children with ALL enrolled in the AIEOP-BFM ALL 2009 study. Patients with B-cell precursor-acute lymphoblastic leukemia (BCP-ALL) were stratified as standard-risk/medium-risk (MR)/high-risk (HR) and those with T-ALL as non-High/HR. PEG-ASNase was administered intravenously at 2500 IU/sqm/dose. All patients received 2 PEG-ASNase doses in induction; thereafter non-HR versus HR patients received 1 versus 6 PEG-ASNase doses, respectively. After the single regular dose of PEG-ASNase at the beginning of delayed intensification, BCP-ALL-MR patients were randomized to receive 9 additional PEG-ASNase doses every 2 weeks (experimental arm [EA]) versus none (standard arm [SA]); HR patients were randomized to receive, in consolidation, 4 weekly PEG-ASNase doses (EA) versus none (SA). The HSR cumulative incidence (CI) was estimated adjusting for competing risks. An HSR occurred in 472 of 6136 (7.7%) patients. T-non- HR/BCP-Standard-Risk, BCP-MR-SA, BCP-MR-EA, HR-SA and HR-EA patients had 1-year-CI-HSR (±SE) rates of 5.2% (0.5), 5.2% (0.5), 4.0% (0.8), 20.2% (1.2), and 6.4% (1.3), respectively. The randomized intensification of PEG-ASNase did not significantly impact on HSR incidence in BCP-MR patients (1-y-CI-HSR 3.8% [0.8] versus 3.2% [0.6] in MR-EA versus MR-SA; P = 0.55), while impacted significantly in HR patients (1-y-CI-HSR 6.4% [1.3] versus 17.9% [1.8] in HR-EA and HR-SA, respectively; P < 0.001). The CI-HSR was comparable among non-HR groups and was not increased by a substantial intensification of PEG-ASNase in the BCP-MR-EA group whilst it was markedly higher in HR-SA than in HR-EA patients, suggesting that, in such a chemotherapy context, a continuous exposure to PEG-ASNase reduces the risk of developing an HSR.

Definition and Prognostic Value of Ph-like and IKZF1plus Status in Children With Down Syndrome and B-cell Precursor Acute Lymphoblastic Leukemia.

Children with Down syndrome have an augmented risk for B-cell acute lymphoblastic leukemia (DS-ALL), which is associated with lower survival than in non-DS-ALL. It is known that cytogenetic abnormalities common in childhood ALL are less frequent in DS-ALL, while other genetic aberrancies (ie, CRLF2 overexpression and IKZF1 deletions) are increased. A possible cause for the lower survival of DS-ALL that we herewith evaluated for the first time was the incidence and prognostic value of the Philadelphia-like (Ph-like) profile and the IKZF1plus pattern. These features have been associated with poor outcome in non-DS ALL and therefore introduced in current therapeutic protocols. Forty-six out of 70 DS-ALL patients treated in Italy from 2000 to 2014 displayed Ph-like signature, mostly characterized by CRLF2 (n = 33) and IKZF1 (n = 16) alterations; only 2 cases were positive for ABL-class or PAX5-fusion genes. Moreover, in an Italian and German joint cohort of 134 DS-ALL patients, we observed 18% patients positive for IKZF1plus feature. Ph-like signature and IKZF1 deletion were associated with poor outcome (cumulative incidence of relapse: 27.7 ± 6.8% versus 13 ± 7%; P = 0.04 and 35.2 ± 8.6% versus 17 ± 3.9%; P = 0.007, respectively), which further worsens when IKZF1 deletion was co-occurring with P2RY8::CRLF2, qualifying for the IKZF1plus definition (13/15 patients had an event of relapse or treatment-related death). Notably, ex vivo drug screening revealed sensitivity of IKZF1plus blasts for drugs active against Ph-like ALL such as Birinapant and histone deacetylase inhibitors. We provided data in a large setting of a rare condition (DS-ALL) supporting that these patients, not associated with other high-risk features, need tailored therapeutic strategies.

Effect of two additional doses of intrathecal methotrexate during induction therapy on serious infectious toxicity in pediatric patients with acute lymphoblastic leukemia.

Although initial central nervous system (CNS) involvement is rarely detected in childhood acute lymphoblastic leukemia (ALL), risk-adapted CNS-directed therapy is essential for all patients. Treatment intensity depends on the initial CNS status. In the AIEOP-BFM ALL 2009 trial, patients with cytomorphologic detection of leukemic blasts in initial cerebrospinal fluid were classified as CNS2 or CNS3 and received five intrathecal doses of methotrexate (MTX) in induction therapy compared to patients with CNS1 status (no blasts detected) who received three doses. The impact of additional intrathecal (IT) MTX on systemic toxicity in induction therapy is unknown. Between June 1st 2010 and February 28th 2017, a total of 6,136 ALL patients aged 1-17 years were enrolled onto the AIEOP-BFM ALL 2009 trial. The effect of three versus five doses of IT MTX during induction therapy on the incidence of severe infectious complications was analyzed. Among 4,706 patients treated with three IT MTX doses, 77 (1.6%) had a life-threatening infection during induction as compared to 59 of 1,350 (4.4%) patients treated with five doses (P<0.001; Odds Ratio 2.86 [95% Confidence Interval 1.99-4.13]). In a multivariate regression model, treatment with additional IT MTX proved to be the strongest risk factor for life-threatening infections (Odds Ratio 2.85 [1.96-4.14]). Fatal infections occurred in 16 (0.3%) and 38 (1.6%) patients treated with three or five IT MTX doses, respectively (P<0.001). As the relevance of additional intrathecal MTX in induction for relapse prevention in CNS2 patients is unclear, doses of intrathecal therapy have been reduced for these patients. (Clinicaltrials.gov identifiers: NCT01117441 and NCT00613457).

A Hematopoietic Stem Cell Transplantation Startup in Iraqi Kurdistan: Results in Thalassemia Patients and Analysis of the Methodology.

In hemoglobinopathy-prone regions, like the Middle East, thalassemia is the most prevalent noncommunicable life-threatening disorder of children and is highly curable by hematopoietic stem cell transplantation (HSCT). Moreover, transplantation is very cost-effective, and HSCT programs can be established directly in middle-income countries (MICs) at a reduced cost while maintaining quality standards and outcomes consistent with international ones. The aim of the present study was to review and verify the efficacy of the applied methodology through the analysis of 47 consecutive matched-related HSCTs in children with thalassemia. In 2016, the first HSCT unit for adults and children with both malignant and nonmalignant diseases was developed in Iraqi Kurdistan, thanks to a capacity building project funded by the Italian Agency for Development Cooperation. Data on clinical activity were obtained from a cohort of patients treated in the newly established HSCT unit. Primary endpoints were overall survival (OS) and thalassemia-free survival (TFS). Startup of the HSCT unit was completed over a 3-year period. Assessing and meeting minimum requirements were crucial for the startup; moreover, a team of international health care professionals (HCPs), all experts in the field of HSCT, conducted the education and training phase, involving all the clinical and nonclinical professionals in the program. At a median follow-up of 2.6 years, the 3-year TFS and OS were 82.8% (SE, 5.5%) and 87.1% (SE, 4.9%), respectively. TFS and graft-versus-host-disease-free composite survival was 80.6% (SE, 5.8%). At present, the HSCT service is completely autonomous, and more than 250 transplants have been done in both adults and children. The minimal essential requirements for an HSCT startup may be affordable in many MICs. Our results for thalassemia are comparable with international data. A twinning program with an international group of experts and a capacity-building approach is crucial for the success of the program, a strategy that allows for rapid development of HSCT units.

Near-tetraploid T-cell acute lymphoblastic leukaemia in childhood: Results of the AIEOP-BFM ALL studies.

BACKGROUND: Near-tetraploidy-defined by DNA index 1.79-2.28 or 81-103 chromosomes-is a rare cytogenetic abnormality observed both in children and adults with T-cell acute lymphoblastic leukaemia (T-ALL) and its prognostic value is not yet determined. PATIENTS AND METHODS: We report a retrospective study conducted in paediatric patients with newly diagnosed T-ALL treated in AIEOP-BFM ALL 2000 and 2009 studies. 31 near-tetraploid T-ALL patients (1.4%) are compared to T-ALL patients without near-tetraploidy. RESULTS: Near-tetraploid karyotype was associated with lower frequency of high-risk features: white blood cells count at diagnosis ≥100,000/µL (19.3% versus 41.0%, p-value < 0.001), PPR (13.3% versus 35.8%, p-value = 0.01) and minimal residual disease high-risk at the end of consolidation phase Induction B (4.03% versus 14.6%, p-value = 0.001). Complete remission was achieved at the end of induction phase (day 33) in 100% near-tetraploid T-ALL patients, compared to 93.2% T-ALL without near-tetraploidy. CONCLUSION: Overall, we found that near-tetraploid T-ALL in newly diagnosed paediatric patients is associated with low-risk presenting features, with favourable treatment response and outcome.

PAX5 fusion genes are frequent in poor risk childhood acute lymphoblastic leukaemia and can be targeted with BIBF1120.

BACKGROUND: Despite intensive risk-based treatment protocols, 15% of paediatric patients with B-Cell Precursor Acute Lymphoblastic Leukaemia (BCP-ALL) experience relapse. There is urgent need of novel strategies to target poor prognosis subgroups, like PAX5 translocated. METHODS: We considered 289 childhood BCP-ALL cases consecutively enrolled in Italy in the AIEOP-BFM ALL2000/R2006 protocols and we performed extensive molecular profiling, integrating gene expression, copy number analyses and fusion genes discovery by target-capture NGS. We developed preclinical strategies to target PAX5 fusion genes. FINDINGS: We identified 135 cases without recurrent genetic rearrangements. Among them, 59 patients (43·7%) had a Ph-like signature; the remaining cases were identified as ERG-related (26%), High-Hyperdiploid-like (17%), ETV6::RUNX1-like (8·9%), MEF2D-rearranged (2·2%) or KMT2A-like (1·5%). A poor prognosis was associated with the Ph-like signature, independently from other high-risk features. Interestingly, PAX5 was altered in 54·4% of Ph-like compared to 16·2% of non-Ph-like cases, with 7 patients carrying PAX5 fusions (PAX5t), involving either novel (ALDH18A1, IKZF1, CDH13) or known (FBRSL1, AUTS2, DACH2) partner genes. PAX5t cases have a specific driver activity signature, extending to multiple pathways including LCK hyperactivation. Among FDA-approved drugs and inhibitors, we selected Dasatinib, Bosutinib and Foretinib, in addition to Nintedanib, known to be LCK ligands. We demonstrated the efficacy of the LCK-inhibitor BIBF1120/Nintedanib, as single agent or in combination with conventional chemotherapy, both ex vivo and in patient-derived xenograft model, showing a synergistic effect with dexamethasone. INTERPRETATION: This study provides new insights in high-risk Ph-like leukaemia and identifies a potential therapy for targeting PAX5-fusion poor risk group. FUNDING: Ricerca Finalizzata-Giovani Ricercatori (Italian Ministry of Health), AIRC, Transcall, Fondazione Cariparo.

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

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

Pharmacological and clinical monitoring in children with acute lymphoblastic leukemia treated with a biogeneric PEG-l-asparaginase product.

BACKGROUND: l-Asparaginase (ASP) plays a crucial role in the treatment of childhood acute lymphoblastic leukemia (ALL). Currently, different ASP products are available in the market, including both native and pegylated drugs. Several biogeneric Escherichia coli ASP (GEN-ASP) products have been developed in response to shortages and expensiveness of the native E. coli ASP innovator compounds, but some concerns have been raised about their quality. Recently, a number of generic pegylated ASP products (GEN-PEG-ASP) have been marketed to substitute for the innovator product (PEG-ASP). METHODS: Clinical courses and serum asparaginase activity (SAA) levels were monitored in 12 children with ALL, who were treated in our institution with two doses of a GEN-PEG-ASP product, given IV at 2500 IU/m2 during the remission induction phase. Results were compared with those obtained in a reference cohort of 35 patients treated in our institution, who received the innovator PEG-ASP product at same dosage and within the same chemotherapy background. RESULTS: Compared to the reference cohort treated with PEG-ASP, SAA levels were significantly lower in the 12 patients receiving GEN-PEG-ASP (p < .0001); a higher proportion of ASP-associated hypersensitivity reactions (2/12 vs. 0/35; p = .061) and silent inactivation (3/12 vs. 0/35; p = .014) were observed in comparison with the reference cohort. CONCLUSIONS: Our results highlighted different pharmacological profiles and different rates of hypersensitivity reactions and silent inactivation in the GEN-PEG-ASP cohort compared to those treated with the innovator product. Our findings suggest that a rigorous clinical attention and a thorough pharmacological monitoring are advisable in patients treated with GEN-PEG-ASP products.

FLT3-ITD in Children with Early T-cell Precursor (ETP) Acute Lymphoblastic Leukemia: Incidence and Potential Target for Monitoring Minimal Residual Disease (MRD).

Early T-cell precursor (ETP) is an aggressive form of acute lymphoblastic leukemia (ALL), associated with high risk of relapse. This leukemia subtype shows a higher prevalence of mutations, typically associated with acute myeloid leukemia (AML), including RAS and FLT3 mutations. FLT3-ITD was identified in 35% cases of adult ETP-ALL, but data in the pediatric counterpart are lacking. ETPs frequently lack immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, used for minimal residual disease (MRD) monitoring. Among 718 T-ALL enrolled in Italy into AIEOP-BFM-ALL2000, AIEOP-ALLR2006, and AIEOP-BFM-ALL2009 consecutive protocols, 86 patients (12%) were identified as ETP and 77 out of 86 children were studied for the presence of FLT3-ITD. A total of 10 out of 77 (13%) ETP cases were FLT3-ITD positive. IG/TR MRD monitoring was feasible only in four cases. FLT3-ITD MRD monitoring was performed using real-time PCR in all FLT3-ITD positive ETP cases. A comparison between IG/TR and FLT3-ITD resulted in comparable findings. Our study demonstrated that the FLT3-ITD prevalence in children was lower (13%) than that reported in adult ETP-ALL. FLT3-ITD can be used as a marker for sensitive molecular MRD monitoring in ETP-ALL when IG/TR markers are not available, potentially selecting those patients who should spare allogeneic hematopoietic stem cell transplantation (HSCT). Finally, the FLT3 pathway is a robust druggable target in this aggressive form of leukemia.

Osteonecrosis in paediatric acute lymphoblastic leukaemia: Incidence, risk factors, radiological patterns and evolution in a single-centre cohort.

Osteonecrosis (ON) is a well-known sequela of paediatric acute lymphoblastic leukaemia (ALL) treatment. Incidence differs substantially among studies and the clinical significance of radiological findings is not fully established. We analysed 256 consecutive patients with ALL treated in our Institution between October 2010 and December 2016. Within the cohort, 41 developed ON, with a mean 5-year cumulative incidence of 18.5 (standard error, SE, 5.7)% overall. The mean (SE) 5-year cumulative incidence of ON was 12.7 (2.1)% after censoring upon stem cell transplantation (SCT) and/or relapse. Patients aged ≥10 years and patients allocated to the high-risk stratum had a 10-fold and fivefold higher risk of ON respectively. The risk of ON was more than double in relapsed patients, whereas no significant impact of gender, immunophenotype and SCT was demonstrated. Multiple lesions (median four joints involved per patient) were detected by magnetic resonance imaging in all but one patient, with the knee being the most affected joint. Lesions affecting convex joint surfaces experienced the worst evolution, whereas most lesions on diaphyses and concave surfaces remained radiologically stable or disappeared during follow-up. ON has a high prevalence in paediatric ALL, presenting with multiple lesions. Lesions involving convex surfaces were at higher risk of radiological deterioration.

Monocyte-macrophage polarization and recruitment pathways in the tumour microenvironment of B-cell acute lymphoblastic leukaemia.

B-cell acute lymphoblastic leukaemia (B-ALL) reprograms the surrounding bone marrow (BM) stroma to create a leukaemia-supportive niche. To elucidate the contribution of immune cells to the leukaemic microenvironment, we investigated the involvement of monocyte/macrophage compartments, as well as several recruitment pathways in B-ALL development. Immunohistochemistry analyses showed that CD68-expressing macrophages were increased in leukaemic BM biopsies, compared to controls and predominantly expressed the M2-like markers CD163 and CD206. Furthermore, the "non-classical" CD14+ CD16++ monocyte subset, expressing high CX3CR1 levels, was significantly increased in B-ALL patients' peripheral blood. CX3CL1 was shown to be significantly upregulated in leukaemic BM plasma, thus providing an altered migratory pathway possibly guiding NC monocyte recruitment into the BM. Additionally, the monocyte/macrophage chemoattractant chemokine ligand 2 (CCL2) strongly increased in leukaemic BM plasma, possibly because of the interaction of leukaemic cells with mesenchymal stromal cells and vascular cells and due to a stimulatory effect of leukaemia-related inflammatory mediators. C5a, a macrophage chemoattractant and M2-polarizing factor, further appeared to be upregulated in the leukaemic BM, possibly as an effect of PTX3 decrease, that could unleash complement cascade activation. Overall, deregulated monocyte/macrophage compartments are part of the extensive BM microenvironment remodelling at B-ALL diagnosis and could represent valuable targets for novel treatments to be coupled with classical chemotherapy.

Droplet Digital PCR Improves IG-/TR-based MRD Risk Definition in Childhood B-cell Precursor Acute Lymphoblastic Leukemia.

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.

Primary immunodeficiencies, autoimmune hyperthyroidism, coeliac disease and systemic lupus erythematosus in childhood immune thrombocytopenia.

AIM: To evaluate the cumulative prevalence of coeliac disease, systemic lupus erythematosus, autoimmune hyperthyroidism and primary immunodeficiencies in children with either newly diagnosed/persistent or chronic immune thrombocytopenia (ITP). METHODS: Monocentric retrospective analysis of the clinical and biochemical features of 330 consecutive patients with ITP referred to our Pediatric Hematology Unit between January 2009 and December 2018. RESULTS: The prevalence of systemic lupus erythematosus (0.3%), coeliac disease (0.3%) and autoimmune hyperthyroidism (0.6%) was not increased compared to general paediatric population. Of note, the prevalence of underlying primary immunodeficiencies was 2.4%, remarkably higher than the general paediatric population (P = .005). All the patients diagnosed with immunodeficiency developed either bi-/trilinear cytopenia or splenomegaly. CONCLUSION: Whilst autoimmune and immunological screening is already recommended at the onset of immune thrombocytopenia, we recommend that primary immunodeficiencies be regularly screened during follow-up, especially in case of additional cytopenia or lymphoproliferation.