PAX5-altered acute lymphoblastic leukemia (PAX5-alt ALL) is a recently recognized molecular subtype of B-ALL characterized by a distinct transcriptional signature and frequent PAX5 fusions (PAX5-r). While PAX5-alt ALL has been associated with intermediate outcomes, clinical data specifically investigating pediatric PAX5-r ALL remain limited. We collected and analyzed 159 pediatric cases of PAX5-r ALL treated between 2001-2024 within AIEOP-BFM ALL studies across Italy, Germany and Austria, revealing high-risk clinical features. Comparative analyses of patients consecutively enrolled in the AIEOP-BFM ALL 2017 study (PAX5-r, n=96 vs. non-PAX5-r, n=1948) confirmed higher rates of hyperleukocytosis at diagnosis (22.9% vs. 8%, p<0.001) and enrichment of IKZF1plus profile (14.7% vs. 7.8%, p=0.0015) in PAX5-r patients. No relevant differences in terms of minimal/measurable residual disease (MRD)-based treatment response and risk-group stratification were observed. PAX5-r patients had a 4-year EFS and OS of 72.6±5.7% and 95.4±2.3%, respectively. Four-year EFS were 100%, 63.4±9.7% and 63.3±10% for standard-risk, medium-risk (MR) and high-risk (HR) groups, respectively, indicating that the poor prognostic impact of PAX5-r applies only when end-of-induction MRD is positive (MR/HR). Whole transcriptome sequencing revealed high FLT3 median expression in PAX5-r ALL and high-throughput drug screening of patient-derived xenografts (PDXs) showed marked sensitivity to several FLT3 inhibitors. Gilteritinib showed potent ex vivo cytotoxicity and synergism with dexamethasone in PAX5-r PDX models. Collectively, this study investigated clinical and biological features of pediatric PAX5-r ALL highlighting its MRD-dependent unfavorable prognosis and identifying FLT3 overexpression as a novel, potential therapeutic target.

Background Targeted therapies and immunotherapies are increasingly used in pediatric oncology; however, many innovative agents are prescribed off-label or through compassionate use programs due to limited pediatric approvals and restricted access to clinical trials. Real-world data on these access pathways remain limited. Methods This retrospective multicenter observational study was promoted by the Italian Association of Pediatric Hematology and Oncology (AIEOP) Working Group on New Drugs. Prescriptions made between 2018 and 2023 for patients aged ≤25 years receiving targeted therapies or immunotherapies through off-label or compassionate use were collected from 19 AIEOP pediatric oncology centers, covering approximately 80% of national pediatric cancer cases. Variables included diagnosis, indication, access modality, regulatory status, molecular rationale, treatment duration, discontinuation. Results A total of 562 prescriptions involving 70 drugs were collected. Off-label prescriptions accounted for 77% (n=431) and compassionate use for 23% (n=131). Treatments were mainly for relapsed (46%) or refractory (35%) disease. The most frequent diagnoses were acute lymphoblastic leukemia (n=119, 21%) and gliomas (n=76, 14%). Molecular alterations supporting treatment selection were reported in a subset, including FLT3, BCR::ABL1, BRAF, and MAPK alterations. Median treatment duration was 133 days (interquartile range (IQR), 42–399). Discontinuation was mainly due to disease progression (n=216, 47%), completion of planned therapy (n=175, 38%), or toxicity (n=33, 7%). Lack of clinical trials was the main driver for access (n=438, 78%). Median authorization time was 9.5 days (IQR, 5–15)., with bureaucratic complexity as the main barrier. Conclusions Off-label and compassionate use represented a common access pathway for innovative therapies in Italian pediatric oncology, particularly in relapsed or refractory disease. These findings highlight persistent challenges in pediatric drug development, clinical trial availability, and regulatory pathways.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, with most cases arising from B-cell precursors expressing the CD19 marker. CD19 negativity in B-lineage ALL (B-ALL) is very rare de novo and poses diagnostic and therapeutic challenges. Sometimes, de novo CD19-negative B-ALL is associated with hypercalcemia, which is a potentially life-threatening metabolic disorder in children, rarely occurring in cancers. Most often it is reported in solid tumors, and few cases are reported in pediatric acute leukemia. CD19-negative B-ALL relapse is also an increasing dramatic event, secondary to immunotherapy. We describe a ten-month-old infant presenting with hypercalcemia, anemia, and osteolytic bone lesions. Bone marrow analysis revealed CD10-positive and CD19-negative B-ALL. The patient achieved complete remission but later experienced two relapses and died of respiratory failure after a second allogeneic hematopoietic stem cell transplantation (HSCT). Only nine cases of de novo CD19-negative B-ALL have been reported so far. Many are associated with hypercalcemia and osteolytic lesions. However, here we highlight the clinical impact of the more common secondary form of CD19-negative B-ALL as a relapse of CD19-positive ALL, right after the administration of targeted immunotherapy.

The human microbiota, comprising bacteria, fungi, and viruses, primarily resides in the gut, skin, and oral and nasal cavities. The gut microbiota represents the largest and most functionally relevant microbial community, playing a crucial role in digestion, metabolism and immunity. Microbiota composition and diversity have been associated with several diseases such as cancer, affecting drug efficacy, toxicity and clinical outcome. Interactions between the gut microbiota and the host immune system may contribute to the aetiology and progression of childhood acute lymphoblastic leukaemia (ALL). In this review, we summarized the general features of the gut microbiota in cancer patients and its specific role in aetiology, treatment and prognosis of paediatric ALL. Profiling the gut microbiota may help to shed the light on the mechanism of onset and to support the development of personalized treatment strategies and follow-up in children with ALL, contributing to precision medicine.

The previous Italian national trial (AIEOP-AML-2002/01) on children with acute myeloid leukemia (AML) achieved a 3-year overall (OS) and event-free survival (EFS) of 72.3

We report a case of multiple lineage switches (rare events mostly occurring in infants) in a 10-year-old female with KMT2A::AFF1-positive acute lymphoblastic leukemia (ALL). Although she underwent standard chemotherapy, immunotherapy, and hematopoietic stem cell transplantation, the leukemic clone continuously switched on acute myeloid leukemia (AML) or ALL and maintained immunoglobulin/T-cell receptor rearrangements in addition to showing a high therapy-escaping grade. As other genes are involved in this event, and after considering the selective pressure imposed by the applied treatment, characterizing the cell-of-origin that is capable of proliferating toward both lineages while attempting to design targeted therapies is mandatory.

Background: PAX5-altered acute lymphoblastic leukemia (PAX5alt ALL) is a recently defined molecular subtype of BCP-ALL, which displays a specific gene expression and is frequently associated with PAX5 aberrations, including rearrangements (PAX5r). Poor/intermediate outcomes were reported for PAX5alt ALL. However, evidence on clinical features and prognosis of pediatric PAX5r ALL remains limited. Since FLT3 is directly repressed by wild-type PAX5, we hypothesized a potential role for FLT3 in sustaining blast survival in PAX5r ALL and investigated the efficacy of FLT3 targeting in this ALL subtype. Methods: Clinical data of all newly diagnosed PAX5r ALL patients (pts) (<18 years) enrolled in the consecutive AIEOP-BFM ALL trials (ALL 2000, NCT0061345; ALL 2009, NCT01117441; ALL 2017, NCT03643276) or observational studies in Italy (AIEOP), Germany and Austria between 2001-2024, were retrospectively collected. PAX5 gene fusions were identified either by cytogenetics, FISH, targeted-RNA-seq or whole-RNA-seq (WTS). FLT3 expression was assessed by WTS in AIEOP pts and in PAX5r patient derived xenografts (PDXs). A high-throughput drug screening (HTS) platform was used to evaluate FLT3 inhibitors (FLT3i) in PAX5r ALL PDXs. Apoptosis experiments using AnnexinV/7AAD staining, were conducted on PAX5r cell line (NALL-1) and on PAX5r PDX blasts in co-culture with human bone marrow stromal cells, to test the ex vivo efficacy of gilteritinib (GILT). Results: Overall, 165 pts with a confirmed PAX5r ALL were included in the study. Sixty-five different fusion gene partners were identified, with JAK2 (n=22), NOL4L (N=18), AUTS2 (n=13) and ETV6 (n=12) as the most frequent ones. Median age was 3.4 yrs (0.4-17.8) and 66.1% of the pts were males. Low incidence of CNS involvement (CNS3, 3.8%) and a high rate of hyperleukocytosis (WBC>100x109/L) (22%) was found. According to NCI criteria, 53.3% had high-risk (HR) status. IKZF1plus profile was found in 15.1% of the pts. A tendency to slow treatment response was observed: 13% of pts had prednisone poor response (PPR), 74.7% were PCR-MRD+ at the end of induction (EOI), with 28.6% showing MRD≥5x10-4, and 31.5% were still PCR-MRD+ at the end of consolidation. Per protocol final risk stratification: 25.8%, 42.9% and 31.3% of the pts were standard-risk (SR), medium-risk (MR) and HR, respectively. Further, we restricted the analyses solely to the consecutive PAX5r pts (n=99) enrolled in the AIEOP-BFM ALL 2017 study and compared their clinical features with all non-PAX5r pts (n=1945) enrolled in the same protocol. ETV6::RUNX1 pts were excluded from the analysis. Lower median age at diagnosis (3.4 vs 4.7 yrs), higher hyperleukocytosis rate (23.2% vs 8%, p<0.001), increasedrate of IKZF1plus profile (14.4% vs 7.8%, p=0.02) and NCI HR status (57.6% vs 39.1%, p<0.001) were observed in the PAX5r group. No relevant differences in terms of treatment response and risk-stratification distribution were found between the two groups, except for a higher frequency of PPR (14.4% vs 5.7%, p=0.001) in the PAX5r ALL. With a median follow-up of 3.6 yrs, 5-yr EFS and OS for the AIEOP-BFM ALL 2009/2017 PAX5r pts (n=116) were 66.8±5.0% and 95.3±2.1%, respectively; 5-yr EFS were 100%, 59.8±9.3% and 43.8±12.7% for standard-risk (SR), medium-risk (MR) and high-risk (HR) groups, respectively, indicating that the poor prognostic impact of PAX5r applies only when EOI MRD is positive (MR/HR). FLT3 expression was profiled in 465 consecutive BCP-ALL AIEOP pts. PAX5r pts (n=16) showed 20% higher median FLT3 expression than the whole cohort (5.3 vs 4.4 relative expression units, RU p=0.04, unpaired t-test), similar to ZNF384r (5.8 RU), KMT2Ar (5.6 RU) and hyperdiploid ALL (5.4 RU). High FLT3 expression was preserved also in PAX5r PDX samples. HTS revealed sensitivity to 8 FLT3i for PAX5r ALL, showing a Drug Sensitivity Score (DSS) ranging from 14.7 to 47.9, with GILT (median DSS=26.7) emerging as a promising agent in terms of efficacy and hematological toxicity in vitro profile. Apoptosis experiments with GILT in NALL-1 and PDX blasts (n=4) showed single-agent cytotoxicity at nanomolar doses (IC50, range 250-550nM) and a synergistic effect in combination with dexamethasone. Conclusions: This multinational retrospective study indicates that PAX5r ALL is frequently associated with HR clinical features and unfavorable outcomes in non-SR pts. FLT3 is highly expressed in PAX5r ALL and ex vivo treatment with GILT showed promising efficacy.

Introduction: Treatment of acute promyelocytic leukemia (APL) has been revolutionized over the past two decades by the introduction of both all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), leading to the use of chemotherapy-free protocols in standard-risk (SR) adult patients. In October 2019, a multicenter European pediatric trial from the International Consortium for Childhood (ICC) APL delivering a non-chemotherapy-based treatment for children with newly diagnosed SR and High Risk (HR) APL was started [NCT04793919]. Here, we report the results of the second planned interim analysis of the ICC-APL-02 study. Methods: ICC-APL-02 Study is an open-label prospective, non-randomized, multicenter trial for children and adolescents (up to the age of 18) with newly diagnosed APL delivering a risk-stratified treatment [according to the modified pediatric criteria (Testi, Blood 2018)] based on the use of ATRA and ATO, plus gemtuzumab ozogamicin (GO) only in HR patients. During induction, ATO was given at the dose of 0.15 mg/kg/day iv and ATRA at 25 mg/m2/day orally. Treatment started on day 1 and was continued until achievement of hematological complete remission (CR), for at least 28 days and for a maximum of 60 days. In HR patients only, GO was administered on days +2 and +4 of induction treatment, at the dose of 3 mg/m2 iv (maximum 5 mg/dose). During consolidation, ATO was given at the same dose employed during induction for 5 days/week, 4 weeks on and 4 weeks off, for 4 courses, while ATRA (25 mg/m2/day) was administered 2 weeks on and 2 weeks off for 7 courses. In the HR group only, triple intrathecal therapy was administered at the beginning of the 1st and 3rd consolidation. The primary endpoint was to evaluate the efficacy in terms of event-free survival (EFS) of the treatment regimen. Early death, death in CR, resistant disease and hematological relapse were considered as events. Results: The trial was opened in 52 centers across 5 countries (Italy, France, Czech Republic, Netherlands and Sweden). From 10/2019 to 06/2025, 110 patients were enrolled; 73 were SR (66.4%) and 37 HR (33.6%). Median white blood cell count at diagnosis for SR and HR patients was 2.5x109/L (0.6-9.9) and 18.0x109/L (10.55-151.00), respectively. Median age at diagnosis was 12 years (range 1-17); 45% of patients were females; 49% of patients were FLT3-ITD mutated. Median duration of induction was 37 days (range 28-59), with 36 patients (29 SR and 7 HR) interrupting the treatment for a median of 5 days (range 1-23), due to toxicity which resolved in all cases. During induction, 19 patients (17.3%) developed differentiation syndrome (DS) at a median time of 12 days (range 4-25) from treatment start, which resolved in all patients. Pseudotumor cerebri occurred in 14 patients (12.7%). Four patients experienced clinically-relevant prolongation of the QTc interval, which was successfully managed in all patients (in 3 with temporary ATO discontinuation). One patient developed myocarditis following GO which completely resolved without sequelae; no other drug-related side effect was registered. One 16-year old, FLT3-ITD+ HR patient died of cerebral hemorrhage during induction (day +30). All patients achieved hematological CR at the end of induction. All but one evaluated patients achieved molecular CR after the third consolidation course. The only positive patient (HR) remained MRD+ also after the end of treatment; he obtained the molecular remission with 2 additional doses of GO and was then treated with autologous hematopoietic stem cell transplantation; he is now alive and disease-free. One SR patient experienced molecular relapse 1 year after completing the treatment protocol; he was re-treated with ATRA-ATO and he is now alive with negative MRD. With a median follow-up of 21 months (range 2-68.7), the 2-yr overall and EFS of the whole cohort are 99% (95% CI 93.4-99.9) (SR 100% vs HR 97.1%, p=n.s.) and 96.4% (95% CI 88.6-98.9) (SR 97.4% vs HR 94.2%, p=n.s.), respectively. Nineteen patients are still on treatment. Conclusions: This is the first large prospective pediatric trial delivering a non-chemotherapy-based treatment for children with newly diagnosed SR and HR APL.We confirm that ATRA/ATO treatment is safe and highly effective in SR patients. More importantly, the combination of ATRA, ATO and GO in HR subjects showed an excellent safety profile and efficacy in pediatric patients with de novo APL.

Background/Objectives: Art therapy is a psychotherapeutic technique that involves the creation of tangible visual arts and represents a coping strategy to support children with cancer. Evaluating the effects of such activities on children with cancer is essential for providing evidence of the value that creativity holds within healthcare systems. A dedicated tool for assessing the creative process is the Arts Observational Scale (ArtsObS), focusing on mood and emotional states as key indicators of psychosocial well-being. This study aims to validate a translated version of the ArtsObS in the Italian language. Methods: The translation process followed recommendations for translation and cultural adaptation. The distribution properties of the scores, internal consistency, sensitivity to change, reliability, and convergent validity were assessed through observations conducted by two different evaluators. Results: The ArtsObS in its Italian adaptation is proven to be an adequate tool for capturing changes following an intervention, with good internal consistency and low sensitivity to differences between operators. The analysis supports the reliability of the ArtsObS across different observers. Conclusions: The Italian ArtsObS is a valid and reliable instrument for evaluating the impact of art therapy on pediatric patients’ mood and emotional states. It provides a standardized tool for clinical and research settings to assess creative interventions in pediatric oncology.

Background: Asparaginase (ASPase) plays an important role in the therapy of acute lymphoblastic leukemia (ALL). Serum ASPase activity (SAA) can be modified and even abolished by host immune responses; therefore, current treatment guidelines recommend to monitor SAA during treatment administration. The SAA monitoring schedule needs to be carefully planned to reduce the number of samples without hampering the possibility of measuring pharmacokinetics (PK) parameters in individual patients. Complex modelling approaches, not easily applicable in common practice, have been applied in previous studies to estimate ASPase PK parameters. This study aimed to estimate PK parameters by using a simplified approach suitable for real-world settings with limited sampling. Methods: Our study was based on 434 patients treated in Italy within the AIEOP-BFM ALL 2009 trial. During the induction phase, patients received two doses of pegylated ASPase and were monitored with blood sampling at five time points, including time 0. PK parameters were estimated by using the individually available SAA measurements with simple modifications of the classical non-compartmental PK analysis. We also took the opportunity to develop and validate a series of limited sampling models to predict ASPase exposure. Results: During the induction phase, average ASPase activity at day 7 was 1380 IU/L after the first dose and 1948 IU/L after the second dose; therapeutic SAA levels (>100 IU/L) were maintained until day 33 in 90.1% of patients. The average AUC and clearance were 46,937 IU/L × day and 0.114 L/day/m2, respectively. The database was analyzed for possible associations of PK parameters with biological characteristics of the patients, finding only a limited dependence on sex, age and risk score; however, these differences were not sufficient to allow any dose or schedule adjustments. Thereafter the possibility of further sampling reduction by using simple linear models to estimate the AUC was also explored. The most simple model required only two samplings 7 days after each ASPase dose, with the AUC being proportional to the sum of the two measured activities A(7) and A(21), calculated by the formula AUC = 14.1 × [A(7) + A(21)]. This model predicts the AUC with 6% average error and 35% maximum error compared to the AUC estimated with all available measures. Conclusions: Our study demonstrates the feasibility of a direct estimation of PK parameters in a real-life situation with limited and variable blood sampling schedules and also offers a simplified method and formulae easily applicable in clinical practice while maintaining a reliable pharmacokinetic monitoring.

Systemic viral infections are frequently life-threatening in immunocompromised children. Many viral pathogens are reported to be the cause of morbidity and mortality in these pediatric patients, but scarce evidence is related to respiratory syncytial virus infection (RSV), which is one of the main viral causes of lower respiratory tract infection in infants and young children. Herein we report the experience of the Center of Pediatric Hematology Oncology of Catania regarding RSV infection in pediatric leukemia patients, describing four cases: three with only respiratory involvement and complete recovery (two of them presented mild symptoms and one evolved into severe respiratory failure) and a fourth case with an initial hepatic and pulmonary involvement leading to death. Unfortunately, some viral infections have delayed diagnoses because of lack of awareness and atypical presentation. Therefore, our intent is to highlight the importance of mindfulness of the occurrence of this infection and of its typical and atypical manifestations in order to detect it early and decrease the risk of morbidity and mortality.

Despite the excellent survival rate, relapse occurs in 20% of children with ALL. Deep analyses of cell signaling pathways allow us to identify new markers and/or targets promising more effective and less toxic therapy. We analyzed 61 diagnostic samples collected from 35 patients with B- and 26 with T-ALL, respectively. The expression of CK2, MYC and ERG genes using Sybr-Green assay and the comparative 2-ΔΔCt method using 20 healthy donors (HDs) was evaluated. We observed a statistically significant difference in CK2 expression in non-HR (p = 0.010) and in HR (p = 0.0003) T-ALL cases compared to HDs. T-ALL patients with PTEN-Exon7 mutation, IKZF1 and CDKN2A deletions showed high CK2 expression. MYC expression was higher in pediatric T-ALL patients than HDs (p = 0.019). Surprisingly, we found MYC expression to be higher in non-HR than in HR T-ALL patients. TLX3 (HOX11L2)-rearranged T-ALLs (27%) in association with CRLF2 overexpression (23%) showed very high MYC expression. In B-ALLs, we detected CK2 expression higher than HDs and MYC overexpression in HR compared to non-HR patients, particularly in MLL-rearranged B-ALLs. We observed a strong difference in ERG expression between pediatric T- and B-ALL cases. In conclusion, we confirmed CK2 as a prognostic marker and a therapeutic target.

Despite aggressive multimodal treatment, the outcome of pediatric patients affected by high-risk (HR) neuroblastoma (NB) remains poor. The rational of use of allogeneic hematopoietic stem cell transplantation (allo-HCT) for NB was based on the possible graft versus tumor effect; however, toxicity represents a limitation to its efficacy. We sought to prospectively assess the feasibility and efficacy of allo-HCT using a reduced intensity conditioning regimen in pediatric patients with HR NB in a multicenter phase II trial. Primary endpoints were the rate of neutrophil and platelet engraftment, the 5-year transplant-related mortality (TRM) and the disease-free survival (DFS). Secondary endpoint measures included the incidence of acute and chronic graft-versus-host disease (aGvHD and cGvHD). Fifty-one patients were enrolled in the study. Five-year cumulative incidence (CI) of TRM and DFS were 29.4%±6.4%, and 11.8%±4.5%, respectively. Patients receiving allo-HCT within 1 year from diagnosis or with bone marrow as cell source had higher DFS probability. CI of neutrophil engraftment, platelet engraftment, and grade II-IV aGvHD was 97.9%±2.1%, 93.8%±3.5%, and 47.1%±7.0%. The development of new therapeutic strategies could further improve the disease control.

Ataxia-telangiectasia (A-T) is an autosomal-recessive disorder caused by pathogenic variants (PVs) of the ATM gene. Children with A-T are predisposed to hematological malignancies. We aimed to investigate their characteristics and outcomes in order to generate data-based treatment recommendations. In this multinational, observational study we report 202 patients aged ≤25 years with A-T and hematological malignancies from 25 countries. Ninety-one patients (45%) presented with mature B-cell lymphomas, 82 (41%) with acute lymphoblastic leukemia/lymphoma, 21(10%) with Hodgkin lymphoma and eight (4%) with other hematological malignancies. Four-year overall survival and event-free survival (EFS) were 50.8% (95% CI 43.6-59.1) and 47.9% (95% CI 40.8-56.2), respectively. Cure rates have not significantly improved over the last four decades (p=.76). The major cause of treatment failure was treatment-related mortality (TRM) with a four-year cumulative incidence of 25.9% (95% CI 19.5-32.4). Germline ATM PVs were categorized as null or hypomorphic and patients with available genetic data (n=110) were classified as having absent (n=81) or residual (n=29) ATM kinase activity. Four-year EFS was 39.4% (95% CI 29-53.3) vs 78.7% (95% CI 63.7-97.2), (p<.001), and TRM rates were 37.6% (95% CI 26.4-48.7) vs 4.0% (95% CI 0-11.8), (p=.017), for those with absent and residual ATM kinase activity, respectively. Absence of ATM kinase activity was independently associated with decreased EFS (HR=0.362, 95% CI 0.16-0.82; p=.009) and increased TRM (HR=14.11, 95% CI 1.36-146.31; p=.029). Patients with A-T and leukemia/lymphoma may benefit from de-escalated therapy for patients with absent ATM kinase activity and near-standard therapy regimens for those with residual kinase activity.

Noonan syndrome (NS) is an autosomal dominant disorder that varies in severity and can involve multiple organ systems. In approximately 50% of cases, it is caused by missense mutations in the PTPN11 gene (12q24.13). NS is associated with a higher risk of cancer occurrence, specifically hematological disorders. Here, we report a case of a child who was diagnosed at birth with a transient myeloproliferative disorder (TMD). After two years, the child developed hyperdiploid B-cell precursor acute lymphoblastic leukemia (BCP-ALL), receiving a two-year course of treatment. During her continuous complete remission (CCR), a heterozygous germline mutation in the PTPN11 gene [c.218 C>T (p.Thr73lle)] was identified. At the age of ten, the child presented with massive splenomegaly, hyperleukocytosis, and thrombocytopenia, resulting in the diagnosis of juvenile myelomonocytic leukemia (JMML). After an initial response to antimetabolite therapy (6-mercaptopurine), she underwent haploidentical hematopoietic stem cell transplantation (HSCT) and is currently in complete remission. The goal of this review is to gain insight into the various hematological diseases associated with NS, starting from our unique case.

Introduction T-cell acute lymphoblastic leukemia (T-ALL) has been historically associated with inferior outcome compared to B-cell precursor ALL with slower treatment response and lack of recurrent prognostic cytogenetic alterations. With the introduction of next-generation sequencing analyses, a deeper characterization of childhood ALL has been achieved. However, if for B-ALL the identification of fusion-class genes has clinical implication, the frequency and relevance of gene fusions in T-ALL still remain poor. Here we describe the incidence of gene-class fusions in the pediatric cohort of T-ALL patients treated according to the AIEOP-BFM ALL 2017 protocol and explore the association with presenting patient characteristics and early treatment response. Methods From Dec 1st 2021 to Mar 31st 2024, in AIEOP Italian centers 132 consecutive patients with T-ALL have been enrolled in the trial AIEOP-BFM ALL 2017 Study. According to material availability, Whole transcriptome RNA-seq was performed at disease onset in 128 (97%) patients (Universal RNA-seq kit, Tecan with paired-end sequencing on Nextseq2000). The analysis was performed with 4 independent pipelines with the purpose to detect fusion genes (Dragen RNA, Star, Fusion Catcher, Arriba) and results were evaluable in 123 (96%) patients (5 cases were excluded due to poor library quality). Results Among 123 evaluable patients, at least one fusion gene was identified in 51 (41.5%) patients and validated through real-time PCR. Most common gene-class fusions were STIL::TAL1 (n=10, 8.1%), KMT2A-rearranged (KMT2Ar, n=9, 7.3%), ABL-class (n=8, 6.5%) and MLLT10-class (n=8, 6.5%). Other fusions were identified in 16 (13.0%) patients, with ETV6-class in 4 patients (3.3%) and CDK6-class in 2 patients (1.6%), in addition to single non recurrent fusion/cases. All gene-class fusions were mutually exclusive. Patient characteristics at diagnosis were evaluated in association with the presence of gene-class fusions. ABL-class and MLLT10-class fusions were more common in females and patients with MLLT10-class fusion tended to be older (age ≥ 10 years). Patients with STIL::TAL1 fusion were more likely to have hyperleukocytosis (white blood cell counts ≥ 100 × 109/L) and central nervous system disease (CNS3). We investigated whether the presence of gene-class fusion was associated with treatment response in term of prednisone poor response (PPR), high level of bone marrow disease (≥10% of blasts) measured by flow-cytometry (FCM-MRD) at induction day 15, end of induction (EOI) PCR-MRD, end of consolidation (EOC) PCR-MRD and final risk group classification. Patients carrying a KMT2Ar were characterized by higher proportion of PPR, FCM-MRD with ≥10% of blasts and higher EOI MRD levels: of note, approximately half (44.4%) of them had EOI MRD ≥5x10-3. The presence of MLLT10-class fusion was associated with high EOI MRD, with 50% of patients having levels ≥5x10-3, as well as for STIL::TAL1 patients (40% of patients with ≥5x10-3). No association was found at EOC between PCR-MRD levels and gene-class fusions. Interestingly, all patients carrying KMT2Ar except one were classified as HR while a lower proportion of HR patients was found among patients with ABL-class fusion. Indeed, the presence of ABL-class fusion was not associated with any unfavorable early treatment responses. Conclusion Our study showed a high incidence of gene-class fusions in children and adolescents with newly diagnosed T-ALL. We found that KMT2Ar, STIL::TAL1 and MLLT10-class fusions are associated with high risk features in a prospective cohort of uniformly treated patients with pediatric T-ALL. On the contrary, patients carrying ABL-class fusions did not show adverse characteristics of early treatment response. Despite a longer follow-up is required, prognostic relevant gene-class fusions might integrate current risk stratification and identify patients who might qualify for early interventional studies or targeted therapies.

Human leukocyte antigen (HLA) restriction of conventional T-cell targeting introduces complexity in generating T-cell therapy strategies for patients with cancer with diverse HLA-backgrounds. A subpopulation of atypical, major histocompatibility complex-I related protein 1 (MR1)-restricted T-cells, distinctive from mucosal-associated invariant T-cells (MAITs), was recently identified recognizing currently unidentified MR1-presented cancer-specific metabolites. It is hypothesized that the MC.7.G5 MR1T-clone has potential as a pan-cancer, pan-population T-cell immunotherapy approach. These cells are irresponsive to healthy tissue while conferring T-cell receptor(TCR) dependent, HLA-independent cytotoxicity to a wide range of adult cancers. Studies so far are limited to adult malignancies. Here, we investigated the potential of MR1-targeting cellular therapy strategies in pediatric cancer. Bulk RNA sequencing data of primary pediatric tumors were analyzed to assessMR1expression.In vitropediatric tumor models were subsequently screened to evaluate their susceptibility to engineered MC.7.G5 TCR-expressing T-cells. Targeting capacity was correlated with qPCR-basedMR1mRNA and protein overexpression. RNA expression ofMR1in primary pediatric tumors varied widely within and between tumor entities. Notably, embryonal tumors exhibited significantly lowerMR1expression than other pediatric tumors. In line with this, most screened embryonal tumors displayed resistance to MR1T-targetingin vitro. MR1T susceptibility was observed particularly in pediatric leukemia and diffuse midline glioma models. This study demonstrates potential of MC.7.G5 MR1T-cell immunotherapy in pediatric leukemias and diffuse midline glioma, while activity against embryonal tumors was limited. The dismal prognosis associated with relapsed/refractory leukemias and high-grade brain tumors highlights the promise to improve survival rates of children with these cancers.

EDITORIAL article Front. Oncol., 01 December 2023Sec. Hematologic Malignancies Volume 13 - 2023 | https://doi.org/10.3389/fonc.2023.1293795

Introduction Ataxia telangiectasia (A-T) is a multisystem disorder caused by biallelic germline pathogenic variants (PV) in the ATM gene. An important feature of A-T is an increased predisposition to cancer with a reported incidence of 25%, primarily attributed to hematological malignancies. Patients with A-T and cancer are usually excluded from therapeutic clinical trials, limited information thus exists concerning their treatment outcomes and toxicity profiles and consequently, optimal management strategies are unclear and an unmet need. In this multinational study, we aimed to investigate the characteristics and outcomes of leukemia and lymphoma in a large cohort of children with A-T and to determine risk factors which impact treatment outcome in order to generate consensus and data-based prospective treatment recommendations. Methods This study of patients aged ≤25 years with A-T and hematological malignancies was conducted through the International BFM Study Group. Patient data were collected from medical records, including specific patient comorbidities. Each reported ATM PV identified in the cohort was classified as null (resulting in complete loss of ATM activity) or hypomorphic (allowing residual ATM activity) according to the expected functional activity of the ATM protein and published functional studies. Patients with reported ATM PV were then classified as Group A (two null PV) or Group B (at least one hypomorphic PV). Results We report 202 pediatric and adolescent/young adult patients with A-T and hematological malignancies from 25 countries. The cohort included 82 patients with ALL/LBL (41%), predominantly (85%) of T-cell lineage, 91 with mature B-cell lymphomas (45%), 21 with Hodgkin lymphoma (10%) and 8 with other hematological malignancies (4%) (Fig 1). Of 111 patients with classifiable germline ATM variants, 82 (74%) were classified as Group A and 29 as Group B (26%). The distribution of patients with Group A and Group B germline ATM PV differed considerably between tumor types with 44% of the patients with lymphoblastic leukemia/lymphoma classified as Group B vs. 5% of those with mature B cell lymphomas ( p<.001). In total, 185 patients (92%) treated with curative intent were included in the outcome analyses, 135 (73%) of whom were treated with attenuated therapy regimens. Four-year OS and EFS for the entire cohort were 50.8% (95% CI 43.6-59.1) and 47.9% (95% CI 40.8-56.2), respectively. Surprisingly, cure rates of patients with A-T and malignancy did not appear to improve significantly with therapy modernization over the last four decades with 4-year EFS rates of 41.6% (95% CI 26.6-65), 49.6% (95% CI 38.8-63.4) and 48.0% (95% CI 37.4-61.7) for those treated before 2000, between 2000-2009 and since 2010, respectively ( p=.54). The major cause of treatment failure for the entire cohort was treatment-related mortality (TRM) with a 4-year cumulative incidence of 32.8% (95% CI 19.5-32.4), followed by progressive cancer in 14.5% (95% CI 10-19.8) and second malignancy in 4.9% (95% CI 13.1-85.8) (Fig 2). We identified factors that were significantly associated with survival for this unique patient population. Older age had a significantly deleterious effect upon survival with 4-year EFS rates of 69.1% (95%CI 55.9-85.4), 45.3% (95% CI 34.5-59.4), 39.8% (95% CI 25-63.2), and 33.7% (95% CI 20.8-54.6) for children aged ≤5 years, 5-10 years, 10-15 years and ≥15 years, respectively ( p=.003). The type of germline ATM PV also had a significant impact: 4-year EFS for patients with Group B ATM PV was 78.7% (95% CI 63.7-97.2) vs. 39.4% (95% CI 29-53.3) for Group A ( p<.001). Group A PV were associated with an increased TRM (OR 9.3; 95% CI 1.6-180.1; p=.042) and decreased EFS (HR .371 95% CI 16.6-82.6; p=.009). Conclusions We demonstrate in this first comprehensive international study that leukemia and lymphoma in children with A-T are curable. While the standard treatment stratification system for patients with hematological malignancies without A-T focuses upon cancer relapse/progression as the main cause of treatment failure, TRM was the main cause of therapy failure in patients with A-T and was strongly associated with the underlying germline ATM variant type. This study further fulfills an unmet need for international collaboration and provides a platform for data-based guidelines for a novel risk stratification system and optimal therapy selection for this unique patient population.