From the identification of actionable molecular targets to the generation of faithful neuroblastoma patient-derived preclinical models.

BACKGROUND: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent. METHODS: Within the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models. RESULTS: Eighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as "very high priority", that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient's tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors. CONCLUSIONS: PREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.

TP53 DNA binding domain mutational status and rituximab-based treatment are independent prognostic factors for pediatric Burkitt lymphoma patients stratification.

Not available.

Outcome of primary hemophagocytic lymphohistiocytosis: a report on 143 patients from the Italian Registry.

Primary hemophagocytic lymphohistiocytosis (pHLH) is a severe, life-threatening hyperinflammatory syndrome caused by defects in genes of the granule-dependent cytotoxic pathway. Here we investigated the clinical presentation and outcome in a large cohort of 143 patients with pHLH diagnosed in the last 15 years and enrolled in the Italian registry. The median age at diagnosis was 12 months (IQR 2-81), and ninety-two patients (64%) fulfilled the HLH-2004 criteria. Out of 111 patients who received first-line combined therapy (HLH-94, HLH-2004, Euro-HIT protocols), 65 (59%) achieved complete response (CR) and 21 (19%) partial response (PR). Thereafter, 33 patients (30%) reactivated, and 92 (64%) received HSCT, 78 of whom (85%) survived and were alive at a median follow-up from diagnosis of 67 months. Thirty-six patients (25%) died before HSCT and 14 (10%) after. Overall, 93 patients (65%) were alive after a median follow-up of 30 months. Unadjusted predictors of non-response were age.

Oncological pediatric early warning score: a dedicated tool to predict patient's clinical deterioration and need for pediatric intensive care treatment.

Pediatric oncohematological patients frequently require PICU admission during their clinical history. The O-PEWS is a specific score developed to predict the need for PICU admission of oncohematological children. This study aimed at i) describing the trend of the O-PEWS in a cohort of patients hospitalized in the Pediatric Oncohematology ward and transferred to the PICU of Padua University Hospital, measured at different time-points in the 24 hours before PICU admission and to evaluate its association with mortality and presence of organ failure; ii) investigating the association between the recorded O-PEWS, and PIM3, number of organ failure and the need for ventilation, dialysis and inotropes.This retrospective single-center study enrolled oncohematological children admitted to the PICU between 2017 and 2021. The O-PEWS, ranging between 0 and 15, was calculated on the available medical records and the TIPNet-Network database at 24 (T-24), 12 (T-12), 6 (T-6) and 0 (T0) hours before PICU admission.RESULTS: 101 PICU admissions, related to 80 children, were registered. During the 24 hours prior to PICU admission, the O-PEWS progressively increased in all the patients. At T-24 the median O-PEWS was 3 (IQR 1-5), increasing to a median value of 6 (IQR 4-8) at T0. The O-PEWS was positively associated with mortality, organ failure and the need for ventilation at all the analyzed time-points and with the need for dialysis at T-6.The O-PEWS appears as a useful tool for predicting early clinical deterioration in oncohematological patients and for anticipating the initiation of life-support treatments.

Germline IKAROS dimerization haploinsufficiency causes hematologic cytopenias and malignancies.

IKAROS is a transcription factor forming homo- and heterodimers and regulating lymphocyte development and function. Germline mutations affecting the IKAROS N-terminal DNA binding domain, acting in a haploinsufficient or dominant-negative manner, cause immunodeficiency. Herein, we describe 4 germline heterozygous IKAROS variants affecting its C-terminal dimerization domain, via haploinsufficiency, in 4 unrelated families. Index patients presented with hematologic disease consisting of cytopenias (thrombocytopenia, anemia, neutropenia)/Evans syndrome and malignancies (T-cell acute lymphoblastic leukemia, Burkitt lymphoma). These dimerization defective mutants disrupt homo- and heterodimerization in a complete or partial manner, but they do not affect the wild-type allele function. Moreover, they alter key mechanisms of IKAROS gene regulation, including sumoylation, protein stability, and the recruitment of the nucleosome remodeling and deacetylase complex; none affected in N-terminal DNA binding defects. These C-terminal dimerization mutations are largely associated with hematologic disorders, display dimerization haploinsufficiency and incomplete clinical penetrance, and differ from previously reported allelic variants in their mechanism of action. Dimerization mutants contribute to the growing spectrum of IKAROS-associated diseases displaying a genotype-phenotype correlation.

Italian Precision Medicine in Pediatric Oncology: Moving beyond Actionable Alterations.

Neuroblastoma (NB) is the most common extracranial solid tumor encountered in childhood. Although there has been significant improvement in the outcomes of patients with high-risk disease, the prognosis for patients with metastatic relapse or refractory disease is poor. Hence, the clinical integration of genome sequencing into standard clinical practice is necessary in order to develop personalized therapy for children with relapsed or refractory disease. The PeRsonalizEdMEdicine (PREME) project focuses on the design of innovative therapeutic strategies for patients suffering from relapsed NB. We performed whole exome sequencing (WES) of patient-matched tumor-normal samples to identify genetic variants amenable to precision medicine. Specifically, two patients were studied (First case: a three-year-old male with early relapsed NB; Second case: a 20-year-old male who relapsed 10 years after the first diagnosis of NB). Results were reviewed by a multi-disciplinary molecular tumor board (MTB) and clinical reports were issued to the ordering physician. WES revealed the mutation c.G320C in the CUL4A gene in case 1 and the mutation c.A484G in the PSMC2 gene in case 2. Both patients were treated according to these actionable alterations, with promising results. The effective treatment of NB is one of the main challenges in pediatric oncology. In the era of precision medicine, the need to design new therapeutic strategies for NB is fundamental. Our results demonstrate the feasibility of incorporating clinical WES into pediatric oncology practice.

Metastatic progression in infants diagnosed with stage 4S neuroblastoma. A study of the Italian Neuroblastoma Registry.

PURPOSE: Stage 4S neuroblastoma, a tumor affecting infants, is characterized by the capacity to regress spontaneously and high cure rate. About a third of these infants undergo tumor progression requiring antitumor treatment and 10-15% eventually die. In case of metastatic progression, it may occur either at 4S sites (mainly liver) or sites characterizing stage 4 (mainly bone). Aim of this study was to estimate incidence, presenting features and outcome of infants who progressed to stage 4S or stage 4 sites. PATIENTS: Of 280 Italian infants diagnosed with stage 4S neuroblastoma between 1979 and 2013 and registered in the Italian Neuroblastoma Registry, 268 were evaluable for this study, of whom 57 developed metastatic progression. RESULTS: Progression to stage 4S sites occurred in 29/268 infants (10.8%) (Group A) and to stage 4 in 28/268 (10.4%) (Group B). No significant difference was observed between the two groups at the time of diagnosis. At the time of progression, Group A infants were younger (7.3 vs 14.4 months, P = .001) and had a shorter interval from diagnosis to progression (3.8 vs 9.6 months, P = .001). Survival after progression was worse for Group B infants (45% vs 69%, P = .058) and was associated with age at diagnosis lower than 2 months (P = .005) and adrenal primary tumor site (P = .008). Survival rates increased for both groups along the study period. CONCLUSIONS: Infants who progressed to stage 4 did worse, possibly in relation to older age at progression and longer interval between diagnosis and progression. Large prospective studies of these patients may lead to more effective treatments.

Genomic coamplification of CDK4/MDM2/FRS2 is associated with very poor prognosis and atypical clinical features in neuroblastoma patients.

Neuroblastoma (NB) is the most common extracranial malignant tumor of childhood and is characterized by a broad heterogeneity in clinical presentation and evolution. Recent advances in pangenomic analysis of NB have revealed different recurrent chromosomal aberrations. Indeed, it is now well established that the overall genomic profile is important for treatment stratification. In previous studies, 11 genes were shown to be recurrently amplified (ODC1, ALK, GREB1, NTSR2, LIN28B, MDM2, CDK4, MYEOV, CCND1, TERT, and MYC) besides MYCN, with poor survival of NB patients harboring these amplifications being suggested. Genomic profiles of 628 NB samples analyzed by array-comparative genome hybridization (a-CGH) were re-examined to identify gene amplifications other them MYCN amplification. Clinical data were retrospectively collected. We additionally evaluated the association of FRS2 gene expression with NB patient outcome using the public R2 Platform. We found eight NB samples with high grade amplification of one or two loci on chromosome arm 12q. The regional amplifications were located on bands 12q13.3-q14.1 and 12q15-q21.1 involving the genes CDK4, MDM2, and the potential oncogenic gene FRS2. The CDK4, MDM2, and FRS2 loci were coamplified in 8/8 samples. The 12q amplifications were associated with very poor prognosis and atypical clinical features of NB patients. Further functional and clinical investigations are needed to confirm or refute these associations.

Detection of prognostic factors in children and adolescents with Burkitt and Diffuse Large B-Cell Lymphoma treated with the AIEOP LNH-97 protocol.

Burkitt lymphoma (BL) and Diffuse Large B-Cell Lymphoma (DLBCL) account for most cases of non-Hodgkin lymphoma (NHL) in childhood. We report the clinical characteristics, outcome and prognostic factors in children with BL or DLBCL treated according to the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) LNH-97 protocol. Patients aged up to 18 years that were newly diagnosed with BL/DLBCL were included in the study. Therapy consisted of pre-phase followed by 2-6 high-dose chemotherapy courses tailored according to lactate dehydrogenase (LDH) value and disease stage. A total of 442 patients (379 BL, 63 DLBCL) were enrolled between 1997 and 2014, of whom 18 failed to achieve remission, 6 experienced treatment-related death, 2 developed second malignancy and 20 relapsed. At a median follow-up time of 5 years, overall survival was 93% (±1%) and event-free survival was 90% (±1%). LDH value above the median value had an independently negative prognostic value (P < 0·0001). However, in the subgroup of 128 patients in which minimal disseminated disease (MDD) was analysed, MDD-positivity became the only unfavourable prognostic factor for progression-free survival. Tailored chemotherapy could be extremely effective with limited toxicity. Identification of MDD as a hallmark of a higher risk of treatment failure may provide a target population for treatment intensification by anti-CD20.

Non-Hodgkin lymphoma in children with an associated inherited condition: A retrospective analysis of the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP).

BACKGROUND: Inherited conditions affecting genetic aberration, viral oncogenesis, reduced immune surveillance, and long-lasting antigen stimulation may build the way to lymphomagenesis in humans. METHODS: We extracted from the database of 4 consecutive trials for pediatric non-Hodgkin lymphoma (NHL) all cases with an associated genetic disease. RESULTS: Among 1,430 patients, 34 (2.4%) had an associated inherited condition and a mature B-lineage (n = 28), anaplastic large cell lymphoma (n = 4), or T-lineage (n = 2) NHL. Their median age at the diagnosis was 9.3 years (range, 2.6-17.8 years). In 14 cases (41%) the underlying condition was considered to be a potential cause for undue toxicity if the expected therapy was applied. Thus, treatment modification had been planned in advance. The overall survival was 89% (standard error [SE] 1%), 73% (SE 10%), and 73% (SE 23%) at 3 years for registered patients with no inherited condition associated, with genetic abnormalities and with underlying condition causing an immune deficiency, respectively (P = 0.003). CONCLUSION: In our cohort, patients with NHL with an underlying constitutional condition represent the 2.4% of the cases. In the subset of patients with primary immune deficiency, which may have contributed to lymphomagenesis, allogeneic hematopoietic stem cell transplantation may be required. In the remaining patients, the association with lymphoma remains apparently unexplained and could be not causative. Detailed reporting of such cases may contribute to disclose even rare and fully unexpected association, which may have implications for research in the field of lymphomagenesis.

Inside the Biology of the ß3-Adrenoceptor.

Since the first discovery in 1989, the ß3-adrenoceptor (ß3-AR) has gained great attention because it showed the ability to regulate many physiologic and metabolic activities, such as thermogenesis and lipolysis in brown and white adipose tissue, respectively (BAT, WAT), negative inotropic effects in cardiomyocytes, and relaxation of the blood vessels and the urinary bladder. The ß3-AR has been suggested as a potential target for cancer treatment, both in adult and pediatric tumors, since under hypoxia its upregulation in the tumor microenvironment (TME) regulates stromal cell differentiation, tumor growth and metastases, signifying that its agonism/antagonism could be useful for clinical benefits. Promising results in cancer research have proposed the ß3-AR being targeted for the treatment of many conditions, with some drugs, at present, undergoing phase II and III clinical trials. In this review, we report the scientific journey followed by the research from the ß3-Ars' discovery, with focus on the ß3-Ars' role in cancer initiation and progression that elects it an intriguing target for novel antineoplastic approaches. The overview highlights the great potential of the ß3-AR, both in physiologic and pathologic conditions, with the intention to display the possible benefits of ß3-AR modulation in cancer reality.

Long-term outcome and prognosis of mixed histiocytosis (Erdheim-Chester disease and Langerhans Cell Histiocytosis).

Background: Erdheim-Chester disease (ECD) is a rare histiocytosis that may overlap with Langerhans Cell Histiocytosis (LCH). This "mixed" entity is poorly characterized. We here investigated the clinical phenotype, outcome, and prognostic factors of a large cohort of patients with mixed ECD-LCH. Methods: This retrospective study was performed at two referral centers in France and Italy (Pitié-Salpêtrière Hospital, Paris; Meyer Children's Hospital, Florence). We included children and adults with ECD diagnosed in 2000-2022 who had biopsy-proven LCH, available data on clinical presentation, treatment and outcome, and a minimum follow-up of one year. Outcomes included differences in clinical presentation and survival between mixed ECD-LCH and isolated ECD; we also investigated response to treatments and predictors of survival in the mixed cohort. Survival was analyzed using the Kaplan-Maier method and differences in survival with the long-rank test. Cox regression models were used to evaluate the potential impact of age and gender on survival and to identify predictors of non-response and survival. Findings: Out of a cohort of 502 ECD patients, 69 (14%) had mixed ECD-LCH. Compared to isolated ECD, mixed ECD-LCH occurred more frequently in females (51 vs. 26%, p < 0.001) and in patients with multisystem disease (≥4 sites). Mixed ECD-LCH more frequently involved long bones (91 vs. 79%, p = 0.014), central nervous system (51 vs. 34%, p = 0.007), facial/orbit (52 vs. 38%, p = 0.031), lungs (43 vs. 28%, p = 0.009), hypothalamic/pituitary axis (51 vs. 26%, p < 0.001), skin (61 vs. 29%, p < 0.001), and lymph nodes (15 vs. 7%, p = 0.028); the BRAFV600E mutation was also more frequent in mixed ECD-LCH (81 vs. 59%, p < 0.001). Targeted treatments (BRAF and/or MEK inhibitors) induced response more frequently than conventional therapies (interferon-α, chemotherapy), either as first-line (77 vs. 29%, p < 0.001) or as any line (75 vs. 24%, p < 0.001). After a median follow-up of 71 months, 24 patients (35%) died. Survival probability was comparable between ECD alone and mixed ECD-LCH (log-rank p = 0.948). At multivariable analysis, age at diagnosis (HR 1.052, 95% CI 1.008-1.096), associated hematologic conditions (HR 3.030, 95% CI 1.040-8.827), and treatment failure (HR 9.736, 95% CI 2.919-32.481) were associated with an increased risk of death, while lytic bone lesions with a lower risk (HR 0.116, 95% CI 0.031-0.432). Interpretation: Mixed ECD-LCH is a multisystem disease driven by the BRAFV600E mutation and targeted treatments are effective. Age at diagnosis, bone lesion patterns, associated hematologic conditions, and treatment failure are the main predictors of death in mixed ECD-LCH. Funding: None.

Targeted treatment of solid tumors in pediatric precision oncology.

The treatment of childhood solid cancer has markedly evolved in recent years following a refined molecular characterization and the introduction of novel targeted drugs. On one hand, larger sequencing studies have revealed a spectrum of mutations in pediatric tumors different from adults. On the other hand, specific mutations or immune dysregulated pathways have been targeted in preclinical and clinical studies, with heterogeneous results. Of note, the development of national platforms for tumor molecular profiling and, in less measure, for targeted treatment, has been essential in the process. However, many of the available molecules have been tested only in relapsed or refractory patients, and have proven poorly effective, at least in monotherapy. Our future approaches should certainly aim at improving the access to molecular characterization, to obtain a deeper picture of the distinctive phenotype of childhood cancer. In parallel, the implementation of access to novel drugs should not only be limited to basket or umbrella studies but also to larger, multi-drug international studies. In this paper we reviewed the molecular features and the main available therapeutic options in pediatric solid cancer, focusing on available targeted drugs and ongoing investigations, aiming at providing a useful tool to navigate the heterogeneity of this promising but complex field.

Approaching hemophagocytic lymphohistiocytosis.

Hemophagocytic Lymphohistiocytosis (HLH) is a rare clinical condition characterized by sustained but ineffective immune system activation, leading to severe and systemic hyperinflammation. It may occur as a genetic or sporadic condition, often triggered by an infection. The multifaceted pathogenesis results in a wide range of non-specific signs and symptoms, hampering early recognition. Despite a great improvement in terms of survival in the last decades, a considerable proportion of patients with HLH still die from progressive disease. Thus, prompt diagnosis and treatment are crucial for survival. Faced with the complexity and the heterogeneity of syndrome, expert consultation is recommended to correctly interpret clinical, functional and genetic findings and address therapeutic decisions. Cytofluorimetric and genetic analysis should be performed in reference laboratories. Genetic analysis is mandatory to confirm familial hemophagocytic lymphohistiocytosis (FHL) and Next Generation Sequencing is increasingly adopted to extend the spectrum of genetic predisposition to HLH, though its results should be critically discussed with specialists. In this review, we critically revise the reported laboratory tools for the diagnosis of HLH, in order to outline a comprehensive and widely available workup that allows to reduce the time between the clinical suspicion of HLH and its final diagnosis.

Blockade of IL-10 Signaling Ensures Mifamurtide Efficacy in Metastatic Osteosarcoma.

Osteosarcoma (OS) is the most common primary malignancy of the bone, highly aggressive and metastasizing, and it mainly affects children and adolescents. The current standard of care for OS is a combination of surgery and chemotherapy. However, these treatment options are not always successful, especially in cases of metastatic or recurrent osteosarcomas. For this reason, research into new therapeutic strategies is currently underway, and immunotherapies have received considerable attention. Mifamurtide stands out among the most studied immunostimulant drugs; nevertheless, there are very conflicting opinions on its therapeutic efficacy. Here, we aimed to investigate mifamurtide efficacy through in vitro and in vivo experiments. Our results led us to identify a new possible target useful to improve mifamurtide effectiveness on metastatic OS: the cytokine interleukin-10 (IL-10). We provide experimental evidence that the synergic use of an anti-IL-10 antibody in combination with mifamurtide causes a significantly increased mortality rate in highest-grade OS cells and lower metastasis in an in vivo model compared with mifamurtide alone. Overall, our data suggest that mifamurtide in combination with an anti-IL-10 antibody could be proposed as a new treatment protocol to be studied to improve the outcomes of OS patients.

ß3-adrenergic receptor on tumor-infiltrating lymphocytes sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma.

Neuroblastoma (NB) is a heterogeneous extracranial tumor occurring in childhood. A distinctive feature of NB tumors is their neuroendocrine ability to secrete catecholamines, which in turn, via ß-adrenergic receptors ligation, may affect different signaling pathways in tumor microenvironment (TME). It was previously demonstrated that specific antagonism of ß3-adrenergic receptor (ß3-AR) on NB tumor cells affected tumor growth and progression. Here, in a murine syngeneic model of NB, we aimed to investigate whether the ß3-AR modulation influenced the host immune system response against tumor. Results demonstrated that ß3-AR antagonism lead to an immune response reactivation, partially dependent on the PD-1/PD-L1 signaling axis involvement. Indeed, ß3-AR blockade on tumor-infiltrating lymphocytes (TILs) dampened their ability to secrete IFN-γ, which in turn reduced the PD-L1 expression, caused by TILs infiltration, on NB tumor cells. Further investigations, through a genomic analysis on NB patients, showed that high ADRB3 gene expression correlates with worse clinical outcome compared to the low expression group, and that ADRB3 gene expression affects different immune-related pathways. Overall, results indicate that ß3-AR in NB TME is able to modulate the interaction between tumor and host immune system, and that its antagonism hits multiple pro-tumoral signaling pathways.

Therapeutic Targeting of ALK in Neuroblastoma: Experience of Italian Precision Medicine in Pediatric Oncology.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Patients with relapsed/refractory disease have a poor prognosis, and additional therapeutic options are needed. Mutations and amplifications in the ALK (Anaplastic Lymphoma Kinase) gene constitute a key target for treatment. Our goal, within the Italian project of PeRsonalizEdMEdicine (PREME), was to evaluate the genomic status of patients with relapsed/refractory NB and to implement targeted therapies in those with targetable mutations. From November 2018 to November 2021, we performed Whole Exome Sequencing or Targeted Gene Panel Sequencing in relapsed/refractory NB patients in order to identify druggable variants. Activating mutations of ALK were identified in 8(28.57%) of 28 relapsed/refractory NB patients. The mutation p.F1174L was found in six patients, whereas p.R1275Q was found in one and the unknown mutation p.S104R in another. Three patients died before treatment could be started, while five patients received crizotinib: two in monotherapy (one with p.F1174L and the other with p.S104R) and three (with p.F1174L variant) in combination with chemotherapy. All treated patients showed a clinical improvement, and one had complete remission after two cycles of combined treatment. The most common treatment-related toxicities were hematological. ALK inhibitors may play an important role in the treatment of ALK-mutated NB patients.