The person-to-person transmission landscape of the gut and oral microbiomes.

The human microbiome is an integral component of the human body and a co-determinant of several health conditions1,2. However, the extent to which interpersonal relations shape the individual genetic makeup of the microbiome and its transmission within and across populations remains largely unknown3,4. Here, capitalizing on more than 9,700 human metagenomes and computational strain-level profiling, we detected extensive bacterial strain sharing across individuals (more than 10 million instances) with distinct mother-to-infant, intra-household and intra-population transmission patterns. Mother-to-infant gut microbiome transmission was considerable and stable during infancy (around 50% of the same strains among shared species (strain-sharing rate)) and remained detectable at older ages. By contrast, the transmission of the oral microbiome occurred largely horizontally and was enhanced by the duration of cohabitation. There was substantial strain sharing among cohabiting individuals, with 12% and 32% median strain-sharing rates for the gut and oral microbiomes, and time since cohabitation affected strain sharing more than age or genetics did. Bacterial strain sharing additionally recapitulated host population structures better than species-level profiles did. Finally, distinct taxa appeared as efficient spreaders across transmission modes and were associated with different predicted bacterial phenotypes linked with out-of-host survival capabilities. The extent of microorganism transmission that we describe underscores its relevance in human microbiome studies5, especially those on non-infectious, microbiome-associated diseases.

Nivolumab and brentuximab vedotin with or without bendamustine for R/R Hodgkin lymphoma in children, adolescents, and young adults.

Children, adolescents, and young adults (CAYA) with relapsed/refractory (R/R) classic Hodgkin lymphoma (cHL) without complete metabolic response (CMR) before autologous hematopoietic cell transplantation (auto-HCT) have poor survival outcomes. CheckMate 744, a phase 2 study for CAYA (aged 5-30 years) with R/R cHL, evaluated a risk-stratified, response-adapted approach with nivolumab plus brentuximab vedotin (BV) followed by BV plus bendamustine for patients with suboptimal response. Risk stratification was primarily based on time to relapse, prior treatment, and presence of B symptoms. We present the primary analysis of the standard-risk cohort. Data from the low-risk cohort are reported separately. Patients received 4 induction cycles with nivolumab plus BV; those without CMR (Deauville score >3, Lugano 2014) received BV plus bendamustine intensification. Patients with CMR after induction or intensification proceeded to consolidation (high-dose chemotherapy/auto-HCT per protocol). Primary end point was CMR any time before consolidation. Forty-four patients were treated. Median age was 16 years. At a minimum follow-up of 15.6 months, 43 patients received 4 induction cycles (1 discontinued), 11 of whom received intensification; 32 proceeded to consolidation. CMR rate was 59% after induction with nivolumab plus BV and 94% any time before consolidation (nivolumab plus BV ± BV plus bendamustine). One-year progression-free survival rate was 91%. During induction, 18% of patients experienced grade 3/4 treatment-related adverse events. This risk-stratified, response-adapted salvage strategy had high CMR rates with limited toxicities in CAYA with R/R cHL. Most patients did not require additional chemotherapy (bendamustine intensification). Additional follow-up is needed to confirm durability of disease control. This trial was registered at www.clinicaltrials.gov as #NCT02927769.

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.

Overexpression of H2AFX gene in neuroblastoma is associated with worse prognosis.

BACKGROUND: Neuroblastoma (NB) is the most common solid tumour in childhood, and rises in the sympathetic nervous system. Here, we addressed the in silico analysis of the association between the expression of H2AFX gene involved in DNA damage response, and the survival of a cohort of 786 NB patients. METHODS: In silico gene expression was retrieved from the publicly available dataset summarised by Cangelosi et al., including 13,696 gene expression profiles of 786 NB tumours at onset of disease. The prognostic value of H2AFX (H2A histone family member X) gene expression for event-free survival (EFS) and overall survival (OS) was evaluated by Kaplan-Meier and Cox regression analysis. The main results were validated on another openly accessible in silico database (NRC-283) containing 13,489 gene expressions in 283 NB patients. The expression of H2AFX protein was then tested by immunofluorescence on 48 primary NB samples of different tumour stages. H2AFX activity as an oncogene has been further validated in vitro by silencing the molecule in two NB cell lines, characterised by MYCN amplified or not, and performing cell growth and migration assays. RESULTS: A strong inverse association between H2AFX expression and patients' survival was observed and confirmed by immunofluorescence results on primary NB tissue sections. Cox regression analysis also disclosed H2AFX as an independent predictor of EFS and OS. The gene-silencing experiments strongly suggested an oncogenic role for H2AFX on NB cells, regardless of MYCN amplification. CONCLUSIONS: H2AFX is a prognostic marker for unfavourable NB and could be considered a target for therapeutic interventions.

Reaching the target dose with one single 131 I-mIBG administration in high-risk neuroblastoma: The determinant impact of the primary tumour.

BACKGROUND: 131 I-metaiodobenzylguanidine (131 I-mIBG) effectiveness in children with metastasised neuroblastoma (NB) is linked to the effective dose absorbed by the target; a target of 4 Gy whole-body dose threshold has been proposed. Achieving this dose often requires administering 131 I-mIBG twice back-to-back, which may cause haematological toxicity. In this study, we tried identifying the factors predicting the achievement of 4 Gy whole-body dose with a single radiopharmaceutical administration. MATERIALS AND METHODS: Children affected by metastatic NB and treated with a high 131 I-mIBG activity (>450 MBq (megabecquerel)/kg) were evaluated retrospectively. Kinetics measurements were carried out at multiple time points to estimate the whole-body dose, which was compared with clinical and activity-related parameters. RESULTS: Seventeen children (12 females, median age 3 years, age range: 1.5-6.9 years) were included. Eleven of them still bore the primary tumour. The median whole-body dose was 2.88 Gy (range: 1.63-4.22 Gy). Children with a 'bulky' primary (>30 mL) received a higher whole-body dose than those with smaller or surgically removed primaries (3.42 ± 0.74 vs. 2.48 ± 0.65 Gy, respectively, p = .016). Conversely, the correlation between activity/kg and the whole-body dose was moderate (R: 0.42, p = .093). In the multivariate analysis, the volume of the primary tumour was the most relevant predictor of the whole-body dose (p = .002). CONCLUSIONS: These data suggest that the presence of a bulky primary tumour can significantly prolong the 131 I-mIBG biological half-life, effectively increasing the absorbed whole-body dose. This information could be used to model the administered activity, allowing to attain the target dose without needing a two-step radiopharmaceutical administration.

Neuroblastoma Patients' Outcome and Chromosomal Instability.

Chromosomal instability (CIN) induces a high rate of losses or gains of whole chromosomes or parts of chromosomes. It is a hallmark of most human cancers and one of the causes of aneuploidy and intra-tumor heterogeneity. The present study aimed to evaluate the potential prognostic role of CIN in NB patients at diagnosis. We performed array comparative genomic hybridization analyses on 451 primary NB patients at the onset of the disease. To assess global chromosomal instability with high precision, we focused on the total number of DNA breakpoints of gains or losses of chromosome arms. For each tumor, an array-CGH-based breakpoint instability index (BPI) was assigned which defined the total number of chromosomal breakpoints per genome. This approach allowed us to quantify CIN related to whole genome disruption in all NB cases analyzed. We found differences in chromosomal breakages among the NB clinical risk groups. High BPI values are negatively associated with survival of NB patients. This association remains significant when correcting for stage, age, and MYCN status in the Cox model. Stratified analysis confirms the prognostic effect of BPI index in low-risk NB patients with non-amplified MYCN and with segmental chromosome aberrations.

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.

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.

Association of PARP1 polymorphisms with response to chemotherapy in patients with high-risk neuroblastoma.

The genetic aetiology and the molecular mechanisms that characterize high-risk neuroblastoma are still little understood. The majority of high-risk neuroblastoma patients do not take advantage of current induction therapy. So far, one of the main reasons liable for cancer therapeutic failure is the acquisition of resistance to cytotoxic anticancer drugs, because of the DNA repair system of tumour cells. PARP1 is one of the main DNA damage sensors involved in the DNA repair system and genomic stability. We observed that high PARP1 mRNA level is associated with unfavourable prognosis in 3 public gene expression NB patients' datasets and in 20 neuroblastomas analysed by qRT-PCR. Among 4983 SNPs in PARP1, we selected two potential functional SNPs. We investigated the association of rs907187, in PARP1 promoter, and rs2048426 in non-coding region with response chemotherapy in 121 Italian patients with high-risk NB. Results showed that minor G allele of rs907187 associated with induction response of patients (P = .02) and with decrease PARP1 mRNA levels in NB cell line (P = .003). Furthermore, rs907187 was predicted to alter the binding site of E2F1 transcription factor. Specifically, allele G had low binding affinity with E2F1 whose expression positively correlates with PARP1 expression and associated with poor prognosis of patients with NB. By contrast, we did not find genetic association for the SNP rs2048426. These data reveal rs907187 as a novel potential risk variant associated with the failure of induction therapy for high-risk NB.

Brachiocephalic vein for percutaneous ultrasound-guided central line positioning in children: A 20-month preliminary experience with 109 procedures.

BACKGROUND: Ultrasound-guided (USG) cannulation of the brachiocephalic vein (BCV) is gaining worldwide consensus for central venous access in children. This study reports a 20-month experience with this approach in children. METHODS: All patients who underwent percutaneous USG central venous catheter (CVC) positioning in the BCV between August 2013 and March 2015 have been included. Devices inserted during this period were open-ended, either single or double-lumen tunneled CVC. Our series was divided into three consecutive study periods in order to determine the relative incidence of repositioning and complications. RESULTS: During the study period, a total of 95 patients underwent 109 CVC insertions in the BCV. The median length of CVC duration was 230 days for a total of 23,212 catheter days. No major intraoperative complications occurred. Overall rate of CVC-related postoperative complications requiring repositioning or precocious removal was 0.90 per 1,000 catheter days and involved 21 CVC (19%, 95% confidence interval 13-28). These included 18 dislodgments, two infections, and one malfunction. Double-lumen CVCs represented the only significant risk factor for complications (52% complications-three per 1,000 catheter days). CONCLUSION: USG supraclavicular cannulation of the BCV represents a safe approach for central line placement in children. It proved to be versatile, as it can be used in premature infants as well as in adolescents. Provided it is adopted by operators experienced in USG cannulation, we strongly suggest to resort to this approach as a first-line choice in children undergoing tunnelled central line placement for long-lasting therapy.

Outcome of children with relapsed or refractory neuroblastoma: A meta-analysis of ITCC/SIOPEN European phase II clinical trials.

BACKGROUND: Few randomized trials have been conducted in children with relapsed/refractory neuroblastoma and data about outcomes including progression-free survival (PFS) in these patients are scarce. PROCEDURE: A meta-analysis of three phase II studies of children with relapsed/refractory neuroblastoma conducted in Europe (temozolomide, topotecan-vincristine-doxorubicin and topotecan-temozolomide) was performed. Individual patient data with extended follow-up were collected from the trial databases after publication to describe trial outcomes (response rate, clinical benefit ratio, duration of treatment, PFS, and overall survival [OS]). Characteristics of subjects with relapsed/refractory neuroblastoma were compared. RESULTS: Data from 71 children and adolescents with relapsed/refractory neuroblastoma were collected. Response definitions were not homogeneous in the three trials. Patients were on study for a median of 3.5 months (interquartile range [IQR] 1.9-6.2). Of those, 35.2% achieved a complete or partial response, 26.3% experienced a response after more than two cycles, and 23.9% received more than six cycles. Median PFS from study entry for all, refractory, and relapsed patients was 6.4 ± 1.0, 12.5 ± 6.8, and 5.7 ± 1.0 months, respectively (P = 0.006). Median OS from study entry for all, refractory, and relapsed patients was 16.1 ± 4.3, 27.9 ± 20.2, and 11.0 ± 1.6 months, respectively (P = 0.03). CONCLUSIONS: Baseline data for response rate, clinical benefit ratio, duration of treatment, PFS, and OS were provided. Two subpopulations (relapsed/refractory) were clearly distinct and should be included in the interpretation of all trials. These results should help informing the design of forthcoming studies in relapsed/refractory neuroblastoma.

Performance of 1,3-ß-D-glucan for diagnosing invasive fungal diseases in children.

Plasma 1,3-ß-D-glucan (BDG) is indicated as a tool for early diagnosis of invasive fungal diseases (IFD). However, data on its diagnostic value are scarce in children. Therefore, definition of BDG test performance in paediatrics is needed. BDG was evaluated in children admitted to "Istituto Giannina Gaslini," Genoa, Italy, who developed clinical conditions at risk for IFD. Results were analysed for sensitivity, specificity, predictive values, likelihood ratios, accuracy, informedness and probability of missing one case by a negative test. A total of 1577 BDG determinations were performed on 255 patients (49% males, median age 5.4 years). Overall 46 IFD were diagnosed, 72% proven/probable. The test performance was evaluated for 80 pg/mL, 120 pg/mL, 200 pg/mL, 350 pg/mL, 400 pg/mL cut offs. Sensitivity was always <0.80 and specificity > 0.90 only for cut offs ≥200 pg/mL. Negative predictive value was ≥0.90 for all the cut offs evaluated, while positive predictive value resulted barely 0.50 (8% IFD prevalence). Accuracy was never >0.90, and informedness was at best 0.50. The risk of missing one IFD by a negative result was < 10%. Analyses in haemato-oncological or newborn patients did not show major differences. Detection of serum BDG does not appear a valuable adjunctive diagnostic tool for IFD in paediatrics.

Candida infections in paediatrics: Results from a prospective single-centre study in a tertiary care children's hospital.

To describe the epidemiology of invasive Candida infection in a tertiary care paediatric hospital. Prospective single-centre survey on all Candida strains isolated from normally sterile fluids and urines in the period 2005-2015 . A total of 299 ICI were documented in 262 patients. Urinary tract infection represented the most frequent diagnosis (62%), followed by fungaemia (34%) and peritonitis (4%). Fungaemia was most frequent in children with cancer (59%) or in low birth weight neonates (61%), while urinary tract infections were more frequent in patients with urinary tract malformation. C.albicans was the most frequently isolated species (60%) compared with C. non-albicans, but differences were present according to the site of isolation and underlying conditions. Overall 90-day mortality was 7%, 13% in fungaemias, 8% in peritonitis and 2% in urinary tract infections. The rates of invasive Candida infection increased during the study period. Invasive Candida infection is diagnosed with increasing frequency in children. Site of isolation and aetiology are frequently related with the presence of underlying, favouring conditions. Mortality was not negligible, especially in the presence of more invasive infections and specific underlying conditions.

Neuroblastoma is associated with alterations in gut microbiome composition subsequent to maternal microbial seeding.

BACKGROUND: Neuroblastoma is the most frequent extracranial solid tumour in children, accounting for ∼15% of deaths due to cancer in childhood. The most common clinical presentation are abdominal tumours. An altered gut microbiome composition has been linked to multiple cancer types, and reported in murine models of neuroblastoma. Whether children with neuroblastoma display alterations in gut microbiome composition remains unexplored. METHODS: We assessed gut microbiome composition by shotgun metagenomic profiling in an observational cross-sectional study on 288 individuals, consisting of patients with a diagnosis of neuroblastoma at disease onset (N = 63), healthy controls matching the patients on the main covariates of microbiome composition (N = 94), healthy siblings of the patients (N = 13), mothers of patients (N = 59), and mothers of the controls (N = 59). We examined taxonomic and functional microbiome composition and mother-infant strain transmission patterns. FINDINGS: Patients with neuroblastoma displayed alterations in gut microbiome composition characterised by reduced microbiome richness, decreased relative abundances of 18 species (including Phocaeicola dorei and Bifidobacterium bifidum), enriched protein fermentation and reduced carbohydrate fermentation potential. Using machine learning, we could successfully discriminate patients from controls (AUC = 82%). Healthy siblings did not display such alterations but resembled the healthy control group. No significant differences in maternal microbiome composition nor mother-to-offspring transmission were detected. INTERPRETATION: Patients with neuroblastoma display alterations in taxonomic and functional gut microbiome composition, which cannot be traced to differential maternal seeding. Follow-up research should include investigating potential causal links. FUNDING: Italian Ministry of Health Ricerca Corrente and Ricerca Finalizzata 5 per mille (to MPonzoni); Fondazione Italiana Neuroblastoma (to MPonzoni); European Research Council (ERC-StG project MetaPG-716575 and ERC-CoG microTOUCH-101045015 to NS); the European H2020 program ONCOBIOME-825410 project (to NS); the National Cancer Institute of the National Institutes of Health 1U01CA230551 (to NS); the Premio Internazionale Lombardia e Ricerca 2019 (to NS); the MIUR Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN) Bando 2017 Grant 2017J3E2W2 (to NS); EMBO ALTF 593-2020 and Knowledge Generation Project from the Spanish Ministry of Science and Innovation (PID2022-139328OA-I00) (to MV-C).

Multiple Genes with Potential Tumor Suppressive Activity Are Present on Chromosome 10q Loss in Neuroblastoma and Are Associated with Poor Prognosis.

Neuroblastoma (NB) is a tumor affecting the peripheral sympathetic nervous system that substantially contributes to childhood cancer mortality. Despite recent advances in understanding the complexity of NB, the mechanisms determining its progression are still largely unknown. Some recurrent segmental chromosome aberrations (SCA) have been associated with poor survival. However, the prognostic role of most SCA has not yet been investigated. We examined a cohort of 260 NB primary tumors at disease onset for the loss of chromosome 10q, by array-comparative genomic hybridization (a-CGH) and Single Nucleotide Polymorphism (SNP) array and we found that 26 showed 10q loss, while the others 234 displayed different SCA. We observed a lower event-free survival for NB patients displaying 10q loss compared to patients with tumors carrying other SCA. Furthermore, analyzing the region of 10q loss, we identified a cluster of 75 deleted genes associated with poorer outcome. Low expression of six of these genes, above all CCSER2, was significantly correlated to worse survival using in silico data from 786 NB patients. These potential tumor suppressor genes can be partly responsible for the poor prognosis of NB patients with 10q loss.

Afatinib in paediatric patients with recurrent/refractory ErbB-dysregulated tumours: Results of a phase I/expansion trial.

AIM: This phase I/expansion study assessed the safety, pharmacokinetics and preliminary antitumor activity of afatinib in paediatric patients with cancer. METHODS: The dose-finding part enroled patients (2-<18 years) with recurrent/refractory tumours. Patients received 18 or 23 mg/m2/d afatinib orally (tablet or solution) in 28-d cycles. In the maximum tolerated dose (MTD) expansion, eligible patients (1-<18 years) had tumours fulfilling ≥2 of the following criteria in the pre-screening: EGFR amplification; HER2 amplification; EGFR membrane staining (H-score>150); HER2 membrane staining (H-score>0). The primary end-points were dose-limiting toxicities (DLTs), afatinib exposure, and objective response. RESULTS: Of 564 patients pre-screened, 536 patients had biomarker data and 63 (12%) fulfilled ≥2 EGFR/HER2 criteria required for inclusion in the expansion part. A total of 56 patients were treated (17 in the dose-finding and 39 in the expansion part). DLTs were observed in one of six MTD-evaluable patients receiving 18 mg/m²/d and in two of five MTD-evaluable patients receiving 23 mg/m²/d; 18 mg/m²/d was defined as the MTD. There were no new safety signals. Pharmacokinetics confirmed exposure consistent with the approved dose in adults. One partial response (-81% per Response Assessment in Neuro-Oncology) was observed in a patient with a glioneuronal tumour harbouring a CLIP2::EGFR fusion; unconfirmed partial responses were observed in two patients. In total, 25% of patients experienced objective response or stable disease (95% confidence interval: 14-38). CONCLUSION: Targetable EGFR/HER2 drivers are rare in paediatric cancers. Treatment with afatinib led to a durable response (>3 years) in one patient with a glioneuronal tumour with CLIP2::EGFR fusion.

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.

Maternal and food microbial sources shape the infant microbiome of a rural Ethiopian population.

The human microbiome seeding starts at birth, when pioneer microbes are acquired mainly from the mother. Mode of delivery, antibiotic prophylaxis, and feeding method have been studied as modulators of mother-to-infant microbiome transmission, but other key influencing factors like modern westernized lifestyles with high hygienization, high-calorie diets, and urban settings, compared with non-westernized lifestyles have not been investigated yet. In this study, we explored the mother-infant sharing of characterized and uncharacterized microbiome members via strain-resolved metagenomics in a cohort of Ethiopian mothers and infants, and we compared them with four other cohorts with different lifestyles. The westernized and non-westernized newborns' microbiomes composition overlapped during the first months of life more than later in life, likely reflecting similar initial breast-milk-based diets. Ethiopian and other non-westernized infants shared a smaller fraction of the microbiome with their mothers than did most westernized populations, despite showing a higher microbiome diversity, and uncharacterized species represented a substantial fraction of those shared in the Ethiopian cohort. Moreover, we identified uncharacterized species belonging to the Selenomonadaceae and Prevotellaceae families specifically present and shared only in the Ethiopian cohort, and we showed that a locally produced fermented food, injera, can contribute to the higher diversity observed in the Ethiopian infants' gut with bacteria that are not part of the human microbiome but are acquired through fermented food consumption. Taken together, these findings highlight the fact that lifestyle can impact the gut microbiome composition not only through differences in diet, drug consumption, and environmental factors but also through its effect on mother-infant strain-sharing patterns.

Nucleolin expression has prognostic value in neuroblastoma patients.

BACKGROUND: Neuroblastoma (NB) represents the most frequent form of extra-cranial solid tumour of infants, responsible for 15% of childhood cancer deaths. Nucleolin (NCL) prognostic value in NB was investigated. METHODS: NCL protein expression was retrospectively evaluated in tumour samples of NB patients at diagnosis and after chemotherapy. NCL prognostic value at mRNA level was assessed in a cohort of 20 patients with stage 4 NB (qPCR20, n=20, discovery dataset) and in the MultiPlatform786 including 786 patients of all stages (validation dataset). Overall and event-free survival curves were plotted by Kaplan-Meier method and compared by log-rank test. FINDINGS: NCL protein, down-modulated after chemotherapy in association with features of neuroblastic differentiation,resulted statistically significantly overexpressed in NB tumours and higher in stage 4 compared to stage 1,2,3 patients. In the stage 4 patients cohort qPCR20, patients with high NCLmRNA expression revealed a statisticallysignificant lower survival probability than those with low NCL expression (OS: HR 4.1 95%CI 1.2-13.8;p=0.0215[Log-rank test], EFS: HR 4.1 95%CI 1.2-14.0, p=0.0197[Log-rank test]). In the MultiPlatform786 (n=786), multivariate analysis suggested thatNCL expression has a statistically significant prognostic value even in the model adjusted for established prognostic markers. NCL expression significantly stratified also patients with >18 months and stage 4 tumour (OS: HR 1.8 95%CI 1.2-2.7, p=0.0009[Log-rank test]; EFS: HR 1.7 95%CI 1.1-2.5, p=0.002[Log-rank test]), patients with>18 months stage 4 with MYCN non amplified tumour[EFS: HR 2.3 95%CI 1.2-4.7, p=0.01[Log-rank test]), and patients with MYCN non amplified and MYC high [OS: HR 11.9 95%CI 2.3-62.4, p=0.003[Log-rank test]; EFS: HR 7.2 95%CI 1.6-33.4, p=0.01[Log-rank test]). A statistically significant correlation between NCL and MYCN, MYC, and TERT was found in independent datasets (MultiPlatform786 (n=786) and Agilent394 (n=394). Gene set enrichment analysis revealed a statisticallysignificant positive enrichment of MYC target genes and genes involved in telomerase maintenance. INTERPRETATION: NCL is a novel and independent (adjusting for age, INSS stage, and MYCN status) prognostic marker for NB. FUNDING: IMH-EuroNanoMed II-2015 and AIRC-IG.

Bridging therapeutic opportunities: a survey by the Italian molecular tumor board workgroup of Alliance Against Cancer.

BACKGROUND: Molecular tumor boards (MTBs) match molecular alterations with targeted anticancer drugs upon failure of the available therapeutic options. Special and local needs are most likely to emerge through the comparative analysis of MTB networks, but these are rarely reported. This manuscript summarizes the state-of-art of 16 active Italian MTBs, as it emerges from an online survey curated by Alliance Against Cancer (ACC). MAIN TEXT: Most MTBs (13/16) are exclusively supported through local Institutional grants and meet regularly. All but one adopts a fully virtual or a mixed face-to-face/virtual calling/attendance meeting model. It appears that the ACC MTB initiative is shaping a hub-and-spoke virtual MTB network reminiscent of non-redundant, cost-effective healthcare organization models. Unfortunately, public awareness of MTB opportunities presently remains insufficient. Only one center has a website. Dedicated e-mail addresses are for the exclusive use of the MTB staff. More than half of ACC members consider a miscellanea of most or all solid and hematological malignancies, and more than one-third consider neoplasms arising at any anatomical location. The average number of Staff Members in MTBs is 9. More than 10 staff members simultaneously attend MTB meetings in 13 MTBs. A medical oncologist is invariably present and is in charge of introducing the clinical case either with (45%) or without previous discussion in organ-specific multidisciplinary Boards. All but two MTBs take charge of not only patients with no standard-of-care (SoC) therapy option, but also cases receiving NGS profiling in SoC settings, implying a larger number of yearly cases. All MTBs run targeted NGS panels. Three run whole-exome and/or RNAseq approaches. ESCAT-ESMO and/or Onco-KB levels of evidence are similarly used for diagnostic reporting. Most MTBs (11) provide a written diagnostic report within 15 days. Conclusions are invariably communicated to the patient by the medical oncologist. CONCLUSIONS: MTB networking is crucial not only for molecular diagnosis and therapy assignment, but also for healthcare governance. Survey results show that MTBs review therapeutic opportunities at the crossover between standard-of-care with off-label, the former task being much beyond their scope. Societal and scientific implications of this beyond-the-scope MTB function may be relevant for healthcare in Italy and abroad.