Rationale for irradiation of persisting oligo-skeletal metastases to improve survival of metastatic neuroblastoma patients with a poor response to chemotherapy: A retrospective study.

BACKGROUND: Persistent metaiodobenzylguanidine (mIBG)-positive skeletal metastases post induction in high-risk neuroblastoma correlate with a poor outcome. The aim of this study was to investigate the potential rationale for a prospective randomized study evaluating the impact on event-free survival of the irradiation of residual oligo-skeletal metastases. PROCEDURE: Patients over 1 year with a stage M neuroblastoma treated between 2000 and 2020 at Gustave Roussy were identified. Patients with a positive mIBG scan at diagnosis and persistent skeletal metastases after high-dose chemotherapy (HDC) were included. Data were retrospectively collected and mIBG scans reviewed by two nuclear medicine physicians. RESULTS: Persistent skeletal uptake after HDC was observed in 30/201 patients (15%). Four patients reached a complete response at the end of maintenance treatment and did not relapse (median follow-up [FU] 8 years [1.8-11.8]), while two patients had progressive disease during maintenance. Among the 24 patients with persistent skeletal uptakes at the end of treatment, seven had a persistent response (median FU 8.2 years [4-15.6]). Median SIOPEN (International Society of Paediatric Oncology European Neuroblastoma) scores post consolidation and at the end of treatment were, respectively, 2 [1-6] and 2 [0-4] for patients with persistent responses compared to 4 [1-28] and 2 [1-17] for patients with progressive diseases. Median SIOPEN score at progression was 34 [2-56]. CONCLUSIONS: Our study underlines that only a minority of patients had persistent skeletal mIBG-positive scans after HDC. Recurrence mainly occurred in disease sites present at diagnosis that cleared with chemotherapy. On-therapy control of the disease is the main challenge. These results highlight the complexity of conducting a randomized study exploring this strategy.

Induced pluripotent stem cell-derived macrophages as a platform for modelling human disease.

Macrophages are innate immune cells that are present in essentially all tissues, where they have vital roles in tissue development, homeostasis and pathogenesis. The importance of macrophages in tissue function is reflected by their association with various human diseases, and studying macrophage functions in both homeostasis and pathological tissue settings is a promising avenue for new targeted therapies that will improve human health. The ability to generate macrophages from induced pluripotent stem (iPS) cells has revolutionized macrophage biology, with the generation of iPS cell-derived macrophages (iMacs) providing unlimited access to genotype-specific cells that can be used to model various human diseases involving macrophage dysregulation. Such disease modelling is achieved by generating iPS cells from patient-derived cells carrying disease-related mutations or by introducing mutations into iPS cells from healthy donors using CRISPR-Cas9 technology. These iMacs that carry disease-related mutations can be used to study the aetiology of the particular disease in vitro. To achieve more physiological relevance, iMacs can be co-cultured in 2D systems with iPS cell-derived cells or in 3D systems with iPS cell-derived organoids. Here, we discuss the studies that have attempted to model various human diseases using iMacs, highlighting how these have advanced our knowledge about the role of macrophages in health and disease.

Molecular Recognition in Mechanochemistry: Insights from Solid-State NMR Spectroscopy.

Molecular-recognition events are highly relevant in biology and chemistry. In the present study, we investigated such processes in the solid state under mechanochemical conditions using the formation of racemic phases upon reacting enantiopure entities as example. As test systems, α-(trifluoromethyl)lactic acid (TFLA) and the amino acids serine and alanine were used. The effects of ball-milling and resonant acoustic mixing (RAM) on the formation of racemic phases were probed by using solid-state Nuclear Magnetic Resonance (NMR) spectroscopy. In a mixer mill, a highly efficient and fast racemic phase formation occurred for both TFLA and the two amino acids. RAM led to the racemic phase for TFLA also, and this process was facilitated upon employing pre-milled enantiopure entities. In contrast, under comparable conditions RAM did not result in the formation of racemic phases for serine and alanine.

Surgical Management of Wilms Tumors with Intravenous Extension: A Multicenter Analysis of Clinical Management with Technical Insights.

BACKGROUND: About 5% of Wilms tumors present with vascular extension, which sometimes extends to the right atrium. Vascular extension does not affect the prognosis, but impacts the surgical strategy, which is complex and not fully standardized. Our goal is to identify elements of successful surgical management of Wilms tumors with vascular extensions. PATIENTS AND METHODS: A retrospective study of pediatric Wilms tumors treated at three sites (January 1999-June 2019) was conducted. The inclusion criterion was the presence of a renal vein and vena cava thrombus at diagnosis. Tumor stage, pre and postoperative treatment, preoperative imaging, operative report, pathology, operative complications, and follow-up data were reviewed. RESULTS: Of the 696 pediatric patients with Wilms tumors, 69 (9.9%) met the inclusion criterion. In total, 24 patients (37.5%) had a right atrial extension and two presented with Budd-Chiari syndrome at diagnosis. Two died at diagnosis owing to pulmonary embolism. All patients received neoadjuvant chemotherapy and thrombus regressed in 35.6% of cases. Overall, 14 patients had persistent intra-atrial thrombus extension (58%) and underwent cardiopulmonary bypass. Most thrombi (72%) were removed intact with nephrectomy. Massive intraoperative bleeding occurred during three procedures. Postoperative renal insufficiency was identified as a risk factor for patient survival (p = 0.01). With a median follow-up of 9 years (range: 0.5-20 years), overall survival was 89% and event-free survival was 78%. CONCLUSIONS: Neoadjuvant chemotherapy with proper surgical strategy resulted in a survival rate comparable to that of children with Wilms tumors without intravascular extension. Clinicians should be aware that postoperative renal insufficiency is associated with worse survival outcomes.

Chemo-immunotherapy with dinutuximab beta in patients with relapsed/progressive high-risk neuroblastoma: does chemotherapy backbone matter?

BACKGROUND: Addition of anti-GD2 antibodies to temozolomide-based chemotherapy has demonstrated increased antitumor activity and progression-free survival in patients with relapsed/progressive high-risk neuroblastoma. However, chemo-immunotherapy is not yet approved for this indication. This study presents the chemo-immunotherapy experience in patients with relapsed/progressive high-risk neuroblastoma treated within the off-label use program of the Neuroblastoma Committee of the French Society of Pediatric Oncology (SFCE). METHODS: Dinutuximab beta (dB) was administered alongside temozolomide-topotecan (TOTEM) or temozolomide-irinotecan (TEMIRI) at first disease relapse/progression or topotecan-cyclophosphamide (TopoCyclo) at further relapse/progression. Real-world data on demographics, treatment, antitumor activity and safety was collected from all patients after inclusion in SACHA-France (NCT04477681), a prospective national registry, which documents safety and efficacy data on innovative anticancer therapies prescribed to patients ≤ 25 years old as compassionate or off-label use. RESULTS: Between February 2021 and July 2023, 39 patients with confirmed relapsed/progressive high-risk neuroblastoma (median age 6 years, range 1-24) were treated with dB+TopoCyclo (n = 24) or dB+TOTEM/TEMIRI (n = 15) across 17 centers. In total, 163 chemo-immunotherapy cycles were administered, main toxicities were mild or moderate, with higher incidence of hematological adverse drug reactions with dB+TopoCyclo than dB+TOTEM/TEMIRI. Objective response rate was 42% for dB+TopoCyclo (CI95% 22-63%) and 40% for dB+TOTEM/TEMIRI (CI95% 16-68%). CONCLUSION: Similar objective response rates for dB+TopoCyclo and dB+TOTEM/TEMIRI in patients with relapsed/progressive high-risk neuroblastoma emphasize the importance of chemo-immunotherapy, irrespective of the chemotherapy backbone.

Sweet Cherry Extract as Permeation Enhancer of Tyrosine Kinase Inhibitors: A Promising Prospective for Future Oral Anticancer Therapies.

Although patients would rather oral therapies to injections, the gastrointestinal tract's low permeability makes this method limiting for most compounds, including anticancer drugs. Due to their low bioavailability, oral antitumor therapies suffer from significant variability in pharmacokinetics and efficacy. The improvement of their pharmacokinetic profiles can be achieved by a new approach: the use of natural extracts enriched with polyphenolic compounds that act as intestinal permeability enhancers. Here, we propose a safe sweet cherry extract capable of enhancing oral absorption. The extract was characterized by the HPLC-UV/MS method, evaluated for in vitro antioxidant activity, safety on the Caco-2 cell line, and as a potential permeation enhancer. The sweet cherry extract showed a high antioxidant capacity (ABTS and DPPH assays were 211.74 and 48.65 µmol of Trolox equivalent/g dried extract, respectively), high content of polyphenols (8.44 mg of gallic acid per gram of dry extract), and anthocyanins (1.80 mg of cyanidin-3-glucoside equivalent per g of dry extract), reassuring safety profile (cell viability never lower than 98%), and a significant and fully reversible ability to alter the integrity of the Caco-2 monolayer (+81.5% of Lucifer yellow permeability after 2 h). Furthermore, the ability of the sweet cherry extract to improve the permeability (Papp) and modify the efflux ratio (ER) of reference compounds (atenolol, propranolol, and dasatinib) and selected pyrazolo[3,4-d]pyrimidine derivatives was investigated. The obtained results show a significant increase in apparent permeability across the Caco-2 monolayer (tripled and quadrupled in most cases), and an interesting decrease in efflux ratio when compounds were co-incubated with sweet cherry extract.

Development and validation of derivatization-based LC-MS/MS method for quantification of short-chain fatty acids in human, rat, and mouse plasma.

Short-chain fatty acids (SCFAs), the end products of gut microbial fermentation of dietary fibers and non-digestible polysaccharides, act as a link between the microbiome, immune system, and inflammatory processes. The importance of accurately quantifying SCFAs in plasma has recently emerged to understand their biological role. In this work, a sensitive and reproducible LC-MS/MS method is reported for SCFAs quantification in three different matrices such as human, rat and mouse plasma via derivatization, using as derivatizing agent O-benzylhydroxylamine (O-BHA), coupled with liquid-liquid extraction. First, the instrumental parameters of the mass spectrometer and then the chromatographic conditions were optimized using previously SCFAs derivatives synthetized and used as standards. After that, the best conditions for derivatization and extraction from plasma were studied and a series of determinations were performed on human, rat, and mouse plasma aliquots to validate the overall method (derivatization, extraction, and LC-MS/MS determination). The method showed good performance in terms of recovery (> 80%), precision (RSD <14%), accuracy (RE < ± 10%) and sensitivity (LOQ of 0.01 µM for acetic, butyric, propionic and isobutyric acid) in all plasma samples. The method thus developed and validated was applied to the quantification of major SCFAs in adult and aged mice, germ-free mice and in germ-free recipient mice subjected to fecal transplant from adult and aged donors. Results highlighted how plasma concentrations of SCFAs are correlated with age further highlighting the importance of developing a method that is reliable for the quantification of SCFAs to study their biological role.

Progress towards Adjuvant Development: Focus on Antiviral Therapy.

In recent decades, vaccines have been extraordinary resources to prevent pathogen diffusion and cancer. Even if they can be formed by a single antigen, the addition of one or more adjuvants represents the key to enhance the response of the immune signal to the antigen, thus accelerating and increasing the duration and the potency of the protective effect. Their use is of particular importance for vulnerable populations, such as the elderly or immunocompromised people. Despite their importance, only in the last forty years has the search for novel adjuvants increased, with the discovery of novel classes of immune potentiators and immunomodulators. Due to the complexity of the cascades involved in immune signal activation, their mechanism of action remains poorly understood, even if significant discovery has been recently made thanks to recombinant technology and metabolomics. This review focuses on the classes of adjuvants under research, recent mechanism of action studies, as well as nanodelivery systems and novel classes of adjuvants that can be chemically manipulated to create novel small molecule adjuvants.

Biological Evaluation and In Vitro Characterization of ADME Profile of In-House Pyrazolo[3,4-d]pyrimidines as Dual Tyrosine Kinase Inhibitors Active against Glioblastoma Multiforme.

The therapeutic use of tyrosine kinase inhibitors (TKIs) represents one of the successful strategies for the treatment of glioblastoma (GBM). Pyrazolo[3,4-d]pyrimidines have already been reported as promising small molecules active as c-Src/Abl dual inhibitors. Herein, we present a series of pyrazolo[3,4-d]pyrimidine derivatives, selected from our in-house library, to identify a promising candidate active against GBM. The inhibitory activity against c-Src and Abl was investigated, and the antiproliferative profile against four GBM cell lines was studied. For the most active compounds endowed with antiproliferative efficacy in the low-micromolar range, the effects toward nontumoral, healthy cell lines (fibroblasts FIBRO 2-93 and keratinocytes HaCaT) was investigated. Lastly, the in silico and in vitro ADME properties of all compounds were also assessed. Among the tested compounds, the promising inhibitory activity against c-Src and Abl (Ki 3.14 µM and 0.44 µM, respectively), the irreversible, apoptotic-mediated death toward U-87, LN18, LN229, and DBTRG GBM cell lines (IC50 6.8 µM, 10.8 µM, 6.9 µM, and 8.5 µM, respectively), the significant reduction in GBM cell migration, the safe profile toward FIBRO 2-93 and HaCaT healthy cell lines (CC50 91.7 µM and 126.5 µM, respectively), the high metabolic stability, and the excellent passive permeability across gastrointestinal and blood-brain barriers led us to select compound 5 for further in vivo assays.

Tenebrio molitor as a Simple and Cheap Preclinical Pharmacokinetic and Toxicity Model.

The progression of drugs into clinical phases requires proper toxicity assessment in animals and the correct identification of possible metabolites. Accordingly, different animal models are used to preliminarily evaluate toxicity and biotransformations. Rodents are the most common models used to preliminarily evaluate the safety of drugs; however, their use is subject to ethical consideration and elevated costs, and strictly regulated by national legislations. Herein, we developed a novel, cheap and convenient toxicity model using Tenebrio molitor coleoptera (TMC). A panel of 15 drugs-including antivirals and antibacterials-with different therapeutic applications was administered to TMC and the LD50 was determined. The values are comparable with those already determined in mice and rats. In addition, a TMC model was used to determine the presence of the main metabolites and in vivo pharmacokinetics (PK), and results were compared with those available from in vitro assays and the literature. Taken together, our results demonstrate that TMC can be used as a novel and convenient preliminary toxicity model to preliminarily evaluate the safety of experimental compounds and the formation of main metabolites, and to reduce the costs and number of rodents, according to 3R principles.

Number of lymph nodes sampled in SFCE/SIOP 2001 patients with Wilms tumour: Is the goal of more than six achievable?

AIM: The number of lymph nodes (LN) that should be sampled during nephrectomy for Wilms tumour (WT) remains controversial but of utmost importance for staging purposes. The aim of this French national retrospective study of patients enrolled in SIOPWT2001 trial was to analyse the number of LN sampled according to their site and to determine if the number of six asked by the International Society of Paediatric Oncology - Renal Tumour Study Group (SIOP-RTSG) UMBRELLA protocol is achievable. METHODS: We reviewed the data collected on central pathology review forms from 2002 to 2014 for only unilateral WT. LN were divided whether they were clearly identified by surgeons at nephrectomy or only found by pathologists on the nephrectomy specimen. RESULTS: A total of 539 patients (240 male/299 female) were included (458 localized/81 metastatic). Median age at surgery was 41.3 months [0-189]. The number of LN sampled was 0, 1-6, ≥7 and unknown in 69 (12.8%), 293 (54.3%), 160 (29.7%) and 17 (3.2%) cases, respectively. The number of patients with sampled LN were higher if LN were identified by both the pathologist and the surgeon (n = 231, 42.8%) (p = < .001). At least one invaded LN (LN+) was found in 66 patients (12.2%), more than half being found among patients having LN sampled by both pathologist and surgeon (p < .001). The mean number of identified LN was six if no LN+ was detected on final histological analysis, while it was 11 in case of LN+ (p < .001). CONCLUSIONS: The aim of sampling more than six LN is achievable, but only with the active collaboration of both surgeons and pathologists.

Divergent HLA variations and heterogeneous expression but recurrent HLA loss-of- heterozygosity and common HLA-B and TAP transcriptional silencing across advanced pediatric solid cancers.

The human leukocyte antigen (HLA) system is a major factor controlling cancer immunosurveillance and response to immunotherapy, yet its status in pediatric cancers remains fragmentary. We determined high-confidence HLA genotypes in 576 children, adolescents and young adults with recurrent/refractory solid tumors from the MOSCATO-01 and MAPPYACTS trials, using normal and tumor whole exome and RNA sequencing data and benchmarked algorithms. There was no evidence for narrowed HLA allelic diversity but discordant homozygosity and allele frequencies across tumor types and subtypes, such as in embryonal and alveolar rhabdomyosarcoma, neuroblastoma MYCN and 11q subtypes, and high-grade glioma, and several alleles may represent protective or susceptibility factors to specific pediatric solid cancers. There was a paucity of somatic mutations in HLA and antigen processing and presentation (APP) genes in most tumors, except in cases with mismatch repair deficiency or genetic instability. The prevalence of loss-of-heterozygosity (LOH) ranged from 5.9 to 7.7% in HLA class I and 8.0 to 16.7% in HLA class II genes, but was widely increased in osteosarcoma and glioblastoma (~15-25%), and for DRB1-DQA1-DQB1 in Ewing sarcoma (~23-28%) and low-grade glioma (~33-50%). HLA class I and HLA-DR antigen expression was assessed in 194 tumors and 44 patient-derived xenografts (PDXs) by immunochemistry, and class I and APP transcript levels quantified in PDXs by RT-qPCR. We confirmed that HLA class I antigen expression is heterogeneous in advanced pediatric solid tumors, with class I loss commonly associated with the transcriptional downregulation of HLA-B and transporter associated with antigen processing (TAP) genes, whereas class II antigen expression is scarce on tumor cells and occurs on immune infiltrating cells. Patients with tumors expressing sufficient HLA class I and TAP levels such as some glioma, osteosarcoma, Ewing sarcoma and non-rhabdomyosarcoma soft-tissue sarcoma cases may more likely benefit from T cell-based approaches, whereas strategies to upregulate HLA expression, to expand the immunopeptidome, and to target TAP-independent epitopes or possibly LOH might provide novel therapeutic opportunities in others. The consequences of HLA class II expression by immune cells remain to be established. Immunogenetic profiling should be implemented in routine to inform immunotherapy trials for precision medicine of pediatric cancers.

DEAD-Box Helicase DDX3X as a Host Target against Emerging Viruses: New Insights for Medicinal Chemical Approaches.

In recent years, globalization, global warming, and population aging have contributed to the spread of emerging viruses, such as coronaviruses (COVs), West Nile (WNV), Dengue (DENV), and Zika (ZIKV). The number of reported infections is increasing, and considering the high viral mutation rate, it is conceivable that it will increase significantly in the coming years. The risk caused by viruses is now more evident due to the COVID-19 pandemic, which highlighted the need to find new broad-spectrum antiviral agents able to tackle the present pandemic and future epidemics. DDX3X helicase is a host factor required for viral replication. Selective inhibitors have been identified and developed into broad-spectrum antivirals active against emerging pathogens, including SARS-CoV-2 and most importantly against drug-resistant strains. This perspective describes the inhibitors identified in the last years, highlighting their therapeutic potential as innovative broad-spectrum antivirals.

The role of irinotecan-bevacizumab as rescue regimen in children with low-grade gliomas: a retrospective nationwide study in 72 patients.

INTRODUCTION: At least half of children with low-grade glioma (LGG) treated with first line chemotherapy experience a relapse/progression and may therefore need a second-line chemotherapy. Irinotecan-bevacizumab has been recommended in this setting in France after encouraging results of pilot studies. We performed a retrospective analysis to define the efficacy, toxicity and predictors for response to the combination on a larger cohort. METHODS: We reviewed the files from children < 19 years of age with progressive or refractory LGG treated between 2009 and 2016 in 7 French centers with this combination. RESULTS: 72 patients (median age 7.8 years [range 1-19]) received a median of 16 courses (range 3-30). The median duration of treatment was 9 months (range 1.4-16.2). 96% of patients experienced at least disease stabilization. The 6-month and 2-year progression-free survivals (PFS) were 91.7% [IC 95% 85.5-98.3] and 38.2% [IC 95% 28.2-51.8] respectively. No progression occurred after treatment in 18 patients with a median follow-up of 35.6 months (range 7.6-75.9 months). Younger patients had a worse PFS (p = 0.005). Prior chemoresistance, NF1 status, duration of treatment, histopathology or radiologic response did not predict response. The most frequent toxicities related to bevacizumab included grades 1-2 proteinuria in 21, epistaxis in 10, fatigue in 12 and hypertension in 8 while gastro-intestinal toxicity was the most frequent side effect related to irinotecan. CONCLUSIONS: Bevacizumab-irinotecan has the potential of disease control clinically and radiographically in children with recurrent LGG whatever their previous characteristics; in many cases however these responses are not sustained, especially in younger children.

High-dose chemotherapy followed by autologous stem cell rescue in Wilms tumors: French report on toxicity and efficacy.

BACKGROUND: Heterogeneous data have been reported on high-dose chemotherapy (HDCT) with autologous stem cell rescue (ASCR) in Wilms tumors (WTs). We aimed to define its safety and efficacy in the French cohort, and to compare this management to current international recommendations. METHODS: Data prospectively collected from children, adolescents, and young adults with WT treated with HDCT/ASCR between 2000 and 2016 in French centers were retrospectively analyzed. Toxicity was reported according to CTCAE v4.03. RESULTS: Fifty-four patients received HDCT/ASCR (first line, n = 13; recurrence, n = 41). Their median age at the time of ASCR was 5.3 years (range 2.2-21.6). Main nonhematological acute grades 3-4 toxicities were digestive and renal. No significant difference of toxicity rate was observed among HDCT regimens and schedules. Two patients died shortly after ASCR (renal and multiorgan failure), and one heavily pretreated patient died of late respiratory failure. The selection criteria applied to define those patients eligible for HDCT/ASCR retrospectively matched to those currently used in the International Society of Pediatric Oncology (SIOP) UMBRELLA protocol for 38 patients, with encouraging survival rates compared to published data. The objective response rate to HDCT was 21%, with a disease control rate after HDCT of 85%. After a median follow-up of 7 years, the 5-year event-free survival (EFS) and overall survival (OS) were 54% (95% CI: 32%-76%) and 62% (95% CI: 31%-82%) for frontline patients, and 57% (95% CI: 39%-71%) and 69% (95% CI: 52%-81%) at recurrence. CONCLUSION: HDCT was feasible and showed encouraging results in well-defined settings. Data from the current prospective protocol will help to better evaluate HDCT impact on survival.

Long-term follow-up of high-risk neuroblastoma survivors treated with high-dose chemotherapy and stem cell transplantation rescue.

Intensive treatments including high-dose chemotherapy (HDC) with autologous stem cell rescue have improved high-risk neuroblastoma (HRNB) survival. We report the long-term health status of 145 HRNB survivors, alive and disease-free 5 years post HDC. Median follow-up was 15 years (range = 5-34). Six patients experienced late relapses, 11 developed second malignant neoplasms (SMNs), and 9 died. Event-free and overall survivals 20 years post HDC were 82% (95% CI = 70%-90%) and 89% (78%-95%), respectively. Compared with the French general population, the standardized mortality ratio was 19 (95% CI = 8.7-36.1; p < 0.0001) and the absolute excess risk was 37.6 (19.2-73.5). Late effects were observed in 135/145 patients (median = 3 events/patient); 103 had at least one severe event. SMNs arose at a median of 20 years post HDC and included carcinoma (n = 5), sarcoma (2), acute myeloid leukemia (2), melanoma (1), and malignant glioma (1). Non-oncologic health events included dental maldevelopment (60%), severe hearing loss (20% cumulative probability at 15 years), hepatic focal nodular hyperplasia (14%), thyroid (11%), cardiac (8%), and renal (7%) diseases and growth retardation (height-for-age z-score ≤ -2 for 21%). Gonadal insufficiency was near-universal after busulfan (40/43 females, 33/35 males). Severe late effects are frequent and progressive in HRNB survivors needing systematic very long-term follow-up.

Phase II and biomarker study of programmed cell death protein 1 inhibitor nivolumab and metronomic cyclophosphamide in paediatric relapsed/refractory solid tumours: Arm G of AcSé-ESMART, a trial of the European Innovative Therapies for Children With Cancer Consortium.

PURPOSE: AcSé-ESMART is a European multicentre, proof-of-concept multiarm phase I/II platform trial in paediatric patients with relapsed/refractory cancer. Arm G assessed the activity and safety of nivolumab in combination with metronomic cyclophosphamide +/- irradiation. EXPERIMENTAL DESIGN: Following a Phase II Simon two-stage design, nivolumab was administered intravenously at 3 mg/kg every 2 weeks of a 28-day cycle, oral cyclophosphamide at 25 mg/m2 twice a day, 1 week on/1 week off. The primary endpoint was objective response rate. Irradiation/radioablation of primary tumour or metastasis could be administered as per physician's choice. Biomarker evaluation was performed by tumour immunohistochemistry, whole exome and RNA sequencing, and immunophenotyping of peripheral blood by flow cytometry. RESULTS: Thirteen patients were treated with a median age of 15 years (range: 5.5-19.4). The main histologies were high-grade glioma, neuroblastoma, and desmoplastic small round cell tumour (DSRCT). The safety profile was similar to those of single-agent nivolumab, albeit haematologic toxicity, mainly lymphocytopenia, was commonly reported with the addition of cyclophosphamide +/- irradiation. Two patients with DSRCT and ependymoma presented unconfirmed partial response and prolonged disease stabilisation. Low mutational load with modest intratumour CD3+ T-cell infiltration and immunosuppressive tumour microenvironment were observed in the tumour samples. Under combined treatment, no positive modulation of circulating T cells was displayed, while derived neutrophil-to-lymphocyte ratio increased. CONCLUSIONS: Nivolumab in combination with cyclophosphamide was well tolerated but had limited activity in this paediatric setting. Metronomic cyclophosphamide did not modulate systemic immune response that could compensate limited T-cell infiltration and the immunosuppressive tumour microenvironment. CLINICALTRIALS. GOV IDENTIFIER: NCT2813135.

Outcome of Stage IV Completely Necrotic Wilms Tumour and Local Stage III Treated According to the SIOP 2001 Protocol.

OBJECTIVE: Wilms tumour (WT) patients with a localised completely necrotic nephroblastoma after preoperative chemotherapy are a favourable outcome group. Since the introduction of the SIOP 2001 protocol, the SIOP- Renal Tumour Study Group (SIOP-RTSG) has omitted radiotherapy for such patients with low-risk, local stage III in an attempt to reduce treatment burden. However, for metastatic patients with local stage III, completely necrotic WT, the recommendations led to ambiguous use. The purpose of this descriptive study is to demonstrate the outcomes of patients with metastatic, completely necrotic and local stage III WT in relation to the application of radiotherapy or not. METHODS AND MATERIALS: all metastatic patients with local stage III, completely necrotic WT after 6 weeks of preoperative chemotherapy who were registered in the SIOP 2001 study were included in this analysis. The pattern of recurrence according to the usage of radiation treatment and 5 year event-free survival (EFS) and overall survival (OS) was analysed. RESULTS: seven hundred and three metastatic WT patients were registered in the SIOP 2001 database. Of them, 47 patients had a completely necrotic, local stage III WT: 45 lung metastases (11 combined localisations), 1 liver/peritoneal, and 1 tumour thrombus in the renal vein and the inferior vena cava with bilateral pulmonary arterial embolism. Abdominal radiotherapy was administered in 29 patients (62%; 29 flank/abdominal irradiation and 9 combined with lung irradiation). Eighteen patients did not receive radiotherapy. Median follow-up was 6.6 years (range 1-151 months). Two of the 47 patients (4%) developed disease recurrence in the lung (one combined with abdominal relapse) and eventually died of the disease. Both patients had received abdominal radiotherapy, one of them combined with lung irradiation. Five-year EFS and OS were 95% and 95%, respectively. CONCLUSIONS: the outcome of patients with stage IV, local stage III, completely necrotic Wilms tumours is excellent. Our results suggest that abdominal irradiation in this patient category may not be of added value in first-line treatment, consistent with the current recommendation in the SIOP-RTSG 2016 UMBRELLA protocol.