Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Target actionability review to evaluate CDK4/6 as a therapeutic target in paediatric solid and brain tumours.

BACKGROUND: Childhood cancer is still a leading cause of death around the world. To improve outcomes, there is an urgent need for tailored treatment. The systematic evaluation of existing preclinical data can provide an overview of what is known and identify gaps in the current knowledge. Here, we applied the target actionability review (TAR) methodology to assess the strength and weaknesses of available scientific literature on CDK4/6 as a therapeutic target in paediatric solid and brain tumours by structured critical appraisal. METHODS: Using relevant search terms in PubMed, a list of original publications investigating CDK4/6 in paediatric solid tumour types was identified based on relevancy criteria. Each publication was annotated for the tumour type and categorised into separate proof-of-concept (PoC) data modules. Based on rubrics, quality and experimental outcomes were scored independently by two reviewers. A third reviewer evaluated and adjudicated score discrepancies. Scores for each PoC module were averaged for each tumour type and visualised in a heatmap matrix in the publicly available R2 data portal. RESULTS AND CONCLUSIONS: This CDK4/6 TAR, generated by analysis of 151 data entries from 71 publications, showed frequent genomic aberrations of CDK4/6 in rhabdomyosarcoma, osteosarcoma, high-grade glioma, medulloblastoma, and neuroblastoma. However, a clear correlation between CDK4/6 aberrations and compound efficacy is not coming forth from the literature. Our analysis indicates that several paediatric indications would need (further) preclinical evaluation to allow for better recommendations, especially regarding the dependence of tumours on CDK4/6, predictive biomarkers, resistance mechanisms, and combination strategies. Nevertheless, our TAR heatmap provides support for the relevance of CDK4/6 inhibition in Ewing sarcoma, medulloblastoma, malignant peripheral nerve sheath tumour and to a lesser extent neuroblastoma, rhabdomyosarcoma, rhabdoid tumour and high-grade glioma. The interactive heatmap is accessible through R2 [r2platform.com/TAR/CDK4_6].

Target Actionability Review: a systematic evaluation of replication stress as a therapeutic target for paediatric solid malignancies.

BACKGROUND: Owing to the high numbers of paediatric cancer-related deaths, advances in therapeutic options for childhood cancer is a heavily studied field, especially over the past decade. Classical chemotherapy offers some therapeutic benefit but has proven long-term complications in survivors, and there is an urgent need to identify novel target-driven therapies. Replication stress is a major cause of genomic instability in cancer, triggering the stalling of the replication fork. Failure of molecular response by DNA damage checkpoints, DNA repair mechanisms and restarting the replication forks can exacerbate replication stress and initiate cell death pathways, thus presenting as a novel therapeutic target. To bridge the gap between preclinical evidence and clinical utility thereof, we apply the literature-driven systematic target actionability review methodology to published proof-of-concept (PoC) data related to the process of replication stress. METHODS: A meticulous PubMed literature search was performed to gather replication stress-related articles (published between 2014 and 2021) across 16 different paediatric solid tumour types. Articles that fulfilled inclusion criteria were uploaded into the R2 informatics platform [r2.amc.nl] and assessed by critical appraisal. Key evidence based on nine pre-established PoC modules was summarised, and scores based on the quality and outcome of each study were assigned by two separate reviewers. Articles with discordant modules/scores were re-scored by a third independent reviewer, and a final consensus score was agreed upon by adjudication between all three reviewers. To visualise the final scores, an interactive heatmap summarising the evidence and scores associated with each PoC module across all, including paediatric tumour types, were generated. RESULTS AND CONCLUSIONS: 145 publications related to targeting replication stress in paediatric tumours were systematically reviewed with an emphasis on DNA repair pathways and cell cycle checkpoint control. Although various targets in these pathways have been studied in these diseases to different extents, the results of this extensive literature search show that ATR, CHK1, PARP or WEE1 are the most promising targets using either single agents or in combination with chemotherapy or radiotherapy in neuroblastoma, osteosarcoma, high-grade glioma or medulloblastoma. Targeting these pathways in other paediatric malignancies may work as well, but here, the evidence was more limited. The evidence for other targets (such as ATM and DNA-PK) was also limited but showed promising results in some malignancies and requires more studies in other tumour types. Overall, we have created an extensive overview of targeting replication stress across 16 paediatric tumour types, which can be explored using the interactive heatmap on the R2 target actionability review platform [https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?option=imi2_targetmap_v1].

Systematic target actionability reviews of preclinical proof-of-concept papers to match targeted drugs to paediatric cancers.

BACKGROUND: Children with cancer are in urgent need of new therapies, as approximately 25% of patients experience a relapse and 20% succumb to their disease. Moreover, the majority of survivors suffer from clinically relevant health problems. Repurposing of targeted agents developed for adult indications could provide novel therapeutic options for paediatric cancer patients. To prioritise targeted drugs for paediatric clinical development, we applied a systematic review methodology to develop a Target Actionability Review (TAR) strategy. These TARs assess the strength and completeness of published preclinical proof-of-concept (PoC) data by structured critical appraisal of and summarising the available scientific literature for a specific target (pathway) and the associated drugs in paediatric tumours. METHODS: A sensitive literature search in PubMed was performed and relevant papers were identified. For each paper, the individual experimental findings were extracted, marked for paediatric tumour type and categorised into nine separate PoC data modules. Each experimental finding was scored for experimental outcome and quality independently by two reviewers; discrepancies were assessed by a third reviewer and resolved by adjudication. Scores corresponding to one PoC module were merged for each tumour type and visualised in a heat map matrix in the publicly available R2 data portal [r2.amc.nl]. RESULTS AND CONCLUSIONS: To test our TAR methodology, we conducted a pilot study on MDM2 and TP53. The heat map generated from analysis of 161 publications provides a rationale to support drug development in specific paediatric solid and brain tumour types. Furthermore, our review highlights tumour types where preclinical data are incomplete or lacking and for which additional preclinical testing is advisable.

Outcomes of metastatic non-rhabdomyosarcoma soft tissue sarcomas (NRSTS) treated within the BERNIE study: a randomised, phase II study evaluating the addition of bevacizumab to chemotherapy.

PURPOSE: We analysed the cohort of paediatric patients with metastatic non-rhabdomyosarcoma soft tissue sarcomas (NRSTS) treated in the BERNIE protocol, i.e. open-label, multicentre, randomised phase II study evaluating the role of bevacizumab (BO20924/ITCC-006; ClinicalTrials.gov: NCT00643565). METHODS: Eligible patients were randomised 1:1 to add or not add bevacizumab to nine courses of intensive multi-drug chemotherapy, followed by 12-month maintenance chemotherapy (plus surgery and radiotherapy). The primary end-point was event-free survival (EFS); secondary objectives were objective response rate (ORR) and overall survival (OS). RESULTS: From 2008 and 2013, 49 NRSTS patients (out of 154 cases) were treated, 26 in the standard arm and 23 in the bevacizumab arm. ORR was seen in 10 out of 36 evaluable cases (27.7%), i.e. 4/18 standard arm cases and 6/18 bevacizumab arm cases. Two-year EFS was 27.3% (95% confidence interval [CI] 13.9-42.5) for all NRSTS patients, i.e. 34.9% (95% CI 14.6-56.2) for bevacizumab arm and 22.9% (95% CI 7.1-43.9) for standard arm (p-value = 0.19). Three-year OS (median follow-up 48.6 months) was 35.2%, with no differences in the two arms. Time to event and time to death were 16.3 and 17.2 months for bevacizumab arm and 2.1 and 7.6 months for standard arm, respectively. Patients not receiving any local treatment on primary disease had a worse outcome as compared to others. Treatment results were better for patients receiving surgical resection and worse for those who did not receive any specific treatment. CONCLUSION: The addition of the anti-angiogenic agent to the standard chemotherapy did not show statistically significant improvement in survival in metastatic NRSTS.

Declining free thyroxine levels over time in irradiated childhood brain tumor survivors.

OBJECTIVE: The incidence of cranial radiotherapy (cRT)-induced central hypothyroidism (TSHD) in childhood brain tumor survivors (CBTS) is reported to be low. However, TSHD may be more frequent than currently suspected, as its diagnosis is challenging due to broad reference ranges for free thyroxine (FT4) concentrations. TSHD is more likely to be present when FT4 levels progressively decline over time. Therefore, we determined the incidence and latency time of TSHD and changes of FT4 levels over time in irradiated CBTS. DESIGN: Nationwide, 10-year retrospective study of irradiated CBTS. METHODS: TSHD was defined as 'diagnosed' when FT4 concentrations were below the reference range with low, normal or mildly elevated thyrotropin levels, and as 'presumed' when FT4 declined ≥ 20% within the reference range. Longitudinal FT4 concentrations over time were determined in growth hormone deficient (GHD) CBTS with and without diagnosed TSHD from cRT to last follow-up (paired t-test). RESULTS: Of 207 included CBTS, the 5-year cumulative incidence of diagnosed TSHD was 20.3%, which occurred in 50% (25/50) of CBTS with GHD by 3.4 years (range, 0.9-9.7) after cRT. Presumed TSHD was present in 20 additional CBTS. The median FT4 decline in GH-deficient CBTS was 41.3% (P < 0.01) to diagnosis of TSHD and 12.4% (P = 0.02) in GH-deficient CBTS without diagnosed TSHD. CONCLUSIONS: FT4 concentrations in CBTS significantly decline over time after cRT, also in those not diagnosed with TSHD, suggesting that TSHD occurs more frequently and earlier than currently reported. The clinical relevance of cRT-induced FT4 decline over time should be investigated in future studies.

Risk and associated risk factors of hospitalization for specific health problems over time in childhood cancer survivors: a medical record linkage study.

Childhood cancer survivors (CCS) experience higher hospitalization rates compared to the general population for neoplasms, circulatory diseases, endocrine/nutritional/metabolic diseases and eye disorders. We studied trends in hospitalization rates and associated patient and treatment-specific risk factors for diagnosis subgroups among these four diseases. We performed medical record linkage of a ≥5-year CCS cohort with national registers, and obtained a random reference sample matched on age, gender and calendar year per CCS. For each diagnosis subgroup we compared hospitalization rates and trends over time in CCS and the reference population. Further, we analyzed risk factors for hospitalizations within the four CCS diagnosis groups. We used multivariate Poisson regression for all models. We retrieved hospitalization data from 1382 CCS and 26,583 reference persons. CCS had increased hospitalization rates for almost all diagnosis subgroups examined. Hospitalization rates for endocrine/nutritional/metabolic diseases appeared to increase with longer time since primary cancer diagnosis up to 30 years after primary cancer diagnosis. Survivors initially treated with radiotherapy had increased hospitalization rates for neoplasms (P < 0.001), those initially treated with anthracyclines (2.5 [1.1-5.5]) and radiotherapy to thorax and/or abdomen (9.3 [2.4-36.6]) had increased hospitalization rates for diseases of the circulatory system, and those initially treated with radiotherapy to head and/or neck had increased hospitalization rates for endocrine/nutritional/metabolic diseases (6.7 [3.5-12.7]) and diseases of the eye (3.6 [1.5-8.9]). Our study highlights that long-term health problems resulting in hospitalizations are still clinically relevant later in life of CCS. The identified treatment-related risk factors associated with hospitalizations support targeted follow-up care for these risk groups of CCS.

Prevalence and Risk Factors of Early Endocrine Disorders in Childhood Brain Tumor Survivors: A Nationwide, Multicenter Study.

Purpose To evaluate the prevalence of, and risk factors for, early endocrine disorders in childhood brain tumor survivors (CBTS). Patients and Methods This nationwide study cohort consisted of 718 CBTS who were diagnosed between 2002 and 2012, and who survived ≥ 2 years after diagnosis. Patients with craniopharyngeoma or a pituitary gland tumor were excluded. Results of all endocrine investigations, which were performed at diagnosis and during follow-up, were collected from patient charts. Multivariable logistic regression was used to study associations between demographic and tumor- and treatment-related variables and the prevalence of early endocrine disorders. Results After a median follow-up of 6.6 years, 178 CBTS (24.8%) were diagnosed with an endocrine disorder. A total of 159 CBTS (22.1%) presented with at least one endocrine disorder within the first 5 years after diagnosis. The most common endocrine disorders were growth hormone deficiency (12.5%), precocious puberty (12.2%), thyroid-stimulating hormone deficiency (9.2%), and thyroidal hypothyroidism (5.8%). The risk of hypothalamic-pituitary dysfunction (n = 138) was associated with radiotherapy (odds ratio [OR], 15.74; 95% CI, 8.72 to 28.42), younger age at diagnosis (OR, 1.09; 95% CI, 1.04 to 1.14), advanced follow-up time (OR, 1.10; 95% CI, 1.02 to 1.18), hydrocephalus at diagnosis (OR, 1.77; 95% CI, 1.09 to 2.88), and suprasellar (OR, 34.18; 95% CI, 14.74 to 79.29) and infratentorial (OR, 2.65; 95% CI, 1.48 to 4.74) tumor site. Conclusion The prevalence of early endocrine disorders among CBTS is high. The observation that 22.1% of CBTS developed at least one endocrine disorder within the first 5 years after diagnosis stresses the importance of early and regular assessment of endocrine function in CBTS who are at risk for endocrine damage.

Prevalence of Symptomatic and Asymptomatic Thrombosis in Pediatric Oncology Patients With Tunneled Central Venous Catheters.

BACKGROUND: Pediatric oncology patients with tunneled central venous catheters (CVCs) are at increased risk to develop venous thromboembolic events (VTEs), but the true prevalence of (a)symptomatic VTE is unknown. Aim of this study was to evaluate the prevalence of (a)symptomatic VTE in pediatric oncology patients with tunneled CVCs. PROCEDURE: All patients were included in the Aristocaths study: a randomized controlled multicenter trial investigating the prophylactic effect of 70% ethanol locks on CVC-associated bloodstream infections (CABSIs) were eligible for this study. We assessed the following outcomes: (i) symptomatic VTE and (ii) asymptomatic CVC-related VTE (using ultrasound [US]). Follow-up was 6 months, unless patients developed one of the following events: VTE, CABSI, CVC removal, or death. RESULTS: We included 305 patients (hematologic malignancy, n = 148; solid tumor, n = 157), median age 9 years (range, 1-18 years). Symptomatic VTE was detected in 8 of 305 patients (2.6%; 95% confidence interval [CI]: 1.1-5.1%), which was related to the CVC in three patients. Patients (185/305) were evaluated with US: 11 of 185 (5.9%; 95% CI: 3.0-10.4%) patients had asymptomatic CVC-related VTE. CONCLUSIONS: Prevalence of both symptomatic VTE and asymptomatic CVC-related VTE was low compared to other studies, which may be explained by the inclusion of patients with solid tumors, reduction of CABSI by ethanol, use of tunneled CVCs, and use of US.

Varicella vaccination in pediatric oncology patients without interruption of chemotherapy.

BACKGROUND: Morbidity and mortality from primary varicella-zoster virus (VZV) infection is increased in immunocompromised children. Vaccination of VZV-seronegative cancer patients with live-attenuated varicella vaccine is safe when chemotherapy is interrupted. However, VZV vaccination without interruption of chemotherapy would be preferable. OBJECTIVE: To vaccinate VZV-seronegative pediatric oncology patients with live-attenuated VZV vaccine without interrupting their chemotherapy. STUDY-DESIGN: We performed a single-center prospective cohort study. RESULTS: Thirty-one patients with either a hematological malignancy (n=24) or a solid tumor (n=7) were vaccinated early during their course of chemotherapy. VZV IgG seroconversion occurred in 14 of the 31 patients (45%) after one vaccination. Only 20 patients were revaccinated after 3 months. These were patients who did not seroconvert (5 patients) and patients who serocoverted (15 patients) to induce or sustain seropositivity. Of these 20 patients the final seroconversion rate was 70%. Seven out of the 31 patients (23%) developed a mild rash of which 5 were treated with antivirals and recovered completely without interrupting chemotherapy, and 2 recovered untreated. Of these 31 immunized patients 26 were available for cellular testing. After one vaccination 20 of 26 patients (77%) tested positive for VZV-specific CD4(+) T cells, of which 7 patients had remained VZV-seronegative. After the second vaccination 11 of 11 patients showed VZV-specific CD4(+) T cells to sustain positivity, although 4 remained VZV-seronegative. CONCLUSIONS: This study indicates that live-attenuated VZV vaccine can be safely administered to closely monitored pediatric oncology patients without interruption of chemotherapy and adaptive immunity was induced despite incomplete seroconversion.

Changes in body mass index in long-term childhood cancer survivors.

BACKGROUND: Previous studies have reported changes in the body mass index (BMI) with time in childhood cancer survivors (CCSs) during follow-up. The limitations of these studies include that they described only a subgroup of survivors or used questionnaires with self-reported heights and weights. The goal of this study was to examine BMI in a large cohort of long-term CCSs and relate this to the BMI at diagnosis, age, sex, tumor type, treatment, and endocrine defects. METHODS: All patients treated for childhood cancer at the Emma Children's Hospital/Academic Medical Center between 1966 and 1996 who had survived for at least 5 years were eligible for inclusion. For 893 CCSs with a mean follow-up of 14.9 years, the BMI at the late effects outpatient clinic was compared with the BMI for the general Dutch population. RESULTS: For girls, an increased prevalence of obesity was found. Risk factors for developing a high BMI at follow-up were a younger age and a high BMI at diagnosis and treatment with cranial radiotherapy. A significantly increased prevalence of severe underweight was found in all adult subgroups except for females aged 26 to 45 years. An association was found between a low BMI at diagnosis and a low BMI at follow-up. No treatment-related variables could be related to changes in BMI. CONCLUSIONS: The BMI at diagnosis is one of the most important predictors for the BMI at follow-up, and this suggests an important genetic or environmental cause. Adult CCSs are at high risk for developing severe underweight at follow-up. Future studies should focus on the causes and clinical consequences of underweight.

Paratesticular neuroblastoma: a case against metastatic disease?

Paratesticular lesions of neuroblastoma are in contrast to testicular infiltration extremely rare. Six cases were reported in literature. It is debated, whether localizations are either metastatic lesions or part of multicentric disease. We present a 9-month-old boy with stage 4 neuroblastoma, in which an unsuspected scrotal localization was already present at birth. This lesion had not grown, in contrast to the other sites. The case illustrates that neuroblastoma is a multicentric tumor, originating in the adrenal area and paratesticular sympathic remnants. The lesions differed in clinical behavior. It further illustrates that multicentric localizations exhibit different clinical behavior.

Phenotypic abnormalities: terminology and classification.

Clinical morphology has proved essential for the successful delineation of hundreds of syndromes and as a powerful instrument for detecting (candidate) genes (Gorlin et al. [2001]; Syndromes of the Head and Neck; Oxford: Oxford University Press. 1 p]. The major approach to reach this has been careful clinical evaluations of patients, focused on congenital anomalies. A similar careful physical examination performed in patients, who have been treated for childhood cancer, may allow detection of concurrent patterns of anomalies and provide clues for causative genes. In the past, several studies were performed describing the prevalence of anomalies in patients with cancer. However, in most studies, it was not possible to indicate the biologic relevance of the recorded anomalies, or to judge their relative importance. Are the detected anomalies common variants, and should they thus be regarded as normal, or are they minor anomalies or true abnormalities, indicating a possible developmental cause? Classification of items in the categories of common variants (disturbances of phenogenesis with a prevalence >4%), minor anomalies (disturbances of phenogenesis with a prevalence