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We describe the case of a 14-year-old girl with osteosarcoma who was treated with high-dose methotrexate (12 g/m 2 ). Twenty-four hours after the infusion, her plasma methotrexate concentration was elevated at 937 μmol/L (normal < 10 µmol/L). She exhibited severe signs of methotrexate toxicity, including encephalopathy, acute liver failure (ALF), and acute kidney injury. In this case report, we highlight the severe and rare adverse effects secondary to methotrexate administration and the efficacity of molecular adsorbent recirculating system and continuous venovenous hemodiafiltration to recover from multiple organ failure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Corbisier, Von Bueren, Breunis and Grazioli.)

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children and adolescents, represents an aberrant form of skeletal muscle differentiation. Both skeletal muscle development, as well as regeneration of adult skeletal muscle are governed by members of the myogenic family of regulatory transcription factors (MRFs), which are deployed in a highly controlled, multi-step, bidirectional process. Many aspects of this complex process are deregulated in RMS and contribute to tumorigenesis. Interconnected loops of super-enhancers, called core regulatory circuitries (CRCs), define aberrant muscle differentiation in RMS cells. The transcriptional regulation of MRF expression/activity takes a central role in the CRCs active in skeletal muscle and RMS. In PAX3::FOXO1 fusion-positive (PF+) RMS, CRCs maintain expression of the disease-driving fusion oncogene. Recent single-cell studies have revealed hierarchically organized subsets of cells within the RMS cell pool, which recapitulate developmental myogenesis and appear to drive malignancy. There is a large interest in exploiting the causes of aberrant muscle development in RMS to allow for terminal differentiation as a therapeutic strategy, for example, by interrupting MEK/ERK signaling or by interfering with the epigenetic machinery controlling CRCs. In this review, we provide an overview of the genetic and epigenetic framework of abnormal muscle differentiation in RMS, as it provides insights into fundamental mechanisms of RMS malignancy, its remarkable phenotypic diversity and, ultimately, opportunities for therapeutic intervention.

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma and can emerge throughout the whole body. For patients with newly diagnosed RMS, prognosis for survival depends on multiple factors such as histology, tumour site, and extent of the disease. Patients with metastatic disease at diagnosis have impaired prognosis compared to those with localised disease. Appropriate staging at diagnosis therefore plays an important role in choosing the right treatment regimen for an individual patient. Fluorine-18-fluorodeoxyglucose ( 18 F-FDG) positron emission tomography (PET) is a functional molecular imaging technique that uses the increased glycolysis of cancer cells to visualise both structural information and metabolic activity. 18 F-FDG-PET combined with computed tomography (CT) could help to accurately stage the extent of disease in patients with newly diagnosed RMS. In this review we aimed to evaluate whether 18 F-FDG-PET could replace other imaging modalities for the staging of distant metastases in RMS., Objectives: To determine the diagnostic accuracy of 18 F-FDG-PET/CT imaging for the detection of bone, lung, and lymph node metastases in RMS patients at first diagnosis., Search Methods: We searched MEDLINE in PubMed (from 1966 to 23 December 2020) and Embase in Ovid (from 1980 to 23 December 2020) for potentially relevant studies. We also checked the reference lists of relevant studies and review articles; scanned conference proceedings; and contacted the authors of included studies and other experts in the field of RMS for information about any ongoing or unpublished studies. We did not impose any language restrictions., Selection Criteria: We included cross-sectional studies involving patients with newly diagnosed proven RMS, either prospective or retrospective, if they reported the diagnostic accuracy of 18 F-FDG-PET/CT in diagnosing lymph node involvement or bone metastases or lung metastases or a combination of these metastases. We included studies that compared the results of the 18 F-FDG-PET/CT imaging with those of histology or with evaluation by a multidisciplinary tumour board as reference standard., Data Collection and Analysis: Two review authors independently performed study selection, data extraction, and methodological quality assessement according to Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2). We analysed data for the three outcomes (nodal involvement and lung and bone metastases) separately. We used data from the 2 × 2 tables (consisting of true positives, false positives, true negatives, and false negatives) to calculate sensitivity and specificity in each study and corresponding 95% confidence intervals. We did not consider a formal meta-analysis to be relevant because of the small number of studies and substantial heterogeneity between studies., Main Results: Two studies met our inclusion criteria. The diagnostic accuracy of 18 F-FDG-PET/CT was reported in both studies, which included a total of 36 participants. We considered both studies to be at high risk of bias for the domain reference standard. We considered one study to be at high risk of bias for the domain index test and flow and timing. Sensitivity and specificity of 18 F-FDG-PET/CT for the detection of bone metastases was 100% in both studies (95% confidence interval (CI) for sensitivity was 29% to 100% in study one and 40% to 100% in study two; 95% CI for specificity was 83% to 100% in study one and 66% to 100% in study two). The reported sensitivity of 18 F-FDG-PET/CT for the detection of lung metastases was not calculated since only two participants in study two showed lung metastases, of which one was detected by 18 F-FDG-PET/CT. Reported specificity was 96% in study one (95% CI 78% to 100%) and 100% (95% CI 72% to 100%) in study two. The reported sensitivity for the detection of nodal involvement was 100% (95% CI 63% to 100% in study one and 40% to 100% in study two); the reported specificity was 100% (95% CI 78% to 100%) in study one and 89% (95% CI 52% to 100%) in study two., Authors' Conclusions: The diagnostic accuracy of 18 F-FDG-PET/CT for the detection of bone, lung, and lymph node metastases was reported in only two studies including a total of only 36 participants with newly diagnosed RMS. Because of the small number of studies (and participants), there is currently insufficient evidence to reliably determine the diagnostic accuracy of 18 F-FDG-PET/CT in the detection of distant metastases. Larger series evaluating the diagnostic accuracy of 18 F-FDG-PET/CT for the detection of metastases in patients with RMS are necessary., (Copyright © 2021 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.)

Background: Ewing sarcomas are solid tumours of the bone and soft tissue, that usually affect children, adolescents, and young adults. The incidence is about three cases per million a year, with a peak incidence at 12 years of age. Metastatic disease is detected in about 20 % to 30% of people, and is typically found in the lungs, bone, bone marrow, or a combination of these. Presence of metastatic disease at diagnosis (primary metastatic disease) is the most important adverse prognostic factor, and is associated with a five-year survival lower than 30%. High-dose chemotherapy (HDC) followed by autologous haematopoietic cell transplantation (AHCT) is used in various solid tumours with unfavourable prognoses in children, adolescents, and young adults. It has also been used as rescue after multifocal radiation of metastases. The hypothesis is that HDC regimens may overcome the resistance to standard multidrug chemotherapy and improve survival rates., Objectives: To assess the effects of high-dose chemotherapy with autologous haematopoietic cell transplantation compared with conventional chemotherapy in improving event-free survival, overall survival, quality-adjusted survival, and progression-free survival in children, adolescents, and young adults with primary metastatic Ewing sarcoma, and to determine the toxicity of the treatment., Search Methods: We searched CENTRAL, MEDLINE, Embase, conference proceedings from major international cancer-related conferences, and ongoing trial registers until January 2020. We also searched reference lists of included articles and review articles., Selection Criteria: We included randomised controlled trials (RCTs) or (historical) controlled clinical trials (CCTs) comparing the effectiveness of HDC and AHCT with conventional chemotherapy for children, adolescents, and young adults (younger than 30 years at the date of diagnostic biopsy) with primary metastatic Ewing sarcoma., Data Collection and Analysis: We used standard methodological procedures expected by Cochrane., Main Results: We identified one RCT, which investigated the effects of HDC with AHCT versus conventional chemotherapy with whole lung irradiation (WLI) in people with Ewing sarcoma metastasised to the lungs only at diagnosis. Only a selection of the participants were eligible for our review (N = 267: HDC with AHCT group N = 134; control group N = 133). There may be no difference in event-free survival between the two treatment groups (hazard ratio (HR) 0.83, 95% confidence interval (CI) 0.59 to 1.17; low-certainty evidence). We downgraded one level each because of study limitations and imprecision. Overall survival and toxicity were not reported separately for the participants eligible for this review, while quality-adjusted survival and progression-free survival were not reported at all. We did not identify any studies that addressed children, adolescents, and young adults with Ewing sarcoma with metastases to other locations., Authors' Conclusions: In people with Ewing sarcoma with primary metastases to locations other than the lungs, there is currently no evidence from RCTs or CCTs to determine the efficacy of HDC with AHCT compared to conventional chemotherapy. Based on low-certainty evidence from one study (267 participants), there may be no difference in event-free survival between children, adolescents, and young adults with primary pulmonary metastatic Ewing sarcoma who receive HDC with AHCT and those who receive conventional chemotherapy with WLI. Further high-quality research is needed. Results are anticipated for the EuroEwing 2008R3 study, in which the effects of HDC with treosulfan and melphalan followed by AHCT on survival, in people with Ewing sarcoma with metastatic disease to bone, other sites, or both were explored. Achieving high-quality studies in a selection of people with rare sarcoma requires long-term, multi-centre, international participant inclusion., (Copyright © 2021 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.)

Background: Ewing sarcoma is a solid tumour, which is the second most common primary bone malignancy in children, often occurring in the long bones and pelvis. An incidence rate of 4.5 per million a year is reported, with a peak incidence of 11 per million at the age of 12 years. Despite more intensive chemotherapy, 30% to 40% of young people with Ewing sarcoma will have recurrence of the disease. Less than 30% of young people with a recurrence of Ewing sarcoma are alive at 24 months, and less than 10% are alive at 48 months. High-dose chemotherapy (HDC), followed by autologous haematopoietic cell transplantation (AHCT), is used in a variety of paediatric groups with diverse solid tumours. The hypothesis is that HDC regimens may overcome resistance to standard polychemotherapy, and this way may eradicate minimal residual disease, leading to improved survival after a first recurrence of disease., Objectives: To assess the efficacy of HDC with AHCT versus conventional chemotherapy in improving event-free survival, overall survival, quality-adjusted survival, and progression-free survival in children, adolescents, and young adults with first recurrence of Ewing sarcoma, and to determine the toxicity of the treatment., Search Methods: We searched CENTRAL, MEDLINE, Embase, conference proceedings from the SIOP, ASPHO, CTOS, ASBMT, EBMT, and EMSOS, and two trial registries in January 2020. We also searched reference lists of relevant articles and review articles., Selection Criteria: We planned to include randomised controlled trials (RCTs) or (historical) controlled clinical trials (CCTs) comparing the effectiveness of HDC plus AHCT with conventional chemotherapy for children, adolescents, and young adults (up to 30 years old at the date of diagnostic biopsy) with a first recurrence of Ewing sarcoma., Data Collection and Analysis: We used standard methodological procedures expected by Cochrane., Main Results: We did not identify any eligible studies., Authors' Conclusions: Since we did not identify any eligible studies, we are unable to draw any conclusions about the efficacy and toxicity of HDC with AHCT versus conventional chemotherapy in children, adolescents, and young adults with a first recurrence of Ewing sarcoma. Further high-quality research is urgently needed., (Copyright © 2021 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.)

Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood. Results of clinical trials, with three-year event-free and overall survival as primary outcomes, often take 7 to 10 years. Identification of an early surrogate biomarker, predictive for survival, is therefore crucial. We conducted a systematic review to define the prognostic value of early tumor size response in children with IRSG group III rhabdomyosarcoma. The search included MEDLINE/EMBASE from inception to 18 November 2020. In total, six studies were included, describing 2010 patients, and assessed by the Quality in Prognosis Studies (QUIPS) instrument. Four studies found no prognostic value for tumor size response, whereas two studies reported a prognostic effect. In these two studies, the survival rate of patients with progressive disease was not separately analyzed from patients with stable disease, potentially explaining the difference in study outcome. In conclusion, our findings support that early progression of disease is associated with poorer survival, justifying adaptation of therapy. However, in patients with non-progressive disease, there is no evidence that the degree of response is a prognostic marker for survival. Because the vast majority of patients do not have progressive disease, early tumor size response should be reconsidered for assessment of treatment efficacy. Therefore, at present, early surrogate biomarkers for survival are still lacking.

Amino acids are integral components of cancer metabolism. The non-essential amino acid asparagine supports the growth and survival of various cancer cell types. Here, different mass spectrometry approaches were employed to identify lower aspartate levels, higher aspartate/glutamine ratios and lower tricarboxylic acid (TCA) cycle metabolite levels in asparagine-deprived sarcoma cells. Reduced nicotinamide adenine dinucleotide (NAD + )/nicotinamide adenine dinucleotide hydride (NADH) ratios were consistent with redirection of TCA cycle flux and relative electron acceptor deficiency. Elevated lactate/pyruvate ratios may be due to compensatory NAD + regeneration through increased pyruvate to lactate conversion by lactate dehydrogenase. Supplementation with exogenous pyruvate, which serves as an electron acceptor, restored aspartate levels, NAD + /NADH ratios, lactate/pyruvate ratios and cell growth in asparagine-deprived cells. Chemicals disrupting NAD + regeneration in the electron transport chain further enhanced the anti-proliferative and pro-apoptotic effects of asparagine depletion. We speculate that reductive stress may be a major contributor to the growth arrest observed in asparagine-starved cells.

Cancer therapy is currently shifting from broadly used cytotoxic drugs to patient-specific precision therapies. Druggable driver oncogenes, identified by molecular analyses, are present in only a subset of patients. Functional profiling of primary tumor cells could circumvent these limitations, but suitable platforms are unavailable for most cancer entities. Here, we describe an in vitro drug profiling platform for rhabdomyosarcoma (RMS), using a living biobank composed of twenty RMS patient-derived xenografts (PDX) for high-throughput drug testing. Optimized in vitro conditions preserve phenotypic and molecular characteristics of primary PDX cells and are compatible with propagation of cells directly isolated from patient tumors. Besides a heterogeneous spectrum of responses of largely patient-specific vulnerabilities, profiling with a large drug library reveals a strong sensitivity towards AKT inhibitors in a subgroup of RMS. Overall, our study highlights the feasibility of in vitro drug profiling of primary RMS for patient-specific treatment selection in a co-clinical setting.

The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the FCGR2/3 locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the FCGR2/3 locus in >4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the FCGR2C -ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the FCGR2C -ORF haplotype showed strong LD with, among others, rs201218628 ( FCGR2A -Q27W, r 2 = 0.63). LD between these two variants was weaker ( r 2 = 0.17) in Africans, whereas the FCGR2C -ORF haplotype was nearly absent in Asians (minor allele frequency <0.005%). The FCGR2C -ORF haplotype and rs1801274 ( FCGR2A -H131R) were in weak LD ( r 2 = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the FCGR2C -ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the FCGR2/3 locus with knowledge of LD and ethnic variation.

Background and Purpose: Survival after relapse of head and neck rhabdomyosarcoma (HNRMS) after prior external beam radiotherapy (EBRT) is poor, since options for adequate local treatment are often lacking. In this study we describe our experience with salvage AMORE in patients with relapsed HNRMS after prior EBRT., Materials and Methods: Patients with relapsed HNRMS after prior EBRT in which salvage AMORE treatment was considered feasible were analysed; this includes patients with parameningeal, head and neck non-parameningeal and orbital localization. AMORE treatment consisted of Ablative surgery, MOuld technique brachytherapy and surgical REconstruction., Results: In total 18 patients received salvage AMORE treatment; nine patients had relapsed parameningeal (PM) RMS, two patients had relapsed head and neck non-parameningeal RMS (HN-nonPM) and seven patients had relapsed orbital RMS. Local control rate was 67% and 5-year overall survival was 54% (95% confidence interval: 31-78%); 3/9 patients with PM RMS, 0/2 patients with HN-nonPM RMS and 6/7 patients with orbital RMS were alive after a median follow-up of 8.6 years. One patient with PM RMS survived more than 5 years after which he died from a secondary cancer. Six patients developed a local relapse (of which one patient also developed a distant metastasis) and two patients developed distant metastases., Conclusions: Salvage AMORE treatment is a feasible and effective local therapy approach even after prior EBRT. Since salvage AMORE treatment is sometimes the only curative option in patient with relapsed HNRMS, we encourage physicians to consider salvage AMORE treatment for patients with relapsed HNRMS after prior EBRT., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Early response to induction chemotherapy is used in current European guidelines to evaluate the efficacy of chemotherapy and subsequently to adapt treatment in pediatric patients with rhabdomyosarcoma (RMS). However, existing literature on the prognostic value of early radiologic response on survival is contradictory; here the prognostic value is analyzed with data from the International Society of Pediatric Oncology (SIOP) Malignant Mesenchymal Tumor 95 (MMT-95) study., Methods: This study examined 432 Intergroup Rhabdomyosarcoma Study Grouping III (macroscopic residue) patients enrolled in the SIOP MMT-95 study with a response assessment after 3 courses of chemotherapy (a 2-dimensional assessment). Patients with progressive disease (PD) after 3 courses of chemotherapy were excluded (n = 7). Failure-free survival (FFS) and overall survival (OS), calculated with the Kaplan-Meier method, were compared for 3 groups (complete response [CR]/partial response [PR], objective response [OR], and no response [NR]). The prognostic impact of early response was assessed through the calculation of Cox proportional hazards., Results: After 3 courses of chemotherapy, 85.2% of the patients had CR/PR, 8.6% had OR, and 6.3% had NR. For all patients, the 5-year FFS and OS rates were 60% (95% confidence interval [CI], 56%-65%) and 74% (95% CI, 70%-78%), respectively. However, a Cox proportional hazards regression analysis revealed no significant difference in FFS or OS between the response groups. The adjusted hazard ratios for an OR and NR were 1.09 (95% CI, 0.63-1.88) and 0.81 (95% CI, 0.39-1.67), respectively, for FFS and 0.91 (95% CI, 0.47-1.76) and 1.27 (95% CI, 0.61-2.64), respectively, for OS., Conclusions: No evidence was found for the idea that early radiologic response to chemotherapy is prognostic for survival for patients with RMS. Treatment adaptation based on early response (except for patients with PD) should, therefore, no longer be incorporated into future studies. Cancer 2018;124:1016-24. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Background: The differential diagnosis of vaginal blood loss in childhood is broad, and includes irritation of the mucous membranes, trauma, tumours, foreign bodies and sexual abuse. Physical and additional examination is often initially difficult; however, prompt detection of a rhabdomyosarcoma, a soft-tissue tumour principally diagnosed in childhood, is vitally important., Case Description: A 3-year-old girl with a history of vaginal blood loss and an introital mass was referred to the gynaecologist. Treatment with oestriol and triamcinolone cream did not lead to healing. Pathological examination of a biopsy taken under general anaesthetic indicated an embryonic rhabdomyosarcoma. Chemotherapy, surgical resection and brachytherapy lead to persistent remission of the tumour., Conclusion: Because rhabdomyosarcoma is rare and can present atypically, diagnosis can be delayed. Early recognition is, however, essential and this condition should be placed high in the differential diagnosis by vaginal blood loss or vaginal abnormality in childhood.

Purpose: Head and neck rhabdomyosarcoma (HNRMS) survivors are at increased risk of developing pituitary dysfunction as an adverse event of radiotherapy. Our aim was to investigate the frequency and risk factors for pituitary dysfunction in these survivors. Secondly, we aimed to compare the prevalence of pituitary dysfunction between survivors treated with external beam radiation therapy (EBRT) and survivors treated with the ablative surgery, moulage technique after loading brachytherapy, and surgical reconstruction (AMORE) procedure., Methods: Eighty HNRMS survivors treated in London (EBRT based) and Amsterdam (AMORE based: AMORE if feasible, otherwise EBRT) in the period 1990-2010 and alive ≥ 2 years post-treatment were evaluated. Survivors were evaluated in multidisciplinary late-effects clinics, with measurement of linear growth, determination of thyroid function, and growth hormone parameters. Additional data, such as baseline characteristics, anthropometrics, pubertal stage, and the results of additional laboratory investigations, were retrieved from patient charts., Results: Pituitary dysfunction was diagnosed in 24 in 80 (30%) survivors, after a median follow-up time of 11 years. Median time to develop pituitary dysfunction after HNRMS diagnosis was 3.0 years. Risk factors were EBRT-based therapy (odds ratio [OR] 2.06; 95% confidence interval [CI] 1.79-2.46), parameningeal tumour site (OR 1.83; 95% CI 1.60-2.17) and embryonal RMS histology (OR 1.49; 95% CI 1.19-1.90)., Conclusions: Radiotherapy used for the treatment of HNRMS confers a significant risk of the development of pituitary dysfunction. AMORE-based treatment in children with HNRMS resulted in less pituitary dysfunction than treatment with conventional EBRT. Our findings underscore the importance of routine early endocrine follow-up in this specific population., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped >4000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

Background: The aim of this study was to evaluate the incidence, disease presentation, treatment and cardiac outcome of Kawasaki disease (KD) in The Netherlands., Methods: The national Dutch Pediatric Surveillance Unit was used to prospectively register new KD cases from 2008 through 2012. Questionnaires were sent to pediatricians to obtain clinical information., Results: Nationwide 341 cases were reported during the 5-year study period, of which 319 questionnaires (93.0%) were returned. The mean incidence of KD was estimated to be 5.8/100,000 children <5 years of age. The median age at disease onset was 2.4 years (range 0.1-14.6 years) and 79.2% of cases were <5 years of age. The male-to-female ratio was 1.5 to 1. Incomplete KD was diagnosed in 22.3% of cases and these cases were significantly younger than complete cases [median: 1.1 (0.1-13.7) vs. 2.8 (0.2-14.6) years, P < 0.001]. In total, 308 patients (96.6%) received intravenous immunoglobulins (IVIG). Retreatment with IVIG was given in 71 (23.1%) and additional steroid treatment in 17 patients (5.5%). During the acute phase, coronary artery aneurysms developed in 43 cases (13.5%). Multivariate logistic regression analysis showed that male gender, delay of treatment (>10 days) and IVIG retreatment were independent risk factors for coronary artery aneurysms development., Conclusions: This prospective study of KD in The Netherlands revealed a mean annual incidence of 5.8/100,000 children <5 years of age. Clinicians should consider the diagnosis of KD in young (male) children with persistent inexplicable fever to start IVIG treatment within 10 days to prevent development of coronary artery aneurysms.

The balance between activating and inhibitory signals from the different FcγRs for IgG ensures homeostasis of many inflammatory responses. FCGR2C is the product of an unequal crossover of the FCGR2A and FCGR2B genes encoding the activating FcγRIIa (CD32a) and inhibitory FcγRIIb (CD32b), respectively. A single nucleotide polymorphism (SNP) in exon 3 of FCGR2C results in either expression of the activating FcγRIIc (CD32c) (FCGR2C-open reading frame [ORF]) or its absence because of a stop codon (FCGR2C-Stop). Two additional variations in FcγRIIb/c expression on leukocytes have now been identified. In case of "nonclassical" FCGR2C-ORF alleles, FcγRIIc expression was unexpectedly absent, because of novel splice site mutations near exon 7 leading to another stop codon. In some individuals with FCGR2C-Stop alleles FcγRIIb was detected on NK cells, which normally are devoid of this protein. Individuals with these nonclassical FCGR2C-Stop alleles carried a deletion of FCGR2C-FCGR3B that extends into the promoter region of the adjacent FCGR2B gene and probably deletes a negative regulatory element in the FCGR2B promoter in NK cells. FcγRIIb expression on NK cells effectively inhibited killing mediated by FcγRIIIa (CD16a) in Ab-dependent cytotoxicity tests. Our findings demonstrate a more extensive and previously unnoticed variation in FcγR expression with relevance to immunity and inflammation.

Objective: In Kawasaki disease (KD), a pediatric vasculitis of medium-sized arteries, the coronary arteries are most commonly affected. Angiopoietins and vascular endothelial growth factor (VEGF) play an important role in maintaining vascular homeostasis. Recently, we identified ANGPT1 and VEGFA as susceptibility loci for KD. This study was undertaken to fine-map these associations and to gain further insight into their role in this vasculitis of unknown etiology to further the search for improved diagnostic and therapeutic options., Methods: A total of 292 single-nucleotide polymorphisms (SNPs) located in VEGF and ANGPT and their receptors were genotyped in 574 families, including 462 trios. For replication, 123 cases and 171 controls were genotyped., Results: A significant association with KD susceptibility was observed with 5 SNPs in the ANGPT1 gene (most significantly associated SNP +265037 C>T; Pcombined=2.3×10(-7) ) and 2 SNPs in VEGFA (most significantly associated SNP rs3025039; Pcombined=2.5×10(-4) ). Both ANGPT1 +265037 C>T and VEGFA rs3025039 are located in 3' regulatory regions at putative transcription factor binding sites. We observed significantly down-regulated transcript levels of angiopoietin 1 (Ang-1) in patients with acute KD compared to patients with convalescent KD. In patients with acute KD, high serum protein levels of VEGF and Ang-2 were observed compared to patients with convalescent KD and to both controls with and controls without fever. Immunohistochemistry demonstrated VEGF and angiopoietin expression in the coronary artery wall in autopsy tissue., Conclusion: Our data support the hypothesis that dysregulation of VEGF and angiopoietins contributes to the disruption of vascular homeostasis in KD., (Copyright © 2012 by the American College of Rheumatology.)

Kawasaki disease is a systemic vasculitis of unknown etiology, with clinical observations suggesting a substantial genetic contribution to disease susceptibility. We conducted a genome-wide association study and replication analysis in 2,173 individuals with Kawasaki disease and 9,383 controls from five independent sample collections. Two loci exceeded the formal threshold for genome-wide significance. The first locus is a functional polymorphism in the IgG receptor gene FCGR2A (encoding an H131R substitution) (rs1801274; P = 7.35 × 10(-11), odds ratio (OR) = 1.32), with the A allele (coding for histadine) conferring elevated disease risk. The second locus is at 19q13, (P = 2.51 × 10(-9), OR = 1.42 for the rs2233152 SNP near MIA and RAB4B; P = 1.68 × 10(-12), OR = 1.52 for rs28493229 in ITPKC), which confirms previous findings(1). The involvement of the FCGR2A locus may have implications for understanding immune activation in Kawasaki disease pathogenesis and the mechanism of response to intravenous immunoglobulin, the only proven therapy for this disease.

Background: Transforming growth factor (TGF)-β is a multifunctional peptide that is important in T-cell activation and cardiovascular remodeling, both of which are important features of Kawasaki disease (KD). We postulated that variation in TGF-β signaling might be important in KD susceptibility and disease outcome., Methods and Results: We investigated genetic variation in 15 genes belonging to the TGF-β pathway in a total of 771 KD subjects of mainly European descent from the United States, the United Kingdom, Australia, and the Netherlands. We analyzed transcript abundance patterns using microarray and reverse transcriptase-polymerase chain reaction for these same genes, and measured TGF-β2 protein levels in plasma. Genetic variants in TGFB2, TGFBR2, and SMAD3 and their haplotypes were consistently and reproducibly associated with KD susceptibility, coronary artery aneurysm formation, aortic root dilatation, and intravenous immunoglobulin treatment response in different cohorts. A SMAD3 haplotype associated with KD susceptibility replicated in 2 independent cohorts and an intronic single nucleotide polymorphism in a separate haplotype block was also strongly associated (A/G, rs4776338) (P=0.000022; odds ratio, 1.50; 95% confidence interval, 1.25 to 1.81). Pathway analysis using all 15 genes further confirmed the importance of the TGF-β pathway in KD pathogenesis. Whole-blood transcript abundance for these genes and TGF-β2 plasma protein levels changed dynamically over the course of the illness., Conclusions: These studies suggest that genetic variation in the TGF-β pathway influences KD susceptibility, disease outcome, and response to therapy, and that aortic root and coronary artery Z scores can be used for phenotype/genotype analyses. Analysis of transcript abundance and protein levels further support the importance of this pathway in KD pathogenesis.

Aim: To describe the patient characteristics, management and cardiovascular sequelae of Kawasaki disease (KD) in patients taking part in a multidisciplinary follow-up in the Emma Children's Hospital during the period January 1999-June 2010., Design: Retrospective, observational study., Methods: We included 392 patients who were diagnosed with complete or incomplete KD. Clinical and outpatient statuses were used to collect clinical data., Results: The median age at onset of the disease was 3.2 years (range: 0.1-16.4). The male-to-female ratio was 1.6 : 1. Complete KD was diagnosed in 83.9% of patients. Patients with incomplete KD were younger than those with complete KD: 2.2 versus 3.4 years (both SD: 3.0; p < 0.01). 357 patients (91.1%) were treated with intravenous immunoglobulins; 65 patients (16.6%) received a second intravenous dose. Coronary artery aneurysms were diagnosed in 83 patients (21.2%). Male gender, age < 1 year, incomplete presentation and late start of treatment (> 10 days after start of fever) were shown to be independent risk factors for developing aneurysms. These abnormalities normalized in 50 of the 83 patients. 2 patients died of the disease within a year. 5 patients underwent coronary artery bypass grafting during the follow-up period., Conclusion: Kawasaki disease is a rare form of vasculitis seen in children, in which aneurysms of the coronary artery can develop. Clinicians should be alert to the possibility of KD in cases of persistent inexplicable fever, especially in young children, even in the absence of complete clinical disease. A timely start to treatment reduces the risk of developing coronary artery aneurysms.

Meningococcal disease is an infection caused by Neisseria meningitidis. Genetic factors contribute to host susceptibility and progression to disease, but the genes responsible for disease development are largely unknown. We report here a genome-wide association study for host susceptibility to meningococcal disease using 475 individuals with meningococcal disease (cases) and 4,703 population controls from the UK. We performed, in Western European and South European cohorts (consisting of 968 cases and 1,376 controls), two replication studies for the most significant SNPs. A cluster of complement factor SNPs replicated independently in both cohorts, including SNPs within complement factor H (CFH) (rs1065489 (p.936D

Human Fcgamma receptors (FcgammaRs) are glycoproteins that bind the Fc region of IgG. The genes encoding the low-affinity FcgammaRs are located on chromosome 1q23-24. Beside single nucleotide polymorphisms (SNPs), gene copy number variation (CNV) is now being recognized as an important indicator for inter-individual differences. Recent studies on identifying CNV in the human genome suggest large areas at chromosome 1q23-24 to be involved, and CNV in this region has been associated with manifestations of systemic autoimmune disease. To study both SNPs and CNV of the low-affinity FcgammaRs in one assay, we have developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay. A novel CNV for FCGR3A was observed. Similar to FCGR3B and FCGR2C, a gene-dosage effect of FCGR3A was found, that seemed to correlate nicely with the FcgammaRIIIa expression on NK cells. Next, we delineated the approximate boundaries of CNV at the FCGR locus. Variation in co-segregation of neighboring FCGR genes was limited to four variants, with patterns of Mendelian inheritance. No CNV of the FCGR2A and FCGR2B genes was observed in over 600 individuals. In conclusion, we report a novel CNV of the FCGR3A gene that correlates with FcgammaRIIIa expression and function on NK cells. Only FCGR3A, FCGR2C and FCGR3B show CNV, in contrast to FCGR2A and FCGR2B., (Copyright 2009 Wiley-Liss, Inc.)

Kawasaki disease (KD) is a pediatric vasculitis that damages the coronary arteries in 25% of untreated and approximately 5% of treated children. Epidemiologic data suggest that KD is triggered by unidentified infection(s) in genetically susceptible children. To investigate genetic determinants of KD susceptibility, we performed a genome-wide association study (GWAS) in 119 Caucasian KD cases and 135 matched controls with stringent correction for possible admixture, followed by replication in an independent cohort and subsequent fine-mapping, for a total of 893 KD cases plus population and family controls. Significant associations of 40 SNPs and six haplotypes, identifying 31 genes, were replicated in an independent cohort of 583 predominantly Caucasian KD families, with NAALADL2 (rs17531088, p(combined) = 1.13 x 10(-6)) and ZFHX3 (rs7199343, p(combined) = 2.37 x 10(-6)) most significantly associated. Sixteen associated variants with a minor allele frequency of >0.05 that lay within or close to known genes were fine-mapped with HapMap tagging SNPs in 781 KD cases, including 590 from the discovery and replication stages. Original or tagging SNPs in eight of these genes replicated the original findings, with seven genes having further significant markers in adjacent regions. In four genes (ZFHX3, NAALADL2, PPP1R14C, and TCP1), the neighboring markers were more significantly associated than the originally associated variants. Investigation of functional relationships between the eight fine-mapped genes using Ingenuity Pathway Analysis identified a single functional network (p = 10(-13)) containing five fine-mapped genes-LNX1, CAMK2D, ZFHX3, CSMD1, and TCP1-with functional relationships potentially related to inflammation, apoptosis, and cardiovascular pathology. Pair-wise blood transcript levels were measured during acute and convalescent KD for all fine-mapped genes, revealing a consistent trend of significantly reduced transcript levels prior to treatment. This is one of the first GWAS in an infectious disease. We have identified novel, plausible, and functionally related variants associated with KD susceptibility that may also be relevant to other cardiovascular diseases., Competing Interests: The authors have declared that no competing interests exist.

Blockade of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), a down-regulator of T-cell activation, can cause cancer regression in patients with metastatic melanoma. However, not all patients respond well to the therapy and some develop severe autoimmune reactions. We hypothesized that common genetic variation in the CTLA4 gene could contribute to response to CTLA-4 blockade and the occurrence of autoimmune reactions. We investigated 7 common single nucleotide polymorphisms, SNPs, (rs733618, rs4553808, rs11571317, rs5742909, rs231775, rs3087243, and rs7565213) in 152 white melanoma patients who received CTLA-4 blockade. Three SNPs were associated with response to therapy: proximal promoter SNPs, rs4553808 [P=0.002; odds ratio (OR) 3.39; 95% confidence interval (CI), 1.62-7.10] and rs11571327 (P=0.02; OR 2.89; 95% CI, 1.23-6.83) and the nonsynonymous SNP rs231775 (Thr17Ala, P=0.009; OR 0.39; 95% CI, 0.18-0.82). A haplotype analysis including the 7 SNPs suggested that the common haplotype, TACCGGG could be associated with no response (P=0.02) whereas the haplotype TGCCAGG (P=0.06; OR 4.13; 95% CI, 1.17-14.5) could be associated with response to the treatment. No significant association was observed for occurrence of severe autoimmune reactions (grade III/IV) either by single SNP or haplotype analyses. Our results suggest that genetic variation in CTLA4 could influence response to CTLA-4 blockade therapy in metastatic melanoma patients, but further studies are necessary to confirm the observed associations.

Gene copy number variation (CNV) and single nucleotide polymorphisms (SNPs) count as important sources for interindividual differences, including differential responsiveness to infection or predisposition to autoimmune disease as a result of unbalanced immunity. By developing an FCGR-specific multiplex ligation-dependent probe amplification assay, we were able to study a notoriously complex and highly homologous region in the human genome and demonstrate extensive variation in the FCGR2 and FCGR3 gene clusters, including previously unrecognized CNV. As indicated by the prevalence of an open reading frame of FCGR2C, Fcgamma receptor (FcgammaR) type IIc is expressed in 18% of healthy individuals and is strongly associated with the hematological autoimmune disease idiopathic thrombocytopenic purpura (ITP) (present in 34.4% of ITP patients; OR 2.4 (1.3-4.5), P < .009). FcgammaRIIc acts as an activating IgG receptor that exerts antibody-mediated cellular cytotoxicity by immune cells. Therefore, we propose that the activating FCGR2C-ORF genotype predisposes to ITP by altering the balance of activating and inhibitory FcgammaR on immune cells.

Kawasaki disease (KD) is an acute vasculitis occurring in young children. Its aetiology is unknown, but an infectious agent is assumed. Increased levels of proinflammatory cytokines and chemokines have been reported in KD. Genetic variation in these genes and the receptors for these genes could influence the regulation of cytokines and chemokines. In a case-control study of 170 Dutch Caucasian KD patients and 300 healthy Dutch Caucasian controls, common genetic variants in chemokine receptor genes CCR3, CCR2, CCR5, CX3CR1, CXCR1 and CXCR2 were analysed. Of the eight studied single nucleotide polymorphisms (SNPs) in the CCR3-CCR2-CCR5 gene cluster, four showed a significant association with susceptibility to KD. Moreover the CCR5-Delta32 was observed with an allele frequency of 10.7% in the control population compared to 6.5% in the KD patients (P = 0.04). Two haplotypes of the CCR3-CCR2-CCR5 gene-cluster appear to be at risk haplotypes for KD and one a protective haplotype. No association was observed with the studied SNPs in CX3CR1, CXCR1 and CXCR2. In conclusion, in a Dutch cohort of KD patients an association of KD occurrence with common genetic variants in the chemokine receptor gene-cluster CCR3-CCR2-CCR5 was observed.

We tested the hypothesis that the ratio between the activating and inhibitory Fcgamma receptor type II (FcgammaRII) in neutrophils determines their responsiveness to immune complexes. We measured mRNA levels of FcgammaRII isoforms and observed differences in the ratio of FcgammaRIIa to FcgammaRIIb2 mRNA in granulocytes of 50 white and 10 black healthy volunteers, and found 4 discrete groups of ratios (ie, 4:1; 3:1, 2:1, or 1:1). The response to either dimeric IgG or aggregated IgG (aIgG) was assessed. Up-regulation of CD11b on the surface as well as the elastase release was significantly more pronounced in neutrophils with a high FcgammaRIIa/FcgammaRIIb2 mRNA ratio of 4:1 compared with a 2:1 or 1:1 ratio. Individual ratios as well as the functional responsiveness of neutrophils were constant over time, as was tested over 12 months. Neutrophil stimulation with various agents in vitro did not alter the FcgammaRIIa/FcgammaRIIb2 mRNA ratio in the neutrophils of these donors, in clear contrast to the findings in their mononuclear cells. We found a strong association between the 2B.4 haplotype of the FCGR2B promoter with increased transcriptional activity in individuals with 1:1 ratios and the more common low-expression 2B.1 haplotype in individuals with FcgammaRIIa/FcgammaRIIb2 mRNA ratios of 2:1, 3:1, or 4:1.

Objective: To investigate whether common genetic variants in the vascular endothelial growth factor (VEGF) gene are associated with Kawasaki disease (KD) and the subsequent development of coronary artery lesions., Methods: Common genetic variants in the VEGF gene were analyzed in an association study in a Dutch cohort of 170 KD patients and 300 healthy Dutch Caucasian controls. Genotyping was done with 5'-nuclease TaqMan assays and 3'-hybridization-triggered fluorescence minor groove binder Eclipse assays., Results: An association with susceptibility to KD was observed with 2 of the 6 single-nucleotide polymorphisms analyzed in VEGF: -2594 A>C (rs699947) and the 236 bp 3' of STP C>T (rs3025039). Also for an 18-bp deletion in the promoter of VEGF a significant difference in the genotype and allele frequencies was observed between the KD patients and the controls. The haplotype CGCC (based on rs699947, rs2010963, rs25648, and rs3025039) was significantly associated with the development of KD (hap score 3.8; P = 0.0002). VEGF plasma levels were significantly higher in patients with the early phase of KD than in the healthy controls, and there was a trend toward higher VEGF plasma levels in KD patients with the -2594 CC and 236 bp 3' of STP CC genotypes., Conclusion: Our results suggest that polymorphisms of the VEGF gene may play a role in the pathogenesis of KD.

Prospective studies in children with B-precursor acute lymphoblastic leukaemia (ALL) have shown that polymerase chain reaction (PCR)-based detection of minimal residual disease (MRD) using immunoglobin (Ig) and T-cell receptor (TCR) gene rearrangements as targets can be used to identify patients with a high relapse risk. The disadvantage of this approach is that for each patient preferably two different targets have to be identified. The t(12;21)(p13;q22) with the TEL-AML1 fusion gene is present in approximately 25% of children with B-precursor ALL. In these patients, sensitive reverse transcription (RT)-PCR analysis of the TEL-AML1 fusion transcript might be a more simple and less laborious alternative approach. However, it is unknown how stable the mRNA is and whether the number of transcripts per leukaemic cell remains constant during follow-up. We investigated whether the MRD results obtained using RT-PCR of TEL-AML1 transcripts correlated with the clinically validated genomic PCR for Ig and TCR gene rearrangements. Therefore, we used real-time quantitative (RQ)-PCR analysis for both types of targets and assessed the MRD levels in 36 follow-up bone marrow samples (obtained during the first 1.5 years after diagnosis) from 13 patients with B-precursor ALL. In 34/36 bone marrow samples the Ig/TCR RQ-PCR and TEL-AML1 RQ-PCR revealed equal levels of MRD and these results had a strong correlation (P < 0.0001, R2 = 0.84). Therefore, we conclude that the TEL-AML1 RQ-PCR can, in principle, replace Ig/TCR RQ-PCR in B-precursor ALL with t(12;21).