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In the last 10 years, intra-arterial chemotherapy (IAC) has been increasingly used in the clinical management of retinoblastoma. It is reported to provide tumor control even in advanced stage disease that might have previously required enucleation. In our clinical experience, there are three conditions that may impair the use of IAC: (1) significant collaterals to meningeal arteries, (2) technical failure of ophthalmic artery catheterization, or (3) retina blood supply from collaterals different to the ophthalmic artery. In the current study, we assessed the rate of IACs that could not be carried out in our institution due to these three reasons. All patients admitted for IAC in our hospital were retrospectively assessed by chart review. Non-application rate of IAC was assessed and classified according to the three abovementioned criteria. Complication rate of both finalized and interrupted interventions was recorded. Ninety-eight patients (median age 21.4 months, range 5.3 months–10.5 years) were identified. IAC was performed in 69 (70.4%) patients and interrupted in 12 (12.2%) cases because of meningeal collaterals, in 8 (8.2%) because of technical failure to cannulate the ophthalmic artery, and in 9 (9.2%) because of alternative blood supply of the retina. The rather defensive approach that is pursued in our center resulted in an overall non-application rate of IAC around 30%. The relatively high probability of conditions that impair the use of IAC needs to be addressed adequately in the patient conversation prior to the procedure. Our rate of 8% of abstention from IAC due to technical limitations might be reduced by the application of more rigorous therapeutic approaches such as balloon occlusion of the distal internal carotid artery. More research is finally needed to determine if IAC can be safely performed in the presence of meningeal collaterals and via branches of the external carotid artery.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging - with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

OBJECTIVE: Generate recommendations for long-term follow-up for adult survivors of heritable retinoblastoma.DESIGN: We convened a meeting of providers from retinoblastoma centers around the world to review the state of the science and to evaluate the published evidence.SUBJECTS: Retinoblastoma is a rare childhood cancer of the retina. Approximately forty percent of retinoblastoma cases are heritable, due to a germline mutation in RB1. Dramatic improvements in treatment and supportive care have resulted in a growing adult survivor population. Survivors of heritable retinoblastoma, however, have significantly increased risk of subsequent malignant neoplasms, particularly bone and soft tissue sarcomas, uterine leiomyosarcoma, melanomas, and radiotherapy-related central nervous system tumors, which are associated with excess morbidity and mortality. In spite of these risks, no surveillance recommendations for this population are currently in place and surveillance practices vary widely by center.METHODS: Following the Institute of Medicine procedure for clinical practice guideline development, a PubMed, EMBASE, and Web of Science search was performed, resulting in 139 papers; after abstract and full text review, 37 papers underwent detailed data abstraction to quantify risk and evidence regarding surveillance, if available. During an in-person meeting, evidence was presented and discussed, resulting in consensus recommendations.MAIN OUTCOME MEASURES: Diagnosis and mortality from subsequent neoplasm.RESULTS: While evidence for risk of subsequent neoplasm, especially sarcoma and melanoma, was significant, evidence supporting routine testing of asymptomatic survivors was not identified. Skin examination for melanoma and prompt evaluation of signs and symptoms of head and neck disease were determined to be prudent.CONCLUSIONS: This review of the literature confirmed some of the common second cancers in retinoblastoma survivors, but found little evidence for a benefit to currently available surveillance for these malignancies. Future research should incorporate international partners, patients, and family members.

Tumors arising in the pineal region comprise a spectrum of different entities with distinct clinical and histopathological characteristics. Pineocytoma (PC), pineal parenchymal tumors of intermediate differentiation (PPTID) and papillary tumors of the pineal region (PTPR) mainly occur in adult patients and are low to moderately aggressive neoplasms (WHO °I-III). In contrast, pineoblastoma (PB) are high-grade (WHO °IV) malignancies primarily affecting children and adolescents. Especially for patients with unresectable or metastatic disease or at very young age survival outcomes remain poor despite aggressive multimodal treatment regimen. To date, no therapeutically actionable molecular targets have been identified. A subset of PB occur in patients with cancer predisposition syndromes including DICER1 and RB1 germline mutations, the latter in the context of trilateral retinoblastoma (TLRB). We analyzed a cohort of ~230 pineal tumors of different histologies using genome-wide DNA methylation profiling and copy-number analysis, as well as gene panel sequencing, miRNA sequencing and gene expression profiling. Unsupervised clustering based on DNA methylation profiles revealed clear separation of known histopathological entities (PC, PTPR, PPTID) and, furthermore, distinction of subclasses within these groups. Interestingly, several biologically discrete subgroups emerged within the group of histologically diagnosed PBs or pineal primitive embryonal tumors/PNETs, which displayed distinct clinical associations (e.g. age distribution). RB1 alterations were recurrent in a small subgroup (PB_RB) including TLRBs as well as sporadic PB cases (~60%) showing similarities with retinoblastoma. About 45% of cases in the largest PB subgroup (PB_B) harbored alterations within the miRNA processing pathway (affecting DROSHA, DGCR8 or DICER1) suggesting a central role of altered miRNA biogenesis in the development of this group which showed evidence for global reduction of mature miRNA by miRNA-Seq. With this study, we provide a foundation for further clinical, molecular and functional characterization of PB subgroups.

Intraorbitale Meningeome sind extrem seltene Tumoren und machen nur 4 % aller orbitalen Tumoren aus. Im Kindesalter sind sie Raritaten und nur in Einzelfallen beschrieben. Der hier vorgestellte Fall dokumentiert ein primares Meningeom des Nervus opticus mit intraokularer Ausdehnung bei einem 8 Monate alten Kind, das mit Strabismus und Nystagmus auffallig geworden war. Eine MRT-Untersuchung zeigte eine langstreckige Verdickung des Nervus opticus mit einem intraokularen Tumor. Bei unklarem Befund wurde das Auge zur Diagnosesicherung bzw. zum Tumorausschluss enukleiert. Histologisch zeigte sich das typische Bild eines meningothelialen Meningeoms (WHO Grad I) mit ausgedehnter Extension entlang des Nervus opticus und nach intraokular sowie zusatzlich eine Retinadysplasie und epiretinale Membranen. Da eine Assoziation zwischen kindlichen Meningeomen, Retinadysplasie und epiretinalen Membranen mit einer Neurofibromatose Typ 2 besteht, wurden bei dem Kind entsprechende humangenetische Untersuchungen eingeleitet, die schlussendlich zur Diagnose einer Neurofibromatose Typ 2 fuhrten. Dieser Fall dokumentiert eine ausergewohnlich fruhe Manifestation einer Neurofibromatose Typ 2 mit Entwicklung eines intraorbitalen Meningeoms.

In retinoblastoma research tumor-derived cell lines remain an important model to investigate tumorigenesis and new therapy options, due to limited tumor material and lack of adequate animal models. A panel of 10 retinoblastoma cell lines was characterized with respect to mutation, methylation and expression of RB1 and MYCN. These established retinoblastoma cell lines represent the most frequent types of RB1 inactivation and together with the MYCN amplification status, three classes can be distinguished: RB1mut/MYCNnonA, RB1mut/MYCNA and RB1wt/MYCNA. MYCN amplification was identified in five cell lines, whereby two of them, RB522 and RB3823, harbor no aberration in RB1. Targeted sequencing of 160 genes often mutated in cancer identified only few variants in tumor-associated genes other than in RB1. None of these variants was recurrent. mRNA expression analyses of retinal markers, cell cycle regulators and members of the TP53 signaling pathway revealed a high variability between cell lines but no class-specific differences. The here presented thorough validation of retinoblastoma cell lines, including microsatellite analysis for cell line authentication, provides the basis for further in vitro studies on retinoblastoma.

Background Retinoblastoma is the most common malignant cancer of the eye in children. While metastatic retinoblastoma is rare, cure rates for this advanced disease remain below 50%. High-level PLK1 expression in retinoblastomas has previously been shown to be correlated with adverse outcome parameters. PLK1 is a serine/threonine kinase involved in cell cycle regulation at the G2/M transition. PLK1 inhibition has been demonstrated to have anti-tumour effects in preclinical models of several paediatric tumours. Here we assessed its efficacy against retinoblastoma cell lines. Methods Expression of PLK1 was determined in a panel of retinoblastoma cell lines by PCR and western blot analysis. We analysed viability (MTT assay), proliferation (BrdU ELISA), cell cycle progression (PI staining) and apoptosis (Cell death ELISA) in three retinoblastoma cell lines after treatment with two ATP-competitive PLK1 inhibitors, BI6727 or GSK461364. Activation of PLK1 downstream signalling components including TP53 were assessed. Results Treatment of retinoblastoma cells with either BI6727 or GSK461364 reduced cell viability and proliferative capacity, and induced both cell cycle arrest and apoptosis. PLK1 inhibition also induced the p53 signalling pathway. Analysis of key players in cell cycle control revealed that low nanomolar concentrations of either PLK1 inhibitor upregulated CCNB1 and increased activated CDK1 (phosphorylated at Y15) in retinoblastoma cell lines. Conclusions These preclinical data indicate that PLK1 inhibitors could be useful as components in rationally designed chemotherapy protocols to treat patients with metastasised retinoblastoma in early phase clinical trials.

Purpose Intraocular retinoblastoma is curable, but survivors with a heritable predisposition are at high risk for second malignancies. Because second malignancies are associated with high mortality, prognostic factors for second malignancy influence long-term overall survival. This study investigates the impact of all types of eye-preserving therapies on long-term survival in the complete German cohort of patients with heritable retinoblastoma. Patients and Methods Overall survival, disease staging using international scales, time period of diagnosis, and treatment type were analyzed in the 633 German children treated at the national reference center for heritable retinoblastoma. Results The 5-year overall survival of children diagnosed in Germany with heritable retinoblastoma between 1940 and 2008 was 93.2% (95% CI, 91.2% to 95.1%), but long-term mortality was increased compared with patients with nonheritable disease. Overall survival correlated with tumor staging, and 92% of patients were diagnosed with a favorable tumor stage (International Retinoblastoma Staging System stage 0 or I). Despite a 5-year overall survival of 97.4% (95% CI, 96.0% to 98.8%) in patients with stage 0 or I, only 79.5% (95% CI, 74.2% to 84.8%) of these patients survived 40 years after diagnosis. Long-term overall survival was reduced in children treated with eye-preserving radiotherapy compared with enucleation alone, and adding chemotherapy aggravated this effect. Conclusion The benefits of preserving vision must be balanced with the impact of eye-preserving treatments on long-term survival in heritable retinoblastoma, and the genetic background of the patient influences choice of eye-preserving treatment. Germline RB1 genetic analysis is important to identify heritable retinoblastoma among unilateral retinoblastoma cases. Eye-preserving radiotherapy should be carefully considered in patients with germline RB1 mutations. Life-long oncologic follow-up is crucial for all retinoblastoma survivors, and less detrimental eye-preserving therapies must be developed.

Background Survivors of heritable retinoblastoma carry a high risk to develop second cancers. Eye-preserving radiotherapy raises this risk, while the impact of chemotherapy remains less defined. Procedure This population-based study characterizes the impact of all treatment modalities on second cancers incidence and type after retinoblastoma treatment in Germany. Data on second cancer incidence in 648 patients with heritable retinoblastoma treated between 1940 and 2008 at the German national reference center for retinoblastoma were analyzed to identify associations with treatment. Results The cumulative incidence ratio (per 1,000 person years) of second cancers was 8.6 (95% confidence interval 7.0–10.4). Second cancer incidence was influenced by type of retinoblastoma treatment but not by the year of diagnosis or by sex. Radiotherapy and systemic chemotherapy increased the incidence of second cancers (by 3.0- and 1.8-fold, respectively). While radiotherapy was specifically associated with second cancers arising within the periorbital region in the previously irradiated field, chemotherapy was the strongest risk factor for second cancers in other localizations. Soft tissue sarcomas and osteosarcomas were the most prevalent second cancers (standardized incidence ratio 179.35 compared to the German population). Conclusions Second cancers remain a major concern in heritable retinoblastoma survivors. Consistent with previous reports, radiotherapy increased second cancer incidence and influenced type and localization. However, chemotherapy was the strongest risk factor for second malignancies outside the periorbital region. Our results provide screening priorities during life-long oncological follow-up based on the curative therapy the patient has received and emphasize the need for less-detrimental therapies for children with heritable retinoblastoma.

Background Recent years have seen major changes in the diagnosis and treatment of solid intraocular tumors, mainly owing to an improved molecular biological understanding of their pathogenesis, new therapeutic approaches for the local treatment of tumors in children, and long-term follow-up observations in clinical trials. Methods This review is based on pertinent publications retrieved by a selective search in PubMed. Results Retinoblastoma is the most common type of primary intraocular tumor, with approximately 8000 new cases per year around the world, while malignant melanoma of the uvea is the most common primary intraocular tumor in adults, with approximately 7000 new cases per year around the world. Intraocular metastases of malignant tumors are ten times more common, in terms of incidence, than primary intraocular tumors and are therefore the most common intraocular tumors overall. Improved methods of intraocular biopsy, diagnostic imaging, and molecular genetic investigation have led to steady improvement in clinical and predictive diagnostic assessment. In the treatment of retinoblastoma, local techniques including brachytherapy and intra-arterial and intravitreal chemotherapy play a prominent role. Prognostic molecular-genetic testing now enables the highly selective identification of uveal melanomas that have a high potential to metastasize. Cutaneous and uveal melanomas differ both in their clinical behavior and in their basic biological features; to date, effective systemic treatment has been established for melanoma of the skin, but not for metastatic melanoma of the uvea. Intraocular metastases are common and often the initial manifestation of an extraocular tumor, particularly lung cancer. Conclusion Modern diagnostic and therapeutic concepts for intraocular tumors can only be implemented through the close interdisciplinary collaboration of ophthal - mologists, oncologists, radiologists, radiotherapists, pathologists, and human geneticists.

Survivors of childhood cancer frequently suffer from endocrine late effects, which are, at least partly, attributed to toxic effects of chemotherapy. Treatment of retinoblastoma typically involves chemotherapy at a very young age. The authors conducted a cross-sectional study to assess bone health in a pediatric cohort of 33 survivors of retinoblastoma (mean age: 4.4 years) who had undergone chemotherapy treatment at an especially young age (mean age: 0.76 years). Of these patients, 14 had unilateral and 19 bilateral retinoblastoma. Polychemotherapy consisted of treatment with cyclophosphamide, etoposide, vincristine, and carboplatin. Ten patients had undergone external beam radiotherapy. Clinical and biochemical parameters of growth, pubertal development, and bone health were obtained. A vitamin D deficiency was found in 51.7% of the patients, and 13.7% of patients displayed severe vitamin D deficiency. Secondary hyperparathyroidism and altered readings for bone formation or resorption markers were present in 15%. Nine percent reported bone pain or experienced fractures of the long bones after primary diagnosis. No difference between children with bilateral and unilateral disease or irradiated versus nonirradiated children was observed. The parameters of thyroid function, growth, and pubertal development were within age-appropriate norms in almost all children. In conclusion, altered parameters of bone health can be present in survivors of retinoblastoma at a young age and warrant regular follow-up in these children. The endocrine hypothalamic-pituitary axes, however, were not impaired at this early age in this group of survivors of retinoblastoma.

Heritable retinoblastoma can rarely be associated with a midline intracranial neuroblastic tumor, referred to as trilateral retinoblastoma. We present an unusual midline brain tumor in an infant that was identified as ectopic retinoblastoma by histopathology, DNA methylation analysis, and molecular genetic detection of biallelic somatic inactivation of the RB1 gene. There was no ocular involvement, and germline mutation was excluded. In this nonresectable tumor, treatment with systemic chemotherapy including high-dose therapy with autologous stem cell transplantation, but without definite local therapy, resulted in long-lasting tumor control.

In Deutschland erkranken ungefahr 40 Kinder im Jahr an einem Retinoblastom. Bei fruher Diagnose ist das Tumorwachstum auf das Auge beschrankt, und die Gesamtuberlebensraten sind mit uber 95 % gut. Patienten mit metastasiertem Retinoblastom dagegen haben nur sehr geringe Heilungschancen. Bei den ca. 40 % der Patienten, bei denen Retinoblastome in beiden Augen auftreten, ist der Erhalt von moglichst viel verbleibender Sehkraft nach der Therapie von groser Bedeutung. In der konservativen Behandlung werden unter anderem Laserkoagulation, Thermotherapie, Kryotherapie, Brachytherapie oder systemische Chemotherapie eingesetzt. In der Primartherapie hat die systemische Chemotherapie die perkutane Bestrahlung heutzutage fast vollstandig ersetzt. Klinische Fallstudien zeigten zudem die Wirksamkeit der intraarteriellen, intravitrealen und periokularen Gabe von Chemotherapie bei der Behandlung intraokularer Retinoblastome. Da das Retinoblastom erblich sein kann, sind genetische Untersuchungen ein wesentlicher Bestandteil der Diagnostik bei Retinoblastom. Insbesondere bei Patienten mit einseitigem Retinoblastom muss, wenn verfugbar, auch Tumormaterial untersucht werden. Bei den meisten Patienten mit sporadischem einseitigem Retinoblastom kann nur so die Diagnose der nicht ererbten Form sicher gestellt werden. Die genetische Diagnostik hat gezeigt, dass bei fast 50 % der Kinder mit Retinoblastom die erbliche Form der Erkrankung vorliegt. Diese Kinder haben ein erhohtes Risiko fur Tumoren auserhalb des Auges. Solche Zweittumoren und andere Spatfolgen sind wahrend des weiteren Lebens von groser Bedeutung fur die Patienten. Sie erfordern eine multidisziplinare Zusammenarbeit in der Behandlung und Nachsorge. Aktuell werden multizentrische klinische Studien entwickelt, um evidenzbasierte Therapiekonzepte fur das lokalisierte und das metastasierte Retinoblastom zu untersuchen und die Uberlebensrate und die Lebensqualitat der erkrankten Kinder zu verbessern. Dieser Artikel wurde ubersetzt und erganzt und ist zuerst erschienen in Klin Padiatr 2012; 224: 339–347.

Heritable predisposition is an important cause of cancer in children and adolescents. Although a large number of cancer predisposition genes and their associated syndromes and malignancies have already been described, it appears likely that there are more pediatric cancer patients in whom heritable cancer predisposition syndromes have yet to be recognized. In a consensus meeting in the beginning of 2016, we convened experts in Human Genetics and Pediatric Hematology/Oncology to review the available data, to categorize the large amount of information, and to develop recommendations regarding when a cancer predisposition syndrome should be suspected in a young oncology patient. This review summarizes the current knowledge of cancer predisposition syndromes in pediatric oncology and provides essential information on clinical situations in which a childhood cancer predisposition syndrome should be suspected.

Retinoblastoma is the most common malignant cancer of the eye in children. Although metastatic retinoblastoma is rare, cure rates for this advanced disease remain below 50%. High-level polo-like kinase 1 expression in retinoblastomas has previously been shown to be correlated with adverse outcome parameters. Polo-like kinase 1 is a serine/threonine kinase involved in cell cycle regulation at the G2/M transition. Polo-like kinase 1 inhibition has been demonstrated to have anti-tumour effects in preclinical models of several paediatric tumours. Here, we assessed its efficacy against retinoblastoma cell lines.Expression of polo-like kinase 1 was determined in a panel of retinoblastoma cell lines by polymerase chain reaction and western blot analysis. We analysed viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT assay), proliferation (5-bromo-2'-deoxyuridine enzyme-linked immunosorbent assay), cell cycle progression (propidium iodid staining) and apoptosis (cell death enzyme-linked immunosorbent assay) in three retinoblastoma cell lines after treatment with two adenosine triphosphate-competitive polo-like kinase 1 inhibitors, BI6727 or GSK461364. Activation of polo-like kinase 1 downstream signalling components including TP53 were assessed.Treatment of retinoblastoma cells with either BI6727 or GSK461364 reduced cell viability and proliferative capacity and induced both cell cycle arrest and apoptosis. Polo-like kinase 1 inhibition also induced the p53 signalling pathway. Analysis of key players in cell cycle control revealed that low nanomolar concentrations of either polo-like kinase 1 inhibitor upregulated cyclin B1 and increased activated cyclin-dependent kinase 1 (phosphorylated at Y15) in retinoblastoma cell lines.These preclinical data indicate that polo-like kinase 1 inhibitors could be useful as components in rationally designed chemotherapy protocols to treat patients with metastasized retinoblastoma in early phase clinical trials.

Purpose Orbital tumor recurrence is a rare but serious complication in children with retinoblastoma, leading to a high risk of metastasis and death. Therefore, we assume that these recurrences have to be detected and treated as early as possible. Preliminary studies used magnetic resonance imaging (MRI) to evaluate postsurgical findings in the orbit. In this study, we assessed the diagnostic accuracy of high-resolution MRI to detect orbital tumor recurrence in children with retinoblastoma in a large study cohort. Design Consecutive retrospective study (2007–2013) assessing MRI findings after enucleation. Participants A total of 103 MRI examinations of 55 orbits (50 children, 27 male/23 female, mean age 16.3±12.4 months) with a median time of 8 months (range, 0–93) after enucleation for retinoblastoma. Methods High-resolution MRI using orbital surface coils was performed on 1.5 Tesla MRI systems to assess abnormal orbital findings. Main Outcome Measures Five European experts in retinoblastoma imaging evaluated the MRI examinations regarding the presence of abnormal orbital gadolinium enhancement and judged them as "definitive tumor," "suspicious of tumor," "postsurgical condition/scar formation," or "without pathologic findings." The findings were correlated to histopathology (if available), MRI, and clinical follow-up. Results Abnormal orbital enhancement was a common finding after enucleation (100% in the first 3 months after enucleation, 64.3% >3 years after enucleation). All histopathologically confirmed tumor recurrences (3 of 55 orbits, 5.5%) were correctly judged as "definitive tumor" in MRI. Two orbits from 2 children rated as "suspicious of tumor" received intravenous chemotherapy without histopathologic confirmation; further follow-up (67 and 47 months) revealed no sign of tumor recurrence. In 90.2%, no tumor was suspected on MRI, which was clinically confirmed during follow-up (median follow-up after enucleation, 45 months; range, 8–126). Conclusions High-resolution MRI with orbital surface coils may reliably distinguish between common postsurgical contrast enhancement and orbital tumor recurrence, and therefore may be a useful tool to evaluate orbital tumor recurrence after enucleation in children with retinoblastoma. We recommend high-resolution MRI as a potential screening tool for the orbit in children with retinoblastoma to exclude tumor recurrence, especially in high-risk patients within the critical first 2 years after enucleation.

Background Anthracycline cardiomyopathy is of concern in children treated for acute myeloid leukaemia (AML), but there are few data on the incidence and natural history of cardiotoxicity after AML treatment in the United Kingdom, where regimens have included high anthracycline exposure. Procedure Prevalence and predictors of cardiotoxicity were retrospectively reviewed in 124 children treated on the MRC AML 10 and AML 12 trials in a single, large centre from November 1987 to September 2004. Subclinical cardiotoxicity was defined as a shortening fraction of less than 28% and clinical cardiomyopathy as evidence of heart failure, and both were classified as late cardiotoxicity 1 year after completing first line therapy. Results Cumulative survival was 61% at 10 years. The prevalence of early and late cardiotoxicity was 13.7% (95%-CI: 8.2–22.0%) and 17.4% (95%-CI: 10.9–26.8%), respectively. Early cardiotoxicity was a strong predictor (OR = 9.18; 95%-CI: 2.10–40.11; P

Background: Erythropoietin (Epo) was originally defined as a hematopoietic growth factor, but also has potent tissue-protective properties. The cytokine-modulating actions of Epo have received scant attention. We hypothesized that Epo significantly influences the in vitro cytokine production in both neonates and adults. Methods: The effects of Epo were investigated using a standardized in vitro whole blood assay. Production of various cytokines was assessed by means of intracellular cytokine detection (IL-2, -6, -8, IFN-γ and TNF-α) in preterm infants, term neonates and adults. Furthermore, synthesis of IL-4, -5 and -10 in adults was investigated via cytometric bead array. Results: Epo significantly inhibits the production of various cytokines in preterm infants, term neonates and adults. In CD3+ lymphocytes, Epo predominantly decreases the number of IL-2-positive cells in all age groups. Similarly, in CD14+ cells, Epo significantly diminishes the number of IL-6- and TNF-α-producing cells. Furthermore, Epo significantly inhibits the synthesis of IL-4, IL-5 and IL-10 in adults. Conclusion: rhEpo has significant inhibitory potential on the production of various cytokines by leukocytes in preterm and term infants as well as in adults. The described effect likely contributes to the tissue protective properties of Epo.

Children with retinoblastoma carry a high risk to develop second primary malignancies in childhood and adolescence. This study characterizes the type of pediatric second primary malignancies after retinoblastoma treatment and investigates the impact of different treatment strategies and prognostic factors at presentation.All national patients treated for retinoblastoma at the German referral center with a current age of 6-27 years were invited to participate in a study to characterize late effects.Data on pediatric second primary malignancies were recorded from 488 patients. Ten developed a malignancy before the age of 18 years. For children with heterozygous oncogenic RB1 alteration (heritable retinoblastoma), the cumulative incidence to develop a second malignancy at the age of 10 years was 5.2% (95% CI 1.7; 8.7%). This results in an elevated risk for sarcoma (n = 4) (SIR 147.98; 95% CI 39.81; 378.87) and leukemia (n = 4) (SIR 41.38; 95% CI 11.13; 105.95). Neither the functional type of the RB1 alteration nor its origin showed a significant impact. Treatment modality influenced incidence, latency, and type of malignancy. Previous radiotherapy increased the risk for solid tumors and 3 of 91 children developed acute leukemia after chemotherapy. However, 2 of 10 malignancies were diagnosed in patients with heritable retinoblastoma but without previous chemotherapy or external beam radiotherapy.Screening for second primary malignancy is an important part of pediatric oncological follow-up in patients with heritable retinoblastoma. For patients with sporadic unilateral retinoblastoma, genetic information influences treatment decisions and allows tailoring of follow-up schedules.

INTRODUCTION A reliable detection of metastatic risk factors is important for children with retinoblastoma to choose the right therapeutic regimen. First studies using high-resolution magnetic resonance imaging (MRI) with orbit surface coils were promising. The aim of this study was therefore to evaluate the ability of high-resolution MRI to detect metastatic and especially advanced metastatic risk factors in a large group of children with retinoblastoma. METHODS One hundred forty-three consecutive children with retinoblastoma (148 enucleated eyes, 64 girls, 79 boys, mean age 19.7 ± 15.3) who received pretherapeutical high-resolution MRI with orbit surface coils on 1.5 T MR scanner systems between 2007 and 2012 and subsequent primary enucleation within 14 days were included in this retrospective study. Image analysis was performed by two neuroradiologists experienced in ocular imaging in consensus. Histopathology served as gold standard. RESULTS Sensitivity/specificity for the detection of metastatic risk factors using high-resolution MRI with orbit surface coils were 60 %/88.7 % for postlaminar optic nerve infiltration, 65.5 %/95.6 % for choroidal invasion, 100 %/99.3 % for scleral invasion, and 100 %/100 % for peribulbar fat invasion, respectively. The results increased for the detection of advanced metastatic risk factors, 81.8 %/89.1 % for deep postlaminar optic nerve infiltration, 70.6 %/97.6 % for massive choroidal invasion. CONCLUSIONS High-resolution MRI is clinically valuable for the detection of metastatic, especially of advanced metastatic risk factors in children with retinoblastoma.

Background: Vitamin C (ascorbic acid) is an essential water-soluble antioxidant in cells and plasma. Besides metabolic functions, vitamin C is also known to contribute to immune homeostasis. Recently, it has been demonstrated that vitamin C has an inhibitory effect on the expression of pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in adult whole blood cells in vitro. It has been postulated that vitamin C might be an interesting compound for modulation of an over-exuberant immune response, e.g., in patient cohorts susceptible for the development of systemic inflammatory response syndrome such as neonates. It was the aim of this study to investigate the modulatory effects of vitamin C on the production of inflammatory mediators in neonatal cord blood cells. Methods: The intracytoplasmic production of pro-inflammatory cytokines in neonatal cord blood cells stimulated with lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin was assessed by flow-cytometry. Results: In contrast to our previous observations from adult whole blood cells, 20 mM vitamin C mildly stimulated the percentage of neonatal monocytes producing IL-6 after lipopolysaccharide stimulation (e.g., 11.3% increase compared to control, p = 0.005). In the presence of 20 mM vitamin C, even a stronger stimulatory effect was noted for the percentage of IL-8 (e.g., 46.7% increase, p < 0.001) and TNF-α producing neonatal monocytes (e.g., 69.2% increase, p = 0.004; n = 20). In accordance with adult data, the percentage of neonatal lymphocytes producing IL-2 after phorbol 12-myristate 13-acetate/ionomycin stimulation was dose-dependently reduced (e.g., 41.3% inhibition, p = 0.001, 20 mM vitamin C), while the percentage of TNF-α producing lymphocytes was mildly stimulated (e.g., 20.8% increase, p = 0.003, 20 mM vitamin C). Conclusions: Interestingly, vitamin C was demonstrated to enhance pro-inflammatory responses in CD14+ cord blood cells while only intracellular IL-2 production in CD3+ cells was diminished. These data suggest that vitamin C differentially influences intracytoplasmic cytokine production in adults and neonates, and further studies are needed to elucidate the underlying mechanisms of this selective immunomodulation.

Background Retinoblastoma can extend beyond the structures of the eye, where cells can enter the bloodstream and cause metastases. Various types of protocols for adjuvant treatment risk-adapted according to histopathological risk factors are used worldwide. Methods Between 1997 and 2009, 420 children were diagnosed with retinoblastoma at the German Retinoblastoma Referral Centre and risk factors were assessed. Patients with post-laminar optic nerve infiltration or choroid or minor scleral invasion received six courses of adjuvant chemotherapy using vincristine, etoposide, carboplatin and cyclophosphamide (group 1). Patients with microscopic extension beyond the sclera to the resection margin of the optic nerve or potential spread due to vitrectomy received chemotherapy plus orbital radiotherapy (group 2). Neoadjuvant chemotherapy was performed in patients with local extraocular invasion detected on MRI. Results Following this protocol, 42 of the 420 patients and 21 referred from other centres showed high-risk histopathological factors qualifying for adjuvant therapy (57 in group 1 and 6 in group 2). Seven of the 63 patients received neoadjuvant and adjuvant treatment. During a mean follow-up of 5.8 (range 0.4–15.4) years, one of six patients in group 2 developed metastases and died. No patients died from toxicity. The 5-year overall survival was 100% for group 1 and 80% for group 2. Conclusions This retrospective single-site study reveals a 10% incidence of high-risk features in children with retinoblastoma diagnosed at the German Retinoblastoma Referral Centre. Overall survival rates of 98.3% underline the safety of this adjuvant chemotherapy protocol and its efficiency in preventing metastasis.

Background A small number of children with unilateral retinoblastoma later develop retinoblastoma in the contralateral eye (metachronous bilateral retinoblastoma). Methods We analysed the clinical and genetic characteristics of children with sporadic unilateral retinoblastoma to identify risk factors for the development of metachronous bilateral disease. Results Fifteen (3.1%) of 480 children with unilateral retinoblastoma later developed metachronous bilateral retinoblastoma (latency period >30 days). The maximum latency period was 2.3 years after initial diagnosis. Nine (22.5%) of 40 children with a RB1 mutation detectable in blood developed metachronous bilateral disease while all 155 children proved to be without a germline RB1 mutation remained unilaterally affected. Clinically, the risk of developing metachronous bilateral retinoblastoma was higher for age at diagnosis ≤0.5 years compared with >0.5 years (19.6% vs 1.2%), and for multifocal compared with unifocal unilateral retinoblastoma (17.1% vs 2.2%). Conclusions This study shows that an oncogenic RB1 mutation in the blood is a risk factor for metachronous bilateral retinoblastoma. Additional clinical risk factors for metachronous bilateral disease are diagnosis at young age (≤0.5 years) and multifocal unilateral retinoblastoma. Early genetic analysis may identify children at high risk of developing metachronous bilateral disease and may help to preserve vision using risk-adapted follow-up and early treatment.

Retinoblastoma affects approximately 40 children in Germany per year. Most children are diagnosed early with localized intraocular disease, and the overall survival rate exceeds 95%. However, the prognosis of metastasized retinoblastoma remains poor. In 40% of the patients, retinoblastoma occurs bilaterally and, especially for these children, the salvage of the eye and visual function is of major importance. The variety of conservative treatment options for localized retinoblastoma includes laser coagulation, thermotherapy, cryotherapy, brachytherapy and chemotherapy. While systemic chemotherapy has nearly completely replaced external beam radiotherapy in the primary treatment of intraocular retinoblastoma, intra-arterial, intravitreal and periocular application of chemotherapy was also shown to be effective in treating intraocular retinoblastoma in case series. Genetic testing is an integral part of the routine diagnostics of all patients. Available tumor material should be analyzed to detect mutational mosaicism, that affects >10% of children with unilateral retinoblastoma. Genetic testing also identifies children with heritable (50% of patients) retinoblastoma. These children have a genetic predisposition for second malignancies. For this reason, late effects are an increasing concern and the care of patients with retinoblastoma requires a multidisciplinary approach to tailor therapy and long-term follow-up. Multicenter clinical trials are being developed to evaluate evidence-based treatment concepts for localized and metastasized retinoblastoma to improve survival rates and quality of life of children with retinoblastoma.

Evaluation of: Zhang J, Benavente CA, McEvoy J et al. A novel retinoblastoma therapy from genomic and epigenetic analyses. Nature 481(7381), 329–334 (2012). The rate-limiting step in retinoblastoma tumorigenesis is inactivation of both RB1 alleles, but it has remained unclear how this tumor acquires the additional changes that constitute a malignant phenotype. Zhang et al. characterized the genetic and epigenetic alterations in four retinoblastomas using whole-genome analysis techniques. In these samples, the retinoblastoma genome was found to be remarkably stable genetically, although recurrent mutations in BCOR were identified in 13% of patients. However, an approach that integrated the results of ChIP, methylation and expression analysis identified multiple, more frequent alterations of the epigenetic landscape. One of the leading genes on the list the authors obtained was SYK, a kinase epigenetically upregulated. Knockdown of this gene and exposure to small molecules inhibiting the kinase function stopped tumor growth in vitro and in vivo, thus offering a new therapeutic target for the treatment of retinoblastoma.

Acute hyperglycemia is considered as a pro-inflammatory state and is related to an adverse outcome in critically ill adults. Neonates are susceptible to infections and systemic inflammatory response syndrome induced by pro-inflammatory cytokines. This study focuses on the interaction between neonatal glucose homeostasis and the pro-inflammatory cytokine production in term and preterm infants in-vitro.We analyzed the pro-inflammatory cytokine production in whole cord blood of term infants (n = 10), preterm infants32 weeks (n=16) and preterm infants ≤32 weeks of gestational age (n = 13) and in adult controls (n=14) using an in-vitro sepsis-model. Whole blood was pre-incubated with different concentrations of glucose (0-1000 mg/dl) and insulin (0-62.5 IE/l) and stimulated with lipopolysaccharide. The intracytoplasmatic TNF-α, IL-6, and IL-8 response was measured by flow cytometry.In-vitro hyperglycemia induced a dose-dependent increase of IL-8 in all age groups while TNF-α was demonstrated to be stimulated by glucose in cord blood samples of preterm infants ≤32 weeks of gestational age and term infants. In contrast, insulin showed no significant effects on pro-inflammatory cytokine production in-vitro.Acute hyperglycemia may induce pro-inflammatory cytokine responses in neonatal whole blood in-vitro. These data provide a basis for further in-vitro signal transduction studies and in-vivo investigations about the significance of neonatal glucose homeostasis and its impact on long-term outcome of this susceptible patient cohort.

Anthracycline cardiomyopathy is of concern in children treated for acute myeloid leukaemia (AML), but there are few data on the incidence and natural history of cardiotoxicity after AML treatment in the United Kingdom, where regimens have included high anthracycline exposure.Prevalence and predictors of cardiotoxicity were retrospectively reviewed in 124 children treated on the MRC AML 10 and AML 12 trials in a single, large centre from November 1987 to September 2004. Subclinical cardiotoxicity was defined as a shortening fraction of less than 28% and clinical cardiomyopathy as evidence of heart failure, and both were classified as late cardiotoxicity 1 year after completing first line therapy.Cumulative survival was 61% at 10 years. The prevalence of early and late cardiotoxicity was 13.7% (95%-CI: 8.2-22.0%) and 17.4% (95%-CI: 10.9-26.8%), respectively. Early cardiotoxicity was a strong predictor (OR = 9.18; 95%-CI: 2.10-40.11; P0.005) and children who received salvage therapy following relapse showed a trend towards increased late cardiotoxicity (OR = 3.53; 95%-CI: 0.86-14.48; P0.08). Subclinical cardiotoxicity resolved spontaneously in all but one case, but clinical cardiomyopathy always required continuing therapy. Two children died of cardiomyopathy and six remained on medical therapy.Anthracycline cardiotoxicity remains a major concern for survivors of childhood AML and correlates with early cardiotoxicity and treatment intensity. Long-term follow-up is required to fully determine the outcome for children with subclinical cardiotoxicity.

Erythropoietin (Epo) was originally defined as a hematopoietic growth factor, but also has potent tissue-protective properties. The cytokine-modulating actions of Epo have received scant attention. We hypothesized that Epo significantly influences the in vitro cytokine production in both neonates and adults.The effects of Epo were investigated using a standardized in vitro whole blood assay. Production of various cytokines was assessed by means of intracellular cytokine detection (IL-2, -6, -8, IFN-gamma and TNF-alpha) in preterm infants, term neonates and adults. Furthermore, synthesis of IL-4, -5 and -10 in adults was investigated via cytometric bead array.Epo significantly inhibits the production of various cytokines in preterm infants, term neonates and adults. In CD3+ lymphocytes, Epo predominantly decreases the number of IL-2-positive cells in all age groups. Similarly, in CD14+ cells, Epo significantly diminishes the number of IL-6- and TNF-alpha-producing cells. Furthermore, Epo significantly inhibits the synthesis of IL-4, IL-5 and IL-10 in adults.rhEpo has significant inhibitory potential on the production of various cytokines by leukocytes in preterm and term infants as well as in adults. The described effect likely contributes to the tissue protective properties of Epo.