The regulation of germ cell proliferation in the prepubertal marmoset testis: towards a strategy for fertility preservation in boys treated for cancer

BACKGROUND Prepubertal boys treated for cancer may exhibit impaired fertility in later life. A number of chemotherapeutic agents have been identified as being gonadotoxic, and certain treatment regimens, such as that used for Hodgkin's disease, are particularly associated with subsequent infertility. Radiotherapy may also cause gonadal damage, most notably following direct testicular irradiation or total body irradiation.Semen cryopreservation is currently the only method of preserving fertility in patients receiving gonadotoxic therapy. This is only applicable to postpubertal patients and can be problematic in the adolescent age group. At present there is no provision for the prepubertal boy, although there are a number of experimental methods currently being investigated. By harvesting testicular tissue prior to gonadotoxic therapy, restoration of fertility could be achieved following treatment, either by germ cell transplantation or by in vitro maturation ofthe germ cells harvested. An alternative, and potentially more acceptable method of fertility protection is to render the testes quiescent during cytotoxic treatment, thereby reducing the susceptibility of germ cells to subsequent damage.However, attempts to protect gonadal function by suppression ofthe reproductive axis in primates have been unsuccessful, possibly because spermatogonial proliferation may be gonadotrophin independent. The primary aim of this project, using the marmoset monkey as an animal model, was to investigate whether spermatogonial proliferation in this species is independent of gonadotrophins. Secondly, if this were to be confirmed, the aim was to identify factors that do regulate their proliferation, as a step towards devising strategies for fertility preservation in boys treated for cancer.METHODOLOGY AND RESULTS Immunohistochemistry was used on Bouins-fixed marmoset testes from birth to adulthood, including tissue from males treated prepubertally for ten weeks with a GnRH antagonist, to elucidate the role ofgonadotrophins. No difference was found between germ cells from control and GNRH antagonist-treated animals in the immunoexpression ofMAGE, a germ cell-specific marker, Ki67, a marker of cell proliferation, and Histone H3, a marker of cell mitosis, confirming that spermatogonial proliferation is gonadotrophin-independentTo determine potential regulators, spermatogonial immunoexpression ofgrowth factor receptors was investigated. Receptors for several growth factors were identified in spermatogonia. The expression ofreceptors for both Epidermal Growth Factor and Neurturin was pronounced in a proportion of germ cells in late infancy, thus implicating a role for these factors in spermatogonial development. Prepubertal expression ofthese receptors was unaffected by prior treatment with a GnRH antagonist, demonstrating their gonadotrophin independence. Studies using confocal microscopy demonstrated that the spermatogonial population expressing these receptors were not actively proliferating, raising the possibility that these factors act to inhibit proliferation. Manipulation ofthese factors may protect fertility in boys treated for cancer.As survival of stem spermatogonia is essential for future fertility, this study also attempted to identify a marker of stem cells in the primate, in order to investigate their activity prepubertally. GFR-al, the receptor for Glial Cell Line-Derived Neurotrophic Factor, is expressed by A-spermatogonia in the mouse. In marmoset testis, expression ofthis receptor was shown in occasional germ cells, in a pattern consistent with stem cells, at all ages studied. Attempts to co-localise GFR-al with cell cycle markers suggest these cells are not actively proliferating, but further studies are underway to fully investigate these cells, and their relationship with the growth factors already identified in this primate model.CONCLUSIONS These results confirm that spermatogonial proliferation in the prepubertal primate testis is gonadotrophin independent. These studies have also identified growth factor receptors expressed by spermatogonia during infancy, and have demonstrated that these are not regulated by gonadotrophins. In addition, a potential stem cell marker has been identified in the primate testis. Further investigation ofthese growth factors, and their relationship with stem cells within the testis, will contribute to the development of strategies to protect fertility in prepubertal boys undergoing cancer treatment

Multicentre service evaluation of presentation of newly diagnosed cancers and type 1 diabetes in children in the UK during the COVID-19 pandemic

Background: The COVID-19 pandemic led to changes in patterns of presentation to emergency departments. Child health professionals were concerned that this could contribute to the delayed diagnosis of life-threatening conditions, including childhood cancer (CC) and type 1 diabetes (T1DM). Our multicentre, UK-based service evaluation assessed diagnostic intervals and disease severity for these conditions.Methods: We collected presentation route, timing and disease severity for children with newly diagnosed CC in three principal treatment centres and T1DM in four centres between 1 January and 31 July 2020 and the corresponding period in 2019. Total diagnostic interval (TDI), patient interval (PI), system interval (SI) and disease severity across different time periods were compared.Results: For CCs and T1DM, the route to diagnosis and severity of illness at presentation were unchanged across all time periods. Diagnostic intervals for CCs during lockdown were comparable to that in 2019 (TDI 4.6, PI 1.1 and SI 2.1 weeks), except for an increased PI in January–March 2020 (median 2.7 weeks). Diagnostic intervals for T1DM during lockdown were similar to that in 2019 (TDI 16 vs 15 and PI 14 vs 14 days), except for an increased PI in January–March 2020 (median 21 days).Conclusions: There is no evidence of diagnostic delay or increased illness severity for CC or T1DM, during the first phase of the pandemic across the participating centres. This provides reassuring data for children and families with these life-changing conditions

Impact of Tumour Epithelial Subtype on Circulating MicroRNAs in Breast Cancer Patients

While a range of miRNAs have been shown to be dysregulated in the circulation of patients with breast cancer, little is known about the relationship between circulating levels and tumour characteristics. The aim of this study was to analyse alterations in circulating miRNA expression during tumour progression in a murine model of breast cancer, and to detemine the clinical relevance of identified miRNAs at both tissue and circulating level in patient samples. Athymic nude mice received a subcutaneous or mammary fat pad injection of MDA-MB-231 cells. Blood sampling was performed at weeks 1, 3 and 6 following tumour induction, and microRNA extracted. MicroRNA microArray analysis was performed comparing samples harvested at week 1 to those collected at week 6 from the same animals. Significantly altered miRNAs were validated across all murine samples by RQ-PCR (n = 45). Three miRNAs of interest were then quantified in the circulation(n = 166) and tissue (n = 100) of breast cancer patients and healthy control individuals. MicroArray-based analysis of murine blood samples revealed levels of 77 circulating microRNAs to be changed during disease progression, with 44 demonstrating changes .2-fold. Validation across all samples revealed miR-138 to be significantly elevated in the circulation of animals during disease development, with miR-191 and miR-106a levels significantly decreased. Analysis of patient tissue and blood samples revealed miR-138 to be significantly up-regulated in the circulation of patients with breast cancer, with no change observed in the tissue setting. While not significantly changed overall in breast cancer patients compared to controls, circulating miR-106a and miR-191 were significantly decreased in patients with basal breast cancer. In tissue, both miRNAs were significantly elevated in breast cancer compared to normal breast tissue. The data demonstrates an impact of tumour epithelial subtype on circulating levels of miRNAs, and highlights divergent miRNA profiles between tissue and blood samples from breast cancer patients

5-Hydroxyvitamin D concentration in paediatric cancer patients from Scotland:a prospective cohort study

Children with cancer are potentially at high risk of plasma 25-hydroxyvitamin D [25(OH)D] inadequacy and despite UK vitamin D supplementation guidelines their implementation remains inconsistent. Thus, we aimed to investigate 25(OH)D concentration and factors contributing to 25(OH)D inadequacy in paediatric cancer patients. A prospective cohort study of Scottish children aged <18 years, diagnosed with and treated for cancer (patients) between Aug 2010-Jan 2014 was performed, with control data from Scottish healthy children (controls). Clinical and nutritional data were collected at defined periods up to 24 months. 25(OH)D status was defined by the Royal College of Paediatrics and Child Health (2013); inadequacy [<50 nmol/L: deficiency (<25 nmol/L), insufficiency (25-50 nmol/L)], sufficiency (51-75 nmol/L), optimal (>75 nmol/L). Eighty-two patients [median(IQR) age 3.9(1.9-8.8); 56% males)] and 35 controls [median(IQR) age (6.2(4.8-9.1); 49% males] were recruited. 25(OH)D inadequacy was highly prevalent in the controls (63%; 22/35), and in the patients (64%; 42/65) at both baseline and during treatment (33-50%). Non-supplemented children had the highest prevalence of 25(OH)D inadequacy at every stage with 25(OH)D median(IQR) ranging from 32.0 (21.0-46.5) nmol/L to 45.0(28.0-64.5) nmol/L. Older age at baseline [R=-0.46; p<0.001], overnutrition (BMI ≥85th centile) at 3 months [p=0.005; RR=3.1] and not being supplemented at 6 months (p=0.04; RR=4.3) may have contributed to lower plasma 25(OH)D. Paediatric cancer patients are not at higher risk of 25(OH)D inadequacy than healthy children at diagnosis; however prevalence of 25(OH)D inadequacy is still high and non-supplemented children have a higher risk. Appropriate monitoring and therapeutic supplementation should be implemented

Assessment of Plasma Antioxidants, Oxidative Stress and Polyunsaturated Fatty Acids in Paediatric Cancer Patients: A Prospective Cohort Pilot Study

Background: Paediatric cancer patients may have a limited dietary intake, particularly nutrients high in antioxidants, docosahexanoic acid (DHA) and eicosapentanoic acid (EPA). Objective: To investigate the antioxidant status (TAS), antioxidant capacity (TAC), oxidative stress, DHA and EPA of paediatric cancer patients during treatment. Methods: A prospective cohort study of Scottish children aged &lt;18 years, diagnosed with and treated for cancer between April-2013 to Jan-2014 was performed. Clinical data and blood samples were collected at baseline and 6 months. Data were stratified by treatment risk (low, medium and high) and nutritional support. We used Oxygen Radical Absorbance Capacity (ORAC) Antioxidant Assay to measure TAC, thiobarbituric acid reactive substances (TBARS) for lipid peroxidation and high performance liquid chromatography and Inductively Coupled Plasma Mass Spectrometry for TAS. The analyses of DHA and EPA were performed by analysing fatty acidmethyl esters (FAME) using gas-liquid chromatography. The reference ranges used were: Yagi 1998 (1.86-3.94) _mol for lipid peroxidation and Damsgaard.,et al. 2014 for EPA (0.45-0.77) % and DHA (2.22-3.76) %. Results: 20 patients (median (IQR) age 4.2 (1.5-8.5) years; 50% males) were recruited. There were no significant changes in plasma TAS, TAC and EPA, but lipid peroxidation significantly decreased from 7.4 (6.2-9.0) at baseline to 5.3 (4.5-6.4) _mol/MDA at 6 months(p = 0.003). The median (IQR) blood percentage of DHA significantly increased from 1.3 (0.9-1.9) to 1.8 (1.3-2.1) (p = 0.001). Lipid peroxidation was high in 95% (19/20) of patients at baseline and 94% (15/16) at 6 months; whilst DHA and EPA were low in 95%(19/20) and 70% (14/20) at baseline and 87.5% (14/16) and 60% (12/16) at 6 months. Children on high-treatment risk exhibited the highest oxidative stress levels. No statitically significant differences were found between non-supplemented and supplemented children in any of the following parameters (TAS, TAC, oxidative stress, EPA and DHA). Conclusion: There was a high prevalence of oxidative stress, especially in children treated with high-risk protocols and during the initial phases of treatment. Nutritional support does not appear to provide enough TAS, EPA and DHA in this cohort; however, larger high-quality population based studies are warranted to confirm these findings. Keywords: Paediatric cancer; Antioxidants; Oxidative stress; Docosahexanoic acid; Eicosapentanoic acidsch_dieThe Determinants of Nutritional Risk in Paediatric Cancer2pub4313pub

Low Plasma Vitamin D (25-Hydroxycholecalciferol) in Children and Adolescents Diagnosed with Cancer: A Case-Control Study

Introduction: Children and young people with cancer are less likely to spend time outdoors and they may also have a limited dietary intake. In addition, some cancer treatments can increase vitamin D catabolism. Objectives: This study aimed to investigate if there was an increased risk of poor vitamin D status in newly diagnosed childhood cancer patients compared to healthy controls in Scotland. Methods: Plasma 25 (OH) D was measured in children and adolescents during initial cancer treatment and compared to 33 healthy controls. Vitamin D deficiency was classified as plasma 25 (OH) D &lt;25 nmol/l, with a plasma 25 (OH) D of 25-49 nmol/l classified as insufficient. Results: Forty-one patients (median age 3.8 years, IQR 1.9-8.0) were diagnosed with cancer, 63% were male. Twenty-three (56 %) had solid tumours, 18 (44%) had haematological cancers. Median (IQR) plasma 25 (OH) D at recruitment was 37.0 nmol/l (23.7-58.2). Ten patients (24%) had vitamin D deficiency and 17 (41%) patients were classified as insufficient. The median (IQR) plasma 25 (OH) D in the control group (n = 33) was 37.5 nmol/l (29.0-58.0). Six controls (18%) had vitamin D deficiency and 14 (42%) were classified as having insufficient results. Plasma 25 (OH) D did not differ (p &gt; 0.05) between the patients and the controls. Conclusions: Almost three in four Scottish children treated for cancer had vitamin D deficiency or insufficiency; there was no increased risk of poor vitamin D status compared to healthy controls. Assessment of vitamin D status at diagnosis and in response to the course of treatment appears necessary to optimise nutritional management.sch_dieThe determinants of nutritional risk in children and young people with cancer3pub4423pub

Comparison of carboplatin versus cisplatin in the treatment of paediatric extracranial malignant germ cell tumours: A report of the Malignant Germ Cell International Consortium.

PURPOSE: To compare the outcomes of paediatric and adolescent extracranial malignant germ cell tumour (GCT) patients treated with either carboplatin or cisplatin on clinical trials conducted by the Children's Oncology Group (COG) and the Children's Cancer and Leukaemia Group (CCLG). METHODS: The Malignant Germ Cell International Consortium (MaGIC) has created a database of the GCT clinical trials conducted since 1983 by COG (United States, Canada and Australia), which used cisplatin-based regimens, and by CCLG (United Kingdom), which used carboplatin-based regimens. Using the parametric cure model, this study compared the overall 4-year event-free survival (EFS), stratified by age, stage, site and the a-priori defined MaGIC 'risk' groups: standard risk ((SR) 1 (EFS >80%; age 80%, age ≥ 11y) and poor risk (PR) (EFS ≤ 70%, age ≥ 11y). RESULTS: Cisplatin-based therapy was used in 620 patients; carboplatin was used in 163 patients. In the overall multivariate cure model, the two regimens did not differ significantly (cisplatin: 4-year EFS 86%; 95% confidence interval (CI) 83-89% versus carboplatin 4-year EFS 86%; 95% CI 79-90%; p = 0.87). No significant differences were noted in stratified analyses by site, stage, age and MaGIC risk groups: SR1 (p = 0.20), SR2 (p = 0.55) or PR (p = 0.72) patients. CONCLUSIONS: In these trials conducted contemporaneously, there is no significant difference in outcome observed overall, or any subset of patients, who were treated with regimens containing cisplatin versus carboplatin These results suggested sufficient equipoise to justify a randomised trial to evaluate the effectiveness of carboplatin versus cisplatin in the treatment of children, adolescents and young adults with standard risk GCT, which is currently underway

Automatisierte Planung von digitalen Hochgeschwindigkeitsnetzen

Der Ausbau von digitalen Hochgeschwindigkeitsnetzen ist gekennzeichnet durch neuartige Anforderungen an den Planungsprozess. Diese Anforderungen erfordern wiederum den Einsatz von neuartigen Paradigmen, die eine effiziente und zugleich genaue Planung von flächendeckenden Glasfasernetzen ermöglichen. Hierbei können wiederkehrende Planungsaufgaben durch eine gezielte computergestützte Automatisierung effizienter und genauer ausgeführt, als es mit bisherigen Planungskonzepten möglich ist. Dieses Arbeitspapier beschreibt die computergestützte Ausführung eines Planungsprozesses auf Basis von fünf grundlegenden, iterativen Planungsschritten und gibt Empfehlungen für eine effiziente und genaue Planung von Glasfasernetzen. Der hier vorgestellte Ansatz ermöglicht es Netzbetreibern und Investoren, den Ausbau beliebiger Siedlungs- und Gewerbegebiete auf der zuverlässigen Basis von belastbarem Faktenwissen wirtschaftlich zu priorisieren