Dynamic Changes in Microvascular Density Can Predict Viable and Non-Viable Areas in High-Risk Neuroblastoma

Despite aggressive treatments, the prognosis of high-risk NB remains poor. Surgical oncology needs innovative intraoperative devices to help surgeons discriminate malignant tissue from necrotic and surrounding healthy tissues. Changes within the tumor vasculature could be used intraoperatively as a diagnostic tool to guide surgical resection. Here, we retrospectively analyzed the mean vascular density (MVD) of different NB subtypes at diagnosis and after induction chemotherapy using scanned histological samples. One patient was prospectively enrolled, and an ex vivo photoacoustic imaging (PAI) scan was performed on two representative sections to assess its capacity to discriminate different tumor regions. We found that post-chemotherapy, viable areas of differentiating NBs and ganglioneuroblastomas are associated with higher MVD compared to poorly differentiated NBs. Early necrotic regions showed higher MVD than late necrotic and viable regions. Finally, calcified areas showed significantly lower MVD than any other histological component. The acquired PAI images showed a good high-resolution ex vivo 3D delineation of NB margins. Overall, these results suggest that a high-definition preclinical imaging device such as PAI could potentially be exploited to guide surgical resection by identifying different vasculature signatures

Dynamic Changes in Microvascular Density Can Predict Viable and Non-Viable Areas in High-Risk Neuroblastoma

Despite aggressive treatments, the prognosis of high-risk NB remains poor. Surgical oncology needs innovative intraoperative devices to help surgeons discriminate malignant tissue from necrotic and surrounding healthy tissues. Changes within the tumor vasculature could be used intraoperatively as a diagnostic tool to guide surgical resection. Here, we retrospectively analyzed the mean vascular density (MVD) of different NB subtypes at diagnosis and after induction chemotherapy using scanned histological samples. One patient was prospectively enrolled, and an ex vivo photoacoustic imaging (PAI) scan was performed on two representative sections to assess its capacity to discriminate different tumor regions. We found that post-chemotherapy, viable areas of differentiating NBs and ganglioneuroblastomas are associated with higher MVD compared to poorly differentiated NBs. Early necrotic regions showed higher MVD than late necrotic and viable regions. Finally, calcified areas showed significantly lower MVD than any other histological component. The acquired PAI images showed a good high-resolution ex vivo 3D delineation of NB margins. Overall, these results suggest that a high-definition preclinical imaging device such as PAI could potentially be exploited to guide surgical resection by identifying different vasculature signatures

Identification of bacterial pathogens in sudden unexpected death in infancy and childhood using 16S rRNA gene sequencing

Background Sudden unexpected death in infancy (SUDI) is the most common cause of post-neonatal death in the developed world. Following an extensive investigation, the cause of ~40% of deaths remains unknown. It is hypothesized that a proportion of deaths are due to an infection that remains undetected due to limitations in routine techniques. This study aimed to apply 16S rRNA gene sequencing to post-mortem (PM) tissues collected from cases of SUDI, as well as those from the childhood equivalent (collectively known as sudden unexpected death in infancy and childhood or SUDIC), to investigate whether this molecular approach could help identify potential infection-causing bacteria to enhance the diagnosis of infection. Methods In this study, 16S rRNA gene sequencing was applied to de-identified frozen post-mortem (PM) tissues from the diagnostic archive of Great Ormond Street Hospital. The cases were grouped depending on the cause of death: (i) explained non-infectious, (ii) infectious, and (iii) unknown. Results and conclusions In the cases of known bacterial infection, the likely causative pathogen was identified in 3/5 cases using bacterial culture at PM compared to 5/5 cases using 16S rRNA gene sequencing. Where a bacterial infection was identified at routine investigation, the same organism was identified by 16S rRNA gene sequencing. Using these findings, we defined criteria based on sequencing reads and alpha diversity to identify PM tissues with likely infection. Using these criteria, 4/20 (20%) cases of unexplained SUDIC were identified which may be due to bacterial infection that was previously undetected. This study demonstrates the potential feasibility and effectiveness of 16S rRNA gene sequencing in PM tissue investigation to improve the diagnosis of infection, potentially reducing the number of unexplained deaths and improving the understanding of the mechanisms involved

Comment on: "Tumour-agnostic drugs in paediatric cancers", Chisholm et al., BJC 2020.

Funder: NIHR Great Ormond Street Hospital Biomedical Research Centre NIHR Academic Clinical fellowshipFunder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/10000444

Short-wave infrared imaging enables high-contrast fluorescence-guided surgery in neuroblastoma

Fluorescence-guided surgery is set to play a pivotal role in the intraoperative management of pediatric tumors. Short-wave infrared imaging (SWIR) has advantages over conventional near-infrared I (NIR-I) imaging with reduced tissue scattering and autofluorescence. Here, two NIR-I dyes (IRDye800CW and IR12), with long tails emitting in the SWIR range, were conjugated with a clinical-grade anti-GD2 monoclonal antibody (Dinutuximab-beta) to compare NIR-I and SWIR imaging for neuroblastoma surgery. A first-of-its-kind multispectral NIR-I/SWIR fluorescence imaging device was constructed to allow an objective comparison between the two imaging windows. Conjugates were first characterized in vitro. Tissue-mimicking phantoms, imaging specimens of known geometric and material composition, were used to assess the sensitivity and depth penetration of the NIR-I/SWIR device, showing a minimum detectable volume of ~0.9 mm3 and depth penetration up to 3 mm. In vivo, fluorescence imaging using the NIR-I/SWIR device showed a high tumor-to-background ratio (TBR) for both dyes, with anti-GD2-IR800 being significantly brighter than anti-GD2-IR12. Crucially, the system enabled higher TBR at SWIR wavelengths than at NIR-I wavelengths, verifying SWIR imaging enables high-contrast delineation of tumor margins. This work demonstrates that by combining the high-specificity of anti-GD2 antibodies with the availability and translatability of existing NIR-I dyes, along with the advantages of SWIR in terms of depth and tumor signal-to-background ratio, GD2-targeted NIR-I/SWIR-guided surgery could improve the treatment of neuroblastoma patients, warranting investigation in future clinical trials

Integrin‐Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor‐Specific Delivery Achieve MYCN Silencing with Improved Survival

The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor‐targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor‐mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non‐targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide‐dependent indicating integrin‐mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN‐amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor‐targeting, with minimal clearance by the liver and so enable delivery of tumor‐targeted siRNA therapeutics

An integrated single-cell analysis of human adrenal cortex development

The adrenal glands synthesize and release essential steroid hormones such as cortisol and aldosterone, but many aspects of human adrenal gland development are not well understood. Here, we combined single-cell and bulk RNA sequencing, spatial transcriptomics, IHC, and micro-focus computed tomography to investigate key aspects of adrenal development in the first 20 weeks of gestation. We demonstrate rapid adrenal growth and vascularization, with more cell division in the outer definitive zone (DZ). Steroidogenic pathways favored androgen synthesis in the central fetal zone, but DZ capacity to synthesize cortisol and aldosterone developed with time. Core transcriptional regulators were identified, with localized expression of HOPX (also known as Hop homeobox/homeobox-only protein) in the DZ. Potential ligand-receptor interactions between mesenchyme and adrenal cortex were seen (e.g., RSPO3/LGR4). Growth-promoting imprinted genes were enriched in the developing cortex (e.g., IGF2, PEG3). These findings reveal aspects of human adrenal development and have clinical implications for understanding primary adrenal insufficiency and related postnatal adrenal disorders, such as adrenal tumor development, steroid disorders, and neonatal stress

DNA methylation-based classification of glioneuronal tumours synergises with histology and radiology to refine accurate molecular stratification

AIMS: Glioneuronal tumours (GNTs) are poorly distinguished by their histology and lack robust diagnostic indicators. Previously, we showed that common GNTs comprise two molecularly distinct groups, correlating poorly with histology. To refine diagnosis, we constructed a methylation-based model for GNT classification, subsequently evaluating standards for molecular stratification by methylation, histology and radiology. METHODS: We comprehensively analysed methylation, radiology and histology for 83 GNT samples: a training cohort of 49, previously classified into molecularly defined groups by genomic profiles, plus a validation cohort of 34. We identified histological and radiological correlates to molecular classification and constructed a methylation-based support vector machine (SVM) model for prediction. Subsequently, we contrasted methylation, radiological and histological classifications in validation GNTs. RESULTS: By methylation clustering, all training and 23/34 validation GNTs segregated into two groups, the remaining 11 clustering alongside control cortex. Histological review identified prominent astrocytic/oligodendrocyte-like components, dysplastic neurons, and a specific glioneuronal element as discriminators between groups. However, these were present in only a subset of tumours. Radiological review identified location, margin definition, enhancement, and T2 FLAIR-rim sign as discriminators. When validation GNTs were classified by SVM, 22/23 classified correctly, comparing favourably against histology and radiology which resolved 17/22 and 15/21 respectively where data were available for comparison. CONCLUSIONS: Diagnostic criteria inadequately reflect glioneuronal tumour biology, leaving a proportion unresolvable. In the largest cohort of molecularly defined glioneuronal tumours, we develop molecular, histological, and radiological approaches for biologically meaningful classification and demonstrate almost all cases are resolvable, emphasising the importance of an integrated diagnostic approach

Advanced x-ray imaging techniques in tissue engineering: a new construct assessment platform for enabling the regeneration of personalised organs

Tissue engineering (TE) holds promise for generating lab-grown patient specific organs which can provide: (1) effective treatment for conditions that require volumetric tissue transplantation and (2) new platforms for drug testing. Even though volumetric structural information is essential for confirming successful organ maturation, TE protocol designs are currently informed through destructive and 2D construct assessment tools (e.g. histology). X-ray phase-contrast computed-tomography (PC-CT) can generate non-destructive, high resolution, 3D density maps of organ architecture. In this work, PC-CT is used as new imaging tool for guiding two TE protocols currently at the in-vitro testing stage. The first (1) involves cell-repopulation of an oesophageal scaffold, with the aim of using the regenerated construct for treating long-gap oesophageal atresia, whilst for the second (2) a lung-derived scaffold is populated with islets for regenerating a pancreas, with the “repurposed” lung offering a platform for diabetes drug testing. By combing 3D images and quantitative information, we were able to perform comprehensive construct evaluation. Specifically, we assessed volumetrically: (1) the cell-distribution within the regenerated oesophagi and (2) islet integration with the vascular tree of the lung-derived scaffold. This new information was proven to be essential for establishing corresponding TE protocols and enabled their progression to more advanced scale-up models. We are confident that PC-CT will provide the novel insights necessary to further progress TE protocols, with the next step being in-vivo testing. Crucially, the non-destructive nature of PC-CT will allow in-vivo assessments of TE constructs following their implantation into animal hosts, to investigate their successful integration

Pathogenic variants in the human m(6)A reader YTHDC2 are associated with primary ovarian insufficiency

Primary ovarian insufficiency (POI) affects 1% of women and carries significant medical and psychosocial sequelae. Approximately 10% of POI has a defined genetic cause, with most implicated genes relating to biological processes involved in early fetal ovary development and function. Recently, Ythdc2, an RNA helicase and N6-methyladenosine reader, has emerged as a regulator of meiosis in mice. Here, we describe homozygous pathogenic variants in YTHDC2 in 3 women with early-onset POI from 2 families: C. 2567C>G, p.P856R in the helicase-associated (HA2) domain and c.1129G>T, p.E377*. We demonstrated that YTHDC2 is expressed in the developing human fetal ovary and is upregulated in meiotic germ cells, together with related meiosisassociated factors. The p.P856R variant resulted in a less flexible protein that likely disrupted downstream conformational kinetics of the HA2 domain, whereas the p.E377*variant truncated the helicase core. Taken together, our results reveal that YTHDC2 is a key regulator of meiosis in humans and pathogenic variants within this gene are associated with POI