Bacillus pumilus on small non-coding regulatory RNAs in the context of mouse RLS40 lymphosarcoma inhibition. In vitro binase exhibited cytotoxicity towards RLS40 cells via apoptosis induction through caspase-3/caspase-7 activation and decreased the levels of miR-21a, let-7g, miR-31 and miR-155. Intraperitoneal injections of binase in RLS40-bearing mice resulted in the retardation of primary tumour growth by up to 60% and inhibition of metastasis in the liver by up to 86%, with a decrease in reactive inflammatory infiltration and mitosis in tumour tissue. In the blood serum of binase-treated mice, decreases in the levels of most studied miRNAs were observed, excluding let-7g, while in tumour tissue, the levels of oncomirs miR-21, miR-10b, miR-31 and miR-155, and the oncosuppressor let-7g, were upregulated. Analysis of binase-susceptible miRNAs and their regulatory networks showed that the main modulated events were transcription and translation control, the cell cycle, cell proliferation, adhesion and invasion, apoptosis and autophagy, as well as some other tumour-related cascades, with an impact on the observed antitumour effects.The important role of miRNA in cell proliferation and differentiation has raised interest in exogenous ribonucleases (RNases) as tools to control tumour-associated intracellular and extracellular miRNAs. In this work, we evaluated the effects of the RNase binase from  The microRNAs (miRNAs) discovered in animals and plants  are non-Endogenous ribonucleases (RNases) participate in the maintenance of normal homeostasis of cellular RNAs. They regulate miRNA biogenesis, RNA decay, cleave aberrant RNAs and control the turnover of transcripts . Taking Rana pipiens (onconase) [Bacillus amyloliquefaciens [Bacillus pumilis [Currently, the most well-studied exogenous RNases with established antitumour activity are fungal RNases  ,14,15, Bnconase) , bovine nconase) ,19 from efaciens  and bina pumilis ,29,30,31 pumilis .KIT, AML1-ETO and FLT3 [The ability of binase to retard primary tumour growth and inhibit metastasis formation has been demonstrated in several experimental mouse tumour models ,28,32,33and FLT3 ,35, and and FLT3 . Neverth40 lymphosarcoma, was compared with alterations in the miRNA profile caused by binase. Bioinformatic analysis of the possible pathways regulated by the miRNA affected by binase in cells and tumour tissue showed that binase altered the levels of particular miRNAs and modulated the tumour microenvironment, with an influence on the differentiation of tumour microenvironment-related cells.Here, we assessed the effect of binase on small non-coding regulatory RNAs in the context of inhibiting tumour progression. The antitumour potential of binase, which was evaluated in vitro and in vivo against mouse RLSEscherichia coli BL21 cells containing the plasmid pGEMGX1/ent/Bi. Binase was purified as described earlier [Binase (12.3 kDa) was isolated as a homogenous protein from the culture medium of  earlier . The end earlier .40 cells were obtained from the cell collection of the Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS) . RLS40 cells were cultured in complete IMDM medium , 100 units/mL penicillin, 100 μg/mL streptomycin and 2 mM glutamine) under standard conditions (at 37 °C in a humidified atmosphere with 5% CO2).Modified RLSMale 10- to 14-week-old CBA/LacSto  mice were provided by the vivarium of the Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia. The mice were housed in plastic cages  under normal daylight conditions. Water and food were provided ad libitum.All animal procedures were performed in strict accordance with the approved protocol and recommendations for the proper use and care of laboratory animals (ECC Directive 2010/63/EU). The experimental protocols were approved by the Committee on the Ethics of Animal Experiments with the Institute of Cytology and Genetics SB RAS  and all efforts were made to minimise suffering.5 cells/well) in serum-free IMDM medium and binase was added at concentrations ranging from 50–700 µg/mL. Plates were incubated under standard conditions for 48 h. The viability was assessed using a WST-1-based test . WST-1 solution  was added according to the manufacturer’s protocol and the cells were incubated for another 3–4 h under the same conditions. The optical density of the resulting solution (A) was measured at wavelengths of 450 and 620 nm on a multichannel Multiscan RC spectrophotometer . The activity of the mitochondrial dehydrogenases was calculated as the difference in absorbance ΔA = (A450 − A620). The respiratory activity of untreated cells was taken to be 100%. All reported values are the means of three independent measurements  ±  SD.Cells were seeded onto 96-well plates . Binase was added at the concentrations of 5 and 10 µg/mL and the cells were incubated under standard conditions for 24 h, followed by RNA isolation.RLS40 cells were seeded in 24-well plates at a density of 1.5 × 106 cells per well and incubated in antibiotic-free IMDM supplemented with 10% FBS  in the presence of binase (500 µg/mL) for 48 h under standard conditions. The medium was discarded and the cells were gently suspended in a binding buffer at a concentration of 106 cells/mL. Then, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and PI  for 15 min in the dark and immediately analysed using a NovoCyte flow cytometer .RLS40 cells was assessed using a Caspase-Glo® 3/7 Assay kit  according to the manufacturer’s instructions. Briefly, the cells were seeded in white-walled 96-well plates at a density of 0.4 × 105 cells/well and incubated in serum- and antibiotic-free IMDM in the presence of binase (500 µg/mL) for 48 h under standard conditions. Further procedures were performed according to the manufacturer’s protocol. Luminescence was detected using a CLARIOstar plate reader .The caspase-3/-7 activity in RLS® Caspase-3/7 Green Flow Cytometry Assay Kit  analysed using a NovoCyte flow cytometer .The plates were prepared as described in previous paragraph. Further procedures were performed according to the manufacturer’s protocol. The number of caspase-3/-7-positive cells was assessed using CellEvent40 cells were generated via the intramuscular (i.m.) injection of RLS40 cells (107 cells/mL) suspended in 0.1 mL of saline buffer into the right thigh of CBA mice. On day 4 after the tumour implantation, mice with RLS40 were divided into three groups (n = 20 per group): (1) the control group received an intraperitoneal injection (i.p.) of saline buffer (0.1 mL), and (2) and (3) received i.p. binase at the doses of 0.5 mg/kg and 1 mg/kg three times per week, respectively. The total number of injections was seven.Solid tumour RLSThe tumour sizes were monitored every other day using calliper measurements in three perpendicular dimensions. Tumour volumes were calculated as V = length × width × height × (π/6). Mice were sacrificed 1 h after the final injection of binase (on day 18 after the tumour implantation) via cervical dislocation. For the analysis of the miRNA expression profile, tumour and blood samples were taken. For histological and morphometric analysis, liver, kidney, spleen, thymus and tumour samples were collected.40 was carried out using a TRIzol reagent  according to the manufacturer’s protocol. Tumour pieces from the mice of the control and experimental groups were homogenised. Blood serum was prepared from the whole blood of mice via clot formation at 37 °C for 30 min and at 4 °C overnight, followed by clot discarding and centrifugation . Serum samples were pooled according to groups. Total RNA was extracted from the serum samples, RLS40 cells and homogenates of the tumour tissue immediately using a TRIzol reagent  according to the manufacturer’s protocol.The isolation of total RNA from RLSRNA concentration was measured via absorbance at 260 and 280 nm using a Bio Mate 3  spectrophotometer and Qubit . The total RNA integrity and quantity were checked using Agilent 2100 Bioanalyzer .40 cells was analysed using stemloop PCR technology [The expression of miRNA in RLSchnology ,38. cDNAPCR was performed in a total volume of 20 µL using 2× BioMaster HS-qPCR SYBR Blue  and 0.05 µM of forward miRNA-specific primers and universal reverse primer . The PCRTissue samples  were fixed in 10% neutral buffered formalin , dehydrated in ascending ethanols and xylols and embedded in HISTOMIX paraffin . Paraffin sections (5 μm) were sliced on a Microm HM 355S microtome  and stained with haematoxylin and eosin. For the immunohistochemical study, the tumour sections (3–4 μm) were deparaffinised and rehydrated. Antigen retrieval was carried out after exposure in a microwave oven at 700 W. The samples were treated with anti-PCNA , anti-caspase-3-specific  and anti-caspase-7-specific  monoclonal antibodies according to the manufacturer’s protocol. Then, the sections were incubated with secondary horseradish peroxidase (HPR)-conjugated antibodies , exposed to a 3,3′-diaminobenzidine (DAB) substrate and stained with Mayer’s haematoxylin. All images were examined and scanned using an Axiostar Plus microscope equipped with an Axiocam MRc5 digital camera  at magnifications of ×100 and ×400.control − mean metastasis areaexperiment)/mean metastasis areacontrol) × 100%.The percentages of the internal metastases areas were determined relative to the total area of the sections using Adobe Photoshop software. The inhibition of metastasis development was assessed using the metastasis inhibition index (MII), calculated as MII = ) of the unchanged tumour tissue, lymphoid infiltration, necrosis and numerical densities (Nv) of mitoses and PCNA-positive cells in the tumour tissue. Morphometric analysis of the spleen included the evaluation of the volume densities (Vv (%)) of the red pulp, the white pulp and the diameter of the lymphoid follicles (µm). Morphometric analysis of the thymus included the evaluation of the volume densities (Vv (%)) of the cortex and medulla with a subsequent calculation of the cortex/medulla index. The histological study of the liver and kidney included the evaluation of destructive changes (dystrophy and necrosis) of hepatocytes and epitheliocytes in the proximal tubules.Morphometric analysis of the tumour, spleen and thymus sections was performed via point counting using a morphometric grid, which consisted of 100 testing points in a testing area equal to 3.2 × 10structure/Ptest) × 100%, where Pstructure denotes the number of points over the structure and Ptest denotes the total number of test points, which was 100 in this case. The numerical density (Nv) of the studied histological structure indicated the number of particles in the unit tissue volume evaluated as a number of particles in the square unit, which was 3.2 × 106 μm2 in this case.The volume density (Vv (%)) of a studied histological structure represented the volume of the tissue fraction occupied by this compartment and determined from testing points lying over this structure and calculated using the following formula: Vv = . The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALK), total protein, creatinine and blood urea nitrogen (BUN)  were measured.Mus musculus) [p ≤ 0.05 were included in the analysis. Functional grouping and linking of the enriched terms were performed with kappa statistics (kappa score = 0.4).The target genes of binase-susceptible miRNAs were predicted using the CyTargetLinker v. 4.1.0 plugin  based onusculus) . The obtusculus)  in CytosThe key common genes were then analysed from the point of view of their participation in cancer-related pathways and events using the GeneCards  and KEGGt-test  or one-way analysis of variance (ANOVA). Post hoc testing was completed using a post hoc Tukey’s test; p < 0.05 was considered to be statistically significant. The statistical package STATISTICA version 10.0  was used for analysis.All experiments were reproduced in triplicate. Data were statistically processed using Student’s 40 lymphosarcoma is derived from hematopoietic tissue and displays a multidrug resistance phenotype related to human diffuse large B-cell lymphoma; melanoma B-16 arose from neuroectodermal tissue and is related to human metastatic melanoma [40 are able to form primary solid tumour nodes after intramuscular implantation, and these tumours metastasise into the lungs and liver, respectively; after intravenous implantation, melanoma B16 can only form metastases in the lungs.The first study of the ability of binase to retard primary tumour growth and metastasis development was performed using three tumour models of different histological types with strong relevance to human tumours: Lewis lung carcinoma (LLC) with an epithelial origin is related to human non-small cell lung cancer ; mouse Rmelanoma . LLC and40 models and inhibited metastasis development by up to 50% in the case of LLC and RLS40 and up to 70% in B16 melanoma [40 lymphosarcoma, taking into account several specific characteristics of this tumour. First of all, RLS40 forms primary tumour nodes and metastasises to the liver. Since binase, like any xenobiotic, will be metabolised in the liver, this model allowed for tracking a triple burden on the liver, i.e., from the tumour, from metastases, and from metabolism. Second, RLS40 lymphosarcoma is a tumour with a multiple-drug-resistant phenotype that has a disrupted system of ABC transporters. This system allows for chemotherapeutics to be pumped out of cells. Binase, being a protein, allowed us to bypass this system and excluded it from the confrontation with chemotherapeutic therapy. A disrupted ABC transporter system can cause an alteration in the mechanism of the antitumour activity of binase.Previous data showed that intraperitoneal injections of binase (dose range 1–5 mg/kg) resulted in the retardation of primary tumour growth by up to 45% in the LLC and RLSmelanoma . In this40 cells were treated with binase at concentrations ranging from 50 to 700 μg/mL . The most expressed miRNAs were miR-155, let-7g and miR-21a, with expression levels of 1.9, 1.8 and 1.7, respectively  an investigation into the antitumour activity of binase in vitro in tumour cells; (2) the study of the potential of anticancer therapy with binase using the RLS40 tumour model in vivo, paying special attention to the dose dependence of the antitumour effects and binase toxicity; (3) a search for correlations between the cytotoxic (in vitro) and antitumour (in vivo) effects of binase and changes in the miRNA profile, both in tumour cells/tissue and serum caused by the enzyme.Here, we studied the mechanism of the antitumour activity of binase using mouse lymphosarcoma RLS40 cells via the induction of apoptosis and changes in the level of a number of miRNAs. Binase induced apoptosis through the activation of caspase-3/-7, which was observed for caspase-3/-7 in vitro and for caspase-7 both in vitro and in vivo. This is consistent with previously obtained data that indicated the activation of apoptotic responses by binase in human A549 alveolar adenocarcinoma cells [Our data obtained in vitro showed that binase exhibited cytotoxic effects towards RLSma cells  and mousma cells .The study of binase’s effects on tumour progression in mice revealed that therapy with binase essentially retarded the growth of primary tumours and inhibited metastases development in the liver, thus showing excellent results, especially because binase was used as a monotherapy for a tumour with a multiple-drug-resistant phenotype . Interes40 cells, binase decreased the expression levels of four out of six tested miRNAs, whereas in the tumour tissue, binase caused an increase in the levels of five out of six miRNAs. Such contradictory effects of binase can be explained in several ways. First of all, the dose used in vitro was many times higher than the dose used in vivo: 0.5 mg/mL used in vitro corresponded to the dose of 0.5 g/kg in comparison with the dose of 0.5–1 mg/kg used in vivo. In previous work, we demonstrated the pronounced antitumour and antimetastatic potential of binase for doses of just 0.5–1 mg/kg [40 tumour tissue, this may be associated with the combined systemic effects of binase in vivo. Binase cleaves miRNAs in the bloodstream, leading to a decrease in their levels and a deficiency regarding building a pre-metastatic niche. The observed upregulation of miRNA expression in the tumour tissue may be related to both a deficiency in miRNA in the bloodstream, as well as with the acceleration of miRNA biogenesis in the tumour tissue due to the cleavage of pre-miRNAs, pri-miRNAs and mature miRNAs. Thus, a feedback system was released that replenished the level of miRNAs required for the formation of a pre-metastatic niche and tumour microenvironment. Furthermore, we cannot exclude the fact that since the measurement of the miRNA levels was done at the end of the experiment when the tumour sizes in the experimental group were significantly smaller compared to the control group, clones of cells with an unaltered expression of miRNA were maintained, which were necessary for the tumour to progress, and all other cells died due to RNA degradation and the induction of apoptosis.We considered the ability of binase to affect miRNAs using a panel of six miRNAs earlier evaluated as a result of an NGS analysis of the miRNA profile in the serum and tumour tissue of mice with Lewis lung carcinoma . As we e–1 mg/kg . Since t–1 mg/kg , and its–1 mg/kg , we can –1 mg/kg  and matu40 was in accordance with data of the decrease of the levels of most miRNAs in the bloodstream of LLC-bearing mice after the treatment with bovine pancreatic RNase A [40 model. The reasons are: (1) the pyrimidine-X cleavage specificity of RNase A and the guanine-X cleavage specificity of binase; (2) targets of RNase A are extracellular RNAs, whereas targets for binase are both intracellular and extracellular RNAs. Some evidence has been obtained showing that binase cleaves cellular non-coding RNAs [hsa-miR-17 and down-regulated hsa-miR-30c, which resulted in NF-κB inhibition and an increase in the chemosensitivity of tumour cells [Binase’s ability to decrease the miRNA levels in the bloodstream of mice with RLS RNase A . In the ing RNAs . Severalur cells . Anotherur cells . Interesur cells . Since Rur cells .Kras, a verified target of binase [Acvr1b, Ccnd2 and Gaa (sensitive to RNase A) [p21 (sensitive to RNases L and MC2) [Analysis of the obtained miRNA networks showed that modulation of the expression of binase-susceptible miRNAs can affect a wide spectrum of processes in cells, including events related to cell transformation, proliferation, apoptosis, cancer-related pathways, angiogenesis and adhesion. Other processes found to be triggered by binase were inflammation and some housekeeping functions, such as ribosome biogenesis and transport. Interestingly, the analysis of common genes for external miRNA networks and genes for internal miRNA-networks revealed f binase , and a rRNase A)  and p21 and MC2) ,55. Addiand MC2)  and the and MC2) . Moreoveand MC2) ,34 and tand MC2) .Our findings using a bioinformatics approach not only verified the tight linkage between the effect of binase on the expression levels of selected miRNAs and events mediating cell transformations but also showed that binase-susceptible miRNAs may be involved in the mechanisms underlying the other activities of this enzyme, with an impact on the formation of a pre-metastatic niche and tumour microenvironment. Additionally, binase can probably affect the differentiation of tumour microenvironment-related cells, cancer stem cells and tumour-associated macrophages or fibroblasts, and may inhibit vascularisation of the tumour site. These data provide a reasonable foundation for further investigations into binase as a modulator of antitumour responses during the various steps of tumour progression.


Tumour budding in colorectal cancer has become an important prognostic factor. Represented by single cells or small tumour cell clusters at the invasion front of the tumour mass, these tumour buds seem to reflect cells in a ‘hybrid’ state of epithelial–mesenchymal transition, and evidence indicates that the presence of these entities is associated with lymph node metastasis, local recurrence and distant metastatic disease. The International Tumour Budding Consensus Conference (ITBCC) has highlighted a scoring system for the reporting of tumour budding in colorectal cancer, as well as different clinical scenarios that could affect patient management. Other organs are not spared: tumour budding has been described in numerous gastrointestinal and non-gastrointestinal cancers. Here, we give an update on ITBCC validation studies in the context of colorectal cancer and the clinical implications of tumour budding throughout the upper gastrointestinal and pancreatico-biliary tract. Tumour budding occurs in a large variety of cancers from different organs.2 The frequency of high-grade tumour budding is difficult to estimate, due to the use of various scoring systems, but it might be found in approximately 40% of colorectal cancers (CRC),3 oesophageal cancers and gastric cancers8  Fig.  or at thrs8 Fig. , as well. 9 Fig.  and chol16 Double staining for the epithelial marker cytokeratin and the mesenchymal marker vimentin, highlights a small number of tumour buds that co-express both proteins, adding weight to the hypothesis that a ‘hybrid’ EMT phenotype exists in a subset of these cells.17 EMT has been linked to therapy resistance and cancer-cell stemness,19 so it follows that the detection of tumour budding in preoperative biopsy samples of patients with, for example, either rectal or oesophageal cancers, is associated with a poor response to neoadjuvant therapy and overall clinical outcome.21 Mounting data suggest that the presence of tumour budding is an unfavourable prognostic factor across all tumour types in which it is found and is tightly associated with lymph node metastasis, local recurrence and distant metastatic disease.Evidence indicates that tumour buds might adopt the properties of cells undergoing epithelial–mesenchymal transition (EMT), suggesting that these cells have a more invasive and migratory potential.22 although RNA sequencing studies clearly underline changes in mRNA and microRNAs involved in transforming growth factor-β (TGF-β) and WNT signalling pathways.23 Downstream of these pathways, repressors of the cell–cell adhesion molecule E-cadherin are overexpressed, as are markers of extracellular matrix degradation and migration. The presence of tumour buds in areas of desmoplastic stroma strongly suggests an interplay between tumour cells and cancer-associated fibroblasts, which presents an exciting area for future investigation.The reason for the occurrence of tumour budding is not known. DNA sequencing studies show no difference in the mutational profile of driver genes in tumour buds in comparison with the main tumour mass,24 tumour budding has increased in popularity as an important prognostic factor in CRC. The first guideline for reporting tumour budding was published in 2017 for CRC following the 2016 International Tumour Budding Consensus Conference (ITBCC),1 and is now included in the College of American Pathologists (CAP) protocol. The ITBCC method was selected due to the large evidence base supporting its clinical utility, and is based on the Japanese Guidelines for Colorectal Cancer Reporting.25 The evidence-based guidelines describe clinical scenarios in which tumour budding should be assessed using a three-tier scoring system. In detail, the haematoxylin and eosin (H&E) slide with the greatest degree of budding at the invasion front (PTB) is selected, then ten individual fields at medium power (10× objective) are used to identify the ‘hotspot’ and tumour buds are counted using a ×20 objective. Normalisation of the count to an area of 0.785 mm2 (depending on the eyepiece field number) is performed, and the budding categories are defined: BD1 (1–4 buds), BD2  and BD3 (≥10 buds). Subsequent validation of these guidelines not only in CRC, but also in lung, gastric and PDAC, has underlined the usefulness of this standardised scoring approach.27 H&E is the standard stain, but pan-cytokeratin staining can also be used in conjunction with a number of approaches to assess tumour budding  followed by evaluation of buds in one or ten hotspots, to more subjective scoring methods based on the observed severity of the budding throughout the slide (e.g. low/moderate/severe). The actual tumour budding counts have been used in both pan-cytokeratin and H&E for analysis with clinical endpoints, and cut-offs for classification into ‘low’ or ‘high’ have been suggested at four, five or ten buds, depending on the clinical scenario and the type of material (biopsy sample or resection). Both have advantages and disadvantages. On the one hand, pan-cytokeratin helps to facilitate the visual count and results in a higher number of true tumour buds being detected, but also might lead to overestimation of non-bud-type objects, such as cell fragments. The nucleus, which needs to be observed to classify a cell as a bud, can be masked by strong cytokeratin staining. These issues contribute to interobserver variability, especially on a single-object level.95 H&E is a standard staining method and can be implemented in all pathology laboratories, but leads to difficulties in areas of peritumoural inflammation, and is challenging for the discrimination of tumour budding versus activated fibroblasts—again, a source of interobserver variation.A variety of different methods have previously been used to assess tumour budding, both intra- and peri-tumourally using either pan-cytokeratin or H&E-stained slides.28 however, it should be kept in mind that the sum of all the molecular features probably leads to a particular morphological picture that has defined histopathological characteristics. One of those histological characteristics in CRC is tumour budding, a phenomenon that is indicative of tumour progression and adverse prognosis.29 There are now enough data in the literature, as well as the ITBCC guidelines, to justify implementing the assessment of tumour budding into routine clinical practice for CRC.30 In CRC, the presence of tumour budding along with other established biomarkers might support clinicians in four potential clinical scenarios , lymphatic invasion , poor histological differentiation  and tumour budding . The conclusion was therefore quite similar to that proposed by Ueno et al.31—namely, that the absence of lymphatic invasion and budding, submucosal invasion ≤1 mm and poor histological differentiation was each associated with a low risk of lymph node metastases.32 In 2017, Cappellesso et al. focused specifically on the role of tumour budding in a meta-analysis of 41 studies and 10137 patients with pT1 CRC.33 They found tumour budding to be strongly associated with the risk of nodal metastases and, when comparing a positive tumour-budding status  with a negative tumour-budding status , the prevalence of lymph node positivity resulted in an odds ratio (OR) value of 6.44 .33 The ITBCC states that tumour budding is an independent predictor of lymph node metastases in pT1 CRC patients, and therefore strongly recommends that tumour budding is reported, along with other histopathological predictors of lymph node metastasis, such as poor differentiation, lymphovascular invasion and the depth/level of submucosal invasion,1 in patients with pT1 CRC.The clinical management of pT1 CRC includes the decision as to whether patients with early invasive cancer should undergo a wait-and-see approach or if they should be considered for oncological resection. There is therefore a major need for robust and reproducible biomarkers that correlate with the presence or absence of lymph node metastases. In 2004, Ueno et al. investigated a panel of clinicopathological parameters, including tumour location, tumour diameter, macroscopic tumour configuration (sessile vs. pedunculated), tumour grade, vascular invasion, tumour budding and width and depth of submucosal invasion.35 In the past 10 years, numerous studies and meta-analyses have reported tumour budding to be an independent factor of poor survival and recurrence in patients with stage II CRC, with outcomes similar to those of patients with stage III CRC.44 The ITBCC therefore recommended in 2016 that tumour budding be included among the high-risk factors in stage II CRC.1 This recommendation was supported by the 2019 World Health Organisation (WHO) classification of tumours of the digestive system, which reports tumour budding—along with perineural invasion, intramural and extramural vascular invasion, lymphatic invasion and tumour deposits—as a high-risk factor with an OR of 4.51 .45 In 2019, Ueno et al. validated the ITBCC scoring system in a multicentre stage II colon cancer cohort from 123 institutions (n = 991).46 The 5-year relapse-free survival (RFS) rate was 90.9% in patients with tumours classified as BD1, 85.1% in those with BD2 and 74.4% in those with BD3 (P < 0.001). There also was a significant correlation between the budding grade and recurrence in the liver, lungs, lymph nodes and peritoneum (P < 0.001–0.01). Multivariable analysis revealed that budding had an independent impact on RFS. The study concluded that tumour budding should be routinely reported in stage II colon cancer.46The updated European Society for Medical Oncology (ESMO) guidelines for the management and treatment recommends a follow-up for patients with low-risk stage II colon cancer, while adjuvant therapy with fluoropyrimidine should be considered for patients who have high-risk factors, such as T4 , number of examined lymph nodes <12, primary tumour perforation or occlusion, tumour grade 3 or absence of microsatellite instability (MSI).47 This observation led to a systematic assessment of PTB and ITB,48 and to the finding of a potential prognostic and predictive role for ITB. ITB is highly associated with PTB, and therefore a surrogate for the tumour-budding status of the whole tumour, as well as being associated with lymph node and distant metastases, local recurrence, poor survival and tumour regression grade.53 Therefore, the assessment of ITB in biopsy samples might have important clinical implications, especially in the preoperative management of rectal cancer patients. Patients who present with high-grade ITB along with the already-implemented clinical factors in preoperative biopsy samples might be considered for neoadjuvant therapy. Although the ITBCC recognises the ITB approach, more data are definitely necessary prior to its implementation in daily practice.1In 1989, Morodomi et al. described the presence of tumour buds in biopsy samples from patients with rectal cancer and the association of this phenomenon with lymph node metastases.54 The most frequently used biomarkers in clinical practice are molecular, and report on the RAS, BRAF and MSI status,54 whereas current histopathological features reported for clinical management are metastatic size, percentage of fibrosis and necrosis, resection status and tumour regression grade.55 Several studies have shown the prognostic potential of the histological growth pattern—desmoplastic, pushing and replacement—of the tumour–liver interface of CRLM.62 Tumour budding, a morphological feature of the tumour microenvironment at the invasive front, might therefore also be an important factor in disease progression in stage IV CRC patients. Similar to the primary tumour, tumour buds can be detected at the invasive front (PMB) or within the main metastasis body (IMB)63 but, in contrast to the primary tumour, there is still a major challenge for scoring tumour budding in CRLM. Indeed, the detection of tumour buds can be difficult in cases without desmoplastic stromal reaction or a strong reactive perimetastatic ductular proliferation.63 In addition, pan-cytokeratin immunohistochemical staining can sometimes obscure important morphological features, and therefore a budding score based on H&E staining is recommended.63 In a 2018 study, tumour budding assessed in CRLM from 229 patients with stage IV CRC was a prognostic factor, but not an independent predictor of survival.64The ESMO consensus guidelines for the management of patients with metastatic CRC highlight the importance of a multidisciplinary management, including oncology, surgery, radiology and pathology.In summary, there are currently not enough data to make any firm conclusions on the prognostic or predictive role of tumour budding in CRLM, and further retrospective and prospective studies on large multicentric cohorts are needed.65 Similar results have been observed in numerous studies  underlining significantly poorer 3-year or 5-year survival rates after oesophagectomy in SCC patients with high-grade budding. Nakanishi and colleagues published comparable results, with 5-year survival rates of 49% versus 15% in 74 patients with low-grade versus high-grade budding, respectively, receiving preoperative chemotherapy.72 The results of these studies have been reviewed by Koelzer et al.73 High-grade tumour budding has also been found to be associated with lymph node metastasis in oesophageal lesions involving the muscularis mucosae (T1a-MM) to those of the upper third of the submucosa (T1b-SM1) using both H&E and cytokeratin staining.74 These results suggest that budding could be a useful histomorphological feature in patients with primary resected oesophageal SCC in the neoadjuvant setting and in early-stage cancers.The first study on tumour budding in oesophageal squamous cell carcinoma (SCC) dates from the early 2000s. Investigating tumour budding by H&E in a small cohort of 56 patients, which included surgically treated individuals with stage I–III oesophageal SCC, Roh et al. found a marked reduction in the 3-year survival rate in patients with high-grade versus low-grade budding .73 High-grade tumour budding is described in 28–51.7% of cases, albeit using different scoring systems and based on either H&E or pan-cytokeratin staining.75 Its presence is associated with higher TNM stage, lymph node metastases, poor disease-free survival and poor overall survival. These results again highlight the correlation between tumour budding and disease course, and might be useful to guide follow-up.Although only a handful of studies have evaluated tumour budding in oesophageal adenocarcinoma, similar results have been reported.7 Seven cohorts encompassing data from 2178 patients were analysed; the method used to analyse budding was based on H&E staining, and the cut-off values for ‘high-grade’ budding varied across studies, from five or ten buds to the median number of buds in the particular cohort. As a first step, the presence of high-grade tumour budding showed a positive correlation with tumour stage  as well as with undifferentiated tumour status . High-grade tumour budding was significantly associated with lymphatic vessel invasion and lymph node metastasis , as well as with poor 5-year overall survival in a pooled analysis of 1833 patients . These results were confirmed in a subgroup analysis of intestinal-type cancers but not in diffuse-type adenocarcinoma.76 In their evaluation of tumour budding in 621 radical gastrectomies for submucosal early gastric carcinoma,77 Du et al. identified high-grade tumour budding as a predictor of lymph node metastases . Moreover, when Ulase et al. applied the ITBCC tumour-budding score to 456 surgically resected gastric cancers, they found that the BD score was significantly associated with sex, Laurén phenotype, pT-, pN- and pM classification, as well as perineural invasion and survival times.26Guo and colleagues published a review summarising the evidence of tumour budding in gastric cancer in 2019.In summary, tumour budding shows prognostic potential in oesophageal adenocarcinomas, SCC and gastric cancers, and might be predictive in the neoadjuvant setting. Although a standardised scoring system is still missing, the ITBCC approach for CRC might also be applicable in the context of upper gastrointestinal cancers.78 it is subdivided into intrahepatic and extrahepatic cholangiocarcinoma, and the latter can be further subclassified as perihilar or distal cholangiocarcinoma.79 The vast majority of patients with cholangiocarcinoma present with unresectable disease at the time of diagnosis. Consequently, the prognosis for cholangiocarcinoma is poor, with a 5-year OS of 30–40% for localised tumours and a median OS of nearly 12 months for unresectable or metastatic disease.81Cholangiocarcinoma is a rare type of tumour, comprising <1% of all cancers;82 its significance in cholangiocarcinoma is far less clear. However, Ogino et al. demonstrated in 2019 that peritumoral budding in both perihilar and extrahepatic cholangiocarcinoma is associated with adverse clinicopathological features, such as higher T stage, lymphovascular and perineural invasion, lymph node metastases and higher histological grade, which translate into a worse clinical outcome.83 Cholangiocarcinoma patients with high-grade tumour budding had a significantly shorter OS compared with those with low-grade tumour budding. In another cholangiocarcinoma cohort comprising 299 Asian patients, the presence of peritumoral budding was associated with worse OS.84 According to the results of these two retrospective studies, tumour budding might be a potential prognostic factor.Although tumour budding is a well-established prognostic factor in CRC,86 However, the study population was heterogeneous, comprising all T and N stages with or without R0 resection. A preplanned subanalysis indicated that male patients and those with poorly differentiated tumours derived the most benefit from adjuvant capecitabine treatment,85 highlighting the importance of identifying predictive biomarkers to predict which subset of patients with resected cholangiocarcinoma should respond to chemotherapy. Further validation in independent datasets—preferably from Phase 3 studies—is needed to finally confirm both the prognostic and predictive values of tumour budding in cholangiocarcinoma. In addition, no data demonstrating whether patients with high-grade, tumour-budding cholangiocarcinoma might benefit from adjuvant chemotherapy exist yet. Therefore, the predictive impact of tumour budding in cholangiocarcinonoma is still unclear.Based on the results of the adjusted intention-to-treat and per-protocol analysis of the previously published BILCAP trial, a randomised clinical trial comparing adjuvant chemotherapy with capecitabine with expectant treatment following resection, the American Society of Clinical Oncology (ASCO) guidelines recommended adjuvant treatment with capecitabine in resected cholangiocarcinoma patients.87 In 2019, a meta-analysis demonstrated that patients with PDAC exhibiting high-grade tumour budding had a higher all-cause mortality rate compared with those who showed low-grade tumour budding .88 Due to the aggressive tumour biology and the inherent poor prognosis of PDAC, adjuvant chemotherapy with either FOLFIRINOX  or gemcitabine combined with capecitabine is recommended in all patients after curative resected PDAC, regardless of the pathological stage.90 However, patients who may not qualify for a doublet or triplet therapy, treatment with gemcitabine alone is a reasonable option. In a retrospective analysis of the CONKO-001 trial, designed to compare adjuvant gemcitabine with observation in patients with PDAC undergoing complete, curative-intent tumour resection, the presence of tumour budding was associated with decreased OS, irrespective of whether or not the patients were treated with adjuvant gemcitabine.91 In this study, no further subclassification into PTB or ITB has been carried out. In contrast to the situation for stage II colon cancer, in which, although still unproven, the presence of high-grade tumour budding might contribute to the treatment strategy, tumour budding in PDAC has not yet had an impact on adjuvant treatment decisions. However, just as new treatment strategies targeting PDAC might continue to evolve in the near future, the role of tumour budding on clinical decision-making might be redefined.PDAC also has a poor prognosis, with a 5-year OS of ~5%.83 , American Joint Committee on Cancer (AJCC) and the WHO. Grading in SCC of the oesophagus is based on the degree of cytological atypia, mitotic activity and the presence of keratinisation, whereas in colorectal adenocarcinoma, grading is determined according to solid areas.Tumour budding is emerging as a promising morphological biomarker not only in CRC, but also in other gastrointestinal cancers, such as oesophageal, gastric, PDAC and cholangiocarcinomas94 Alternatively, the development of a digitally supported scoring system for tumour budding could be time-saving as well as beneficial for increasing reproducibility.96 In addition, although enough data exist for tumour budding in pT1 CRC and stage II CRC patients for implementation into clinical practice, more studies are needed for the clinical scenarios that involve preoperative biopsies and the assessment of tumour budding in CRLM. Finally, although the predictive value of tumour budding is still not clear, the investigation of potential target molecules expressed by tumour buds might offer a promising approach for an anti-budding therapy to specifically target the tumour cells that seem to be responsible for local and distant metastases and consequently for tumour progression and decreased survival.For CRC, four aspects of tumour budding need to be addressed in order to optimise its clinical application. Interobserver variability in the assessment of budding is still suboptimal, especially among non-gastrointestinal pathologists, leading potentially to the up- or downgrading of budding. Accordingly, tumour-budding courses are needed to improve the reproducibility of tumour budding in CRC, similar to the assessment by immunohistochemistry of programmed death ligand 1 (PD-L1) in lung cancer.


A 52-year-old woman presented with shortness of breath and cough. An endobronchial sialolipoma was found at the left entrance of the main bronchus. Sialolipoma is an exceedingly rare type of lipoma reported of the minor salivary glands, especially within the bronchus.A 52-year-old woman presented with shortness of breath and cough with 6 months´ evolution. Endobronchial endoscopy revealed a tumour at the left entrance of the main bronchus. The entire removal of the tumour was removed using a cryoprobe device. Pathological examination showed a tumour consistent with the diagnosis of sialolipoma due to the presence of mature adipose cells blended with acinar, ductal, basal, and myoepithelial cells. The patient had a favourable outcome.The infrequent tracheobronchial presentation of this tumour can be challenging for correct diagnosis. Tracheobronchial tree obstructions can be caused by endobronchial tumours despite their clinical and biological behaviour. Tumour removal is the primary treatment to support airway function and to improve the respiratory status.Benign tumours of the endobronchial tree are rare, with endobronchial lipomas are being the least common.Lipomas are common benign mesenchymal tumours originated in the human body , 2, and Nagao T et al. , proposeThis tumour typically occurs in adults (90%), with fewer cases reported in infants , 4.These tumour can also developed inside the bronchus though there are few endobronchial cases reported in the literature. Indeed, despite a wide investigation, we only found a case reported by Kajiwara N et al. . HoweverThe aetiopathogenesis is unclear.A 52-year-old woman presented complaining of shortness of breath and cough with 6 months of evolution. The patient reported the use of tobacco for 25 years and a medical history of hypertension. A chest X-ray was performed revealing no lung lesions. There were no relevant findings during a physical examination. Endobronchial endoscopy showed scarce serous secretions in the trachea and major bronchi. An endobronchial tumour was found at the entrance of the left main bronchus shown. A flexible cryoprobe was used for tumour extraction and argon plasma coagulation was applied to control bleeding Fig. .Fig. 1TThe patient was discharged a few days after the procedure, and she remains well to date.Gross examination showed a round shape, smooth surface, solid and whitish-pink tumour histologically consistent with a sialolipoma due to the presence of mature adipose cells blended with acinar, ductal, basal, and myoepithelial cells Figs. , 4 and 5Lipomas are common benign subcutaneous or deep soft tissue tumours composed of lobules of mature adipose tissue. They can occur at any age, with most diagnoses occurring in the adults with some cases reported in infants , 2.Sialolipoma is a rare salivary gland lesion characterized by benign salivary gland parenchyma interspersed with mature adipose tissue.One of the hypotheses regarding the pathogenesis correlates the tumour with salivary gland dysfunction and the substitution of normal glandular tissue by mature adipose tissue, atrophy of the glandular structures, chronic ductal epithelial changes (oncocytic metaplasia), fibrosis, and lymphocytic infiltration .Histological examination in our case revealed a tumour consisting of mature adipose cells mixed with acinar, ductal, basal, and myoepithelial cells inside the left main bronchus. Therefore, the diagnosis of endobronchial sialolipoma was established. A diagnosis of endobronchial lipoma diagnosis was roled out because this entity is characterized by a uniform appearance of mature univacuolated adipocytes of uniform size and shape, without intermingled epithelial ductal-acinar components. In contrast, very few cases inside the bronchus have been reported worldwide .Sialolipoma can be found within both major and minor salivary glands –8. In coRegarding differential diagnosis, bronchial lipomas, neuroectodermic tumours, hamartomas, carcinomas, and other tumours must be considered, and definitive diagnosis may be a challenge. Thus, the possibility of an endobronchial tumour should be considered for patients with respiratory symptoms, asthmatic patients who are not responsive to bronchodilators, and those with a medical history of recurrent pneumonia.Chest tomography and magnetic resonance imaging can show exhaustive details of the tumour characteristics, determining the base to be narrow or broad and extension across the cartilaginous rings, and demonstrating extramural invasion, or the presence of distal lesions. Additionally, the tumour position, size, surface appearance and degree of mobility can be accurately confirmed .In the case we present, endobronchial endoscopy was used for detection of the lesion.Bronchoscopic resection is considered the first choice of treatment; however, surgical removal is indicated in cases associated with malignant tumour, in patients with destructive peripheral lung disease, with extrabronchial tumour, with evident technical difficulties for bronchoscopic procedures .Sialolipoma is a recently described variant of lipoma. The rare clinical presentation of this tumour can be challenging for a correct diagnosis because of the need to rule out the much more frequent malignancies. Computed tomography or magnetic resonance are the main procedures employed; however, definitive diagnosis is achieved by bronchoscopy and biopsy of the tumour. Therefore, the treatment of choice is endoscopic resection.


Zhou CC, Wang J, Bu H, Zhongguo Fei Ai Za Zhi, 2020, 23(2): 65-76.In the version of this article initially published, error appeared on page 65. The author address should be “400037 重庆，重庆新桥医院（朱波）”. And another error appeared on page 66. The first sentence in part 1 should be “年龄标化率（age standardized rate, ASR）31.5/10万和14.6/10万”.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049793


Lymphatic disorders have received an increasing amount of attention over the last decade. Sparked primarily by improved imaging modalities and the dawn of lymphatic interventions, understanding, diagnostics, and treatment of lymphatic complications have undergone considerable improvements. Thus, the current review aims to summarize understanding, diagnostics, and treatment of lymphatic complications in individuals with congenital heart disease.The altered hemodynamics of individuals with congenital heart disease has been found to profoundly affect morphology and function of the lymphatic system, rendering this population especially prone to the development of lymphatic complications such as chylous and serous effusions, protein-losing enteropathy and plastic bronchitis.Although improved, a full understanding of the pathophysiology and targeted treatment for lymphatic complications is still wanting. Future research into pharmacological improvement of lymphatic function and continued implementation of lymphatic imaging and interventions may improve knowledge, treatment options, and outcome for affected individuals. The lymphatic vasculature is widely distributed throughout the entire body. In health, it functions as a unidirectional drainage and transport system originating in the interstitial space and terminating with the return of the lymphatic fluid back into the great veins of the neck or thorax. The initial lymphatic capillaries are composed of a single layer of lymphatic endothelial cells with interstitially anchored filaments preventing collapse. For initial uptake and transport, the fluid is dependent on favorable pressure gradients to reach the pre-collecting and collecting lymphatic vessels. From here, an increasing concentration of smooth muscle cells intertwiningly weaved around the lymphatic vessel contract in order to propel the fluid forward –3. The iDue to the structural and functional heterogeneity of congenital heart disease (CHD), incidence and prevalence of lymphatic complications are immensely varying within subtypes of malformations. Being often referred to as the forgotten circulation, the etiology behind the development of lymphatic complications remains poorly understood. Generally, the incidence of effusions and other lymphatic complications tend to increase with increasing venous congestion and more severe congenital heart malformations. Accordingly, individuals with tetralogy of Fallot and univentricular heart disease are excessively overrepresented with regards to lymphatic complications in CHD. Sparked primarily by improved lymphatic imaging modalities and the dawn of the field of lymphatic interventions, understanding, diagnostics, and treatment of lymphatic complications have undergone considerable improvements over the last decade. Thus, the current review aims to summarize understanding, diagnostics, and treatment of lymphatic complications in individuals with CHD.In CHD, hemodynamic changes arising from structural abnormalities or their correction may result in increased hydrostatic pressure in parts of the circulation. In the capillary beds, this causes increased filtration of fluid to the interstitial space increasing the necessity for lymphatic removal. Additionally, the increased central venous pressure (CVP) commonly found in some subgroups impedes the emptying of the thoracic duct, adding additional congestion to the already pressured lymphatic system. Consequently, many individuals with congenital heart defects are especially prone to the development of lymphatic complications Fig. . Thus, eProlonged pericardial or pleural effusions and chylothorax and chylopericardium may all, given the origin of the fluid, be considered as lymphatic complications. Prolonged effusions are most commonly observed following surgical repair of either tetralogy of Fallot or univentricular heart disease . DependiProlonged effusions are reported in between 7.3 and 13.5% of operated tetralogy of Fallot patients and may be viewed as a disruption of the fluid equilibrium , 21. TheSimilarly, during the creation of a bidirectional cavopulmonary connection or total cavopulmonary connection, the blood flow and the hydrostatic pressure in the pulmonary capillary beds tend to increase, augmenting filtration and risk of effusions, the incidence following correction being 12.6% and 37%, respectively . In the Chylothorax and chylopericardium are other postoperative lymphatic complications. They are common following traumatic perforation of the thoracic duct or its tributaries during surgery, with leakage accumulating postoperatively. However, it is also observed more sporadically years after surgery. Here, the proposed mechanism is a combination of increased intralymphatic pressure due to increased uptake and impeded return and structural weakness in the lymphatic vessels. Perforation of the endothelial weakness provides a way of decompression for the congested lymphatic system and reroutes fluid into compartments of lower pressure.Chylothorax occurs with an incidence of 7.4% and 9–24% following surgical repair of tetralogy of Fallot and “surgical palliation” of single ventricles, respectively , 32, 33.The clinical manifestations of effusions and chylothorax may progress from asymptomatic to the experience of dyspnea, coughing, and chest discomfort with gradually increasing amounts of accumulating fluid , 38. PerFollowing symptoms, effusions or chylothorax are normally diagnosed with the finding of fluid during ultrasonography or radiographic examination. Commonly, measurement of triglycerides has been used to categorize the fluid as chyle leaking from the lymphatic system. However, as argued above, also fluid filtered out in the lung and transuded into the pleural space may be considered lymphatic fluid. Thus, the content of triglycerides and ratio of triglycerides or cholesterol in pleural fluid compared with serum should, to a greater extent, be viewed as a way of determining the origin of the lymphatic fluid. Thus, triglycerides above the threshold of 110 mg/dl indicate intestinal origin of the lymphatic fluid, confining the leakage to the thoracic duct or its main tributaries. Similarly, low levels of triglycerides indicate an origin of the lymphatic fluid, before the mixing with the lipid-rich fluid flowing in the central thoracic duct –40. InfoWhile prolonged effusions may resolve following a new steady state between filtration and lymphatic removal, the volume of accumulated fluid may deem treatment necessary. Treatment of effusions and chylothorax evolves around two primary goals: short-term relief of respiratory symptoms by drainage of fluid and long-term prevention of recurrence by treating the underlying cause.Thoracocentesis and placement of a chest tube will secure immediate relief of respiratory symptoms and maintain continuous drainage. Nonsurgical conservative options for chylothorax include a no-fat diet in order to reduce production of chyle. Medium-chain triglycerides may be added, as these are absorbed directly into the portal vein, bypassing the lymphatic system , 44, 45.In patients unresponsive to conservative nutritional therapy, pharmacological agents such as somatostatin or the synthetic analog octreotide have been reported efficient in treatment , 47. TheTraditionally, surgical treatment of chylous leakage has been reliant on thoracic duct ligation or pleurodesis performed via either video-assisted thoracoscopy or open thoracotomy, with pleuroperitoneal shunting reserved for the most persisting cases , 53–55. PLE is characterized by an abnormal and occasionally profound intestinal loss of lymphatic fluid and protein. In addition to CHD, PLE may be observed secondarily to several other diseases ranging from congestive heart failure to infection and inflammatory bowel disease. In CHD, incidence of PLE has been reported as affecting from 4 to 15% of individuals following Fontan palliation –61.The pathophysiology behind the development of PLE is incompletely characterized and with a variety of factors contributing, although the combination and impact of each may vary between cases. Under normal circumstances, the liver and intestines are responsible for the production of 25–50% of the lymphatic fluid flowing in the thoracic duct . In indiOther potential mechanisms thought to be involved in the development of PLE in CHD include diminished mesenteric blood flow causing cellular apoptosis and increased permeability of the intestinal wall . AdditioWhile PLE often manifests when the protein leakage is substantial and resulting in severe morbidity, it may also exist in a subclinical version. In addition, the loss of protein may be transient with alternating asymptomatic and affected periods. Symptomatic progression of PLE is related to the loss of protein and range from the development of chronic diffuse edema to diminished tissue integrity and impaired wound healing . ImpaireMeasurement of an elevated α-1 antitrypsin clearance in a 24-h stool collection is the golden standard for diagnosis of PLE . HoweverThe efficacy of pharmacological treatment of PLE is largely case-based, and the lack of randomized controlled trials is universal. Primary treatment aims at decreasing fluid overload through diuretic therapy and preventing hypoproteinemia and low serum oncotic levels through a high-protein, low-fat diet or parenteral nutrition and intravenous albumin , 78. TraInvasive procedures may secure a more permanent symptomatic relief and should be considered on a case-by-case basis. Late fenestration with the creation of a right to left shunt may increase cardiac output and diminish symptoms at the expense of cyanosis , 90. A lPB is characterized by the formation of thick rubbery casts in the lumen of the airways. Not being exclusively associated with structural CHD, PB may also be observed alongside everything from asthma to various lymphatic diseases, sickle cell disease, and cystic fibrosis. Although rare, and only reported in < 5% of Fontan patients, PB causes severe morbidity in the form of chronic coughing, hypoxemia, expectoration of casts, and asphyxiation .The underlying pathophysiology behind PB in CHD patients is not fully understood. However, recent findings attribute a central role to a pressured and morphologically changed lymphatic system ultimately resulting in lymphatic leakage into the airways –98. SimiChronic coughing, wheezing, dyspnea, pleuritic chest pain, fever, or respiratory symptoms unresponsive to bronchodilators may lead the clinician to consider a possible PB diagnosis –102. CheThe overall treatment of PB should have two primary goals: an initial improvement of respiratory symptoms and hemodynamics and secondary targeting of venous and lymphatic congestion. Because of the rarity and heterogeneity of the condition, the evidence behind treatment for plastic bronchitis is scarce. Removal of any obstruction under diagnostic bronchoscopy may initially relieve respiratory symptoms . OptimizIn addition to pharmacotherapy, dietary changes in form of a low-fat diet in combination with ligation of the thoracic duct have been shown to relieve symptoms or cause resolution in some patients and may be considered . Until rDespite symptomatic relief on specific symptomatic treatment, the detrimental pathophysiology of the Fontan circulation is largely unaltered. Thus, continued pharmacological treatment with pulmonary vasodilators and diuretics may be deemed necessary, or additional surgical interventions to optimize hemodynamics and lymphodynamics may be considered. Late catheter-based fenestration redirects flow and lowers venous congestion . As in pOverall, the cause of interstitial accumulation of fluid or edema can be described as an imbalance between potentially four different factors; the hydrostatic- and the oncotic pressure both intravascularly and in the interstitial space, the permeability of the endothelial glycocalyx, and finally the function of lymphatic vessels in removing fluid and protein. Any disturbance to the equilibrium of these forces resulting in a greater microvascular filtration or an attenuation of the lymphatic removal may cause accumulation of fluid and edema , 111. EdEdema in patients with CHD is thought to be primarily revolving around circulatory pressure changes and a decrease in cardiac function. However, individuals with concurrent Turner or Noonans syndrome may have inherent genetic defects impairing normal lymphatic vessel development .AccordinWhatever the cause, the diagnosis of peripheral edema is predominantly based on patient history and clinical findings. Lymphoscintigraphy, lymphangiography, and NIRF imaging may support the diagnosis and help visualize the abnormal route of the lymphatic flow. In addition, duplex ultrasound may reveal chronic venous insufficiency, a common disposition leading to peripheral edema .Treatment and prevention are cause-dependent; optimizing cardiac function and preventing further renal and hepatic deterioration may be relevant within patients with CHD. Persisting edema may be treated with exercise, decongestive physiotherapy, compression stockings, or pneumatic compression therapy , 117.Although the recent decade has seen a sparked interest in research into the function of the lymphatic system and its involvement in various complications, a full understanding of the pathophysiology behind the majority of lymphatic complications is still wanting. Particularly within the field of CHD, research is challenged by the heterogeneous nature of the primary heart defects, and the low numbers treated at each center. Accordingly, targets for the current pharmacological treatments are speculative, based on a limited number of patients, and the lack of randomized placebo-controlled trials is universal.Future continued implementation of lymphatic interventions and increased experience with pre-operative lymphatic risk stratification may improve patient selection and reduce risk of postoperative development of lymphatic complications . Targete


On the basis of the results of previous national and international trials and studies, the Renal Tumour Study Group of the International Society of Paediatric Oncology (SIOP–RTSG) has developed a new study protocol for paediatric renal tumours: the UMBRELLA SIOP–RTSG 2016 protocol (the UMBRELLA protocol). Currently, the overall outcomes of patients with Wilms tumour are excellent, but subgroups with poor prognosis and increased relapse rates still exist. The identification of these subgroups is of utmost importance to improve treatment stratification, which might lead to reduction of the direct and late effects of chemotherapy. The UMBRELLA protocol aims to validate new prognostic factors, such as blastemal tumour volume and molecular markers, to further improve outcome. To achieve this aim, large, international, high-quality databases are needed, which dictate optimization and international harmonization of specimen handling and comprehensive sampling of biological material, refine definitions and improve logistics for expert review. To promote broad implementation of the UMBRELLA protocol, the updated SIOP–RTSG pathology and molecular biology protocol for Wilms tumours has been outlined, which is a consensus from the SIOP–RTSG pathology panel. In this Consensus Statement, the pathology panel of the International Society of Paediatric Oncology–Renal Tumour Study Group (SIOP–RSTG) present the pathology and molecular biology protocol for Wilms tumours in the UMBRELLA SIOP-RTSG 2016 protocol. Other renal non-Wilms tumours are rare entities and include mesoblastic nephroma, clear cell sarcoma of the kidney, rhabdoid tumour of the kidney, renal cell carcinoma and few other, even rarer tumour types1. The Renal Tumour Study Group of the International Society of Paediatric Oncology (SIOP–RTSG) has developed a new study for all renal tumours of childhood, the UMBRELLA SIOP–RTSG 2016 protocol (the UMBRELLA protocol)3, on the basis of the experiences from SIOP–2001 and the UK Improving Population Outcomes for Renal Tumours of Childhood (IMPORT) 2013 studies4. The aim of the UMBRELLA protocol is to collect all clinical, biological and outcomes data from children with primary renal tumours in a comprehensive data registry, with central review of diagnostics , standardized biobanking and precise treatment recommendations for the most common paediatric renal tumours. Molecular biology research within the protocol is primarily focused on the validation of the prognostic value of 1q gain, which might lead to a more personalized treatment approach (Box TP53 (refs7) or several of the newly identified driver candidates for stratification and inclusion of liquid biopsies might help to reach this goal10.Paediatric renal tumours account for ~6% of all paediatric malignant tumours, of which ~90% are Wilms tumours (nephroblastoma)12, one of the main focuses of the UMBRELLA protocol is assessment of the independent prognostic value of the absolute volume (rather than the percentage) of the blastemal component that survives preoperative chemotherapy15. Furthermore, in order to optimize pathological diagnostics and treatment, rapid central pathology review will be obligatory to enable feedback on institutional pathological diagnosis in a clinically relevant time frame. The UMBRELLA protocol addresses both Wilms tumour and non-Wilms tumours; in this Position Paper, we focus on the recommendations for Wilms tumours.The UMBRELLA protocol includes updated guidelines for pathologists for the handling and processing of tissue as well as criteria that are important for postoperative histological classification, staging and treatment stratification. These recommendations were established by a consensus of pathology experts within the SIOP–RTSG (chaired by G. M. Vujanić and I. Leuschner). As well as the aim of validating 1q gain as a prognostic markerBox 1 Relevant aims of the SIOP–RTSG UMBRELLA 2016 protocolPathology aimsClassify and stage tumours accurately in order to optimize treatment and avoid undertreatment or overtreatment.Monitor and give appropriate feedback on local pathological diagnosis and stage.Accurately assess the blastema in postchemotherapy specimens.Correlate blastema volume and biomarkers with event-free and overall survival.To optimize pathological diagnostics by rapid central review in order to:Biobanking aimsTo store biological material from all participating centres.To show the feasibility of storing serial blood and urine samples and tumour and germline material at diagnosis and at specific time points during treatment for international collaborative studies.To establish national biobanks (if not yet present) and generate a study-wide virtual biobank for translational research.To test the feasibility of returning biomarker results to treatment centres within a clinically relevant time frame.Molecular biology aimsTo assess genomic 1q gain as a prognostic marker in Wilms tumour.MYCN gain and 17p loss encompassing the TP53 locus).To analyse biomarkers with potential prognostic relevance  in blood, and/or plasma, and/or urine and to support research on epigenetics, tumour cell culture, and/or organoids and xenografts.SIOP–RTSG, Renal Tumour Study Group of the International Society of Paediatric Oncology.14, the UMBRELLA protocol mandates preoperative chemotherapy on the basis of clinical and radiological diagnoses in most patients. Thus, pathological diagnosis is mainly based on pretreated nephrectomy specimens. Pretreatment biopsy is not routinely recommended. Percutaneous cutting needle biopsy (tru-cut biopsy) can be considered in young children with stage IV disease and in children >10 years of age, as the frequency of non-Wilms tumours  is increased in these age groups16. Biopsies are particularly not advised in children <6 months of age and in fully cystic tumours; immediate surgery is recommended in these children. If performed, open wedge biopsy automatically upstages tumours to stage III, irrespective of other findings, as it is regarded as an  breach of the capsule (rupture). However, percutaneous cutting needle biopsies do not upstage tumour.As in previous SIOP trials and studies17. Most importantly, a guide block of the selected samples from the tumours is made by the pathologist to assist tumour staging17 , and they are more difficult to assess. Currently, treatment of these tumours depends on other parameters, such as their histological type and response to preoperative chemotherapy, and the best way for a pathologist to manage them is to adopt two methods of analysis: one method is to assess all nodules together and calculate the percentages of nonviable and viable components as if they represented one nodule, and the other method is to assess them individually . The treatment decision should be made at multidisciplinary team meetings and in consultation with national or international lead experts.Histological assignment to the different risk groups is based on quantification of chemotherapy-induced changes, the percentages of the different viable Wilms tumour components  and the presence or absence of anaplasia18 Table . In the 18. These blastemal-type Wilms tumours were treated with more intensive postoperative treatment than other tumour types in the SIOP–2001 trial, resulting in improved outcomes15. This improvement highlights the need to identify high-risk, chemotherapy-resistant blastema to avoid unnecessary failure of first-line therapy, especially as doxorubicin is omitted in treatment of stage II and stage III intermediate-risk tumours19. A retrospective analysis of the SIOP–2001 trial data showed that a threshold of ~20 ml residual blastemal volume in localized Wilms tumour could be used to improve stratification14. An accurate recognition of blastema needs more objective criteria than are currently available to minimize interobserver variation and to enable institutional pathologists to make a correct diagnosis. The SIOP–RTSG agreed that the blastemal volume threshold at which the risk of relapse rises dramatically needs to be established before its implementation as a prognostic factor. Thus, one of the aims of the UMBRELLA protocol is to optimize the definition of high-risk, blastemal-type Wilms tumour.The most important viable component to recognize in pretreated Wilms tumours is blastema, which is the most undifferentiated tumour component and is composed of primitive, undifferentiated cells that are arranged in no particular pattern. Blastemal-type Wilms tumour (in which >66% of the viable tumour consists of blastema in a tumour that is >33% viable) confers a worse prognosis and is, therefore, classified as a high-risk tumour according to the SIOP–2001 working classificationIn the UMBRELLA protocol, institutional pathologists will not be required to calculate the volume of blastema in tumours, as it is not currently a criterion for treatment stratification. However, the estimated percentages of the nonviable and viable components  will be recorded, and blastemal volume will then be calculated in the protocol database. This calculation will be based on both pathological and imaging measurements of the tumour.24. Anaplasia can occur in any component of Wilms tumours, and it can be focal or diffuse22. Importantly, in order to diagnose anaplasia, all three criteria need to be present: large atypical tripolar and/or multipolar mitotic figures; marked nuclear enlargement, with nuclear diameters at least three times those of adjacent cells; and hyperchromatic tumour cell nuclei22. Although the criteria for anaplasia are well established, in many circumstances diagnosis can be challenging, resulting in a considerable number of undiagnosed or overdiagnosed patients25. This misdiagnosis illustrates the value of rapid central pathology review for correct postoperative treatment stratification.Anaplasia has been recognized as a high-risk Wilms tumour feature for many years, and it confers a worse prognosis22. By consensus of the SIOP–RTSG pathology panel, the size of the anaplastic focus does not exceed 15 mm (which was previously undefined). Wilms tumour with focal anaplasia is regarded as an intermediate-risk tumour in the UMBRELLA protocol, but the tumour should still be subclassified according to other components . If focal anaplasia is present in blastemal-type Wilms tumour, it is regarded as a high-risk tumour.Focal anaplasia is defined as the presence of one or two foci showing the abovementioned nuclear criteria with sharp demarcation within the primary intrarenal tumour and without evidence of anaplasia or prominent nuclear atypia (nuclear unrest) in other areas22.Diffuse anaplasia is defined as nonlocalized or multifocal anaplasia, focal anaplasia with marked nuclear unrest in the rest of the tumour, or anaplasia outside of the kidney . If anaplasia is present in a biopsy sample or other incomplete tumour sample, the diagnosis of diffuse anaplasia is warranted26. These patients are treated with preoperative chemotherapy for 6–12 weeks and nephron-sparing surgery (NSS) is considered by the surgical panel, taking into account tumour response to chemotherapy, to spare as much renal function as possible. Each tumour is subclassified and staged separately according to the histological classification and SIOP staging criteria. For multifocal tumours, the approach described above should be used.Synchronous bilateral Wilms tumours (stage V) occur in ~5–8% of patients, and these children are more likely to have an underlying genetic predisposition30. The two main types of nephrogenic rests are perilobar and intralobar rests. The term nephroblastomatosis is used to describe multiple, multifocal, or diffuse nephrogenic rests28. Panlobar nephroblastomatosis encompasses complete replacement of the renal lobe or cortex by nephrogenic rests and is recognized as a high-risk factor for developing Wilms tumour33. Prolonged chemotherapy is recommended in patients with nephroblastomatosis in the UMBRELLA protocol. Most patients are treated with NSS, as this therapy enables disease-free survival while sparing a maximum of healthy parenchyma in both kidneys33.Nephrogenic rests are foci of embryonal cells that persist after 36 weeks of gestation and are considered precursors of Wilms tumours. Nephrogenic rests are identified in 35–40% of patients with unilateral and >90% of patients with bilateral Wilms tumours and are often associated with different syndromes or anomalies such as overgrowth syndromes (hemihypertrophy and Beckwith–Wiedemann syndrome), Denys–Drash syndrome and Wilms tumour, aniridia, genitourinary abnormalities and mental retardation (WAGR) syndrome34. Molecular studies have shown that cystic nephroma and cystic partially differentiated nephroblastoma are unrelated tumours35. Intermediate-risk or high-risk Wilms tumours can also present with numerous cysts, especially after preoperative chemotherapy, but they also contain solid, nodular areas34. The presence of numerous cysts in these tumours is of no prognostic significance34.Cystic partially differentiated nephroblastoma is a distinct variant of Wilms tumour that usually occurs in children <3 years of age and is composed entirely of cysts with septa without tumour nodules inside them14. Histological classification after immediate nephrectomy is based only on the presence or absence of diffuse anaplasia: if absent, the Wilms tumour is classified as intermediate risk and if present, the tumour is high risk17.In rare instances, for example in children <6 months of age, immediate surgery is recommended in the UMBRELLA protocol, as opposed to preoperative chemotherapy. This course of action leads to a different histological classification, as chemotherapy-induced changes are not present. Of note, the presence of substantial amounts of viable blastema is not of prognostic significance in primarily operated Wilms tumours14. The histological staging and treatment stratification of Wilms tumours after preoperative chemotherapy has changed to some extent in the UMBRELLA protocol on basis of the results of the previous trials and studies and by consensus of the SIOP–RSTG pathology panel23. More detailed definitions have been introduced to aid in correct staging by local pathologists , metastatic (stage IV), or bilateral (stage V) disease in order to decide on duration of preoperative chemotherapyBox 2 Staging criteria in the SIOP UMBRELLA protocolStage ITumour is limited to the kidney.Tumour is present in the perirenal fat but is surrounded by a fibrous (pseudo)capsule. The (pseudo)capsule might be infiltrated by viable tumour, which does not reach the outer surface.Tumour might show protruding (botryoid) growth into the renal pelvis or the ureter but does not infiltrate their walls.The vessels or the soft tissues of the renal sinus are not involved by tumour. Intrarenal vessel involvement might be present.Stage IIViable tumour is present in the perirenal fat and is not covered by a (pseudo)capsule, but is completely resected (resection margins are clear).Viable tumour infiltrates the soft tissues of the renal sinus.Viable tumour infiltrates blood and/or lymphatic vessels of the renal sinus or of the perirenal tissue, but it is completely resected.Viable tumour infiltrates the wall of the renal pelvis or of the ureter.Viable tumour infiltrates the vena cava or adjacent organs  but is completely resected.Stage IIIViable tumour is present at a resection margin. Nonviable tumour or chemotherapy-induced changes present at a resection margin are not regarded as stage III.Abdominal lymph node involvement is present by either viable or nonviable tumour.Preoperative or intraoperative tumour rupture, if confirmed by microscopic examination (viable tumour at the surface of the specimen at the area of the rupture).Viable or nonviable tumour thrombus is present at resection margins of ureter, renal vein, or vena cava inferior .Viable or nonviable tumour thrombus, which is attached to the inferior vena cava wall, is removed piecemeal by a surgeon.Wedge or open tumour biopsy before preoperative chemotherapy or surgery.Tumour implants (viable or nonviable) are found anywhere in the abdomen.Tumour (viable or nonviable) has penetrated through the peritoneal surface.Stage IVHaematogenous metastases  or lymph node metastases outside the abdominopelvic region.Stage VBilateral renal tumours at diagnosis. Each side should be substaged according to the above criteria.Stage I tumours are confined within the (pseudo)capsule and, even if the tumours are outside of the kidney in the perirenal fat but surrounded by the (pseudo)capsule and completely removed, they are regarded as stage I. The vessels or soft tissues of the renal sinus are not involved. The presence of necrotic tumour or chemotherapy-induced changes in the renal sinus, renal veins and/or within the perirenal fat is not a reason for upstaging. Botryoid tumour growth into the renal pelvis is also not a reason for upstaging the tumour. Furthermore, infiltration of the soft tissues between the kidney and the adrenal gland or of the adrenal gland itself does not cause upstaging of the tumour if the external capsule of the adrenal gland is intact. However, the presence of viable tumour in the lymphatic or blood vessels in this area is regarded as stage II. Tumour adhesion to the liver capsule is not regarded as infiltration of an adjacent organ; the tumour is upstaged  only if clear infiltration of the liver parenchyma is present. Tumour attachment to other retroperitoneal organs, such as the colon, should be assessed in the same manner. Fine needle aspiration or percutaneous cutting needle biopsy (tru-cut biopsy) does not upstage the tumour.Stage II tumours are defined by viable tumour infiltration of the renal sinus (soft tissues and any vessels), and/or perirenal fat and/or adjacent organs  with no clearly identifiable (pseudo)capsule but with clear resection margins. The presence of a viable tumour thrombus in the renal vein or inferior vena cava is also stage II if completely resected in one piece.18. Now, in the UMBRELLA protocol, the presence of nonviable tumour or chemotherapy-induced changes only at a resection margin is not regarded as stage III. The finding of viable or nonviable tumour thrombus bulging out at the resection margin of the renal vein or inferior vena cava needs to be discussed with the surgeon. If the surgeon confirms they resected the vein away from the thrombus, then protruding thrombus at the vascular resection margin does not upstage the tumour to stage III. If the thrombus is removed with force, owing to attachment to the vascular wall, or by a piecemeal resection, stage III must be considered at a multidisciplinary meeting. The UMBRELLA protocol now explicitly states that the presence of prechemotherapy tumour rupture at diagnosis on imaging studies is only considered as pathological stage III if viable tumour is seen microscopically at the rupture site of the nephrectomy specimen. If not, the tumour is staged on the basis of the other pathological criteria, but the final treatment stage must be decided after discussion at a multidisciplinary team and/or tumour board meeting.Stage III tumours are defined according to a number of criteria Box . Several36. Accumulation of foamy macrophages within the subcapsular or interfollicular sinus should not be regarded as a metastasis. Nonviable metastasis is regarded as replacement of normal lymph node architecture with foamy macrophages and/or chemotherapy-induced changes.The presence of chemotherapy-induced changes (even without viable tumour) in a lymph node is regarded as proof of tumour metastasis and, therefore, the tumour is assigned stage III. Mature tubules can be found in lymph nodes, often associated with Tamm–Horsfall protein deposits, but this finding should not be considered as lymph node metastasis37. For example, patients with Denys–Drash syndrome often demonstrate diffuse mesangial glomerulosclerosis, and Beckwith–Wiedemann syndrome is associated with nephrolithiasis, medullary dysplasia and other renal abnormalities38.The renal parenchyma should also be examined for the presence of nephrogenic rests and for indications of an underlying predisposing syndrome39. Histopathological examination in NSS includes evaluation of the complete circumference of the lesion. Small lesions are embedded completely. The minimal distance of the Wilms tumour to the resection margin will be measured. A safe rim has been defined as a margin of at least 1 mm, as is generally accepted in tumour pathology. The finding of nephrogenic rests at the resection margin is not regarded as a positive resection margin and does not upstage the Wilms tumour.Whenever NSS is pursued, assessing the resection margins very carefully is extremely important. Often, tumour nodules are resected with a small rim of renal parenchyma, especially in patients who have multiple nodules within one kidney or bilateral tumours. Procedures are encoded as NSS (A), which is partial nephrectomy , and NSS (B), which is enucleation Histopathological assessment clearly states one of the following findings — safe rim of renal parenchyma on resection margin (except nephrogenic rests), intact (pseudo)capsule along the resection margin, or tumour breach and/or rupture.25. In the SIOP–2001 trial, rapid central pathology review was introduced in some parts of the trial, and it proved to be feasible and extremely valuable, avoiding undertreatment and overtreatment caused by misdiagnosis and/or mis-staging by institutional pathologists25. The SIOP–2001 study also resulted in an increase in submissions for central pathology review from 76%  to 100% in the UK part of the SIOP–2001 trial25.As in the previous SIOP trials and studies, all renal tumours and their assessment by the institutional pathologists will be reviewed by their national and/or regional pathology panel Fig. . The resDiscrepancies in diagnosis and staging between institutional pathologists and central pathology review are likely to remain, but introduction of rapid central pathology review in the UMBRELLA protocol in more countries than it was available to in SIOP–2001 will enable clinicians to modify treatment in a timely manner, if required. Rapid central pathology review should be undertaken before postoperative treatment has started . All participating centres will be required to send their samples to the dedicated review pathologist for central pathology review. As >50 countries will be participating in the UMBRELLA study, central pathology review will be provided by national and/or regional pathology panels  that were identified in the previous SIOP–2001 trial and to demonstrate feasibility of testing in a clinically relevant time frame40.Two of the primary aims of the UMBRELLA protocol are biobanking and biomarker analysis Box ; Fig. 2.Achieving comprehensive collection of biomaterials in a uniform manner across the broad panel of participating countries will be challenging, but success in this goal will be key for future large-scale molecular profiling and implementation of biomarker testing. To achieve this aim, a standard for biobanking and for the submission of samples to the reference biological laboratories has been developed. Virtually linked biobanks will be created at a national level. These biobanks will be instrumental to reach the aims of the study, including evaluation of novel genomic driver candidates, informative microRNA (miRNA) patterns, epigenome analyses and panel sequencing that will be performed in national laboratories. Individual approaches will also cover in vitro and in vivo models and other non-Wilms renal tumours.Sample collection requirements for the UMBRELLA protocol are divided into an essential minimal set and optional extensions to answer specific research questions. For baseline diagnostics, EDTA blood samples and tumour tissue together with healthy kidney tissue (if available) will be collected from primary tumours and relapses and stored at –80 °C. Wherever possible, additional materials including blood samples for miRNA analysis and liquid biopsy testing of circulating tumour DNA, as well as urine for proteomic analyses, should be collected at multiple time points. This collection will facilitate parallel and future translational research, which are detailed in the aims of the UMBRELLA protocol.Blood samples will be asked for at diagnosis, after 2 weeks of chemotherapy and before surgery. In addition, collection of samples at follow-up time points of 1 week after surgery and at the end of treatment will be requested. Separation of plasma and the cellular fraction by centrifugation within <2 hours after venepuncture is preferred to enable liquid biopsy analysis of circulating tumour DNA, as this process might provide an attractive option for upfront diagnostics or follow-up monitoring. For miRNA studies, blood collected in PAXgene tubes, serum samples and urine samples are needed.41. Samples of ~1 cm3 each need to be selected from three or more spatially distinct sites within the tumour, as well as from macroscopically different lesions (if present) to capture potential heterogeneity. To make sure that representative and viable tissue has been selected in all samples, frozen sections or touch imprints can be prepared for evaluation of cellular content, viability and proportion of tumour cells. Besides tumour tissue, collecting matched normal tissue for comparative analyses from all patients is advised.To guarantee correct histological staging, tumour sampling is performed by the institutional pathologist. Given the phenotypic and genetic heterogeneity of Wilms tumours, extensive sampling is neededSnap freezing of tissue in liquid nitrogen must be done as soon as possible after excision to ensure minimal sample degradation. Duration from resection until freezing of the samples will be recorded for quality control. Importantly, all surgically removed metastases or relapses must be collected using the same protocol to facilitate identification of drivers of therapy resistance and metastatic potential. Biobanking of frozen materials will be complemented by surplus formalin-fixed, paraffin-embedded (FFPE) blocks that are also a valuable resource for research, including the molecular analysis of microdissected nephrogenic rests, tumour subcompartments and anaplastic foci.Clinical data and biological samples will be collected on a national basis in the paediatric oncology centres where the children are treated. To achieve inclusion of all registered patients, each country will set up a network for the provision of biological samples. Such a network for submission and central storage of frozen tumour samples from ~80% of children is already established in the UK, Germany, the Netherlands, France and Italy. Sample collection in this virtual biobank, including medical records, informed consent for biobanking by parents and (if applicable) children, will be documented in the web-based SIOP UMBRELLA database.The current proposal aims to further increase inclusion of multiple samples, especially from heterogeneous Wilms tumours, and to enable additional countries to provide the required samples and data. Depending on each national coordinating centre, frozen blood and urine samples can be either directly sent or stored on site until tissue samples are available for batched collection and shipping to the national biobank. Participating countries that lack appropriate infrastructure are encouraged to collect samples and forward batches to larger, host biobanks that can provide the necessary storage capacity and expertise in subsequent laboratory work-up.14. The results from our previous SIOP studies are equally encouraging but not yet definitive enough to mandate a change in clinical approaches14. With the even larger community of participating countries in the UMBRELLA protocol than in the SIOP–2001 study, we expect to collect sufficient materials for studies to provide a definitive answer on whether or not we need to include certain biomarkers in the SIOP protocol as well. The two primary biomarkers under study — blastemal volume and 1q gain — must be considered in the context of any co-dependencies with existing risk factors (tumour stage and histology) and the treatment given to prove their additional value for clinical decision making. On the basis of currently available data and agreed statistical parameters (power of 80% and a significance threshold of α = 0.05), up to 380 (for blastema volume) or 850 (for 1q gain) samples will be needed to test an association with outcome. This analysis entails a prospective duration of 5 years to collect the samples. In the case of 1q gain, testing will be performed on frozen and FFPE material to increase numbers, as several of the participating countries will not be able to guarantee successful provision of high-quality frozen samples.Children’s Oncology Group (COG) protocols in the USA already include molecular markers in clinical decision making12), but the number of targets is limited. Single nucleotide polymorphism arrays or focused next-generation sequencing (NGS) panels might provide improved resolution to identify critical subregions, including copy number neutral allele loss.No final decision has yet been made on the best approach to test for 1q gain and other copy number alterations. Multiplex ligation-dependent probe amplification analysis proved to be cost-effective and reliable in SIOP–2001 (ref.42. MYCN gain was found to be associated with adverse outcomes in a retrospective SIOP–RTSG study published in 2015 (ref.40). Loss of p53 function might be associated with poor outcome in anaplastic tumours, which was suggested to be associated with the volume of anaplastic regions and their incomplete removal7.Several additional genomic copy number biomarkers beyond 1q gain have potential prognostic relevance in Wilms tumour . Evaluating whether aberrations in any of these genes, alone or in combination, have a considerable effect on clinical course and survival will be interesting. With the exception of TP53, mutations in individual known Wilms tumour genes have not generally been associated with adverse outcome. However, combined SIX1 or SIX2 mutations and microRNA processing pathway mutations did confer a worse outcome in at least one of two studies9. Thus, a planned targeted NGS panel for Wilms tumour will facilitate these biomarker studies.Comprehensive NGS analysis of two Wilms tumour cohorts has yielded a large number of potential driver genes mutated in Wilms tumour, including The secondary molecular aims include a much broader scope of studies, covering exploratory analyses of potential novel biomarkers, including circulating nucleic acids detectable in blood and urine, for diagnosis and prognosis. Liquid biopsies, especially, offer the potential to provide a global view on the frequently heterogeneous tumours, reducing the risk of overlooking relevant tumour subclones.On the national level, multiple efforts are underway to explore the role of miRNAs in blood and tumour tissue as biomarkers. A range of initiatives to molecularly characterize all subtypes of Wilms tumour and non-Wilms tumour and their associated nephrogenic rests using whole-genome, epigenomic and proteomic approaches have been planned. This characterization extends to in vitro and in vivo cell models, in which tumours are used to establish cell cultures, organoid cultures, or xenograft models that should support functional validation of newly discovered molecular aberrations and biomarkers and screening for novel therapeutic options.Last but not least, the comprehensive collection of paediatric renal tumours will also aid in the assembly of sufficiently large cohorts to analyse rare entities such as mesoblastic nephroma, cystic partially differentiated nephroblastoma, clear cell sarcoma of the kidney, rhabdoid tumour of the kidney, or renal cell carcinoma, for which the molecular basis is often not known. This analysis might also uncover genetic alterations that can be assayed by liquid biopsies, generating future options for upfront noninvasive discrimination of these tumours and paving the way for improved therapeutic approaches that might lead to either omission of chemotherapy or a change to more appropriate drug regimens.Successful implementation of the UMBRELLA protocol will have the potential to shape future therapeutic approaches and to improve outcomes through several measures. The UMBRELLA protocol provides guidelines for clinical practice and will hopefully stimulate further international collaboration with harmonization of treatment protocols and research. The UMBRELLA study will provide increased patient cohorts, and the rapid central pathology review, the standardization of specimen handling and the improved collection of biological samples by pathologists will provide large numbers of specimens with increased homogeneity compared with other studies collected prospectively. These improved procedures will be of utmost importance to validate biomarkers such as 1q gain, to find driver gene mutations, to evaluate the prognostic significance of blastemal volume, and to unravel tumour heterogeneity with the ultimate goal to improve treatment stratification and to delineate novel therapeutic targets.Supplementary Information