Bone metastases from chondroblastoma: a rare pattern of metastatic disease in an adult.

Chondroblastoma is a rare benign tumor, typically presenting in the first two decades. Systemic metastases in chondroblastoma are extremely rare and it is the rarity of these metastases which lead the World Health Organisation to re-classify this lesion from "intermediate" to "benign" in its updated classification of bone tumors in 2020. We present an unusual case of a 55 year-old male patient who presented with multiple FDG-avid bone lesions on a background of conventional chondroblastoma of the rib excised at another institution 11-years previously. Two of these lesions were also histologically-proven as conventional chondroblastoma at biopsy. This case highlights that, although rare, metastases can be seen in patients with chondroblastoma. To our knowledge, this is the only case with an unusual pattern of metastases limited to bone.

MYH13, a superfast myosin expressed in extraocular, laryngeal and syringeal muscles.

MYH13 is a unique type of sarcomeric myosin heavy chain (MYH) first detected in mammalian extraocular (EO) muscles and later also in vocal muscles, including laryngeal muscles of some mammals and syringeal muscles of songbirds. All these muscles are specialized in generating very fast contractions while producing relatively low force, a design appropriate for muscles acting against a much lower load than most skeletal muscles inserting into the skeleton. The definition of the physiological properties of muscle fibres containing MYH13 has been complicated by the mixed fibre type composition of EO muscles and the coexistence of different MYH types within the same fibre. A major advance in this area came from studies on isolated recombinant myosin motors and the demonstration that the affinity of actin-bound human MYH13 for ADP is much weaker than those of fast-type MYH1 (type 2X) and MYH2 (type 2A). This property is consistent with a very fast detachment of myosin from actin, a major determinant of shortening velocity. The MYH13 gene arose early during vertebrate evolution but was characterized only in mammals and birds and appears to have been lost in some teleost fish. The MYH13 gene is located at the 3' end of the mammalian fast/developmental gene cluster and in a similar position to the orthologous cluster in syntenic regions of the songbird genome. MYH13 gene regulation is controlled by a super-enhancer in the mammalian locus and deletion of the neighbouring fast MYH1 and MYH4 genes leads to abnormal MYH13 expression in mouse leg muscles.

Impact of prostate radiotherapy on survival outcomes in clinically node-positive prostate cancer: A multicentre retrospective analysis.

PURPOSE: To evaluate clinical outcomes for cN1M0 prostate cancer treated with varied modalities. MATERIALS AND METHODS: Men with radiological stage cN1M0 prostate cancer on conventional imaging, treated from 2011-2019 with various modalities across four centres in the UK were included. Demographics, tumour grade and stage, and treatment details were collected. Biochemical and radiological progression-free survival (bPFS, rPFS) and overall survival (OS) were estimated using Kaplan Meier analyses. Potential factors impacting survival were tested with univariable log-rank test and multivariable Cox-proportional hazards model. RESULTS: Total 337 men with cN1M0 prostate cancer were included, 47% having Gleason grade group 5 disease. Treatment modalities included androgen deprivation therapy (ADT) in 98.9% men, either alone (19%) or in combinations including prostate radiotherapy (70%), pelvic nodal radiotherapy (38%), docetaxel (22%), or surgery (7%). At median follow up of 50 months, 5-year bPFS, rPFS, and OS were 62.7%, 71.0%, and 75.8% respectively. Prostate radiotherapy was associated with significantly higher bPFS (74.1% vs 34.2%), rPFS (80.7% vs 44.3%) and OS (86.7% vs 56.2%) at five years (log rank p < 0.001 each). On multivariable analysis including age, Gleason grade group, tumour stage, ADT duration, docetaxel, and nodal radiotherapy, benefit of prostate radiotherapy persisted for bPFS [HR 0.33 (95% CI 0.18-0.62)], rPFS [HR 0.25 (0.12-0.51)], and OS [HR 0.27 (0.13-0.58)] (p < 0.001 each). Impact of nodal radiotherapy or docetaxel was not established due to small subgroups. CONCLUSION: Addition of prostate radiotherapy to ADT in cN1M0 prostate cancer yielded improved disease control and overall survival independent of other tumour and treatment factors.

Slow myosin heavy chain 1 is required for slow myofibril and muscle fibre growth but not for myofibril initiation.

Slow myosin heavy chain 1 (Smyhc1) is the major sarcomeric myosin driving early contraction by slow skeletal muscle fibres in zebrafish. New mutant alleles lacking a functional smyhc1 gene move poorly, but recover motility as the later-formed fast muscle fibres of the segmental myotomes mature, and are adult viable. By motility analysis and inhibiting fast muscle contraction pharmacologically, we show that a slow muscle motility defect persists in mutants until about 1 month of age. Breeding onto a genetic background marking slow muscle fibres with EGFP revealed that mutant slow fibres undergo terminal differentiation, migration and fibre formation indistinguishable from wild type but fail to generate large myofibrils and maintain cellular orientation and attachments. In mutants, initial myofibrillar structures with 1.67 â€‹µm periodic actin bands fail to mature into the 1.96 â€‹µm sarcomeres observed in wild type, despite the presence of alternative myosin heavy chain molecules. The poorly-contractile mutant slow muscle cells generate numerous cytoplasmic organelles, but fail to grow and bundle myofibrils or to increase in cytoplasmic volume despite passive movements imposed by fast muscle. The data show that both slow myofibril maturation and cellular volume increase depend on the function of a specific myosin isoform and suggest that appropriate force production regulates muscle fibre growth.

Isolation, Culture, and Analysis of Zebrafish Myofibers and Associated Muscle Stem Cells to Explore Adult Skeletal Myogenesis.

Adult skeletal musculature experiences continuous physical stress, and hence requires maintenance and repair to ensure its continued efficient functioning. The population of resident muscle stem cells (MuSCs), termed satellite cells, resides beneath the basal lamina of adult myofibers, contributing to both muscle hypertrophy and regeneration. Upon exposure to activating stimuli, MuSCs proliferate to generate new myoblasts that differentiate and fuse to regenerate or grow myofibers. Moreover, many teleost fish undergo continuous growth throughout life, requiring continual nuclear recruitment from MuSCs to initiate and grow new fibers, a process that contrasts with the determinate growth observed in most amniotes. In this chapter, we describe a method for the isolation, culture, and immunolabeling of adult zebrafish myofibers that permits examination of both myofiber characteristics ex vivo and the MuSC myogenic program in vitro. Morphometric analysis of isolated myofibers is suitable to assess differences among slow and fast muscles or to investigate cellular features such as sarcomeres and neuromuscular junctions. Immunostaining for Pax7, a canonical stemness marker, identifies MuSCs on isolated myofibers for study. Furthermore, the plating of viable myofibers allows MuSC activation and expansion and downstream analysis of their proliferative and differentiative dynamics, thus providing a suitable, parallel alternative to amniote models for the study of vertebrate myogenesis.

Skeletal Muscle Regeneration in Zebrafish.

Muscle regeneration models have revealed mechanisms of inflammation, wound clearance, and stem cell-directed repair of damage, thereby informing therapy. Whereas studies of muscle repair are most advanced in rodents, the zebrafish is emerging as an additional model organism with genetic and optical advantages. Various muscle wounding protocols (both chemical and physical) have been published. Here we describe simple, cheap, precise, adaptable, and effective wounding protocols and analysis methods for two stages of a larval zebrafish skeletal muscle regeneration model. We show examples of how muscle damage, ingression of muscle stem cells, immune cells, and regeneration of fibers can be monitored over an extended timecourse in individual larvae. Such analyses have the potential to greatly enhance understanding, by reducing the need to average regeneration responses across individuals subjected to an unavoidably variable wound stimulus.

Where Are We Now with Liver Transplantation in Neuroendocrine Neoplasms? The Place of Liver Transplantation for Grades 1 and 2 Well-Differentiated Unresectable Liver Metastatic Neuroendocrine Tumours.

PURPOSE OF REVIEW: This review outlines the role of liver transplantation in selected patients with unresectable neuroendocrine tumour liver metastases. It discusses the international consensus on eligibility criteria and outlines the efforts taking place in the UK and Ireland to develop effective national liver transplant programmes for neuroendocrine tumour patients. RECENT FINDINGS: In the early history of liver transplantation, indications included cancer metastases to the liver as well as primaries of liver origin. Often, liver transplantation was a salvage procedure. The early results were disappointing, including in patients with neuroendocrine tumours. These data discouraged the widespread adoption of liver transplantation for neuroendocrine tumour liver metastases (NET LM). A few centres persisted in performing liver transplantation for patients with NET LM and in determining parameters predictive of good outcomes. Their work has provided evidence for benefit of liver transplantation in a selected group of patients with NET LM. Liver transplantation for NET LM is now accepted as a valid indication by many professional bodies, including the European Neuroendocrine Tumour Society (ENETS) and the United Network for Organ Sharing (UNOS). It is nevertheless rarely utilised. The UK and the Republic of Ireland are commencing a pilot programme of liver transplantation in selected patients. This programme will help develop the expertise and infrastructure to make liver transplantation for NET LM a routine procedure.

A cohort profile of the Graham Roberts study cohort.

Purpose: The Graham Roberts Study was initiated in 2018 and is the first Trials Within Cohorts (TwiCs) study for bladder cancer. Its purpose is to provide an infrastructure for answering a breadth of research questions, including clinical, mechanistic, and supportive care centred questions for bladder cancer patients. Participants: All consented patients are those aged 18 or older, able to provide signed informedconsent and have a diagnosis of new or recurrent bladder cancer. All patients are required to have completed a series of baseline questionnaires. The questionnaires are then sent out every 12 months and include information on demographics and medical history as well as questionnaires to collect information on quality of life, fatigue, depression, overall health, physical activity, and dietary habits. Clinical information such as tumor stage, grade and treatment has also been extracted for each patient. Findings to date: To date, a total of 125 bladder cancer patients have been consented onto the study with 106 filling in the baseline questionnaire. The cohort is made up of 75% newly diagnosed bladder cancer patients and 66% non-muscle invasive bladder cancer cases. At present, there is 1-year follow-up information for 70 patients, 2-year follow-up for 57 patients, 3-year follow-up for 47 patients and 4-year follow-up for 19 patients. Future plans: We plan to continue recruiting further patients into the cohort study. Using the data collected within the study, we hope to carry out independent research studies with a focus on quality of life. We are also committed to utilizing the Roberts Study Cohort to set up and commence an intervention. The future studies and trials carried out using the Roberts Cohort have the potential to identify and develop interventions that could improve the prevention, diagnosis, and treatment of bladder cancer.

Blood loss in total en bloc spondylectomy for primary spinal bone tumours: a comparison of estimated blood loss versus actual blood loss in a single centre over 10 years.

Background: Total en bloc spondylectomy (TES) is a widely accepted surgical technique for primary spinal bone tumours but is frequently accompanied by substantial peri-operative blood loss. Prior studies have reported estimated blood loss (EBL) can reach up to 3,200 mL. The aim of this study is to estimate the blood loss during TES procedures performed in the last ten years at our tertiary referral centre and compare EBL with actual blood loss (ABL). Methods: We performed a retrospective review of all cases managed surgically with TES referred to our centre between 2005 and 2015. We recorded the oncological characteristics of each tumour and surgical management in terms of resection margins, operative duration and instrumentation. Data relating to peri-operative blood loss was also recorded including an estimation of total blood loss, the use of cell salvage where applicable and transfusion rates. Results: A total of 21 patients were found to meet our inclusion criteria. There were 11 men and 10 women, with a median age of 40 years. The mean total ABL was 3,310 mL. Total operation time ranged from 6.53 to 19.7 h. Compared to ABL, in 59% of cases EBL had been underestimated by an average of 78% by volume. The EBL of the remaining 41% cases had been overestimated by 43%. This was not statistically significant (P=0.373). Cell salvage was used in 62% patients with a mean blood loss of 2,845 mL (884-4,939 mL) and transfusion of 3.8 units (0-12 units) versus 4,069 mL (297-8,335 mL) and 9.3 units (0-18 units) in those not managed with cell salvage. There was no significant difference in ABL between the cell salvage and non-cell salvage groups. Conclusions: We report one of the largest case series in TES for primary bone tumours. EBL is not a reliable predictor for ABL. A large blood loss should be anticipated and use of cell salvage is recommended.

Clonal behaviour of myogenic precursor cells throughout the vertebrate lifespan.

To address questions of stem cell diversity during skeletal myogenesis, a Brainbow-like genetic cell lineage tracing method, dubbed Musclebow2, was derived by enhancer trapping in zebrafish. It is shown that, after initial formation of the primary myotome, at least 15 muscle precursor cells (mpcs) seed each somite, where they proliferate but contribute little to muscle growth prior to hatching. Thereafter, dermomyotome-derived mpc clones rapidly expand while some progeny undergo terminal differentiation, leading to stochastic clonal drift within the mpc pool. No evidence of cell-lineage-based clonal fate diversity was obtained. Neither fibre nor mpc death was observed in uninjured animals. Individual marked muscle fibres persist across much of the lifespan indicating low rates of nuclear turnover. In adulthood, early-marked mpc clones label stable blocks of tissue comprising a significant fraction of either epaxial or hypaxial somite. Fusion of cells from separate early-marked clones occurs in regions of clone overlap. Wounds are regenerated from several local mpcs; no evidence for specialised stem mpcs was obtained. In conclusion, our data indicate that most mpcs in muscle tissue contribute to local growth and repair and suggest that cellular turnover is low in the absence of trauma.

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Surgical Correction of Carcinoid Heart Disease Improves Liver Function and 5-Hydroxyindoleacetic Acid Levels.

Introduction: Carcinoid heart disease (CHD) is a consequence of neuroendocrine tumors releasing 5-hydroxytryptamine (5-HT) into the systemic circulation, affecting right heart valves, causing fibrosis, and eventually right heart failure. The aim of this study was to determine the effect of valve-replacement on kidney function, liver function, and 5-hydroxyindoleacetic acid (5-HIAA) levels. Methods: A Retrospective study of 17 patients with CHD who had undergone heart-valve replacement surgery between 2010 and 2019, from the Queen Elizabeth Hospital Birmingham. 5-HIAA levels, liver, and kidney function were measured in addition to hepatic inferior vena cava (IVC) diameter and its relationship to carcinoid symptoms. Results: Eleven patients were male and six were female. At time of surgery, average age was 66.6 ± 8.1 years and average BMI was 25.8 ± 5.5 Kg/cm2. Three out of 17 patients had one valve replaced, 13/17 had two replaced (tricuspid and pulmonary), and 1/17 had three replaced (tricuspid, pulmonary and aortic). There was a 31% average decline in 5-HIAA [799.8 (343.6-1078.0) to 555.3 (275.8-817.9), p = 0.011], a 35% decline in bilirubin [20 (16-29) to 13 (10-19), p = < 0.001], and a 15% reduction in the short and long axes of the IVC after valve-replacement surgery [20.0 (18.0-25.0) and 36.5 (29.0-39.8) to 17.0 (14.5-19.3) and 31.0 (26.5-34.3) respectively, p = < 0.001 and 0.002 respectively]. Conclusion: Valve replacement surgery improves 5-HIAA levels alongside improved liver function and hepatic IVC diameter. These findings are consistent with resolution of congestive hepatopathy, and therefore enhanced clearance of 5-HIAA. This suggests that valve-replacement surgery can indirectly have beneficial outcomes on hepatic function and is also associated with a drop in the circulating levels of tumor derived serotonin.

Cellular and molecular pathways controlling muscle size in response to exercise.

From the discovery of ATP and motor proteins to synaptic neurotransmitters and growth factor control of cell differentiation, skeletal muscle has provided an extreme model system in which to understand aspects of tissue function. Muscle is one of the few tissues that can undergo both increase and decrease in size during everyday life. Muscle size depends on its contractile activity, but the precise cellular and molecular pathway(s) by which the activity stimulus influences muscle size and strength remain unclear. Four correlates of muscle contraction could, in theory, regulate muscle growth: nerve-derived signals, cytoplasmic calcium dynamics, the rate of ATP consumption and physical force. Here, we summarise the evidence for and against each stimulus and what is known or remains unclear concerning their molecular signal transduction pathways and cellular effects. Skeletal muscle can grow in three ways, by generation of new syncytial fibres, addition of nuclei from muscle stem cells to existing fibres or increase in cytoplasmic volume/nucleus. Evidence suggests the latter two processes contribute to exercise-induced growth. Fibre growth requires increase in sarcolemmal surface area and cytoplasmic volume at different rates. It has long been known that high-force exercise is a particularly effective growth stimulus, but how this stimulus is sensed and drives coordinated growth that is appropriately scaled across organelles remains a mystery.

Isolation of Myofibres and Culture of Muscle Stem Cells from Adult Zebrafish.

Skeletal muscles generate force throughout life and require maintenance and repair to ensure efficiency. The population of resident muscle stem cells (MuSCs), termed satellite cells, dwells beneath the basal lamina of adult myofibres and contributes to both muscle growth and regeneration. Upon exposure to activating signals, MuSCs proliferate to generate myoblasts that differentiate and fuse to grow or regenerate myofibres. This myogenic progression resembles aspects of muscle formation and development during embryogenesis. Therefore, the study of MuSCs and their associated myofibres permits the exploration of muscle stem cell biology, including the cellular and molecular mechanisms underlying muscle formation, maintenance and repair. As most aspects of MuSC biology have been described in rodents, their relevance to other species, including humans, is unclear and would benefit from comparison to an alternative vertebrate system. Here, we describe a procedure for the isolation and immunolabelling or culture of adult zebrafish myofibres that allows examination of both myofibre characteristics and MuSC biology ex vivo. Isolated myofibres can be analysed for morphometric characteristics such as the myofibre volume and myonuclear domain to assess the dynamics of muscle growth. Immunolabelling for canonical stemness markers or reporter transgenes identifies MuSCs on isolated myofibres for cellular/molecular studies. Furthermore, viable myofibres can be plated, allowing MuSC myogenesis and analysis of proliferative and differentiative dynamics in primary progenitor cells. In conclusion, we provide a comparative system to amniote models for the study of vertebrate myogenesis, which will reveal fundamental genetic and cellular mechanisms of MuSC biology and inform aquaculture. Graphic abstract: Schematic of Myofibre Isolation and Culture of Muscle Stem Cells from Adult Zebrafish.

mRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation.

BACKGROUND: During skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation. METHODS: Using isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells. RESULTS: Quiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta. CONCLUSIONS: Taken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.

Knockout of zebrafish desmin genes does not cause skeletal muscle degeneration but alters calcium flux.

Desmin is a muscle-specific intermediate filament protein that has fundamental role in muscle structure and force transmission. Whereas human desmin protein is encoded by a single gene, two desmin paralogs (desma and desmb) exist in zebrafish. Desma and desmb show differential spatiotemporal expression during zebrafish embryonic and larval development, being similarly expressed in skeletal muscle until hatching, after which expression of desmb shifts to gut smooth muscle. We generated knockout (KO) mutant lines carrying loss-of-function mutations for each gene by using CRISPR/Cas9. Mutants are viable and fertile, and lack obvious skeletal muscle, heart or intestinal defects. In contrast to morphants, knockout of each gene did not cause any overt muscular phenotype, but did alter calcium flux in myofibres. These results point to a possible compensation mechanism in these mutant lines generated by targeting nonsense mutations to the first coding exon.